The Pennsylvania State University

The Graduate School

College of Medicine

A JOURNEY TO THE CENTER OF THE CELL:

INSIGHTS INTO SUBNUCLEAR TRAFFICKING

OF THE ROUS SARCOMA VIRUS GAG

POLYPROTEIN

A Dissertation in

Genetics

by

Timothy Lewis Lochmann

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2011

The dissertation of Timothy Lewis Lochmann was reviewed and approved* by the following:

Leslie J. Parent Associate Professor of Medicine, and Microbiology and Immunology Dissertation Adviser Chair of Committee

David J. Spector Professor of Microbiology Member of the Genetics Executive Committee

Jianming Hu Professor of Microbiology and Immunology

Sergei A. Grigoryev Associate Professor of Biochemistry and Molecular Biology

Christopher Herzog Assistant Professor of Pharmacology

*Signatures are on file in the Graduate School.

ii Abstract

The assembly of retrovirus particles is directed by the retroviral Gag polyprotein.

The Rous sarcoma virus (RSV) Gag polyprotein is synthesized in the cytoplasm. During the early steps of assembly, Gag undergoes transient nuclear trafficking mediated by nuclear localization signals (NLSs) in the matrix (MA) and nucleocapsid (NC) domains, and a nuclear export signal (NES) present within the p10 domain. Gag nuclear trafficking has been linked to efficient packaging of viral genomic RNA. However, whether Gag undergoes intranuclear trafficking and what host factors Gag may interact with in the nucleus remains poorly understood. The ability to mutate the NES and inhibit nuclear export makes RSV a useful model to study the roles of Gag within the nucleus and how Gag interacts with nuclear factors during retrovirus infection.

Experiments aimed at identifying Gag-Gag interactions within the nucleus revealed that Gag proteins accumulated within punctate nuclear foci. These subnuclear foci were not virus-like particles forming within the nucleus. The formation of Gag-containing foci was dependent upon the presence of NC, which is an RNA binding domain. Further studies revealed that Gag nuclear puncta were anchored within the nucleoplasm at discrete locations, but individual Gag molecules move between the subnuclear structures and the nucleoplasm. These results suggest that the Gag proteins are retained at a particular subnuclear structure, perhaps being anchored there by an interaction with a cellular protein or RNA molecule.

In addition to nucleoplasmic foci, Gag also accumulated within nucleoli in an NC- dependent manner. Similar to the nuclear puncta, Gag proteins moved in and out of nucleoli. Using site-directed mutagenesis, the amino acids involved in nucleolar localization of NC were identified. Furthermore, mutations that eliminated nucleolar localization of NC also prevented Gag from accumulating within nucleoli. These results indicate that NC plays a key role in the nucleolar localization and subnuclear trafficking of the Gag protein.

iii However, whether these nuclear accumulations of Gag serve a purpose during viral replication is unknown.

To determine whether nucleolar localization was involved in retroviral replication, proviral vectors were used to examine how mutations to the basic residues within NC affected virus particle release, gRNA packaging, and infectivity. In general, mutations that reduced NC nucleolar localization did not affect particle release, but gRNA packaging and infectivity were impaired in some viral mutants. The presence of a minimal number of positively charged residues within NC is not linked to gRNA packaging, although the location of the basic residues was important. Elimination of NC nucleolar localization reduced the ability of the virus to spread in infectivity assays. Together, these results suggest that NC nucleolar localization is important for infectivity, but not gRNA packaging or virus particle release.

To further examine the role of nucleoli in retroviral infection, a gain-of-function approach was used. A heterologous nucleolar localization signal (NoLS), from either the

HIV-1 Rev protein or the HSV ICP27 protein, was inserted into an NC mutant deficient in nucleolar localization. In addition to being NoLSs, both the Rev and ICP27 sequences were also RNA-binding domains (RBDs). Both sequences were able to restore NC nucleolar localization, and the presence of these heterologous sequences caused nucleolar accumulation of Gag without a nuclear export mutation. Surprisingly, although the nucleolar localization signal from ICP27 did not hinder viral assembly, the NoLS from Rev greatly decreased viral budding. Both NoLSs/RBD restored incorporation of gRNA into virus particles. However, a virus containing either NoLS was noninfectious. These data demonstrate that insertion of a heterologous NoLS/RBD binding domain is sufficient to restore incorporation of gRNA to a packaging deficient virus.

iv

Table of Contents

List of Figures ...... vii

List of Tables ...... viii

Abbreviation list ...... ix

Acknowledgements ...... xi

Chapter 1: Literature review ...... 1 1.1 Introduction to dissertation ...... 2 1.2 Classification of retroviruses and genome organization ...... 4 1.3 Organization of retroviral particles ...... 8 1.4 Early events of the retroviral life cycle ...... 11 1.4.1 Attachment, fusion, and entry ...... 11 1.4.2 Uncoating and reverse transcription ...... 14 1.4.3 Nuclear entry of the PIC ...... 15 1.4.4 Integration of the viral DNA ...... 16 1.5 Late events of the retroviral life cycle ...... 16 1.5.1 Transcription and splicing of viral RNA ...... 16 1.5.2 Nuclear export of viral RNA ...... 19 1.5.3 Translation of viral proteins ...... 20 1.5.4 Assembly of immature virus ...... 20 1.5.5 RNA packaging ...... 22 1.5.5.1 Packaging of viral RNA ...... 22 1.5.5.2 Packaging of cellular RNA ...... 23 1.5.6 Budding ...... 25 1.5.7 Maturation ...... 26 1.6 Nuclear trafficking of the RSV Gag polyprotein ...... 26 1.7 The many roles of the retroviral nucleocapsid (NC) ...... 28 1.7.1 Structure and function of NC ...... 28 1.7.2 Role of NC in viral entry ...... 33 1.7.2.1 NC and reverse transcription ...... 33 1.7.2.2 Role of NC in PIC nuclear entry ...... 34 1.7.3 Role of NC in viral assembly ...... 35 1.7.4 Role of NC in gRNA packaging ...... 37 1.7.4.1 NC zinc fingers ...... 37 1.7.4.2 NC basic residues ...... 38 1.8 The nucleolus ...... 39 1.8.1 Signals and mechanisms of nucleolar trafficking ...... 42 1.8.2 Viruses and nucleoli ...... 47 1.9 Live cell imaging techniques ...... 50 1.9.1 Fluorescence recovery after photobleaching (FRAP) ...... 51 1.9.2 Fluorescence resonance energy transfer (FRET) ...... 54 1.10 Conclusion ...... 59

Chapter 2: Characterization of Subnuclear Foci Formed by Gag proteins Restricted to the Nucleus ...... 61 2.1 Abstract ...... 62 2.2 Introduction ...... 62 2.3 Materials and Methods ...... 64 v 2.4 Results ...... 68 2.5 Discussion ...... 86

Chapter 3: Gag Nucleolar Localization and Mapping of the Nucleolar Localization Signal within the NC Domain ...... 90 3.1 Abstract ...... 91 3.2 Introduction ...... 91 3.3 Materials and Methods ...... 94 3.4 Results ...... 98 3.5 Discussion ...... 121

Chapter 4: Effect of NC Nucleolar Localization Signal Mutations on Viral Genomic RNA Packaging and Infectivity ...... 126 4.1 Abstract ...... 127 4.2 Introduction ...... 127 4.3 Materials and Methods ...... 129 4.4 Results ...... 134 4.5 Discussion ...... 150

Chapter 5: Insertion of Heterologous Nucleolar Localization Signals Restores Viral gRNA Packaging to a Nucleolar Deficient Gag Protein ...... 159 5.1 Abstract ...... 160 5.2 Introduction ...... 160 5.3 Materials and Methods ...... 162 5.4 Results ...... 167 5.5 Discussion ...... 178

Chapter 6: Dissertation Discussion ...... 184 6.1 Introduction ...... 185 6.2 Discussion of Gag subnuclear localization ...... 185 6.2.1 Gag nuclear foci ...... 185 6.2.2 Gag nucleolar localization ...... 189 6.2.3 Interplay between Gag nuclear foci and nucleolar Gag ...... 191 6.2.4 Nucleolar restoration mutants ...... 194 6.3 Biological implications of Gag subnuclear localization ...... 196 6.3.1 Nuclear foci in viral replication ...... 196 6.3.2 Nucleoli in viral replication ...... 197 6.4 Summary of conclusion ...... 200

Appendix: Effect of ψ-Containing RNA on RSV Gag Cytoplasmic Trafficking ...... 203 A.1 Abstract ...... 204 A.2 Introduction ...... 204 A.3 Materials and Methods ...... 206 A.4 Results ...... 208 A.5 Discussion ...... 217

Reference List ...... 219

vi List of Figures

Chapter 1 Fig. 1.1. Organization of the RSV genome and Gag polyprotein ...... 6 Fig. 1.2. Schematic diagrams of an immature and a mature RSV particle ...... 9 Fig. 1.3. Early events in retroviral infection ...... 12 Fig. 1.4. Late events in RSV infection ...... 17 Fig. 1.5. Comparison of retroviral NC proteins ...... 29 Fig. 1.6. Structure of the nucleolus ...... 40 Fig. 1.7. Fluorescence recovery after photobleaching (FRAP) ...... 52 Fig. 1.8. Fluorescence resonance energy transfer (FRET) ...... 56

Chapter 2 Fig. 2.1. Intranuclear distribution patterns of Gag proteins ...... 69 Fig. 2.2. Intranuclear Gag-Gag interactions assessed by acceptor photobleaching FRET analysis ...... 72 Fig. 2.3. Volumetric analysis of Gag nuclear foci ...... 75 Fig. 2.4. Transmission electron microscopy of L219A.Gag-YFP ...... 78 Fig. 2.5. Time-lapse tracking of Gag nuclear foci ...... 81 Fig. 2.6. Mobility of Gag nuclear foci assessed by FRAP ...... 84

Chapter 3 Fig. 3.1. Nucleolar localization of retroviral NC and Gag proteins ...... 100 Fig. 3.2. Acceptor photobleaching FRET of Gag and NC in nucleoli ...... 103 Fig. 3.3. Nucleolar Gag mobility assessed by FRAP analysis ...... 106 Fig. 3.4. Localization of RSV NC mutants ...... 110 Fig. 3.5. Localization of nuclear export deficient Gag and Gag NC mutants ...... 113 Fig. 3.6. Localization of HIV NC mutants ...... 116 Fig. 3.7. Interaction of Rev-independent HIV Gag with NC and Rev in nucleoli ...... 119

Chapter 4 Fig. 4.1. Schematic diagrams of YFP-RSV.NC and basic residue mutants ...... 135 Fig. 4.2. Localization profile of NC and basic residue mutants ...... 138 Fig. 4.3. Viral budding of NC basic amino acid mutants ...... 141 Fig. 4.4. Effect of NC basic residue mutations on gRNA packaging ...... 144 Fig. 4.5. Infectivity of virus containing NC basic amino acid mutations ...... 148

Chapter 5 Fig. 5.1. Localization of NC restoration mutants using confocal microscopy ...... 168 Fig. 5.2. Localization of RS.V8 Gag restoration mutants ...... 171 Fig. 5.3. Budding analysis of proviral nucleolar restoration mutants ...... 173 Fig. 5.4. Effect of NC nucleolar restoration mutations on gRNA packaging ...... 176 Fig. 5.5. Infectivity of virus containing nucleolar restoration mutations ...... 179

Chapter 6 Fig. 6.1. Two models of Gag subnuclear trafficking ...... 192

Appendix Fig. A.1. DNA representation of Gag and mCherry constructs ...... 209 Fig. A.2. Cytoplasmic Gag FRAP analysis ...... 212 Fig. A.3. Frequency of Gag nucleolar accumulation ...... 215

vii List of Tables

Chapter 1 Table 1.1. Retroviral NC protein properties ...... 31 Table 1.2. Nucleolar localization signals (NoLSs) ...... 44 Table 1.3. Viral proteins targeted to nucleoli and their known functions ...... 48

Chapter 3 Table 3.1 Primer list for NC basic amino acid mutations ...... 96

Chapter 4 Table 4.1. Primers for additional NC basic amino acid mutations ...... 131 Table 4.2. Summary of NC basic amino acid mutations and effects on virus replication .. 151

Chapter 5 Table 5.1. Primers for insertion of heterologous NoLS into RSV NC ...... 164

viii Abbreviation List

Fig. figure

vRNA viral RNA gRNA viral genomic RNA

RSV Rous sarcoma virus

HIV human immunodeficiency virus

HTLV human T-cell leukemia

MLV murine leukemia virus

MMTV mouse mammary tumor virus

HSV herpes simplex virus tRNA transfer RNA snoRNA small nucleolar RNA

RNA ribonucleic acid

DNA deoxyribonucleic acid

CA capsid

NC nucleocapsid

MA matrix

SP spacer

PR protease

RT reverse transcriptase

IN integrase

EM electron microscopy

RTC reverse transcription complex

SU surface

TM transmembrane

PIC pre-integration complex

NLS nuclear localization signal ix NES nuclear export signal

NoLS nucleolar localization signal

NoRS nucleolar retention signal

RRE Rev-responsive element

LTR long terminal repeat

UTR untranslated region

M membrane binding domain

I RNA interaction domain

MI multimerization interface

L late domain

SA splice acceptor

SD splice donor

VLP virus-like particle nt nucleotide

FRAP florescence recovery after photobleaching

FRET fluorescence resonance energy transfer

FLIP fluorescence loss in photobleaching

RPA ribonuclease protection assay

x Acknowledgements

Throughout the compilation of this dissertation, I have thought back to what has brought me here today. I’d like to thank all of my undergraduate teachers who inspired me to love science and the scientific method during my time at San Diego Mesa College and the

University of California, San Diego. Through them I found my passion for science and a desire to learn what no one else knows through research and experimentation.

I would like to thank the Penn State College of Medicine and the Genetics Program for seeing my potential and giving me the opportunity to expand my training and learn how to think as a scientist. I joined the laboratory of Dr. Leslie Parent knowing I would get a broad training in multiple scientific disciplines, but I had no idea how broad that training would be. I’ve learned microscopy imaging techniques that had never been performed at this institution. My experiments and experience have ranged from cell and molecular biology, to virology, to biochemistry. I’ve greatly matured as a scientist in these past five years in Leslie’s lab. Leslie taught me how to continually push myself to continue learning, to never get complacent in experiments, and how to find the next question to answer. I cannot imagine getting a more varied or better laboratory experience anywhere else.

My parents have always been supportive of me, no matter what I’ve chosen to do.

From when I wanted to become an M.D., to changing my mind and going for a Ph.D., they’ve always been there for me. So thank you Mom and Dad for everything you’ve done for me; my only hope of repaying you is trying to make you proud of what I do.

Thanks to all my friends who have come and gone during my graduate studies, I enjoyed our discussions of science and politics over drinks and dinners. Thanks to my lab mates, especially Scott and Eileen who trained me when I was learning how to do the bench work. Finally and most importantly, I want to thank Andy. She’s been the one who has had to tolerate me nearly 24/7 over the past few years. More importantly, she believed in me even when I stopped believing in myself. Thank you for all you do to make my life happy; you complete me and I love you for it.

xi

CHAPTER 1

Literature Review

1 1.1 Introduction to the dissertation

The Rous sarcoma virus (RSV), discovered by Peyton Rous, was the first retrovirus identified (354). That discovery was made nearly 100 years ago, yet much of our knowledge about how a retrovirus assembles new virus particles has come to light only within the past few decades. Even with this explosion of new research and insights into retroviral assembly, there are still more questions to answer. We know that the virus requires several trafficking events after integration of the proviral DNA. These events include transcription of the viral RNA (vRNA), export of unspliced vRNA for translation of

Gag (the main structural protein of the virus), selection of the viral genomic RNA (gRNA) for packaging into virions, and assembly of new virus particles at the plasma membrane. Many of the studies performed to identify and understand these events came from experiments with RSV, the primary virus used in this dissertation.

Although the assembly pathway of retroviruses first appeared simple, recent data has shed light on the complexity that exists in forming an infectious virion. Previously, it was thought that Gag was translated and assembled into virions entirely within the cytoplasm and at the plasma membrane. Within the past 20 years, the first observation that Gag contains transient nuclear trafficking properties was reported (368). Indeed, only in the past decade the RSV Gag protein was treated with the drug, Leptomycin B (LMB), and found to cause a redistribution of Gag from the cytoplasm to the nucleus, indicating a nuclear trafficking step for Gag during assembly (366). This observation raises a puzzling question: if the gRNA incorporated into virus particles can also function as the template for Gag translation, and assembly occurs within the cytoplasm and at the membrane; why, then, would Gag traffic through the nucleus?

The reasons for Gag nuclear trafficking were pursued through a genetic approach. A mutant of Gag, Myr1E.Gag, contained the membrane-binding domain of Src and bypassed nuclear trafficking step (363). Furthermore, the loss of nuclear trafficking prevented the virus particles from efficiently packaging gRNA. Only upon addition of an exogenous 2 nuclear localization signal (NLS) was the incorporation of gRNA restored to near-wildtype levels (151). The data collected from these experiments strongly suggested that Gag required nuclear trafficking to efficiently select and package gRNA.

Understanding how Gag traffics through the nucleus has revealed other insights into the functions and mechanisms of the protein. A CRM1-dependent nuclear export signal (NES) was identified through sequence comparisons and confirmed by genetic mapping (366).

Mutation of one or more of the essential hydrophobic residues within the NES caused Gag to accumulate within the nucleus, similar to treatment with LMB. It follows that if the Gag protein requires an NES, there may be signals to transport it into the nucleus. Indeed, two domains of Gag were found to contain nuclear localization properties: MA and NC (67).

Thus, multiple signals appeared to be involved in Gag trafficking, further increasing the complexity of RSV assembly.

Assembly and trafficking signals may compete for function as the NLS found in MA overlaps the membrane-binding domain, the NES in p10 coincides with the dimerization interface contained within the p10-CA domains, and the NLS in NC is at the same location as the RNA interaction domain. It appears that stepwise regulations are necessary for these signals to work in concert. A recent report identified several regulation mechanisms involving Gag and multiple cellular proteins (172). First, multiple import factors can bind to the NLSs within MA and NC. Upon introduction of RNA, the import factors are released, and the NES potentially undergoes a conformational change. This structural alteration allows the CRM1-RanGTP export complex to directly bind to the p10 domain, allowing a

Gag-RNA complex to exit the nucleus. Although these findings help elucidate the mode of nuclear import and export, they fail to address a key issue in nuclear trafficking: What is Gag doing in the nucleus? More specifically, does Gag undergo intranuclear regulation events?

The studies presented in this dissertation begin to address several questions: 1) Are there subnuclear trafficking events during retroviral assembly? 2) What signals direct Gag during intranuclear trafficking? 3) Does disruption of these signal affect viral relplication? 4) 3 Can insertion of heterologous trafficking sequences work in place of the endogenous signals found within Gag?

Early observations of NC within nucleoli of infected cells led me to focus on this domain of Gag as a target for subnuclear trafficking (350). By approaching these questions through the use of genetics, cell biology, and molecular biology, I was able to create mutants that interfere with the normal trafficking patterns of NC and Gag. Furthermore, these mutants allowed me to study the effects of disrupting the normal intranuclear trafficking pathways on assembly, gRNA packaging, and infectivity of the virus. These techniques have allowed for the discovery of several previously unknown trafficking events. Through these studies, I have continued to unravel the mystery of Gag nuclear trafficking.

1.2 Classification of retroviruses and genome organization

Retroviruses have been classified by varying categories: gene product homology, site of assembly, and genome organization. The Retroviridae family has been split into the

Spumaretrovirinae and Orthoretrovirinae subfamilies (60). Spumaretretrovirinae contain the single genus Spumavirus (e.g. Simian foamy virus). The Orthoretrovirinae family is divided into the genera Alpharetroviruses (e.g. RSV), Betaretroviruses (e.g. Mouse mammary tumor virus, MMTV), Gammaretroviruses (e.g. Murine leukemia virus, MLV), Deltaretroviruses (e.g.

Human T-lymphotropic virus 1, HTLV), Epsilonretroviruses (e.g. Walleye dermal sarcoma virus), and Lentiviruses (e.g. Human immunodeficiency virus, HIV).

The site of virus assembly has also been used to identify different groups of retroviruses (426). Type-A refers to endogenous retroviruses that bud into the endoplasmic reticulum, but fail to mature or be released from the cell. Type-B retroviruses, which contain spherical cores, and type-D retroviruses, which have a tube or bar-shaped core, form virus capsids within the cytoplasm before transport to the plasma membrane and subsequent release from the cell. Type-C retroviruses, including RSV and HIV, form virus particles directly at the plasma membrane concurrent with budding. Although there are differences 4 between the types of assembly pathways, they do not appear to be mutually exclusive. A single amino acid substitution within a type-B/D virus Gag protein causes a shift to type-C morphogenesis (348).

Additionally, retroviruses are often classified as “simple” or “complex.” Simple retroviruses encode only primary viral proteins required for replication: Gag, the main structural protein; Pro, the protease required for virus maturation; Pol, containing both the reverse transcriptase and integrase; and Env, the protein responsible for fusion and entry into cells (Fig. 1.1A). RSV, a simple retrovirus, also contains the oncogene v-src, but this viral oncogene is not required for viral replication (194). Complex retroviral genomes, including HTLV and HIV-1, require multiple accessory proteins for successful replication.

These proteins function in transcriptional activation (e.g. HIV-1 Tat) (21); viral RNA (vRNA) export (e.g. HIV-1 Rev) (390); and inhibition of host restriction factors (e.g. HIV-1 Nef, Vpu, and Vpr).

Aside from protein-encoding genes, retroviral genomes include many untranslated

RNA elements required for the viral replication (Fig 1.1B). The 5’ and 3’ ends of the genome contain repeat (R) regions. Immediately inside the R regions are unique 5’ and 3’ sequences (U5 and U3, respectively). The 5’ end also contains a psi (ψ) sequence that is required for packaging vRNA into new virus particles (discussed in section 1.5.5.1). In RSV, two direct repeat elements (DR) flank the src gene, and are responsible for export of unspliced RNA for translation (discussed in section 1.5.2). Additionally, there is a splice donor site (SD) 8-nt after the AUG initiation codon in gag, and multiple splice acceptor sites

(SA) that are responsible for spliced RNAs, env and src (discussed in section 1.5.1).

Additional regulatory elements will be discussed later in this review.

This dissertation focuses on RSV, the prototypical member of the avian sarcoma and leukosis viruses (ASLVs). Therefore the rest of this literature review will explore the current knowledge of RSV replication. However, studies of HIV, MLV, and other

5 Fig. 1.1. Organization of the RSV genome and Gag polyprotein. (A) Schematic of an integrated proviral DNA. At the 5’ and 3’ ends are the long terminal repeats (LTR), consisting of the unique 3’ (U3), repeat (R), and unique 5’ (U5) regions. The provirus encodes five genes. The Gag structural protein is encoded by gag, which includes pro, the viral protease. Following gag is pol, the viral polymerase that encodes both the reverse transcriptase and the integrase proteins. Next, the env gene encodes the envelope (Env) glycoprotein. Finally, src is responsible for the oncogenic protein, v-Src. (B) RSV genome and subgenomic RNA transcripts. The provirus is transcribed into a full-length 5’-capped and 3’-polyadenylated RNA (top). The Ψ sequence, necessary for packaging the gRNA into virus particles, is located near the 5’-terminal end. The unspliced RNA transcript is also involved in translation of Gag and Gag-Pol polyproteins. For translation to occur, the direct repeat (DR) elements are necessary for unspliced RNA nuclear export to the cytoplasm.

Splicing between the splicing donor (SD) and splicing acceptor (SA) sites produce transcripts that encode Env and v-Src (middle and bottom). (C) Schematic layout of the

RSV Gag polyprotein. The RSV Gag polyprotein consists of MA (blue), p2 (pink), p10 (dark pink), CA (gray), SP (black), NC (red), and PR (dark blue). Gag is acetylated at the N- terminus (curvy line) and contains multiple trafficking (listed above the protein) and assembly (listed below the protein) domains. MA contains both an NLS and a membrane binding domain (M). The p2 domain has a late domain (L), which is required for budding, and the p10 domain encodes an NES. Overlapping the p10-CA region is a multimerization interface (MI) that is required for Gag-Gag interactions. The NC domain contains a second

NLS and the RNA interaction domain (I).

6 7 retroviruses will be presented to gain a more complete understanding of how retroviruses function.

1.3 Organization of retroviral particles

A retrovirus particle is approximately 127 nm in diameter (218), and consists of protein, RNA, and lipids. RSV buds from an infected cell as an immature particle (Fig.

1.2A), which is enveloped with a host-derived lipid bilayer containing an average of 80 trimers of the Env protein, each consisting of SU (surface) and TM (trans-membrane) domains (140, 457). Cryo electron microscopy identified Gag and Gag-Pol polyproteins radially arranged inside of the envelope, with the protein N-terminal ends at the membrane and the C-terminal ends towards the center of the particle (58, 145, 437). Although Gag is the predominant structural protein, Gag-Pol comprises approximately 5% of the virus particle (381).

Contained within the virus particle are multiple species of both cell- and virally- derived RNA. The virus specifically packages two copies of the 9 kb plus-strand gRNA, which forms a non-covalent dimer near the 5’ end of the genome (426). Several cellular

RNAs are also specifically packaged into virus particles, including a primer tRNA and small

RNAs (U6, 7SL). Host RNA packaging will be discussed in further detail (section 1.5.5.2).

After release from the infected cell, the immature virus particle must undergo maturation of the Gag polyprotein, or the virion will remain noninfectious. The viral protease

(PR) cleaves Gag (Fig. 1.1C) into matrix (MA), capsid (CA), and nucleocapsid (NC), which form the mature virus particle (Fig. 1.2B) (238). The viral polymerase, Pol, is processed into reverse transcriptase (RT) and integrase (IN). Smaller peptides of Gag, including p2, p10 and the spacer peptide (SP), are also released during Gag maturation(427). It is thought that they remain free within the mature virus particle, but their location and role is unknown

(218).

8 Fig. 1.2. Schematic diagrams of an immature and a mature RSV particle. (A) Immature virus particle. The viral glycoproteins (pink pentagons) surround the virus particle, and are embedded in the lipid bilayer envelope (gray circle). Within the virion are the Gag structural proteins, laid out with the N-terminal end at the membrane and the C-terminal end towards the interior. Gag consists of the matrix (MA) domain (blue rectangles), p2/p10 domains

(pink rectangles), capsid (CA) domain (gray ovals), nucleocapsid (NC) domain (red rectangles), and protease (PR) domain (blue pentagons). Gag-Pol also contains the polymerase (Pol) domain (orange circle and yellow triangle). Two copies of the gRNA are also incorporated into the virus particle (green lines). (B) Mature virus particle. After processing of the Gag polyprotein into its constituent components by protease, a morphological change occurs in the virion. NC binds to the gRNA near the center of the virus particle (red rectangles on green lines). MA remains associated with the membrane

(blue rectangles), but CA (gray ovals) forms an icosahedral core (capsid) around the NC- gRNA complex. PR (blue pentagons) and the p2 and p10 peptides (not shown) are thought to be free within the particle. Reverse transcriptase (orange circle) and integrase (yellow triangle) are located near the center of the virus particle within the capsid shell.

9

10 After the processing events facilitated by PR, the virion undergoes several morphological changes (Fig. 1.2B). MA remains associated at the viral membrane. NC condenses with the gRNA, at the center of the particle, and CA forms a core around the NC- gRNA complexes. The final morphology of a mature virus particle can drastically differ depending upon the virus. Cryo-electron tomography has revealed RSV viral capsids as variable, irregular polygonal shapes, although HIV forms cone-shaped cores (34, 218).

After the maturation processes are completed, the virus particle is capable of entering and infecting a new cell.

1.4 Early events of the retroviral life cycle

For the purposes of this literature review, the virus life cycle is separated into two distinct phases: early events (virus particle entry through integration of the proviral DNA), and late events (viral RNA transcription through maturation of newly released virus particles). The early events of the retroviral life cycle are presented in the following subsections (Fig. 1.3)

1.4.1 Attachment, fusion, and entry

The first step of RSV infection is the attachment of the viral Env protein to the host receptor (Fig. 1.3, step 1). Env is present on the viral membrane as a trimeric complex of

SU-TM heterodimers (122, 123). These fusogenic glycoproteins, which define the tropism of the virus, bind to specific receptors (Tva, Tvb, or Tvc) found on avian cells (30, 31, 450).

Upon SU binding to the receptor, a conformational change occurs in TM, exposing the fusion peptide that facilitates membrane fusion between virus and cell (103, 156, 181).

Internalization of the virus initiates quickly (5 minutes), but takes approximately 3 hours to complete (157). The majority of retroviruses, including RSV, do not require an acidic environment for fusion and entry (401). Thus, it appears that deposit of the viral capsid into the cytoplasm occurs at the membrane, rather than through endocytotic vesicles (Fig. 1.3, step 2). 11 Fig. 1.3. Early events in retroviral infection. A summary of the steps involved in the early stages of retroviral infection. (1) The virus binds to a cellular receptor via Env. (2) After attachment, the virus fuses to and enters the cell. (3) Uncoating of the viral core occurs to allow formation of the reverse transcription complex (RTC). (4) The RTC undergoes reverse transcription to form the double-stranded DNA provirus. This process also converts the RTC to a pre-integration complex (PIC). (5) After completion of reverse transcription, the PIC is translocated across the nuclear membrane through a nuclear pore complex. (6) The viral

DNA is integrated into the host genome.

12

13 1.4.2 Uncoating and reverse transcription

After release of the viral core into the host cell, the virus must disassemble the capsid core through a process called uncoating (Fig. 1.3, step 3) and reverse transcription

(Fig. 1.3, step 4). Neither the mechanism nor the timing of uncoating is fully understood, but this process is vital for reverse transcription and progression of the reverse transcription complex (RTC) to the pre-integration complex (PIC) (17, 115, 139, 197, 445).

The RTC is a nucleoprotein complex consisting of gRNA, tRNA primer, RT, IN, NC,

CA, MA, and several host proteins (55, 133, 134). Although RT reverse transcribes the gRNA into DNA, other retroviral proteins, including CA and NC, are important for efficient reverse transcription (96, 161, 373, 375, 393). Mutations within CA have been reported to alter reverse transcription, however the exact role of CA in reverse transcription is unknown

(7, 68). NC is also required for many of the steps in reverse transcription (8, 106, 107, 448).

An in-depth look at the role of NC during reverse transcription is discussed in section

1.7.2.1.

Reverse transcription is controlled by a heterodimer of RT subunits (α and β) present within the virion (16). A specific tRNA is used as a primer to initiate reverse transcription

(proline in MLV, lysine in HIV-1). For RSV, the tryptophan tRNA binds to the primer binding site on the gRNA, and RT reverse transcribes viral RNA into a negative-sense DNA until it reaches the 5’ terminus of the gRNA (409). The resulting 100-150 bp DNA fragment is termed the minus-sense strong stop DNA (-sssDNA). The R sequence, present at both the

5’ and 3’ ends of the genome allows the first strand transfer to occur, with movement of the

–sssDNA to the 3’ end of the genome. After the first strand transfer, the minus-sense DNA synthesis continues. Concomitant with reverse transcription, RNase H activity from RT digests the viral RNA. However, a short polypurine tract (PPT) resists RNA degradation, and serves as the primer for plus-strand DNA synthesis. DNA replication stops at the 3’ end of the genome to form the plus-strand strong stop DNA (+sssDNA). Next, the complementary PBS sequences anneal after degradation of the tRNA primer by RNase H. 14 Once annealed, each DNA strand serves as a template for the other, and DNA synthesis is completed. Each end of the viral DNA now contains an identical long terminal repeat (LTR), formed by the U3, R, and U5 regions.

1.4.3 Nuclear entry of the PIC

After reverse transcription is finished, only viral DNA is present in the nucleoprotein complex, and is known as the pre-integration complex (PIC). The PIC must then translocate into the nucleus for integration into the cellular genome (Fig. 1.3, step 5). Although the exact composition of a PIC appears to vary between retroviruses (62, 133, 134, 286), MA,

NC and IN have been detected in PICs and may play a vital role in nuclear entry. For many viruses, such as MLV, the process of nuclear entry is passive (351). These retroviruses must infect mitotically active cells, where the nuclear envelope disassembles, for integration of the provirus to occur. In contrast, lentiviruses and spumaviruses are capable of actively transporting the PIC into the intact nucleus (245, 246). Alpharetroviruses also actively transport the PIC into the nucleus, although import is less efficient than HIV (177).

The active transport of a PIC requires nuclear localization sequences (NLSs) within both the MA and IN proteins (50, 61, 63, 87). In MLV, the NC protein, which also contains an NLS, is colocalized with IN inside of nucleoli during early infection (350). These observations suggest that NC may also play a role in integration events during early infection. However, a role for NC in the import of the PIC has not been established.

Although it remains plausible that multiple NLSs within the PIC ensure a redundant pathway for nuclear entry, it is likely that only one import pathway is required for transporting the PIC across the nuclear membrane. It is possible that the different NLSs are necessary during separate phases of the virus life cycle. Thus, further examination into the role of viral nuclear trafficking signals is needed to better understand the complex pathways of retroviral entry.

15 1.4.4 Integration of the viral DNA

After nuclear entry of the PIC, the viral DNA undergoes one of two fates: integration of the viral DNA into the host genomic DNA (Fig. 1.3, step 6), or entry into a nonproductive path, where the DNA is circularized and lost with subsequent cellular divisions. Integration requires processing of the viral DNA by IN by the removal of two nucleotides from each

LTR. The processed DNA is then preferentially inserted into sites of active transcription within the host genome (56, 57, 292, 371, 383, 424). Viral DNA may also form 1-LTR circles when the LTRs undergo homologous recombination with each other. 2-LTR circles form through non-homologous recombination, directed by cellular proteins (247). Auto- integration products, formed when the viral DNA is integrated into itself, may also occur.

Any of these three circularization fates, which accumulate in IN deficient cells, render the viral DNA nonproductive but serve as an indicator that PIC nuclear import has taken place

(116, 247, 353). Once integration of a linear viral DNA has occurred, the viral DNA is now referred to as a provirus, and the late events of the viral life cycle begin.

1.5 Late events of the retroviral life cycle

The events encompassing transcription of viral RNA to maturation of newly synthesized virus particles are presented in the following subsections (Fig. 1.4).

1.5.1 Transcription and splicing of viral RNA

The integrated provirus contains all the viral genes necessary for virus particle production: gag, pro, pol, and env (Fig. 1.1A). The host-provided RNA polymerase II transcribes the provirus into a single viral mRNA that is 5’ capped and 3’ polyadenylated

(Fig. 1.4, step 1). The LTR provides a strong enhancer of transcription along with a constitutive core promoter (166). Host transcription factors YY1 and TFII-1 interact with the

U3 region of the 5’ LTR to facilitate production of the viral transcript (294). During retroviral

16 Fig. 1.4. Late events in RSV infection. A summary of the events during the late phase of the RSV life cycle. (1) Viral mRNA transcripts are produced from the proviral DNA. Viral

RNAs are either spliced or unspliced. (2) Viral RNAs are exported from the nucleus.

Spliced RNAs utilize normal cellular export pathways. Unspliced RNA uses the direct repeat

(DR) elements found flanking the src gene to facilitate export from the nucleus. (3 and 3a)

Gag and Gag-Pol are translated from the unspliced viral RNA on free ribosomes; the Env protein is translated from one of the spliced RNA transcripts, and is processed and exported through the Golgi apparatus to the plasma membrane. (4) RSV Gag transiently traffics through the nucleus, via two NLSs and one NES within the Gag protein. (4a) Gag presumably selects the gRNA for packaging during the nuclear trafficking step. The smallest assembly unit is a Gag dimer bound to the gRNA. (5) After nuclear export, Gag undergoes multimerization steps (dimers, to tetramers, to hexamers) within the cytoplasm as virus particle assembly is presumably initiated. (6) Gag oligomers are targeted to the plasma membrane and the virus particle begins to bud out of the cell. (7) Budding and release of the immature virus is completed, facilitated by the late domain (L) found within the p2 domain of RSV Gag. (8) The viral protease cleaves the Gag and Gag-Pol proteins into their component proteins. This processing of Gag results in maturation of the virion.

17

18 infection, 10% of the messenger RNA present in the cell is from the viral promoter (234). It is interesting to note that although viral RNA concentration is high, only two copies of the gRNA are packaged in each virus particle, suggesting a complex mechanism of RNA selection.

The unspliced viral RNA transcript is responsible for the production of both the Gag

(Fig. 1.1C) and Gag-Pol polyproteins. The genome contains a splice donor (SD) site within the gag gene, with a counterpart splice acceptor (SA) site in env and src (Fig. 1.1B). Thus, three different RNA species are formed during infection by RSV that encode Gag and Gag-

Pol, Env, and v-Src. Although most mRNAs within a cell would be spliced before translation, only 20-50% of the viral mRNA will be spliced. RNA splicing is mediated by cis- acting negative regulators of splicing and weak signals in the 3’ SA sites (10, 11, 20, 162,

275, 276, 308, 309).

1.5.2 Nuclear export of viral RNA

Although spliced viral RNAs exit the nucleus through normal cellular export pathways

(Fig 1.4, step 2), the unspliced viral RNA presents a problem: unspliced cellular mRNAs are normally retained or degraded within the nucleus (49, 99). Complex retroviruses circumvent these quality control mechanisms with trans-acting accessory proteins that facilitate unspliced RNA export from the nucleus. The HIV-1 Rev protein interacts with the Rev response element (RRE) in unspliced viral RNAs to mediate export from the nucleus (136,

267, 284). Simple retroviruses, including RSV, do not encode accessory proteins within their genome. Thus, simple retroviruses must use a different strategy to export unspliced viral RNA from the nucleus.

RSV bypasses nuclear restriction of unspliced RNA through the use of two direct repeat (DR) elements, which flank the src gene (Fig. 1.1B). The DR sequences function as cis-acting RNA export elements, which utilize TAP and Dbp5 export factors (227, 310, 322).

Deletion or mutation of the DR elements causes an accumulation of unspliced RNA within or 19 around the nucleus. Expressing dominant-negative forms of Tap and Dbp5 also sequesters the unspliced viral RNA within the nucleus. These studies implicate the role of DR elements in RNA export, which results expression of the Gag protein.

1.5.3 Translation of viral proteins

After export of the spliced and unspliced viral RNAs from the nucleus, translation of viral proteins commences (Fig. 1.4, step 3). The full-length viral RNA serves as a template for both the Gag and Gag-Pol polyproteins, which are translated on free ribosomes within the cytoplasm. A ratio of 20 Gag:1 Gag-Pol is maintained by a -1 frameshift event that occurs during translation of the Gag protein (199). RSV is unique among retroviruses, as the viral protease is a part of Gag. Most retroviruses (e.g. MLV, HIV) translate PR as Gag-

Pro-Pol. Thus, the frameshift events also differ between retroviruses: the RSV frameshift occurs after pro, and HIV frameshifting occurs before.

Spliced transcripts are responsible for the translation of Env and v-Src proteins.

Sub-genomic translation occurs in the endoplasmic reticulum, where Env trimerizes and is

N-glycosylated (Fig. 1.4, step 3a). Processing occurs in the Golgi complex, where cellular proteins facilitate separation into the SU and TM subunits (122). In this dissertation, the

RSV provirus RS.V8 was used to produce virus particles, and contains the green fluorescent protein (gfp) gene in place of src.

1.5.4 Assembly of immature virus

RSV, HIV-1, and other type-C retroviruses assemble virus particles at the plasma membrane (Fig. 1.4, step 6). Gag is both necessary and sufficient for the production of virus like particles (VLPs). In HIV-1, myristoylation of the second glycine residue of Gag is essential for membrane targeting (323). In place of myristoyl, RSV Gag is acetylated and requires several basic residues within the membrane binding domain (M) for membrane association to occur (71, 304, 324, 372, 425). Gag-Gag interactions are mediated by a 20 multimerization interface (MI) that overlaps the p10-CA domains (73, 303, 332) and the interaction domain (I) in NC (52, 89, 232). A more detailed discussion of the role of NC in assembly is presented later (section 1.7.3).

Although in electron micrographs Gag is visible primarily at the plasma membrane

(225), several lines of evidence suggest that Gag-Gag interactions occur before membrane localization. In genetic complementation experiments, wildtype Gag proteins restore budding of a Gag mutant lacking an M domain, suggesting Gag-Gag interactions occur prior to the plasma membrane (15, 35, 59). In RSV, wildtype Gag is also able to rescue budding of a mutant Gag protein unable to leave the nucleus, indicating that Gag-Gag interaction occur within the nucleoplasm or that wildtype Gag interacts with the Gag NES mutant prior to nuclear entry of the mutant Gag (213). Furthermore, cytoplasmic extracts from Gag expressing cells contain assembly intermediates that suggest a stepwise manner of assembly (255). Recent studies have demonstrated direct Gag-Gag interactions occurring within the cytoplasm, indicating the presence of at least Gag dimers (225). These studies demonstrate that although culmination of assembly occurs at the plasma membrane, assembly begins within the nucleus or cytoplasm.

RNA is also a necessary component of retroviral assembly. Retroviruses require viral or cellular RNA for both assembly at the membrane, and virion stability after particle release (302). RNA also facilitates immature VLP assembly in vitro (72, 74, 170, 460). This requirement for RNA may be bypassed through the replacement of NC with a protein-protein interaction domain, such as a zip sequence (2, 6, 97, 204). These zip motifs initiate Gag-

Gag interactions without the need for RNA. Indeed, recent studies have suggested that nucleic acids are not necessary for membrane association, but only stability of the virus

(188, 318). However, these experiments failed to take into account the membrane binding ability of MA. Additionally, mutants lacking the NC domain are unable to bud from cells.

Thus, current evidence indicates that RNA is a necessary assembly component during virus particle release. 21 1.5.5 RNA packaging

Although retroviruses must package viral RNA, this step is dispensable for virus-like particle formation. Cellular RNAs are also packaged, and are sufficient for viral assembly to occur in the absence of vRNA (242, 268). The next two sections will discuss gRNA packaging events, followed by the specific packaging of a small subset of cellular RNAs.

1.5.5.1 Packaging of viral RNA

Two copies of the gRNA, linked as a dimer (206), are specifically incorporated into each virus particle. The gRNA is recognized by the NC domain of Gag through a cis-acting

RNA packaging signal (ψ) found in the 5’ untranslated region (Fig. 1.1B). The ψ packaging signal is a 269 nt sequence located between the primer binding site (PBS) and the SA in gag. An in-depth discussion on NC-RNA interactions is presented in section 1.7.4. Deletion of the ψ sequence decreases genome packaging and viral infectivity for all retroviruses

(105, 210). The addition of the ψ sequence to a non-viral RNA is sufficient to direct packaging of that RNA into VLPs (18, 19, 25, 26). Because Gag is translated from the same

RNA it must package, it would seem likely that Gag selects the gRNA in cis at the site of translation. HIV-2 cotranslationally packages the viral genome (169), but HIV-1 is reported to package gRNA in trans (211, 273). The evidence from HIV-1 suggests that there may be multiple pools of RNA, with one pool for translation and another for packaging.

Furthermore, in RSV, the presence of a packaging sequence on an untranslated RNA is packaged at high efficiency, and indicates that packaging at sites of translation is not necessary (25). Thus, gRNA packaging is possible in trans.

Unlike HIV and MLV, the RSV ψ sequence is present within spliced and unspliced viral RNAs. The unspliced vRNA is preferentially incorporated into virions whereas the spliced RNA is not (25), leading to another question: how does the virus differentiate

22 between gRNA and ψ-containing vRNA? A possibility for the selective packaging of gRNA over spliced vRNA is the location of interaction.

In MLV, Gag and gRNA have been observed within endosomal vesicles en route to the plasma membrane (29). Experiments have suggested that HIV-1 Gag interacts with ψ- containing RNA at centrioles (338). In these experiments, Gag did not localize to centrioles in cells lacking ψ RNA. Together, these observations suggest that the ψ sequence may affect Gag trafficking during packaging. These experiments, however, have not definitively identified the initial site of Gag-gRNA interaction. It is possible that these localization steps are intermediaries between ψ selection by Gag and budding at the plasma membrane.

Unfortunately, the precise location where the Gag-gRNA interaction first occurs remains unknown and further research will be required before the location of Gag selection of gRNA is known.

1.5.5.2 Packaging of cellular RNAs

Although gRNA is necessary for infection, it is dispensable during the formation of new virus particles (302). The two copies of gRNA incorporated into wildtype virus particles account for approximately 50% of the total mass of RNA, with the remainder of the virion

RNAs derived from the host. These cellular RNAs including mRNAs, tRNAs, and small

RNAs, such as 7SL and U6 (84, 102, 131, 132, 314, 315, 361). Although a random distribution of incorporated cellular RNA would be expected, several RNA species are specifically enriched whereas others are excluded from the virus particles (314).

In RSV, an RNA encoding a 34 kD protein was found to be specifically packed in certain strains of virus (5), providing the initial evidence that cellular mRNAs are incorporated into virus particles. If gRNA is not present during virus assembly, cellular mRNAs appear to replace the genome in RNA mass (148). In MLV, a genome-deficient virus was found to contain a heterogeneous cellular mRNA population (355), though a small

23 number of specific cellular mRNAs were enriched within virus particles. Together, these studies suggest that some cellular mRNAs and the viral gRNA may share some common traits, possibly structure, location, or sequence. Although no function is known for packaged cellular mRNAs, further study into why these RNA species are incorporated into virus particle may reveal insights into the selectivity of gRNA packaging.

Retroviral virions incorporate between 50-100 copies of various tRNAs (131, 361).

These packaged tRNAs anneal to the primer binding site (PBS) at the 5’UTR of the gRNA and act as the primer during the initial steps of minus strand vDNA synthesis (131, 362).

Although several tRNAs are packaged into virions, different retroviruses maintain a preference for the tRNA used in reverse transcription initiation. The PBS of RSV is specific

Trp Lys Pro for tRNA (361), while HIV and MLV use tRNA! and tRNA , respectively (265, 434).

The primers are specifically packaged, with tRNA concentration in the virion between 5

(MLV) and 20 (RSV) fold higher than that found in the cytoplasm (263, 434). The RT domain within Gag-Pol mediates the specific packaging of tRNA (244, 263, 264, 330). The aminoacyl synthetase (aaRS) for each tRNA is also involved in tRNA incorporation into virus particles, and the appropriate synthetase (e.g. TrpRS for RSV, LysRS for HIV) is also packaged within the virions (81, 82). It is thought that Gag interacts with the aaRS through the CA domain, whereas the tRNA interacts with RT in Gag-Pol (200). Although the RNA- binding NC domain is dispensable for the selective packaging of tRNA, NC is involved in annealing the tRNA to the PBS during maturation of the virus particle (80, 111). Further discussion on the role of NC in tRNA primer annealing is presented in section 1.7.2.1.

In addition to tRNAs, several small, noncoding RNAs are also preferentially packaged into virus particles. One of the first host RNAs identified in RSV was the 7SL RNA

(42, 126). Although 7SL is only one component of the signal recognition particle, a large ribonucleoprotein complex that promotes protein transport into the endoplasmic reticulum

(430), only the 7SL RNA is incorporated into virus particles (315). The U6 small nuclear

RNA (snRNA) is also preferentially packaged into retrovirions (158, 314). In RSV, 24 experiments have revealed that one U6 RNA is present within each virus particle (158). It is interesting to note that U6 is restricted entirely to the nucleus ((438)), suggesting that gRNA and U6 may interact within the nucleoplasm or that U6 is selected within the nucleus. Given that several retroviral Gag proteins, including MLV and RSV, have transient nuclear trafficking steps (15, 363), and that nuclear trafficking is linked to efficient gRNA incorporation in RSV virions (151), it seems likely that Gag selects U6 during selection of the gRNA.

There are several other small RNAs that are specifically enriched in MLV virus particles, including Y and B RNAs (314). These RNAs represent an assortment of cytoplasmic and nuclear localizations. The only common trait between these RNAs is that they are all transcribed using the Pol III polymerase, which synthesizes transcripts in or near nucleoli (269). Additionally, recent studies have identified that the Y RNA may be packaged early during its biogenesis, which occurs in nucleoli (152). These studies all point to the possibility of nucleoli being involved during packaging or assembly.

1.5.6 Budding

After Gag proteins and Gag-RNA complexes have trafficked to the plasma membrane, they assemble and oligomerize in conjunction with the Env protein and begin to bud (Fig. 1.4, step 7). Endosomal components, including the ESCRT machinery, are recruited to the site of assembly via the Gag late (L) domain. The L domain is a short, proline rich sequence (PPPY) within the p2 domain (Fig 1.1C), which facilitates release of the assembled immature virions through membrane fission (153, 325, 443). Loss of the L domain or inhibition of the endosomal sorting pathway causes immature virus particles to accumulate on the plasma membrane (439).

25 1.5.7 Maturation

Either concurrent with or immediately after release of an immature virus particle, the viral protease (PR) processes RSV Gag into the mature protein components: MA, p2a, p2b, p10, CA, NC, and PR, and Pol is processed into RT and IN. In most retroviruses, PR is part of Gag-Pol; however RSV contains PR within Gag and Gag-Pol that results in a higher concentration of PR within virus particles. In RSV, PR must first cleave itself from Gag (66,

435), prior to recognition of specific cleavage sites within Gag. After Gag is processed into the final mature proteins, a significant rearrangement of the virus particle occurs, culminating in the formation of the infectious, mature virus particle (Fig 1.2B). Organization of the mature retrovirus particle is discussed in section 1.3.

1.6 Nuclear trafficking of the RSV Gag polyprotein

In the past decade, our laboratory identified a transient nuclear trafficking step of the

RSV Gag polyprotein (363). Under steady-state conditions, Gag is observed within the cytoplasm and at the plasma membrane. Treatment of cells with the CRM1 inhibitor, leptomycin B (LMB), localizes Gag within the nucleus (363). This observation suggests Gag undergoes transient nuclear trafficking during assembly. A Gag mutant, Myr1E, is strongly targeted to the membrane, and is insensitive to LMB treatment (363). Bypassing nuclear trafficking caused a significant decrease in gRNA packaging, suggesting Gag requires a nuclear event for the efficient selection and incorporation of gRNA into new virus particles.

Insertion of a heterologous NLS into the MA domain of Gag restored nuclear trafficking of the Myr1E mutant. Additionally, gRNA incorporation was restored, supporting the idea that

Gag nuclear trafficking is necessary for packaging the viral genome.

The inhibition of nuclear export with LMB suggested the presence of a nuclear export signal (NES) within Gag. The NES maps to a stretch of hydrophobic amino acids

(LTDWARVREEL) within the p10 domain of Gag (363). Mutation of any or all of the hydrophobic residues within the NES results in a Gag nuclear localization phenotype 26 indistinguishable from LMB treatment (366). Failure of Gag to exit the nucleus results in lower amounts of virus released from the cell (366). These studies demonstrated that sequestering Gag within the nucleoplasm inhibited virus particle release.

Sequence analysis identified putative nuclear localization signals (NLSs) within the

MA and NC domains of Gag (67). Indeed, both MA and NC localize to nuclei under steady- state conditions. Genetic assays identified that the nuclear import factors Transportin SR and Importin 11 interact with the NLS in MA, and the classical Importin α/β import proteins mediated nuclear entry of NC (67). The first 88 amino acids of MA encode several basic residues that encompass the NLS. Although deleting the basic residues in MA prevents

Gag membrane binding and particle release, deletion of the NLS in MA does not noticeably reduce the presence of Gag in nuclei during LMB treatment (67). Deletion of NC, however, causes a reduction in the amount of Gag signal present within the nucleus during LMB treatment (67), suggesting NC contains a stronger NLS than MA. The identification of nuclear trafficking signals in Gag raises a pertinent question: if the gRNA is used as a template for Gag, Gag selects the gRNA, and virion budding occurs at the plasma membrane, why does Gag traffic to the nucleus?

A Gag mutant containing the first 8 amino acids of the Src membrane-binding domain, Myr1E, is insensitive to LMB treatment (363). In context of a virus Myr1E buds more efficiently than wildtype, but incorporates significantly less gRNA in virus particles

(149). These observations indicate that strong targeting of Gag to the membrane is deleterious to viral replication and gRNA packaging. One proposed reason for a decrease in gRNA packaging under strong membrane targeting conditions is that Gag does not have enough time within the cell to find and interact with its gRNA (70). Another possible explanation is that nuclear trafficking is important for efficient gRNA packaging. In support of the nuclear trafficking hypothesis, a heterologous NLS was inserted into the MA domain of the Myr1E.Gag mutant (151). The additional NLS restores nuclear trafficking of the

Myr1E mutant and rescues gRNA packaging. Although the idea of nuclear selection of 27 gRNA is possible, a puzzling problem persists. Gag encodes two NLSs and a single NES, which should compete with each other for nuclear import and export. How does Gag mediate the action of these localization signals?

Some of the mechanisms of signal regulation appear to derive from the overlapping nature of localization and assembly signals within the Gag protein. A recent, in vitro biochemical study has allowed the development of a model for Gag nuclear trafficking.

Experiments demonstrate that Importin 11 and Transportin SR bind directly to the NLS in

MA (172). Additionally, the Importin α/β complex interacts directly with NC. These interactions allow Gag to traffic across the nuclear envelope. After nuclear import, Gag interacts with RNA, which displaces the import factors from the Gag protein. Concomitant with RNA binding, a structural change occurs within Gag. Presumably, this altered structure exposes the NES and facilitates binding of the CRM1-RanGTP complex to Gag. Although other explanations are also possible, these mechanistic steps may represent the step-wise manner in which Gag enters the nucleoplasm, binds the gRNA, and leaves the nucleus.

However, a full understanding of what occurs while Gag is within the nucleus is lacking.

Thus, many questions remain unanswered, including: do Gag proteins undergo subnuclear trafficking events? Chapters 2 and 3 address this question.

1.7 The many roles of the retroviral nucleocapsid (NC)

Although this dissertation delves into the role of Gag nuclear trafficking during assembly, an understanding the NC domain and protein is necessary. The next several sections will discuss the known functions and roles of NC during viral assembly, maturation, and entry.

1.7.1 Structure and function of NC

The NC proteins of retroviruses are small (~100 amino acids), and found to be enriched with basic amino acids (Fig 1.5 and Table 1.1). All orthoretroviruses contain either 28 Fig. 1.5. Comparison of retroviral NC proteins. The images represent retroviral NC proteins used in this dissertation. The identity and apparent molecular weight are also shown. The black boxes represent zinc fingers (Cys-His motifs) and the white boxes represent the flanking sequences enriched in basic amino acids. A detailed comparison is presented in Table 1.1. Abbreviations: RSV, Rous sarcoma virus; MLV, murine leukemia virus; HIV-1, human immunodeficiency virus type 1; MMTV, mouse mammary tumor virus.

29

30 Table 1.1. Retroviral NC protein properties. The table displays general properties of the various NC proteins depicted in Fig. 1.5. The virus, apparent protein weight (kD), protein size (amino acids), N- and C-terminal zinc finger sequences, and basic and acidic residue numbers are given. The conserved CCHC amino acids within each zinc finger are highlighted in bold. MLV contains a single zinc finger and has no C-terminal Cys-His motif

(N.A.). Sequences were obtained from NCBI GenBank using the following accession numbers: RSV, J02342; MLV, J02255; HIV-1, AF324493; MMTV, P11284

31

32 a single (gammaretroviruses, e.g. MLV) (180) or two strictly conserved zinc fingers, which have the sequence C-X2-C-X4-H-X4-C (Cys-His motifs) (Table 1.1) (36, 91). In the context of

Gag, zinc fingers recognize and directly interact with the ψ sequence within the gRNA (9,

456). The regions immediate flanking either Cys-His motif are enriched with basic amino acids, which are involved in general RNA binding and Gag-Gag interactions (232). The zinc fingers form stable structures, whereas the flanking basic regions are largely unstructured

(9, 106, 228, 296, 297, 456). With both the basic amino acids and the zinc fingers, NC can be considered the nucleic acid “workhorse” of retroviruses. Aside from the role of the NC domain in gRNA packaging, the NC protein acts as a nucleic acid chaperone during the early steps of retroviral replication (111, 243, 341). Thus, NC is essential for viral replication and is involved in a myriad of steps during the viral life cycle.

1.7.2 Role of NC in viral entry

The bulk of this dissertation focuses upon the role of NC and Gag during assembly of retroviral particles. However, NC is intimately involved in many aspects of retroviral entry: reverse transcription, nuclear entry of the PIC, and integration (Fig. 1.3, steps 4-6). A brief review of the function of NC during these steps is presented here to facilitate later discussion. It should be noted that many of the studies presented were performed using the

HIV-1 NC protein, but many parallels may be drawn from and applied to RSV NC.

1.7.2.1 NC and reverse transcription

A brief overview of reverse transcription was presented in section 1.4.2. Although the viral RT is capable of performing reverse transcription alone in vitro, it is very inefficient.

NC acts as an RNA chaperone in many instances during reverse transcription, including facilitating the initiation of RT. As discussed previously (section 1.5.5.2), the virus packages primer tRNAs, facilitated by the CA and RT domains of Gag. NC greatly increases the efficiency of tRNA primer annealing to the PBS of the gRNA (27, 111, 176, 214). The 33 annealing is carried out through a partial disruption of the 18 nt of tRNA that binds to the

PBS. The zinc fingers are dispensable for tRNA annealing, meaning this RNA chaperone activity is due to the basic residues within NC (111, 176).

After initiation of reverse transcription, two strand transfers must occur: the minus- and plus-strand transfers (Fig 1.3, step 4, first and second strand transfer). NC greatly enhances both strand transfers and the RNase H activity of RT (191, 327, 448).

Furthermore, NC increases the efficiency of completing the LTR ends and the finish of reverse transcription (212, 423).

In cell cultures, NC has also shown to have a role in the reverse transcription steps determined in vitro. Although mutations to NC affect the efficient synthesis of vDNA (163,

165, 281, 451), it is difficult to determine the precise step in reverse transcription that is inhibited. This mystery is probably due to the unknown composition of RTC (i.e. nucleic acid concentrations, protein concentrations). Together, these studies support the idea that NC is intricately involved throughout the reverse transcription process.

1.7.2.2 Role of NC in PIC nuclear import

NC is capable of strongly binding to nucleic acids, including DNA (223). With the observation of NC in the nucleus during early infection (147, 350, 454), it has been assumed that NC is associated with the PIC. Additionally, NC has been observed associated with vDNA using transmission electron microscopy (289). Furthermore, deletion of the NLSs in

MA and IN do not prevent PIC nuclear entry (142, 143, 252, 253), suggesting NC may play a role in PIC nuclear import. However, no direct evidence of NC facilitating nuclear import of the vDNA has been found and further study into the role of NC-vDNA interactions are needed to clarify a potential role for NC in PIC nuclear entry.

34 1.7.3 Role of NC in viral assembly

NC is primarily a nucleic acid binding protein with many roles in virus replication. An important role for RNA binding of the NC domain within Gag is mediation of virus particle assembly (77, 108, 207, 282, 320). Deletion or mutation of the ψ-packaging sequence does not prevent virus particle assembly (302, 355). Instead, cellular RNAs comprise the RNA component of these mutant virions. Additionally, immature MLV particles treated with

RNase are disrupted, demonstrating RNA as an essential building block for immature virions

(302). Furthermore, deletion of the NC domain drastically decreases the release of virus particles from the plasma membrane, and the particles released are less dense than wildtype (89, 319). Together, these observations demonstrate that the NC domain within

Gag is essential for virus particle release and for proper particle density.

Initial studies show that the zinc fingers in RSV NC are dispensable for particle release (282). Other experiments demonstrate that the Cys-His motifs are essential for virus particle release (231, 233). These contradictory findings are rather puzzling, however the methods used to detect the virus may be the reason for different results. The assay that detected normal particle release of zinc finger deletions used immunoprecipitation, followed by immunoblotting (282). In the study that found a decrease in virion release with the zinc finger deletions, the virus was pelleted via ultracentrifugation (233). Chapter 4 in this dissertation describes a similar result with a NC basic residue mutant (RS.V8 M5).

However, why this difference is seen is unknown and further studies into the structure of these mutant virus particles are required.

The basic residues flanking the Cys-His motifs are also necessary for particle release. In HIV, mutation of at least 6 basic amino acids reduced detectable virus particles to less than 5 percent of wildtype (89). In RSV, mutation of 8 basic amino acids eliminated

Gag-Gag interactions and particle release (232). From these data, it has been proposed that the number of basic residues is important for efficient virus particle release.

35 Although NC appears to facilitate budding of virus particles, recent data suggest that

NC is partially dispensable for retroviral assembly. The NC domain of Gag can be replaced by a protein dimerization domain (Zip domain), which facilitates release of VLPs from the plasma membrane (204). It was proposed that HIV NC acts as a promoter of Gag-Gag dimerization, and other interaction domains such as the multimerization interface, facilitate the final assembly steps of virus particles. Other studies, using fluorescence resonance energy transfer (FRET), observed protein-protein interactions between Gag proteins lacking the NC domain (188). Furthermore, inactivation of the viral protease rescues particle assembly of NC deletion Gag proteins, but assembly is less efficient than wildtype (319).

These lines of evidence suggest NC is not absolutely necessary for virus particle assembly.

However, many of these studies failed to account for the RNA binding capability of

MA (65, 88). The FRET experiments demonstrated a small amount of Gag-Gag interactions within the cell; however no interaction was detectable at the plasma membrane (188). This result implies that although Gag can interact with other Gag proteins, the ability to assemble at the membrane is severely impaired. Furthermore, although the protease mutation partially rescues a ΔNC Gag (319), it is unable to restore budding to a deletion of NC and mutation of MA RNA binding (320). It is possible that NC directs correct alignment and initiation of proteolytic cleavage of the immature virus. However, further study into the relationship between protease and NC are needed to understand how they promote particle release.

Cellular host factors have also been implicated in NC-mediated retroviral assembly.

The host ESCRT machinery, which is involved in the endosomal sorting pathway, interacts with the L domains in p6 of HIV Gag (154, 403). Additionally, NC cooperates with the late domains in p6 to facilitate the release of virus particles (121, 340). In RSV, the C-terminal half of the ESCRT protein Tsg101 blocks budding of virus (205). The nucleolar protein nucleolin also affects Gag binding, presumably through NC. A C-terminal fragment of nucleolin interacts with the NC domain many retroviral Gag proteins, including HIV, MLV, 36 and RSV (24). The C-terminal fragment of nucleolin inhibits MLV virus particle formation, suggesting nucleolin is involved in viral assembly. This idea is supported by work in HIV.

Nucleolin was found to enhance virus particle production when co-expressed with Gag and the ψ-packaging sequence (421). Furthermore, nucleolin was found incorporated into VLPs.

Together, these data indicate that NC-mediated assembly occurs not just through RNA interactions, but also through interaction with cellular proteins.

1.7.4 Role of NC in gRNA packaging

The most important role of the NC domain during retroviral assembly is the selection and incorporation of gRNA into virus particles (39, 120, 230-232, 282, 455). NC specifically interacts with the viral packaging sequence (ψ), located between the PBS and the SD site at the 5’ end of the genome (19, 25, 39, 210, 402). This specificity is transferrable between different retroviruses, as MLV Gag proteins containing the HIV NC package HIV genomes

(455). Conversely, HIV Gag proteins containing a MLV NC domain package MLV RNA (39).

Although small fragments of the ψ-sequence can be packaged (4, 178, 301), more efficient packaging occurs when the RNA contains most of the 5’ UTR. RSV is unique in this aspect, as a portion as small as 82 nt is efficiently incorporated into particles (26). These small ψ- sequences can be used to package heterologous RNAs efficiently into VLPs (25). Although

NC is involved in gRNA packaging, how it functions to select ψ-RNA in the cell remains controversial.

1.7.4.1 NC zinc fingers

The zinc fingers of the NC protein are strictly conserved amongst retroviruses. Each

Cys-His motif binds zinc with high affinity (168), and incorporation of zinc is required for viral infectivity (281, 282, 349). In RSV, early studies deleting of either or both Cys-His boxes observed a large decrease in gRNA packaging, but deletion of the distal Cys-His motif did not entirely disrupt infectivity (282). More recent studies report a minor decrease in 37 packaging when both zinc fingers are deleted (18). Instead, they propose that the zinc fingers prevent degradation of RNA inside the virus particles, as extraction of RNA with proteinase K yields higher amounts of viral RNA than previously reported. The discrepancy between results may also be due to the method of detection, as the previous studies used northern blots, and the newer studies used ribonuclease protection assays. Additionally, the more recent study used a heterologous RNA containing the ψ-sequence. With further research in context of a proviral construct, experiments show deletion of the proximal Cys-

His motif moderately decreases gRNA packaging, and deletion of the distal Cys-His box increased gRNA packaging 4-fold (231, 233). These observations, along with other results(51), suggest that the zinc fingers in NC are not equivalent. This idea is further supported in HIV. Mutations to the second Cys-His motif were not as detrimental to viral infection as mutations in the first motif (164). Together, these results agree that the zinc fingers are necessary for infection. However, the genetic data presented leaves question as to the role of Cys-His motifs in selection of gRNA.

Evidence supporting the role of zinc fingers in packaging comes from structural analysis of the NC protein bound to the viral ψ-sequence. NMR studies of RSV NC bound to the 82 nt minimal packaging sequence determined that only amino acids within the Cys-

His motifs were bound to the viral RNA (456). The first zinc finger bound to a stem-loop structure in the RNA, whereas the second zinc finger bound to an AUG sequence between two stem-loop structures. HIV studies have also found the zinc fingers bound to stem-loops within the HIV ψ-sequence (9, 109). These data indicate that the zinc fingers are not only essential for the selection of gRNA, but are responsible for the direct interaction with the ψ- sequence.

1.7.4.2 NC basic residues

Although the Cys-His motifs directly interact with gRNA, the basic residues in NC are also necessary for gRNA packaging in addition to general RNA binding. In vitro studies of 38 HIV NC correlate the number of basic residues with viral RNA binding (369). However, in vivo experiments suggest that location of charge may be important for the NC domain of

Gag, as specific mutations that were not significantly reduced for RNA binding by NC were extremely deficient in gRNA incorporation (89). These data suggest that the NC protein and the NC domain of Gag have alternate functional aspects, and the idea of an RNA binding inequality between NC and Gag has been proposed previously (98).

The idea that specific basic residues are necessary for interaction with ψ is supported in RSV (230-232). Immediately flanking the C-terminal end of RSV NC is a cluster of basic amino acids, RKR. Mutation of a single basic residue immediately flanking the second Cys-His motif in RSV NC to SKR drastically reduces gRNA packaging (231).

However, mutation of both basic amino acids immediately after the flanking residue (RTL) has little effect on the amount of gRNA incorporated into virus particles (231). These data indicate that a basic residue immediately flanking a zinc finger may alter NC structure.

However, relatively little work has been done exploring the role of specific basic residues in the context of a viral infection. Chapter 4 examines the effect NC basic residue mutants have on gRNA packaging efficiency of RSV.

1.8 The nucleolus

Eukaryotic nuclei contain many subnuclear compartments, including Cajal bodies

(69), nuclear speckles (394), transcription factories (195), and nucleoli (295). The nucleolus is the most prominent of subnuclear structures, visible under light microscopy, and first described more the 150 years ago (429). The main function of nucleoli appeared to be biogenesis of ribosomes: transcription of pre-ribosomal RNA (rRNA) (79), processing of rRNA (155), and assembly of ribosomal subunits (110). Nucleoli are divided into three separate components, which can be distinguished under electron microscopy: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC) (Fig.

1.6) (299). Ribosomal DNA genes are located within the FC, and transcription occurs on the 39 Fig. 1.6. Structure of the nucleolus. (A) Nuclei (gray oval) contain nucleoli. The outer compartment of a nucleolus is the granular component (GC, blue circle). Within the GC are one or more fibrillar centers (FC, red oval). Immediately surrounding the FC is the dense fibrillar component (DFC). (B) Confocal microscopy image of QT6 quail cells expressing the nucleolar protein fibrillarin fused to CFP. All three nucleolar compartments are visible, with the dark center (FC), the bright fluorescence ringing the center (DFC), and the surrounding less intense fluorescence (DFC).

40

41 periphery between the FC and the DFC. Processing of rRNA occurs within the DFC, and ribosomal subunit assembly transpires within the DFC and the GC. Thus the nucleolus appeared a static structure, involved in an important but solitary role.

Recent studies have overturned the idea that nucleoli are involved solely in ribosomal biogenesis. Nucleoli silence genes, regulate the cell cycle, process mRNAs, and are involved in the synthesis of Pol III transcripts, genome replication, and tRNA regulation

(47, 335, 342, 414, 453). Because nucleoli are such large subnuclear structures, they can be isolated from the cell (14). Recent proteomic analysis has identified more than 700 different proteins within the nucleolus (13, 14, 45, 241). Approximately one third of the identified nucleolar proteins are involved in ribosomal biogenesis. The other proteins identified are kinases, ubiquitin-related proteins, transcription factors, chromatin-related factors, chaperones, RNA-modifying enzymes, and proteins involved in DNA replication and repair. Thus, the nucleolus is not static as was first thought. Instead, the nucleolus is proving to be a dynamic structure, changing under different cell conditions (442).

The mechanisms of nucleolar regulation and function are still poorly understood.

However, DNA and RNA viruses hijack the nucleolus during infection. The next two subsections discuss how proteins traffic to nucleoli, followed by how viruses use nucleoli to facilitate their replication.

1.8.1 Signals and mechanisms of nucleolar localization

Because nucleoli have a plethora of functions, they must be able to rapidly change their constituent proteins. Fluorescence recovery after photobleaching (FRAP) demonstrate that although the nucleolus is a permanent compartment within the nucleus, nucleolar proteins continuously exchange between the nucleolus and the nucleoplasm (83). This constant exchange generates a stable subnuclear structure and allows for dynamic changes in protein architecture. Although the ribosomal genes (rDNA) form the central core of nucleoli (208), ribosomal RNA transcription is required to maintain nucleolar structure (300). 42 These observations would suggest that RNA is the driving structural element of a nucleolus.

However, nucleolar proteins are also required for stability. Interfering with the expression of the RNA- and protein- binding nucleolar protein nucleolin leads to disruption of nucleolar structure (422). Furthermore, the nucleolar protein B23 has been implicated in facilitating nucleolar localization of specific proteins, including nucleolin (130, 249, 250). From these data, two competing models for nucleolar localization have arisen: active transport and passive diffusion.

Nuclear localization is driven by an active transport system, using both cellular import factors and ATP/GTP (182, 280). Similarly, at least one nucleolar protein requires

GTP for nucleolar localization (420). Furthermore, nucleolar localization signals (NoLSs) are present within a multitude of cellular proteins (Table 1.2A). Although no consensus sequence is known (13, 14, 241), all reported NoLSs contain arginine/lysine rich motifs.

Each of the reported signals is sufficient for nucleolar localization; however the mechanism for localization is poorly understood.

The lack of a common targeting signal and no definable transport mechanism has led to an alternate model for nucleolar localization. FRAP data demonstrating that proteins shuttle between nucleoli and the nucleoplasm suggests a nucleolar retention model

(83). These experiments have led to the proposal that nucleolar proteins have access to most of nucleus through diffusional processes. Therefore, nucleolar proteins may reach the nucleolus without the need for a NoLS. However, once they reach the nucleolus, they interact with other nucleolar components (rDNA, rRNA, nucleolar proteins), transiently trapping them within nucleoli. In support of this proposed model, small stretches of basic residues (such as RRRRRRR from adenovirus protein V) can be fused to a fluorescent protein (270). This small protein diffuses through the nuclear pore complex and localizes to nucleoli. It is thought that nucleolar localization is often a consequence of RNA binding, as nucleolin requires multiple RNA binding domains for traffic to nucleoli (370). However, proteins such as B23, which binds to nucleolin, have been implicated in facilitating nucleolar 43 Table 1.2. Nucleolar localization signals (NoLSs). (A) Cellular NoLSs. (B) Viral NoLSs.

Multiple lines of sequences (with amino acid locations) indicate multiple NoLSs contributing to nucleolar localization. Basic residues are indicated in bold. Abbreviations: HDV, hepatitis delta virus; IBV, infectious bronchitis virus; MDV, Marek’s disease virus; SFV, semliki forest virus; TEGV, transmissible gastroenteritis virus; HSV-1, herpes simplex virus 1; HIV-1, human immunodeficiency virus type 1.

44 A

Protein NoLS Reference

NF-kappaB inducing kinase RKKRKKK (41)

Human angiongenin protein IMRRRGL (257)

Survivin-deltaEx3 MQRKPTIRRKNLRLRRK (391)

Fibroblast growth factor-2 RSRKYTSWYVALKR (382)

B

Protein NoLS (amino acid position) Reference

(23-42) KKEEQDYKPRKLKRVKKKK Adenovirus protein V (270) (159-182) KRGLKRESGDLAPTVQLMVPKRQRL (315-337) RPRRRATTRRRTTTGTRRRRRRR

Adenovirus protein preVII (93-112) RRYAKMKRRRRRVARRHRRR (235) (127-141) RARRTGRRAAMRARR

Adenovirus protein preMu MRRAHHRRRRASHRRMRGG (236)

HDV antigen RKLKKKIKKLEEDNPWCLGNIKGIIGKKDKDGC (229)

IBV nucleoprotein GNSPAPRQQRPKKEKKLKKQ (186)

MDV MEQ protein RRRKRNRDAARRRRRKQ (256)

(66-83) KPKKKKTTKPKPKTQPKK (135) SFV capsid protein (92-105) KKKDKQADKKKKKP

TEGV nucleoprotein RPSEVAKEQRKRKSRSKSAE (186)

HSV-1 ICP27 protein RGGRRGRRRGRGRGG (277)

HIV-1 Rev protein RQARRNRRRRWRERQRQ (90)

HIV-1 Tat protein ISYGRKKRQRRRAHQN (388)

45 localization (250).

Many nucleolar proteins shuttle between nuclear compartments, which can be easily explained by a diffusion model. The NHPX protein, which binds to small nucleolar RNAs such as U4, localizes in nucleoli and Cajal bodies (240). However, microinjection experiments from these same studies demonstrate a complex pathway for NHPX: localization with splicing speckles, Cajal bodies, and nucleoli. A hypothesis that diffusion between the different nuclear subcompartments accounts for the multiple localizations was proposed as the simplest and most elegant available. The other possibility is that multiple step-wise association events are necessary and require multiple subnuclear localization signals. Indeed, a step-wise localization pathway was found during studies of the U3 small nucleolar RNA. Under steady-state condition, U3 localizes to nucleoli and requires the

PHAX and CRM1 proteins for nuclear export of the RNA (311). When the PHAX protein was disrupted using antibodies, U3 was diffuse throughout the nucleoplasm, and excluded nucleoli (48). Inhibition of CRM1 with LMB revealed that PHAX traffics the U3 RNA to Cajal bodies. Although CRM1 is involved primarily in exporting proteins from the nucleus, these results demonstrate that CRM1 contains intranuclear trafficking capabilities. Furthermore, these studies suggest that complex, step-wise trafficking of proteins occurs within nucleoli.

Although two competing models are presented within this section, it is likely that they are not mutually exclusive. Perhaps some proteins are actively transported to nucleoli or are transported between multiple nuclear compartments. Other nucleolar proteins might passively diffuse through nuclei until they reach the nucleolus and are retained through interactions with ribosomal DNA, nucleolar RNA, or nucleolar proteins. Further studies are needed to understand the mechanism or mechanisms involved in the complex trafficking/association pathways of nucleoli.

46 1.8.2 Viruses and nucleoli

DNA and RNA virus replication generally occurs in the nucleus and cytoplasm, respectively. Yet both DNA and RNA viruses affect nucleoli through morphological changes. Moreover, viral proteins traffic through the nucleolus where they interact with cellular or viral factors (Table 1.3). Viral nucleolar proteins are involved in replication, assembly, cell cycle disruption, transcription, and viral RNA export. These viral proteins also contain NoLSs, which show little sequence similarity other than enrichment of basic amino acids (Table 1.2B). Although a few nucleolar viral proteins have been extensively studied, including the HIV Rev protein and the HSV-1 ICP27 protein, very little is known about the role of nucleolar localization in viral replication.

DNA viruses disrupt nucleoli during infection. Adenovirus abolishes the synthesis of rRNA in infected cells (78, 344). Although the mechanism of this process is unknown, it is thought to occur by preventing nucleolar trafficking of fibrillarin (138). Nucleolin and B23 are also redistributed from nucleoli, which appears to be caused by adenovirus protein V (270).

Although protein V is involved in condensing viral DNA inside the virus capsid (270), the role of nucleolar trafficking is unknown. However, B23 stimulates adenovirus replication (312), suggesting that protein V recruits B23 to sites of virus replication.

RNA viruses appear to replicate primarily or exclusively within the cytoplasm. Yet many of these cytoplasmic viruses interact with nucleoli (135, 229, 321). The question for these viruses is: why would a cytoplasmic virus need a nuclear trafficking step during viral replication? The hepatitis delta virus (HDV) appears to use nucleoli for replication of the viral genome. The only protein expressed from HDV is the delta antigen HDAg, which is expressed in two forms (large and small HDAg), both of which localize to nucleoli via interaction with nucleolin (229, 408). B23 interacts with the HDAgs to modulate genome replication via a Pol I transcription pathway (192, 193, 248).

The HIV-1 Rev protein also traffics through nucleoli (37). Although it is imported into the nucleus through a direct protein-protein interaction with importin β (179, 419), B23 is 47 Table 1.3. Viral proteins targeted to nucleoli and their known functions. (A) Nucleolar proteins of DNA viruses. (B) Nucleolar proteins of RNA viruses. The name of the virus, protein name and known functions are presented. Proteins in bold are discussed within the text. Abbreviations: AAV, adeno-associated virus; HBV, hepatitis B virus; HSV-1, herpes simplex virus 1; HVS, herpes saimiri virus; HCV, hepatitis C virus; HDV, hepatitis delta virus;

HIV-1, human immunodeficiency virus type 1; HTLV, human t-cell lymphotropic virus;

MMTV, mouse mammary tumor virus; SARS-CoV, severe acute respiratory syndrome coronavirus.

48 A

Virus Viral protein Function References

AAV Cap, Rep Assembly (203, 441)

Core V, preVII, preMu, (226, 235, 236, 261, 270, Adenovirus IVa2, UXP, E4orf4 Replication 271, 312, 358, 417)

HBV Capsid Cell cycle arrest (306)

ICP0, ICP4, ICP27, Replication, (85, 159, 189, 239, 258, HSV-1 gamma34.5, UL24, Intronless RNA export 262, 277, 278, 298, 352) UL27.5, US11, VP22

HVS ORF57 Intronless RNA export (54)

B

Virus Viral protein Function References

(129, 184, 384) Translation, replication, HCV Core, NS5B (203, 441) cell cycle arrest

HDV HDAg Replication (192, 193, 229)

Transcription, HIV-1 Rev, Tat (37, 101, 249) unspliced RNA export

Rex, p30, Transcription, HTLV (3, 32, 187, 220, 305) HBZ-SP1 unspliced RNA export

(196) MMTV Rem Unspliced RNA export

SARS-CoV N-protein, 3b Cell cycle arrest, apoptosis (215, 404, 415, 447, 449, 452)

49 thought to localize Rev to nucleoli (130). The role of Rev is to facilitate nuclear export of unspliced viral RNAs via CRM1 nuclear export (137, 266, 267). When the Rev protein specifically interacts with the Rev-responsive element (RRE), it displaces B23 and presumably interacts with CRM1 (130). CRM1 is recruited to nucleoli, along with the export factors Nup98 and Nup214, further implicating nucleoli as the site of Rev function (459).

Additionally, RRE fused to a small nucleolar RNA is efficiently exported from the nucleus by

Rev (64, 285). Further evidence for nucleoli as a site of viral protein-RNA interaction comes from herpesviruses. The herpesvirus saimiri (HVS) ORF57 protein requires nucleolar trafficking for the export of intronless viral mRNAs (54). When the nucleolar localization signal of ORF57 is deleted, viral RNA accumulates within the nucleus. However, with insertion of a heterologous NoLS from the HIV-1 Rev protein, nuclear export of intronless viral RNA is rescued. Together, these experiments suggest nucleoli as a viral nucleoprotein assembly site.

When nucleolar localization of viral proteins was first discovered several decades ago, it was thought that they accumulated within nucleoli due to RNA binding properties.

However, in the past decade, an explosion of research is beginning to define the role of nucleolar localization in viral replication. With the multiple roles and complex trafficking of many viral proteins, more study is still needed to explore the role of nucleoli in both cellular and viral mechanisms.

1.9 Live cell imaging techniques

Understanding the dynamic process of proteins is a daunting task. Although in vitro studies have revealed much for the actions and interactions of proteins, these experiments do not necessarily mimic what occurs within the cell. Thus, new techniques were necessary to study the interactions and trafficking of proteins in vivo. Within this dissertation, two complex microscopy techniques are frequently used: fluorescence recovery after

50 photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The next sections discuss both methods: how they work and what data can be obtained from them.

1.9.1 Fluorescence recovery after photobleaching (FRAP)

Binding interactions and protein kinetics has generally been studied through in vitro biochemical assays. However, with the advent of fluorescent proteins and fluorescent microscope systems capable of rapid image capture, in vivo techniques have been developed to study protein kinetics in live cells. One such technique is called fluorescence recovery after photobleaching (FRAP) (251, 336). In FRAP, live cells expressing fluorophore tagged proteins are permanently photobleached within a specific region of interest (Fig. 1.7A). After bleaching, the region is tracked over a time for recovery. The data gathered from this assay is used to determine the mobility and kinetics of a fluorophore- tagged protein.

Prior to data analysis, several corrections must be performed to compensate for the loss of fluorescence from the photobleaching event, and from fluorescence acquisition loss

(331). The photobleaching event removes a portion of fluorescence permanently. One part of the analysis reports the mobility of a protein, and this report can be skewed if the entire fluorescence is not accounted for. Furthermore, image acquisition at each time point causes partial photobleaching of the entire cell. Image acquisition loss is partially minimized using the lowest laser intensity possible. Both acquisition and bleaching loss can be corrected for mathematically (331). First, the entire cell is monitored for fluorescence change during the FRAP assay (Fig. 1.7B). Each time point is corrected using the equation

T0 It Irel= , where T0 is the initial total cell fluorescence, Tt is the total cell fluorescence at time Tt I0 point (t), I0 is the initial region of interest fluorescence, and It is the region of interest fluorescence at time point (t). The result is the relative fluorescence intensity (Irel), which is a fraction of the initial region of interest fluorescence, plotted against time (Fig. 1.7C).

51 Fig. 1.7. Fluorescence recovery after photobleaching (FRAP). (A) A live cell (nucleus indicated in blue) expressing a fluorescently tagged protein (green) is imaged under confocal microscopy. A region of interest is bleached using a high intensity laser (black box). The same region is tracked over time for recovery of fluorescence (light green box).

(B) Fluorescence loss due to photobleaching and image acquisition. The red line shows photobleaching loss. The green line indicates image acquisition loss. (C) An idealized

FRAP graph is presented. After normalization (see text for description), the fluorescence recovery is plotted as relative fluorescence (Irel) vs. time. There are three important fluorescent highlights (red horizontal lines): starting fluorescence (Fpre), fluorescence after photobleaching (Fpost), and final recovery fluorescence (Fend). These three values are used to calculate the mobile and immobile fractions of the protein (blue vertical lines) (see text for calculation). The half time of equilibration (green vertical line) (t1/2) is also determined from the graph (see text for calculation).

52

53 The normalized graph is now analyzed for mobility and kinetics. If a protein is mobile, a large fraction of the fluorescence is expected to recover within the region of interest. In contrast, immobile proteins will have little to no recovery within the region.

Whether a protein is mobile or immobile is determined by calculating the mobile (Fm) and immobile (Fimm) fractions (Fig. 1.7C, vertical blue lines). The mobile fraction is determined

Iend-Ipost by the equation Fm= , where Iend is the final relative fluorescence intensity, Ipost is the Ipre-Ipost relative fluorescence intensity immediately after photobleaching, and Ipre is the relative fluorescence intensity prior to photobleaching. The immobile fraction is calculated as

Fimm=1-Fm. The mobility of different proteins or the mobility of the same protein at different subcellular sites can then be compared. In this dissertation, protein mobility is compared in chapters 2 and 3.

The kinetic profiles of a fluorescently tagged protein are also determined via the normalized FRAP graph. Using curve-fitting software, a non-linear regression curve is fit to

-K*x the data using the equation Y x =Iend(1-! ), where K is the fitting variable. Half-time is

ln(0.5) calculated using the equation t = . Although it seems likely that protein size would 1/2 -K have an effect on protein kinetics, protein mass does not drastically alter the half-time (e.g.

27 kD, 0.011 s; 100,000 kD, 0.173 s) (346). Thus, half-time changes in the magnitude of seconds are due to binding differences, not changes in mass. By mutational analysis or insertion of binding partners, the difference in trafficking may be observed under different conditions (76, 217, 379). The effect of the ψ sequence on Gag trafficking in the cytoplasm is examined in the appendix of this dissertation.

1.9.2 Fluorescence resonance energy transfer (FRET)

Colocalization analysis through fluorescence microscopy only reveals whether two proteins are within the same general vicinity (46). However, the resolution limit of light microscopes is approximately 200 nm, far exceeding the size of most proteins (~5 nm) 54 (287). Thus, even if two proteins are within the same subcellular compartment, whether they directly interact remains unknown. Clearly, a technique that reports protein-protein interactions within context of a living cell would be highly beneficial in the study of protein function.

The answer to this problem came from physical phenomenon, through a process known as resonance energy transfer (141). However, this physical phenomenon was not applied to biological approaches until several decades later with the advent of different colored fluorescent proteins (FPs) (378). This technique is known as fluorescence resonance energy transfer (FRET). FRET is possible when the excitation spectrum of a donor FP overlaps the excitation spectrum of an acceptor FP (Fig. 1.8A). This transfer is nonradiative (i.e. no fluorescence occurs within the donor if energy transfer occurs), but it requires the FPs be extremely close. The efficiency of energy transfer can be calculated as

R6 E= 0 , where r is the distance between the two fluorescent proteins and R is the Förster 6 6 0 R0+r distance of the two FPs (377). The Förster distance is calculated from experimentally derived values that describe the distance at which the two FPs would have a 50% energy transfer. The fluorophore pair used exclusively in this dissertation is CFP/YFP, which have an R0 of 49-52 Å (4.9-5.2 nm) (386). FRET with this fluorophore pair allows detection of proteins within 70 Å (7 nm) of each other, indicating a probable protein-protein interaction.

Although FRET offers a valuable tool to determine the distance between two proteins, practical limitations (e.g. dipole orientation, concentration of donor relative to acceptor) prevent these measurements in vivo. Instead, FRET has been extensively used in monitoring the assembly of protein complexes and the dynamics of protein-protein interactions (12, 124, 125, 225). Therefore, FRET has become a valuable and powerful tool in the evaluation of protein-protein interactions within a cellular environment.

Measurement of FRET can be performed in three different ways: donor quenching, sensitized emission of the acceptor, or acceptor photobleaching. The method exclusively

55 Fig. 1.8. Fluorescence resonance energy transfer (FRET). (A) FRET occurs if the emission spectrum of the donor (solid blue line) overlaps the excitation spectrum of the acceptor (dashed yellow line). The overlapping region is shown in green. (B) Acceptor photobleaching FRET requires the two proteins assayed be fused to either the donor fluorophore (CFP, blue circle) or the acceptor fluorophore (YFP, yellow circle). If the fluorophores are close to each other, the donor will transfer energy to the acceptor, resulting in decreased donor fluorescence and increased acceptor fluorescence. After photobleaching the acceptor fluorophore using a specific laser, the donor no longer has a partner for energy transfer. The result is an increase in CFP fluorescence, indicating FRET had occurred between the fluorophores.

56

57 used in this dissertation is acceptor photobleaching (Fig. 1.8B). Although photobleaching does not allow tracking the dynamics of protein-protein interactions, each cell assayed serves as its own control, allowing a single transfection to be used for each protein pair assayed. In contrast, donor quenching and sensitized emission require three separate transfections: one double transfection for the experiment and each fluorescently fused protein transfected individually. The single transfections act as a control for the bleedthrough of fluorescence between the CFP channel and the YFP channel. Channel bleedthrough occurs if the emission spectrum of the donor overlaps with the emission spectrum of the acceptor. If they do, an apparent fluorescence of the acceptor will appear when the donor is expressed alone. With acceptor photobleaching, only the CFP is measured for the calculation of FRET efficiency, eliminating the possibility of channel bleedthrough.

To assay for FRET using the acceptor photobleaching method, two proteins of interest are fused to the fluorescent proteins: one protein to CFP, and the other protein to

YFP. If no protein-protein interaction occurs, there will be no change in fluorescence intensity of either donor or acceptor. If the two proteins are interacting, and the fluorescent proteins are brought within FRET range, the donor (CFP) will transfer its energy to the acceptor (YFP). In effect, this energy transfer causes the fluorescence intensity of CFP to decrease. However, at this point we have no idea if the transfer is occurring. To detect

FRET, a high-intensity laser specific for YFP (514 nm) is used to irrevocably photobleach the acceptor (YFP) while not affecting the donor (CFP). If the two FPs are close enough for

FRET to occur, CFP will now increase in fluorescence. The fluorescence intensity of CFP must be measured before and after the photobleaching event occurred. The resulting

DonorPre- DonorPost intensity values entered into the equation: FRETEff= , when DonorPost is DonorPre greater than DonorPre. Thus, if there is a concomitant increase in CFP fluorescence and

58 YFP fluorescence decreases, energy transfer has occurred. Such a result indicates that there is a direct protein-protein interaction.

Unfortunately, there are several caveats to FRET interpretations. Even if there is no

FRET signal from an experiment, the proteins may be interacting. The fluorophores may be spatially separated in such a way that energy transfer is impossible. One way to correct for this possibility: if the protein structure is unknown, the fluorescent protein may be fused to either end to facilitate the detection of FRET. In contrast, false positives may also occur.

Given that FPs are often fused to proteins via a flexible linker, it is possible to conceive a scenario where two proteins are in a complex, yet their FPs are close enough for FRET to occur. The concentrations of donor and acceptor fluorescent proteins also affect FRET efficiency (146). Crowding of proteins within the same subcellular compartment may cause the presence of weak FRET signals, due to the intrinsic weak interactions between CFP and

YFP. Additionally, the number of donor molecules contributes more to FRET than the number of acceptor molecules. Thus, in situations where a high concentration of acceptor

FPs share the same space as a low concentration of donor FPs, a FRET signal may be detected amongst proteins that do not interact. However, using a protein that is in the same location and is known to not interact controls for this potential problem. Thus, although many problems present themselves during FRET analysis, these obstacles can be overcome through proper controls and thoughtful experiments.

1.10 Conclusion

After translation by the ribosome, the RSV Gag polyprotein undergoes transient nuclear trafficking before assembly and virus budding at the plasma membrane. Genetic evidence has demonstrated that Gag nuclear trafficking is linked to efficient incorporation of gRNA into virus particles. However, the mechanisms of nuclear trafficking are poorly understood. In the following chapters of this dissertation, I will communicate my seminal

59 work identifying multiple subnuclear trafficking events to answer some of the questions presented within this literature review.

60

Chapter 2

Characterization of Subnuclear Foci Formed by Gag Proteins

Restricted to the Nucleus

Figures 2.1 and 2.2 adapted from:

Kenney SP, Lochmann TL, Schmid CL, Parent LJ. J Virol 2008, Jan; 82 (2):683-691

Copyright 2008 American Society for Microbiology

61 2.1 Abstract

The retroviral Gag polyprotein mediates both retroviral assembly and incorporation of viral genomic RNA (gRNA) into new virus particles. The early steps of assembly, immediately after Gag synthesis, remain poorly understood. Rous sarcoma virus (RSV)

Gag proteins undergo a nuclear trafficking step prior to assembly at the plasma membrane, yet little is known as to what occurs during this nuclear step. We previously identified Gag mutants that are unable to leave the nucleus, and asked whether Gag-Gag interactions occur within the nucleus. Visualization of nuclear-trapped Gag via confocal microscopy revealed the formation of subnuclear foci. Fluorescence resonance energy transfer indicated that these structures are sites of Gag-Gag interactions, and were dependent upon the NC domain. The foci were larger than virus-like particles (VLPs) found at the plasma membrane and did not appear to be virus like particles when examined via transmission electron microscopy. Particle tracking revealed that Gag nuclear puncta were anchored within the nucleoplasm, but fluorescence recovery after photobleaching demonstrated that

Gag proteins were moving between the subnuclear structures and the nucleoplasm. These data suggest that Gag proteins are dynamic within the nucleus and accumulate at distinct locations, possibly through RNA-mediated binding to a host factor.

2.2 Introduction

The Gag polyprotein is both necessary and sufficient to drive assembly and release of retrovirus particles from the plasma membrane (167, 224, 440). The Gag polyprotein consists of the MA (matrix), p2, p10, CA (capsid), and NC (nucleocapsid) proteins. After being translated in the cytoplasm, Gag transiently traffics through the nucleus (363).

Nuclear entry of Gag is mediated by nuclear localization signals (NLSs) within the MA and

NC domains (67, 172). Nuclear export is controlled via a CRM-1 nuclear export sequence

(NES) found within the p10 domain (363, 366). The Gag NES consists of the residues

LTDWARVREEL (366). Mutation of any or all of the hydrophobic residues (LWVL) results in 62 an accumulation of Gag within the nucleus. However, these observations lead to the question of why Gag nuclear trafficking occurs during viral assembly.

Although particle release is known to occur at the plasma membrane, recent studies indicate that Gag-Gag interactions occur in the cytoplasm prior to reaching the plasma membrane. Wildtype Gag restores budding to membrane-binding deficient Gag mutants, suggesting Gag proteins interact before arriving at the plasma membrane (15, 35, 59, 317).

Cytoplasmic extracts from cells expressing Gag contain higher-order Gag complexes, which form in a stepwise manner from monomers, to dimers, to multimers (255). Finally, recent experiments using of fluorescently tagged proteins, using fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) analyses have shown that direct Gag-Gag interactions occur within the cytoplasm (225). These lines of evidence begin to shed light on a much more complex pathway of Gag movement from translation to the membrane.

Gag trafficking is more complex than direct transport from the ribosome to budding at the plasma membrane. Under steady-state conditions, Gag is found localized to the cytoplasm and plasma membrane and nuclei appear devoid of Gag (363). However, several retroviral Gag proteins transiently localize to the nucleus, including RSV, murine leukemia virus (MLV), and foamy virus (15, 363, 368). RSV appears unique in that it contains a CRM1-dependent nuclear export signal (NES) within the p10 domain (363). The ability to sequester Gag within the nucleoplasm allows for an examination into the role of

Gag nuclear trafficking. Due to this NES, treatment with CRM1 inhibitory drug, leptomycin B

(LMB), results in the accumulation of Gag within the nucleus (363). Furthermore, mutation of hydrophobic residues within the NES cause Gag to become trapped within the nucleoplasm (366). Gag proteins containing an NES mutation are severely inhibited in virus particle release, indicating that nuclear trafficking is involved in an early step of virus particle assembly. These results suggest that nuclear trafficking of RSV Gag compartmentalizes the

63 steps pertaining to early stages of assembly, culminating in the final steps of plasma membrane interaction and release of virus particles from the cell.

Although what occurs while Gag is present in the nucleus is not well known, bypassing the nucleus reduces gRNA incorporation into new virus particles (363). Insertion of a heterologous nuclear localization signal (NLS) restores packaging to near-wildtype levels (151). These results indicate that Gag nuclear trafficking is linked to incorporation of the viral genome into virus particles. However, it is not known why Gag nuclear trafficking is needed, or where the selection of viral RNA by Gag occurs.

Both DNA and RNA viruses commonly localize to subnuclear components to hijack or interfere with cellular processes during the course of infection (185, 345, 458). Herpes simplex virus replicates and forms new virus capsids within discrete nucleoplasmic foci

(ND10 bodies) (127, 128, 259, 392). The Rev protein from Human immunodeficiency virus type 1 interacts with the viral RNA encoding the Rev-response element within nucleoli to facilitate export of unspliced transcripts (130, 328, 390). Recent studies have also found that a Rev mutant localizes to splicing speckles, suggesting Rev interacts with multiple subnuclear bodies (100). Furthermore, the foamy virus Gag protein forms discrete foci within the nucleus, which are tethered to the host chromatin (416). Together, these studies suggest that viral proteins within the nucleus have multifaceted mechanisms to assist in making new virus particles.

Because Gag-Gag dimers are considered the smallest assembly unit (255), we asked whether Gag dimers form within the nucleus. This chapter focuses on the identification of Gag-Gag interactions within the nucleus. Furthermore, we characterized the properties of these Gag subnuclear foci.

2.3 Materials and Methods

Expression vectors and cells

64 RSV Gag expression plasmid, pGag ΔPR, was described previously (366). The Gag p10 NES mutations encoding L219A, LWVL-A, and NES-A in pGag-GFP (71, 364) were transferred into pGag-CFP and pGag-YFP expression vectors (gifts from V. Vogt, Cornell

University)(225) using SstI-SdaI fragment exchange. pGag.ΔNC-CFP and pGag.ΔNC-

YFP were created using SacI-ApaI fragment exchange between pΔNC-GFP (a gift from J.

Wills, Penn State College of Medicine) (70) and pGag-CFP or pGag-YFP. Clones were screened using restriction endonuclease digestion and confirmed with automated DNA sequencing. A schematic representation of Gag and Gag mutants is presented in Fig. 2.1A.

All experiments were performed using QT6 quail fibroblast cell line (183).

Transfections were performed using the calcium phosphate method (144).

Confocal imaging

0.2 x 106 cells were plated onto 35-mm glass-bottomed dishes (MatTek Corporation) and imaged either live or fixed in 2% paraformaldehyde using a Leica AOBS SP2 confocal microscope at 17 to 24 hours post-transfection. Sequential scanning settings were used to differentiate CFP (excitation at 458 nm, emission at 465-490 nm, and 50% laser power) and

YFP (excitation at 514 nm, emission at 530-600 nm, and 10% laser power) emission spectra.

FRET measurements

Acceptor photobleaching FRET was performed on transfected cells fixed with 4% paraformaldehyde. Prebleach images of both cyan fluorescent protein (CFP) (excitation at

458 nm, emission at 460 to 500 nm, and 20% laser power) and yellow fluorescent protein (YFP) (excitation at 514 nm, emission at 550-600 nm, and 10% laser power) channels were acquired. YFP was specifically photobleached using the 514 nm laser at

100% laser power until the fluorescence intensity was decreased to 10% of the prebleach level. Postbleach images were acquired at the prebleach settings. FRET

donorPost − donorPre efficiency was calculated using the formula FRETEff = , when donorPost 65 donorPost is greater than donorPre. FRET analysis was performed with at least two separate transfections using a minimum of 10 different cells per transfection. Nuclear FRET was performed by bleaching the entire nucleus through a single optical section of the nuclear plane.

Volumetric analysis

Transfected cells on 35-mm glass bottom dishes were imaged live on an Olympus microscope, using Slidebook as the image capture software (Intelligent Imaging Innovations,

Inc.). A z-series containing either the entire cell nucleus (L219A.Gag-YFP) or the entire cell

(Gag-YFP) was captured. The nearest-neighbor deconvolution algorithm was performed on captured z-series to remove out-of-focus light. Volumetric analysis was then carried out on either nuclear foci (L219A.Gag-YFP) or VLPs at the plasma membrane (Gag-YFP) using the

Slidebook software. Briefly, a 3D segmentation mask was created to include as many foci as possible, followed by volumetric analysis using the mask statistics function. Volumetric analysis was performed on at least 3 cells from a single transfection of either L219A.Gag-

YFP or Gag-YFP. A total of 69 nuclear foci (L219A.Gag-YFP) or 238 membrane virus-like particles (Gag-YFP) were analyzed for each construct.

Correlative Light-Electron Microscopy

Correlative Light-Electron microscopy was performed as previously described (347).

Briefly, number 1.5 coverslips were seeded with cells expressing either L219A.Gag-YFP were fixed with 2% paraformaldehyde. A gridded mesh was placed under the coverslip and fluorescence was observed using the Leica AOBS SP2 confocal microscope. Cells presenting a punctate phenotype of Gag were identified, and their grid location was noted.

The cells were processed using negative staining, and embedded in Quetol 651 resin. After a second grid was placed in register with the first, the individual grid points containing the cells identified using confocal microscopy were extracted and thin-sectioned by microtome and imaged as previously described (71)

Particle tracking 66 The Leica AOBS SP2 confocal microscope was used to capture 3D time-lapse images of live, transfected cells on 35-mm glass bottom dishes. The cells were imaged on a live cell stage set at 38.5°C and 5% CO2. The 514 nm laser was set to the lowest possible intensity for image capture (10% or less). A z-stack approximately 4µm in size, encompassing the entire nucleus, was imaged in 0.2 µm steps for a total of approximately

20 z-sections per cell. Each of these 3D captures occurred approximately every minute for

2 to 10 minutes. The captured data was imported into Imaris analysis software (Bitplane) and examined for particle movement. Briefly, particles of were automatically identified in 3D space for each time point. Only particles that could be identified across the entire 10 minute time-lapse were included within the analysis. After focal drift compensation was performed via the software, a line representing the movement of each individual particle was then superimposed onto the 3D time-lapse. The Imaris software reported statistics for individual and average particle movement. A total of three cells were captured, over a time period of 2 to 10 minutes. Only a single 10-minute time-lapse was analyzed using the Imaris software and a total of 25 nuclear foci within this 3D time-lapse were identified for particle tracking.

FRAP analysis

Fluorescence recovery after photobleaching (FRAP) was performed at 38.5°C on live cells transfected with either pL219A.Gag-YFP or pGag-YFP. All assays were performed at a scan speed of 800 Hz with an acquisition time of 0.8 or 1.6 seconds. Five pre-bleach images were acquired using the YFP channel settings at 10% laser power. YFP in nuclear foci (L219A.Gag-YFP) or at the plasma membrane (Gag-YFP) was specifically photobleached using the 514 nm laser at 100% power over four time-points. Recovery was monitored for approximately 60 to 200 seconds at 10% laser power. As a control for photobleaching and fluorescence lost during acquisition, the fluorescence of the entire cell was monitored during the experiment. The background from both the region of interest and the total cell fluorescence was subtracted. Corrections for photobleaching and normalization of the data was performed as previously described (331). Briefly, the relative 67 T0It fluorescence intensity was calculated by IRel= , where T0 is the total cell intensity before TtI0 bleaching, Tt is the total cell intensity at time-point t, I0 is the intensity of the region of interest before bleaching, and It is the intensity of the region of interest at time-point t. The mobile

IE-I0 fraction of each region was determined using Fm= , where IE is the fluorescence in the II-I0 bleached region after full recovery, I0 is the fluorescence immediately after bleaching, and II is the pre-bleach fluorescence. A minimum of 3 assays was performed on Gag at the plasma membrane or within nuclear foci.

2.4 Results

Subnuclear localization of LMB-treated wildtype Gag and p10 NES mutant Gag proteins

Using FRET analysis, RSV Gag-Gag interactions are observed within the cytoplasm and at the plasma membrane (225). However, whether Gag-Gag interactions occur within the nucleus has not been explored. To address this possibility, we co-transfected cells with

Gag-CFP and Gag-YFP and treated with LMB for 1.5 hours. Drug-treated cells were then imaged by confocal microscopy. Two distinct phenotypes were observed within the transfected cells: Gag was diffuse throughout the nucleoplasm (Fig. 2.1B, panel a), or within discrete punctate foci within the nucleoplasm (Fig. 2.1B, panel a’).

As an alternative to LMB drug treatment, a mutant of Gag containing substitution of the p10

NES with alanines (NES-A) was fused to YFP and to CFP and were both co-transfected into cells without LMB treatment. NES-A.Gag-YFP appeared diffuse in one subset of cells and formed punctate foci in a second subset of cells, suggesting that LMB treatment does not induce the formation of Gag nuclear foci (Fig. 2.1B, panels b and b’). Deletion of the NC domain (Gag.ΔNC) eliminated Gag nuclear foci, indicating that the puncta are NC dependent (Fig. 2.1B, panel c). Next, we tested whether the nuclear foci were protein- protein or protein-RNA interaction dependent using Gag.Zip-YFP/CFP constructs, which

68 Fig. 2.1. Intranuclear distribution patterns of Gag proteins. (A) Schematic representation of Gag and Gag mutants used in this study. The gray box indicates the p10 domain, which contains the mapped NES amino acids as shown below. The essential hydrophobic residues are in bold. Mutations of the NES are listed below the wildtype, where a single hydrophobic residue (L219A), the entire NES (NES-A), or all of the hydrophobic residues (LWVL-A) are mutated to alanine. Schematic diagrams of fluorescent proteins are listed, with “XFP” representing either CFP or YFP fusions. Expression of both CFP and YFP was performed for FRET analysis in Fig. 2.2. Gag. ΔNC contains a deletion of amino acid residues 495 to 577 in the NC domain, removing most of NC. Gag.Zip has the leucine zipper domain of the human CREB binding protein (Zip) substituted in place of the NC domain. (B)

Fixed QT6 cells were imaged through the nuclear plane using confocal microscopy. Diffuse intranuclear patterns are shown on the left, punctate distributions are on the right, and cells having different subnuclear distribution patterns were derived from a single transfection.

Cells treated with LMB are indicated as “+LMB”. Punctate foci were not observed for ΔNCGag-YFP/CFP or Gag.Zip-YFP/CFP constructs.

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70 contain a protein dimerization domain (leucine zipper) in place of the NC domain (204). The

Gag.Zip constructs bind in a protein-protein dependent manner in the absence of RNA.

Expression and visualization of these constructs with LMB treatment also demonstrated a lack of nuclear Gag aggregations, indicating that a protein-protein interaction domain cannot be substituted for the NC domain (Fig. 2.1B, panel d).

Analysis of intranuclear Gag-Gag interactions through FRET

The formation of Gag nuclear foci may be due to the accumulation of protein via

Gag-Gag interactions. To identify whether Gag-Gag interactions were occurring within the nuclear foci, we used acceptor photobleaching FRET analysis (209). A single confocal z- section was imaged in cells co-expressing either wildtype Gag-YFP/CFP or NES mutant

Gag-YFP/CFP. Representative raw data images for FRET between NES-A.Gag-YFP and

NES-A.Gag-CFP are shown (Fig. 2.2A). A region of interest (ROI) encompassing the entire nucleus was specifically photobleached using a laser specific for YFP (514-nm at 100% intensity). As expected, successful photobleaching of the nucleus in the YFP channel was observed (Fig. 2.2A, right). A simultaneous increase in fluorescence intensity of CFP (Fig.

2.2A, left) was also seen, indicating that FRET had occurred (Fig. 2.2A, left).

Average FRET efficiency values were collected from at least 10 different cells in two separate transfections. Data was gathered for both the diffuse and punctate localization patterns of Gag (Fig. 2.2B). Cells co-expressing free CFP and YFP were used as a negative control, and should not interact. As expected, a low FRET efficiency between CFP and YFP was detected within the nucleus (0.6%). Cells co-transfected with wildtype Gag-

CFP and Gag-YFP were treated with LMB to elicit Gag relocalization to the nucleus. FRET efficiencies were collected for both the diffuse (2.0%) and punctate (9.4%) localization phenotypes. FRET assays were also performed on the NES Gag mutants L219A (diffuse,

1.4%; punctate, 9.4%), LWVL-A (diffuse, 3.4%; punctate, 8.5%), and NES-A (diffuse, 4.9%, punctate, 12.8%). Although the formation of nuclear foci was not present, the GagΔNC-

CFP/YFP pair did not eliminate Gag-Gag interactions as indicated by FRET efficiency higher 71 Fig. 2.2. Intranuclear Gag-Gag interactions assessed by acceptor photobleaching

FRET analysis. (A) FRET analysis was performed on cells coexpressing NES-A.Gag-CFP and NES-A.Gag-YFP. Confocal images through the nuclear plane were obtained prior to photobleaching (top). The nuclear region of interest, indicated by the white circle, was subjected to bleaching of the YFP (acceptor) fluorophore using a high-intensity laser at a wavelength of 514 nm, resulting in a significant loss of the signal in the postbleach NES-

A.Gag-YFP image (lower right). The increased intensity of the NES-A.Gag-CFP fluorescence in the postbleach image (lower left) resulted from the decreased transfer of energy from the CFP fluorophore (donor) to YFP. (B) QT6 cells expressing the indicated proteins demonstrating punctate (P) or diffuse (D) phenotypes (as shown in Fig. 1) were analyzed using FRET. The FRET efficiency (percent) for each condition was calculated according to the equation shown in Materials and Methods. FRET experiments were performed a minimum of 10 times from at least two separate transfections. The mean FRET efficiency was calculated and graphed, with error bars representing standard errors of the means.

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73 than the CFP/YFP negative control (4.9%). The FRET efficiency for Gag.Zip-CFP/YFP expressing cells remained high (11.1%), indicating that Gag-Gag interactions were occurring within the nucleoplasm in the absence of nuclear foci. The high FRET efficiency without the formation of nuclear foci with Zip containing Gag proteins supports the idea that the NC domain is necessary for the formation of nuclear foci.

Comparison of Gag nuclear foci to VLP formations at the plasma membrane

Although RSV does not normally form particles in the nucleus, we considered that these foci of Gag could be virus-like particles (VLPs) within the nucleoplasm. We expected if the Gag nuclear foci were VLPs forming in the nucleus, they would have a size similar to

VLPs at the membrane. To determine whether the nuclear foci were VLPs, we acquired 3- dimensional images of cells expressing Gag-YFP or L219A.Gag-YFP, using a spinning disc

Olympus microscope. Nearest-neighbor deconvolved images with the top and side views of

Gag-YFP (Fig. 2.3A, panels a and b), and L219A.Gag-YFP (Fig. 2.3A, panels c and d) are shown. After deconvolution of the z-series, we performed volumetric analysis on both plasma membrane (Gag-YFP) and nuclear foci (L219A.Gag-YFP), using the Slidebook acquisition software.

To perform the size measurement, an individual cell from a captured image was selected. A segment mask was layered over the fluorescence data (Fig. 2.3A, a’-d’), and adjusted until individual Gag foci could be seen within the mask. Volumetric data was obtained using the mask statistic module within Slidebook. Only Gag foci or VLPs that had clearly defined borders were selected for analysis.

A total of three cells were analyzed using the volumetric assay, with 238 particles counted for Gag-YFP, and 69 particles counted for L219A.Gag-YFP (Fig. 2.3B). The volumes of VLPs at the plasma membrane were an average of 0.12 µm3. This size does not correspond to the expected volume of an RSV particle (approximately 0.001 µm3, r = 0.0635

µm), but there are multiple explanations for this discrepancy. The radial distribution of fluorescence from YFP may be misleading for an accurate measurement of particle 74 Fig. 2.3. Volumetric analysis of Gag nuclear foci. (A) Deconvolved, 3-dimensional images of Gag-YFP (top view, panel a; side view, panel b), and L219A.Gag-YFP (top view, panel c; side view, panel d) are shown. The Slidebook software was used to create a segment mask, identifying multiple particles for analysis (panels a’-d’). Due to image cropping for volumetric analysis, the Gag-YFP and L219A.Gag-YFP images are not to scale.

The scale bar presented represents approximately 25µm. (B) The average of 238 (Gag-

YFP) or 69 (L219A.Gag-YFP) are graphed, with bars representing the standard error.

Statistical analysis was performed using student’s t-test (*, P<0.0001).

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76 volumes. Additionally it is likely that conventional light microscopy techniques are not precise enough to accurately estimate the true volume of a virus particle. However, the relative sizes of membrane and nuclear puncta can be compared. Accumulations of

L219A.Gag in the nucleus had an average volume of 0.29 µm3, and the L219A.Gag nuclear foci are significantly larger than the membrane particles (P<0.0001). These results suggest that L219A.Gag nuclear foci are not normally sized VLPs or not VLPs at all. It is possible that the aggregates are groups of VLPs or VLPs that are larger than VLPs found at the plasma membrane.

Analysis of Gag nuclear foci by electron microscopy

If the Gag proteins were forming large aggregates or Gag complexes, we would expect to find a large electron-dense aggregation by electron microscopy (71). Alternatively, if VLPs were forming within the nucleus, we would expect to find electron dense spheres similar to what is seen at the plasma membrane or with released virions. Therefore we used electron microscopy to determine whether Gag was capable of forming particles inside the nucleus, or if the nuclear foci were accumulations of the Gag protein.

One problem that arises with the formation of Gag nuclear foci is the dependence on high protein concentrations. We used a method called correlative light-EM microscopy (Fig.

2.4A) (347). This method allows us to identify specific cells under confocal microscopy and image the same cells using electron microscopy. Briefly, cells expressing L219A.Gag-YFP were fixed in 2% paraformaldehyde and mounted onto a slide, with a wire mesh placed on the coverslip. Fluorescent and light images were observed, identifying cells of interest within the grid pattern. The cells were then processed for electron microscopy and sealed with resin. A second mesh was placed on top of the resin, in register with the second, and the identified regions were thin-sectioned using a microtome. Images were then obtained via transmission electron microscopy.

Using Gag-YFP as a control, we observed particle formation at the plasma membrane (Fig. 2.4B). However, we were unable to detect any electron dense structures 77 Fig. 2.4. Transmission electron microscopy of L219A.Gag-YFP. (A) The schematic diagram depicts the correlative electron microscopy technique used to obtain images. (1) A coverslip containing cells expressing L219A.Gag-YFP are fixed in 2% paraformaldehyde and mounted on a glass slide, and a mesh grid is placed on the coverslip. (2) Confocal images are acquired, and grids containing Gag nuclear foci phenotypes are identified. (3)

Coverslip is placed in a 35 mm dish, stained for EM, and embedded in resin. (4) The coverslip is excised from the resin. (5) A second mesh is placed on the resin, in register with the mesh on the coverslip. (6) The coverslip is removed, and the selected regions within the mesh are processed for transmission electron microscopy. (B) Electron micrograph of a Gag-YFP particle released from the membrane. Image is at 16,500x magnification. Scale bar depicts 1 µm. Black arrowhead indicates VLP released from the membrane. (C) Electron micrograph of a nucleus transfected with L219A.Gag-YFP. Image is at 16,500x magnification, with 1 µm scale bar. Yellow arrowhead identifies nucleoli.

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79 within the nucleus of a cell expressing L219A.Gag-YFP nuclear foci (Fig 2.4C). This observation supports the idea that the punctate formations of Gag within the nucleus are not

VLPs. Nor are they large, electron-dense aggregations of Gag proteins.

Particle tracking of Gag nuclear foci

Unable to identify any virus-like particles within the nucleus, we next considered two possibilities: that the Gag nuclear foci were protein aggregates, either freely diffusing or trafficking through the nucleus, or that these punctate spots were anchored in place by protein or nucleic acid. To test these potential models, we performed particle tracking on

L219A.Gag-YFP nuclear foci. First, we captured 3-dimensional time-lapse images using confocal microscopy. Live cells expressing L219A.Gag-YFP were placed onto a live-cell stage and kept at physiological conditions (38.5°C and 5% CO2). A single cell nucleus containing Gag nuclear foci was identified for capturing images. For the representative experiment, a z-stack consisting of 20, 0.2µm-thick optical sections were captured every minute for 10 minutes. Both a top and side view of the entire nucleus are shown (Fig. 2.5a and a’, respectively). Although a noticeable decrease in fluorescence is seen after imaging

(Fig. 2.5b and b’), the loss of signal does not affect the analysis. After acquisition, the data set was imported into Imaris imaging software and analyzed for particle movement. The software was able to identify 20 separate nuclear foci for analysis during the entire 10 minute time period. Each particle was tracked for movement and entire motion paths were plotted (Fig. 2.5c and c’). A closer view of several individual particles is also shown (Fig.

2.5d and d’). The nucleus of the imaged cell was approximately 15µm x 4µm x 4µm, and the average overall displacement of the nuclear foci was 0.33µm. A total of three cells were imaged for 3D time-lapse (imaging times ranging from 2 to 10 minutes). These results suggest that the nuclear foci were immobile, similar to transcription factories (405).

However, only one cell (Fig. 2.5) was analyzed using the Imaris software. The results found through particle tracking suggest that the nuclear foci of Gag are not moving through the

80 Fig. 2.5. Time-lapse tracking of Gag nuclear foci. QT6 cells expressing L219A.Gag3h-

YFP were observed using time-lapse 3D confocal microscopy. A series of single optical slice images through the nucleus of a transfected cell were captured every 1.6 seconds for

10 minutes. After acquisition, the images were reconstructed as a 3-dimensional time- course using Imaris imaging software. The top and side views are show for both t=0min (a, top; a’, side) and t=10min (b, top; b’, side). The decrease in fluorescence intensity from start to end is caused by image acquisition photobleaching. Particle tracking within Imaris calculated and mapped out the movement of individual puncta visible from t=0min to t=10min. The particle tracking is displayed as the start point of an individual Gag focus (c and c’, white box), and the motion path the particle traversed during the time-lapse (colored tail; blue to yellow). A selected region (c and c’, black box) was magnified to better see individual particle motion paths (d and d’). The nucleus size was 15µm x 4µm x 4µm. The average displacement of the tracked foci was 0.33 ± 0.11µm.

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82 cell, but are anchored in place.

Mobility of Gag proteins within nuclear foci assessed by FRAP

Although the Gag nuclear foci were tethered within the nucleoplasm, it did not preclude the possibility that Gag proteins travel between the nucleoplasm and the nuclear foci. Cellular proteins and RNAs are found to shuttle between nuclear subcompartments

(nucleoli, splicing speckles) and the nucleoplasm (83, 334). If Gag were forming nonfunctional aggregations within the nucleoplasm, or VLPs, we would expect no mobility between nuclear foci and the nucleoplasm because VLPs generally show little protein mobility (160).

To test these possibilities, we used fluorescence recovery after photobleaching

(FRAP). FRAP has been frequently used to determine whether a protein is shuttling between two distinct subnuclear compartments (83, 331, 334). To examine the mobility of

Gag within nucleoli, QT6 cells were transfected with L219A.Gag-YFP and imaged using fluorescent confocal microscopy. Five pre-bleach images were taken with 0.8 seconds between each frame. A single nuclear focus was specifically photobleached using the

514nm laser at 100% intensity for four frames. Fluorescence recovery within the punctate spot was then measured for 51 seconds, which resulted in full recovery. A representative example of nucleolar FRAP is depicted (Figure 2.6A). A dramatic decrease in nucleolar fluorescence is seen between the pre-bleach and at the start of recovery (t=0s, white arrowhead). A noticeable recovery of fluorescence intensity is visible at the end of the assay (t=51s, white arrowhead). The graph of a normalized FRAP experiment is depicted, demonstrating a significant amount of recovery (Fig. 2.6A, graph).

For an immobile protein FRAP comparison, cells were transfected with Gag-YFP and a region encompassing punctate foci at the membrane was bleached (Fig. 2.6B, white arrowhead). Fluorescent puncta at the membrane have been previously reported to be budding virus-like particles, and represent an immobile Gag protein population (224). These large Gag complexes are in the final stages of budding and release, therefore the mobility of 83 Fig. 2.6. Mobility of Gag nuclear foci assessed by FRAP. (A) QT6 cells expressing

L219A.Gag-YFP were imaged using confocal microscopy. A single nuclear focus was specifically photobleached (t=0s, white arrowhead), and monitored for 51 seconds. During that time fluorescence recovery was observed (t=51s, white arrowhead). The acquired data was normalized as described in materials and methods and graphed. (B) QT6 cells expressing Gag-YFP were imaged as in part A. A region of membrane foci was selected for photobleaching (t=0, white arrowhead). Little fluorescence recovery is visible at the end of the assay (t=195s). (C) The average of 3 nuclear foci and 4 membrane FRAP assays are shown. Bars represent the standard error of the mean. Statistical analysis was performed using student’s t-test. (*, P=0.0012).

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85 Gag within these structures are limited or nonexistent (160). As expected, very little recovery was observed at the membrane (Fig 2.6B, t=195s).

The FRAP data was corrected and normalized as described in the materials and methods. The relative intensity of both punctate foci and membrane experiments are plotted

(Fig. 2.6A and B). As seen graphed, Gag proteins within the nuclear foci are highly mobile, recovering much of the fluorescence depleted through photobleaching. In contrast, the membrane recovers little fluorescence. The mobile fraction of punctate or membrane Gag was calculated as described in the materials and methods. A minimum of 2 cells was averaged (Fig. 2.6C). L219A.Gag-YFP in nucleoli (0.61) was found to be more mobile than

Gag-YFP at the plasma membrane (0.15). The difference of mobility between foci in the nucleus and at the membrane was significant (P=0.0012). These data demonstrate that

Gag is shuttling between the tethered nuclear foci and the nucleoplasm.

2.5 Discussion

The assembly of retroviral particles is a complex process, and current understanding is limited by the lack of distinct steps in Gag subcellular trafficking (407). RSV Gag is unique in that it transiently traffics to the nucleus (67, 363, 366). Genetic data indicate that nuclear trafficking is linked to the efficient packaging of gRNA (151). Yet the mechanisms and precise functions of this nuclear step of Gag during viral assembly remain poorly understood.

Previous studies have identified that RNA is an essential structural component of retrovirus particles (302, 385). Furthermore, biochemical analysis demonstrated that a Gag homodimer is the smallest assembly unit, and required for binding of ψ-containing RNAs

(171). The confocal microscopy and FRET analysis presented here demonstrate Gag-Gag interactions within the nucleoplasm, indicating that Gag binds to RNA within the nucleus.

However, the results do not distinguish between Gag binding RNA within the nucleus and

86 Gag binding prior to nuclear import. Further studies are needed to distinguish between the two possibilities.

Interestingly, when either wildtype Gag treated with LMB or a Gag nuclear export mutant was overexpressed within cells, we observed the formation of Gag nuclear foci.

However, we found the punctate phenotype of nuclear Gag in a subset of cells. Why punctate foci do not form in every cell may be due to the amount of protein present within the nuclei. To test this possibility, cell sorting will separate the high-intensity fluorescent cells from the low-intensity fluorescent cells. If high protein concentration is the reason for nuclear foci, we would expect that the high-intensity cells would all contain Gag nuclear foci whereas we would observe a diffuse phenotype for low-intensity cells. Another possibility is that the Gag nuclear foci are cell cycle dependent. To test whether nuclear foci are formed at a specific point in the cell cycle, cells expressing the L219A.Gag-YFP will be synchronized and observed over time to watch for changes in Gag localization.

The Gag nuclear punctate foci are dependent upon the NC domain, as demonstrated by the NC deletion. Furthermore, the FRET data demonstrate that insertion of the protein multimerization Zip domain restores Gag-Gag interactions within the nucleus, but does not form nuclear puncta. It is unlikely that nuclear foci represent VLPs forming within the nucleus, although in vitro studies have demonstrated the necessity of the NC domain in Gag and the presence of RNA for VLP formation (89, 302, 431, 460). Additionally, in vivo studies have also identified the NC domain as a necessary component of virus particle release (52,

89, 320). However, the volumetric data in this report refutes the possibility of nuclear VLPs.

The Gag nuclear foci are significantly larger than VLPs that form at the membrane.

Additionally, we were unable to identify any electron dense Gag aggregates or particles by electron microscopy. Together, these data demonstrate that Gag is not forming VLPs within the nucleus. However, further studies involving immunogold staining will be performed to confirm the presence of Gag within imaged nuclei.

87 If Gag is not forming particles or electron-dense aggregates within the nucleoplasm, what are the punctate foci? Particle tracking identified that the foci were tethered within the nucleoplasm, which is reminiscent of transcriptional complexes (387). Additionally, FRAP analysis demonstrated that unlike VLPs located the plasma membrane, the Gag proteins were shuttling between the nuclear foci and the nucleoplasm. This result is possibly due to the Gag proteins associated with a subnuclear structure, either via RNA- or protein- mediated interactions. Interest in viral RNA trafficking has led our laboratory to label proviral DNA with MS2 loops (86). Thus, a fluorescently tagged MS2 protein binds to and effectively labels the retroviral RNA, and the localization of the RNA is observed (40). Our laboratory has visualized retroviral RNA nuclear foci immediately adjacent to or colocalizing with the Gag nuclear foci. It is possible that this observation links the Gag nuclear foci to active sites of transcription. Gag regulation of splicing is unlikely, as viral RNA transcripts contain negative regulators of splicing and do not appear to require any viral proteins for splicing inhibition (11, 20). It is also possible that Gag interacts with these intranuclear compartments to facilitate interaction with the gRNA for packaging. Although unspliced viral

RNA is exported for translation via the Tap/Dbp5 nuclear export pathway, it is unknown whether this pathway is involved in gRNA packaging (227). Given that Gag nuclear trafficking is necessary for efficient incorporation of gRNA into virus particles, subnuclear localization events may facilitate Gag during selection of ψ-containing RNAs.

The nucleus is a complex organelle with many compartmentalized functions, including Cajal bodies, nuclear speckles, transcription factories, and nucleoli (395). Nuclear bodies are frequently hijacked during viral replication. DNA viruses including SV40, Ad5, and HSV-1, use PML bodies (ND10) as sites for viral replication and transcription (272). In

SV40, viral DNA is localized to PML bodies (201). The Ad5 E4ORF3 protein alters the function of PML bodies (198). HSV-1 transcription occurs at the PML bodies (392). Of the

HSV-1 proteins involved in transcription and viral RNA regulation, the ICP27 protein is of particular interest. ICP27 associates with PML bodies and nuclear speckles (SC35) to 88 prevent splicing of viral transcripts (127, 174, 175, 254). The ICP27 protein mediates nuclear export of the intronless viral RNA (219, 360). Thus the splicing speckles and PML bodies appear to be a staging area for the expression and export of intronless herpesvirus

RNA. The Gag nuclear foci resemble these transcription factories/nuclear speckles/PML body complexes. The work reported here is the first to identify the RSV Gag protein localized to a subnuclear body in context of a NES mutant or under LMB treatment.

However, further investigation is needed to confirm the hypotheses presented in this discussion and to identify whether Gag nuclear foci serve a function during retroviral assembly.

89

Chapter 3

Gag Nucleolar Localization and Mapping of the Nucleolar

Localization Signal within the NC Domain

90 3.1 Abstract

The assembly of retrovirus particles is directed by the retroviral Gag polyprotein.

The Rous sarcoma virus (RSV) Gag polyprotein is synthesized in the cytoplasm. During the early steps of assembly, Gag undergoes transient nuclear trafficking, which is required for efficient packaging of viral genomic RNA. However, the specifics of what occurs during Gag nuclear trafficking are poorly understood. Although RSV is not the only retrovirus that is found within the nucleus, it is the only retrovirus encoding a Gag protein with a known nuclear export sequence (NES). This NES makes RSV a useful model to study Gag-nuclei interactions and thereby better understand early assembly events within retroviral assembly.

Using fluorescent confocal microscopy and fluorescence resonance energy transfer, we asked whether nuclear-trapped Gag localized to any known subnuclear bodies, and found a population of Gag within nucleoli. Fluorescence recovery after photobleaching demonstrated that this nucleolar Gag was highly mobile, suggesting a dynamic relationship between nucleoli and Gag. Given that NC is the only domain of Gag that localizes to nucleoli, we next identified the amino acid sequences necessary for nucleolar localization of

NC through site-directed mutagenesis. We also found a correlation between NC nucleolar accumulation and the subnuclear trafficking of Gag. These results suggest that NC plays a key role in the subnuclear trafficking steps of Gag. Finally, we identified the sequences necessary for nucleolar localization in HIV NC. Surprisingly, we found that a Rev- independent HIV Gag was partially colocalized to nucleoli when coexpressed with either HIV

NC or Rev, which suggests a potential role for HIV Gag in nucleoli. These findings are the first to identify nucleolar trafficking of retroviral Gag proteins.

3.2 Introduction

Nucleoli are distinct subnuclear compartments historically known for their key role in ribosomal biogenesis. The nucleolus itself can be divided into three separate subregions.

Ribosomal RNA transcription occurs near the center of nucleoli, termed the fibrillar center 91 (FC). Immediately surrounding the fibrillar center is the dense fibrillar component (DFC), where processing of ribosomal RNA and ribosomal subunits occurs. Surrounding the FC and DFC is the granular component (GC). The GC is the site for ribosomal subunit assembly. Thus the nucleolus includes factors that promote RNA modifications, protein modifications, and ribonucleoprotein complex formation that must occur during the creation of ribosomes. However, recent studies in nucleolar proteomics have revealed a more versatile role for nucleoli (45). Proteomic studies have identified more than 700 proteins in nucleoli (13, 14). These data suggest that nucleoli are a diverse subnuclear body involved in DNA replication and repair, pre-mRNA processing, and cell-cycle control.

Many viral proteins localize to nucleoli, and nucleoli have been called the “gateway to viral infection” (185). Because a large portion of these viral proteins bind RNA, nucleolar localization was once thought to be an artifact due to the high RNA concentration of nucleoli.

However, further studies have demonstrated an essential link between viruses and the nucleolus. Several viruses, including poliovirus, arterivirus, and HIV, deregulate the cell cycle through disruption of normal nucleolar function (293, 428, 446). For other viruses, trafficking to nucleoli or interacting with nucleolar proteins is linked to viral replication. For example, the influenza A virus NP protein is involved in the replication and packaging of the viral genome and is found localized to nucleoli (321). The herpesvirus saimiri ORF57 protein localizes to nucleoli (54). Nucleolar trafficking of ORF57 is required for the nuclear export of intronless viral RNA. When the nucleolar retention signal (NoRS) is abrogated in

ORF57, nuclear export of viral RNA is prevented. Restoration of nucleolar trafficking to

ORF57 via the addition of a heterologous NoLS rescued the nuclear export of the viral RNA.

These data suggest that RNA and DNA viruses commandeer nucleoli and use them as a site for viral protein-RNA interaction for RNA export and packaging.

Retroviruses also interact with nucleoli during infection. The HIV-1 Rev protein, which is responsible for the nuclear export of unspliced viral RNAs, accumulates within nucleoli under steady-state conditions (90, 328). Multimerization of Rev is required for 92 proper function and was found to occur within nucleoli (101). Additionally, the nucleolar protein B23 is bound to Rev until an RNA containing the Rev response element (RRE) displaces B23, suggesting that the site of interaction between unspliced viral RNA and Rev occurs within nucleoli (130). Rev also interacts with the U16 small nucleolar RNA fused to the RRE and exports it to the cytoplasm, further supporting the functional significance of

Rev nucleolar localization (64). Studies involving a ribozyme, specific for cleavage of HIV-1

RNA, fused to a small nucleolar RNA examined the possibility of viral RNA trafficking through nucleoli (285). The ribozyme inhibited HIV-1 replication, suggesting that retroviral

RNA traffics through nucleoli. Furthermore, the nucleolar protein nucleolin has been implicated in enhancing retroviral assembly. When expressed together, Gag, nucleolin, and an RNA containing the psi packaging sequence greatly increase the rate of budding for HIV virus-like particles (421). Taken together, these studies with HIV Rev suggest that nucleoli serve as an RNA-viral protein interaction site.

Research from our laboratory has demonstrated a link between nuclear trafficking of the Rous sarcoma virus (RSV) Gag during assembly and genomic RNA (gRNA) packaging

(151, 363). Mutants of Gag that bypass the nucleus package gRNA less efficiently than wildtype virus. Insertion of an exogenous nuclear localization sequence rescues both nuclear trafficking and gRNA packaging. Our data support a model where nuclear trafficking occurs in several ordered steps (172). First, Gag must bind to host import factors.

Once in the nucleus, Gag binds to psi-containing RNA, stimulating CRM1 binding and nuclear export of the viral RNA-Gag complex. In addition to Gag-RNA binding, Gag-Gag interactions occur in the nucleus in an RNA dependent manner (213). In the course of studying Gag interactions within nuclei, we observed the presence of both punctate nuclear foci and an accumulation of Gag within nucleoli. This chapter follows up on those studies and we now ask whether Gag participates in subnuclear trafficking during viral replication.

We reported that the RSV NC protein alone localizes to nucleoli when overexpressed in

QT6 quail cells (67). Therefore, we hypothesized that NC imparts nucleolar trafficking to the 93 full-length retroviral Gag protein. In this report, we observed a highly mobile population of

Gag within nucleoli when Gag was highly overexpressed and trapped within the nucleus.

Additionally, we identified the residues necessary for nucleolar localization of NC and Gag through mutagenesis. Finally, we explored the possibility of NC-mediated nucleolar localization of HIV Gag.

3.3 Materials and Methods

Expression vectors and cells

Prague C RSV Gag and NES mutant (L219A and NES-A) expression vectors containing a CFP or YFP fluorophore have previously been described (213). pYFP-RSV.NC has been previously described (67). pYFP-NC.SKL and pYFP-NC.ΔCH1/ΔCH2 were made by PCR amplification of the nucleocapsid coding region from pGag.SKL and pGag.ΔCH1/ΔCH2 (a gift from M. Linial, Fred Hutchinson Cancer Research Center) (231).

The amplified sequences were inserted into the pEYFP-c1 vector using ApaI-BspEI

(Clontech). The NC fragments encoded by pYFP-L, pYFP-NT/L, and pYFP-L/CT were made through PCR amplification of pYFP-NC.ΔCH1/ΔCH2 and were inserted into pEYFP- c1 via ApaI-BspEI. For pYFP-RSV.NC.M1, M2, M3, M4, M5, M6, SKL, and M3/M4, basic amino acids were mutated to alanine by site-directed mutagenesis (QuikChange,

Stratagene). Gag NC mutants were created by performing site-directed mutagenesis identical to the YFP-RSV.NC mutations described above. Rev-independent HIV Gag was

PCR amplified from a HIV Gag-GFP vector and inserted into a pCFP-N1 vector using KpnI-

BamHI. HIV NC was amplified from HIV Gag and inserted into a pYFP-N1 vector using

HindIII-BamHI. M1, M2, and M1/M2 basic amino acids were mutated to alanine using site directed mutagenesis. Murine leukemia virus pMLV.NC-YFP was created through PCR amplification of NC from MLV Gag and inserted into pYFP-N1 using HindIII-BamHI. pfibrillarin-YFP was created by using PCR amplification of the YFP fluorophore and swapping YFP in place of CFP in pfibrillarin-GFP (a gift from Mark Olson, University of 94 Mississippi Medical Center) (118) using BamHI-NotI. Mutants were screened by endonuclease digestion and positive clones were confirmed through automated sequencing.

Primers and restriction digest sites for screening are presented listed (Table 3.1). All experiments were performed using QT6, 3T3, NMuMG, or HeLa cell lines (22, 33, 183, 357).

Transfections were performed using the calcium phosphate method (QT6) (144) or

Lipofectamine 2000 (HeLa, 3T3, NMuMG; Invitrogen).

Laser scanning confocal microscopy

Live cells were seeded onto 35-mm glass-bottomed dishes (MatTek Corporation) and imaged using a Leica AOBS SP2 confocal microscope at 14 to 24 h post-transfection.

Sequential scanning settings were used to differentiate CFP (excitation at 458nm, emission at 465-490nm, and 50% laser power) and YFP (excitation at 514 nm, emission at 530-

600nm, and 10% laser power) emission spectra. Light images were obtained using the transmitted light channel during fluorescence acquisition.

Retroviral NC sequence alignment

Sequences of the NC proteins (with accession numbers from the NCBI protein database) from RSV (NP_056887.1), HIV (NP_057850.1), MMTV (AAF31472.1), and MLV

(ABD14438) were aligned using ClustalW (413).

FRAP analysis

Fluorescence recovery after photobleaching (FRAP) was performed at 38.5°C on live cells transfected with either pL219A.Gag-YFP or pGag-YFP. All assays were performed at a scan speed of 800Hz with an acquisition time of 0.8 seconds. Five pre-bleach images were acquired using the YFP channel settings at 10% laser power. YFP in nucleoli

(L219A.Gag-YFP) or at the membrane (Gag-YFP) was specifically photobleached using the

514nm laser at 100% power over four time-points. Recovery was monitored for approximately 120 seconds at 10% laser power. As a control for bleaching and acquisition fluorescence loss, fluorescence of the entire cell was monitored during the experiment. The

95 Table 3.1. Primer list for NC basic amino acid mutations. Primers are listed for NC basic acid mutations in both RSV (A) and HIV (B). Forward (F) and reverse (R) primers are listed for PCR amplification. Altered residues are marked in bold; flanking nucleotides of deletion mutants are indicated with underlined residues. Screen site indicates the restriction enzyme used for identifying positive clones. Size comparisons were performed by performing a restriction digest identical to the insertion digest (see text for restriction enzymes used).

96

97 background from both the region of interest and the total cell fluorescence was subtracted.

Corrections for photobleaching and normalization of the data was performed as previously described (331). Briefly, the relative fluorescence intensity was calculated by ,

where T0 is the total cell intensity before bleaching, Tt is the total cell intensity at time-point t,

I0 is the intensity of the region of interest before bleaching, and It is the intensity of the region of interest at time-point t. The mobile fraction of each region was determined using

I E - I0 Fm = , where IE is the fluorescence in the bleached region after full recovery, I0 is the I I - I0 fluorescence immediately after bleaching, and II is the pre-bleach fluorescence. A minimum of 8 assays was performed on Gag at the membrane or within nucleoli.

FRET measurements

Acceptor photobleaching fluorescence resonance energy transfer (FRET) was performed on live cells at 14 to 24 h post-transfection as previously described (213).

Prebleach images of CFP (excitation at 458nm, emission at 460-496nm, and 50% laser power) and YFP (excitation at 514nm, emission at 538-600nm, and 10% laser power) were acquired. Using the 514nm laser at 100% power, YFP was specifically photobleached until fluorescence intensity was reduced to 10% of prebleach levels. Postbleach images were acquired for both the CFP and YFP channels. FRET efficiency was calculated using the

donorPost-donorPre formula FRETEff= , when donorPost is greater than donorPre. FRET analysis donorPost was performed on at least two different sets of transfected cells using a minimum of 10 different cells per transfection. Nucleolar FRET was performed by bleaching the entire nucleus through a single optical slice of the nuclear plane. After photobleaching, several individual nucleoli were selected as regions of interest for FRET measurement.

3.4 Results

Subcellular localization of retroviral NC proteins 98 The NC proteins of both RSV and Murine leukemia virus (MLV) have been observed within nucleoli (67, 350). However, it has not been determined whether nucleolar accumulation of NC is common amongst all retroviruses or whether this localization is restricted to a particular cell type. The subcellular localization of RSV, HIV, MLV, and

MMTV NC proteins, fused to variants of GFP, were examined by confocal microscopy.

Each of the fluorophore-fused NC proteins was separately transfected into QT6 cells.

Additionally HIV NC was transfected into HeLa cells, MMTV NC into NMuMG cells, and MLV into 3T3 cells. Either transmitted light imaging or co-transfection with the nucleolar marker fibrillarin-CFP was used to identify nucleolar localization (data not shown). In all cases, whether in QT6 or more biologically relevant cell lines, NC from each virus localized to nucleoli (Fig. 3.1A); only with MLV NC did we see any difference. In both QT6 and 3T3 cells the YFP-MLV.NC protein was diffuse throughout the nucleoplasm and nucleoli; however,

YFP-MLV.NC appears to be more nuclear in 3T3 cells. These observations suggest that nucleolar localization of the NC protein is common among retroviruses.

To examine the sequence homology between the NC proteins of the four different viruses, we performed a ClustalW alignment (Fig. 3.1B). As previously reported, there was a high degree of identity and similarity among the Cys-His motifs (Fig. 3.1B, gray highlights)

(92). However, few residues outside the Cys-His motifs align other than a few basic residues. Thus, no NLS or NoLS was identified. In considering sequences that may serve as a NoRS, it seems likely that the basic amino acid sequences flanking the Cys-His boxes are responsible.

NC-Gag interactions within nuclei

Although RSV Gag transiently traffics through the nucleus, Gag normally appears to exclude nucleoli when trapped within nuclei. To determine whether Gag might also be interacting with nucleoli, we required a method to detect the presence of small amounts of

Gag within nucleoli. The NC domain mediates Gag-Gag interactions and multimerization

(52, 232). Thus we hoped to use NC as a trap for Gag proteins that may be passing 99 Fig. 3.1. Nucleolar localization of retroviral NC and Gag proteins. (A) Amino acid sequence comparison between the NC proteins of RSV, MLV, HIV, and MMTV. (B)

Nucleolar localization of NC proteins from RSV, HIV, MMTV, and MLV. Top panels represent localization in QT6 cells; bottom panels represent localization in native cell-types

(Human or mouse).

100

101 through nucleoli. To detect possible interactions between Gag and NC, we used FRET analysis through confocal microscopy.

For FRET experiments, Gag-CFP and YFP-NC were co-transfected into QT6 cells.

The cells were then treated with the drug Leptomycin B (LMB), which localizes Gag within nuclei (363). To determine whether LMB treatment was affecting the localization of Gag, two nuclear export mutants, NES-A and L219A were also used (Fig. 3.2A). Using a confocal microscope, CFP and YFP channels were imaged prior to bleaching.

Representative figures of cells expressing wildtype Gag with LMB treatment or NES-A.Gag are shown (Figure 2B). A region encircling the entire nucleus was photobleached using the

514nm laser at 100% intensity. A decrease in the YFP fluorescence intensity was observed

(Fig. 2B, right panels). The nucleoli were examined for an increase in CFP fluorescence.

Compared to the pre-bleach intensity, an increase in CFP intensity was detected (Fig. 2B, left panels).

Nucleolar FRET efficiencies from at least 10 different cells over two separate transfections were obtained (Fig. 3.2C). As a negative control, cells expressing free CFP and YFP produced very low FRET efficiencies (3.7%) within nucleoli. Cells expressing Gag-

CFP and YFP-NC and treated with LMB to cause relocalization of Gag to the nucleus produced a FRET efficiency of 23.7% in nucleoli. Similarly high FRET values were obtained for L219A.Gag-CFP with YFP-NC (22.2%) and NES-A.Gag-CFP with YFP-NC (22.2%). As a negative control, the nucleolar protein fibrillarin was used in place of NC. The FRET efficiencies were similar to the CFP-YFP value when fibrillarin-CFP was co-transfected with

Gag-CFP in LMB treated cells (3.8%), L219A.Gag-CFP (4.5%), or NES-A.Gag (3.4%).

These results demonstrate the interaction of Gag and NC in nucleoli and suggest that Gag transiently traffics through nucleoli or that NC pulls Gag into nucleoli.

Subnuclear localization of Gag

The interaction between NC and Gag within nucleoli suggests that Gag passes through or is retained within nucleoli. However, it does not preclude the possibility that the 102 Fig. 3.2. Acceptor photobleaching FRET of Gag and NC in nucleoli. (A) Schematic diagram of the RSV Gag protein and NES mutations used for this paper. (B) Top set of images represents FRET of Gag-CFP and YFP-NC when treated with LMB. Bottom set of images shows FRET of NES-A.Gag-CFP and YFP-NC. The nucleus, indicated by the dashed white line, was bleached using a 514nm laser. A significant loss of fluorescence intensity can be observed in the YFP channel between the pre- and post-bleach images.

Nucleoli, indicated by the solid white line, were chosen as the regions of interest. Nucleolar fluorescence intensities of the CFP channel were monitored between pre- and post-bleach.

The resulting increase in CFP fluorescence is due to a decrease in energy transfer from the

CFP to YFP fluorophore. (C) The FRET efficiency (percent) for Gag and NC was calculated using the equation from materials and methods. As a negative control, the nucleolar protein fibrillarin (Fib-YFP) was used. A minimum of 10 cells was analyzed for each set of proteins and the mean and standard error bars have been graphed.

103

104 site of Gag and NC interaction occurs outside of nucleoli, and NC transports Gag into nucleoli. To address this possibility, Gag-YFP, L219A.Gag-YFP, or NES-A.Gag-YFP were transfected into QT6 cells. Cells expressing Gag-YFP were treated with LMB. Under these conditions, a subset of cells expressing each Gag-YFP variant formed punctate foci in the nucleus (Fig. 3.3A, left panels). Additionally, we observed the nucleolar accumulation of

Gag (Fig. 3.3A, right panels), which was present in approximately 70% of cells containing nuclear foci. These observations, when taken with the FRET data (Fig. 3.2), demonstrate that the Gag protein localized to nucleoli when concentrated within nuclei.

Mobility of Gag within nucleoli

Although we see L219A mutant Gag protein accumulate within nucleoli, this Gag population could be a nonfunctional aggregation of protein. If Gag proteins were forming larger complexes or inappropriately forming particles within the nucleus, we would expect to see an immobile fraction of Gag within nucleoli. FRAP has been frequently used to examine what fraction of a protein is mobile within a specific subcellular compartment (331). To examine the mobility of Gag within nucleoli, QT6 cells were transfected with L219A.Gag-

YFP and imaged using fluorescent confocal microscopy. Five pre-bleach images were taken with 0.8 seconds between each frame. A single nucleolus was specifically photobleached using the 514nm laser at 100% intensity for four frames. Fluorescence recovery within the nucleolar region was then measured for 120 seconds. A representative example of nucleolar FRAP is depicted (Figure 3.3B, top row). A dramatic decrease in nucleolar fluorescence is seen between the pre-bleach and at the start of recovery (t=0s, white arrowhead). A noticeable recovery of fluorescence intensity is visible at the end of the assay (t=122.613s, white arrowhead). Furthermore, the unbleached nucleolus (white outlined arrowhead) visibly loses a significant amount of fluorescence.

Although L219A.Gag-YFP is highly mobile within nucleoli, we wanted to compare this mobility to a population of Gag that would be relatively immobile, as a negative control. HIV

Gag at the membrane is immobile (160). Thus, RSV VLPs at the membrane should also 105 Fig. 3.3. Nucleolar Gag mobility assessed by FRAP analysis. (A) Images show either punctate or nucleolar localization of either NES mutant Gag or wildtype Gag treated with

LMB. Both the YFP channel and DIC images are shown with white arrows pointing to nucleoli. 4µg of plasmid DNA was used for these transfections. (B) FRAP analysis was performed on either nucleolar localization of L219A.Gag-YFP (top images) or membrane puncta formed by Gag-YFP (bottom images). Confocal images through the nuclear plane were obtained prior to photobleaching (Pre-Bleach). The nucleolus (solid white arrow) or membrane (white box) was photobleached using a high-intensity laser (100% laser power) at a wavelength of 514nm. After bleaching, a significant loss of signal was seen (t=0s). The bleached region was then monitored for recovery of the fluorescence signal over a period of time. An increase in fluorescence of Gag-YFP within nucleoli was observed (t=122.613, top). No fluorescence recovery was apparent at the plasma membrane (t=122.613s, bottom). (C, left) Kinetics of FRAP at nucleolus or membrane. Fluorescence recovery of the bleached region of interest was plotted as relative intensity (see Materials and Methods).

The Gag-YFP within nucleoli was highly mobile whereas Gag-YFP at the plasma membrane underwent very little recovery of fluorescence during the timeframe. (C, right) A bar graph showing the mobile fractions (Fm) of Gag-YFP in nucleoli and Gag-YFP at the membrane.

FRAP assays were performed a minimum of 8 times from at least two separate transfections. Error bars represent standard error of the mean.

106

107 have limited mobility. To compare the mobility of L219A.Gag-YFP in the nucleolus with foci of Gag-YFP at the plasma membrane, cells were transfected with Gag-YFP and a region encompassing punctate foci at the membrane was bleached (Fig. 3.3B, bottom row).

Fluorescent puncta that form upon expression of RSV Gag-GFP at the plasma membrane have been previously shown to be virus-like particles (224). These complexes of Gag are in the final stages of budding and release, therefore we expected the mobility of Gag-YFP within these structures to be minimal. As expected, very little recovery of fluorescence was observed for Gag-YFP at the plasma membrane (Fig 3.3B, bottom row, white box).

The relative recovery of both L219A.Gag-YFP in a nucleolus and Gag-YFP at the membrane were plotted in Figure 3.3C (left panel). Gag within the nucleolus was highly mobile, with a fluorescent recovery of 80%. In contrast, most of the fluorescence lost through bleaching of Gag-YFP at the plasma membrane recovered 10%. The mobile fraction of nucleolar or membrane Gag was calculated as described in the materials and methods. L219A.Gag-YFP in nucleoli was highly mobile (72.1%), whereas Gag-YFP at the membrane was largely immobile (19.6%) (Fig. 3.3C, right panel). Therefore, L219A.Gag-

YFP within nucleoli is a dynamic protein that travels between nucleoli and the nucleoplasm.

Thus the nucleolar Gag proteins are not static aggregation.

Identifying the location of the NoRS

Because the NC domain localized to nucleoli when expressed alone, we hypothesized that the NoLS found within Gag resided within NC. To identify the sequences responsible for the nucleolar localization of NC, regions of NC were expressed individually.

For this purpose, NC was divided into five regions divided by the two Cys-His boxes (CH1 and CH2): the N-terminal (N) region, the linker (L) region, and the C-terminal (C) region.

Although there is no firm sequence consensus that defines a NoRS, they are typically rich in basic amino acid residues (104). It seemed unlikely that CH1 or CH2 would contain a NoRS as there is a single basic amino acid present in both sequences. Indeed, a mutant NC protein with deletion of both Cys-His motifs (YFP-NC.ΔCH1/ΔCH2) localized to 108 nucleoli in QT6 cells (Fig. 3.4B). This observation shows that the Cys-His motifs are not necessary for nucleolar localization.

To determine whether any of the basic regions alone (NT, L, or CT) were sufficient to direct nucleolar localization, each was expressed as a fusion protein with YFP. Both YFP-

NT and YFP-CT diffused throughout the cell (data not shown). YFP-L appeared to be concentrated in the nucleus, but was present equally in the nucleolus and nucleoplasm (Fig.

3.4B). This observation suggested that the L region had some degree of nucleolar localization capability, but it was not as concentrated within nucleoli as full-length NC. We next looked at a combination of the basic regions. When two basic regions were fused together and expressed, we found that YFP-NT/L was present in the nucleolus and nucleoplasm, whereas YFP-L/CT was concentrated within nucleoli (Fig. 3.4B). These results suggest the NoRS may map to the L and CT regions of NC.

Mapping the nucleolar retention signal of NC

To test whether residues within the L and CT regions were responsible for nucleolar localization, site-directed mutagenesis was used to target specific basic clusters within a

YFP-fused full-length NC protein in the L and CT regions (Fig. 3.4A). Within the L region, residues 36-39 (KKRK) were mutated to alanines (M1). When YFP-NC.M1 was expressed in QT6 cells, both nuclear and nucleolar localization of NC was lost (Fig 3.4C, M1), suggesting that the PKKRK sequence might be an NLS that is responsible for nuclear localization of NC. Additionally, the PKKRK sequence is necessary for nucleolar accumulation of NC. Changing the residues 44 and 46 (RER, altered residues are in bold) to alanines (M2) caused YFP-NC.M2 to become diffuse between nucleoli and the nucleoplasm (Fig. 3.4C, M2). Similarly, the mutant YFP-NC.M3 altered residues 61-63

(RKR) to alanine (M3), and also localized to both nucleoli and the nucleoplasm equally (Fig.

3.4C, M3). The mutant YFP-NC.M4, which changed residues 70 and 73 (RPGK, residues altered are in bold) to alanines, was observed primarily within nucleoli.

109 Fig. 3.4. Localization of RSV NC mutants. Schematic diagrams of YFP-RSV.NC are shown with the WT amino acid sequences in the linker, C-terminal domains (A), and N- terminal (B). Altered residues are depicted in bold. (C) YFP is used as a negative control and can be seen localized throughout the nucleoplasm and cytoplasm, while excluding nucleoli. Wildtype NC is seen localized to nucleoli. Removal of the Cys-His motifs does not disrupt nucleolar localization of the NC protein. L and NT/L are diffuse throughout nucleoli and the nucleoplasm. The L/CT fusion protein is localized to nucleoli. M2, M3, SKL, and

M6 are also diffuse through nucleoli and the nucleoplasm. M3/4 and SKL/4 are seen in the nucleoplasm but exclude nucleoli. M1 is seen in the cytoplasm and nucleoplasm, similar to

YFP alone. Mutants M4, M5, and M6 do not significantly disrupt nucleolar retention of NC.

110

111 The previous section found that the L and CT regions together, when expressed as

YFP-L/CT, localized to nucleoli. We asked whether basic residues within the NT region were involved in nucleolar localization of the full-length NC protein. To test whether basic residues within the NT were contributing to nucleolar localization in context of the full-length

NC protein, we altered the basic residues within the NT using site-directed mutagenesis.

YFP-NC.M5 altered residues 5 and 7 to alanines (RER, altered residues in bold). When expressed in cells, the YFP-NC.M5 mutant localized to nucleoli. The YFP-NC.M6 mutant, which alters residues 16 and 18 to alanines (RAR, altered residues in bold), was observed diffuse throughout nucleoli and the nucleoplasm.

Thus, the M2, M3, and M6 mutation, which flank the Cys-His motifs, were observed throughout the nucleoli and nucleoplasm. NC basic mutations not immediately adjacent to

Cys-His motifs (M4 and M5) retained nucleolar localization. However, it was only the double mutant YFP-NC.M3/M4 caused the NC protein to exclude nucleoli. These results indicate that basic residues throughout the NC protein are involved in nucleolar localization.

However, eliminating all basic charge within the CT region of NC completely eliminated nucleolar accumulation of NC. These results indicate that the number of basic residues are important for NC nucleolar localization.

Nucleolar localization of Gag mutants

We next examined the effect of these basic amino acid mutations on the nucleolar localization of Gag. For these studies, all of the NC mutants were examined in the context of the L219A mutant that is defective in nuclear export (Fig. 3.4A). Each Gag plasmid was co-transfected with the nucleolar protein fibrillarin-CFP, which served as a marker for nucleoli. Under steady state conditions, L219A.Gag was observed as three phenotypes: nucleolar (Fig. 3.5A, panel a), nuclear punctate (Fig. 3.5A, panel a’), and nuclear diffuse (Fig

5, panel a’’). All cells expressing the L219A.Gag.M1-YFP mutant (PKRKK->PAAAA) localized strongly to the plasma membrane (Fig. 3.5, panel b’’). In addition to the plasma membrane localization, the M1 mutant was also observed within the cytoplasm and nucleus. 112 Fig. 3.5. Localization of nuclear export deficient Gag and Gag NC mutants. The images show different localization phenotypes of L219A.Gag-YFP or L219A.Gag NC mutants. The phenotypes are separated into nucleolar (left), punctuate (middle), and diffuse (right). In each phenotype subset, Gag localization is shown on the left, and an overlay image with the nucleolar protein fibrillarin-CFP is depicted on the right. The table to the right of the images scores the ability of the mutations to localize as wildtype Gag.

Nucleolar localization was designated as wildtype (+), or excluding nucleoli (-). The ability to form punctuate foci within the nucleoplasm was similarly scored wildtype (+), or no punctuate foci (-).

113

114 Therefore, loss of the PKKRK sequence results in loss of nuclear localization. However, the

NLS within MA is still present within the L219A.Gag.M1-YFP mutant, which likely explains the small degree of Gag within the nucleus (67). These results suggest that PKKRK might be the functional NLS in the NC domain of Gag, although why MA is unable to drive complete nuclear localization in the absence of the putative NC NLS is unknown.

L219A.Gag mutants M2, M3, and M4 were all observed with nucleolar, nuclear punctate, and nuclear diffuse phenotypes (Fig. 3.5, panels c-e, c’- e’, and c’’- e’’). The

M3/M4 L219A.Gag mutant appeared nuclear diffuse, but nuclear foci and nucleolar phenotype were not observed (Fig 3.5, panels f’’- h’’). The localization of NC mutants within the NT region, however, did not have similar distributions in the context of L219A.Gag-YFP.

Both M5 and M6 demonstrated nucleolar localization in NC, whereas within Gag, neither of these mutants was seen in nucleoli or within punctate foci. Thus, these results indicate that loss of all basic residues in the CT of NC eliminates both nucleolar localization and the formation of nuclear foci in the context of L219A.Gag. Furthermore, mutation of a single basic amino acid cluster within the NT region of NC also eliminates both nucleolar accumulation and nuclear foci in the context of L219A.Gag-YFP.

Nucleolar localization of HIV NC and basic residue mutations

Analogous to RSV NC, the HIV NC protein encodes basic regions flanking the Cys-His motifs. Additionally, there is a PRKK sequence in HIV NC strikingly similar to the putative

NLS within RSV NC. Therefore, due to the similarity between RSV and HIV, we determined what basic residues are responsible for nucleolar localization of HIV NC. The HIV NC protein was divided into five regions based on the two Cys-His motifs (Fig. 3.6A). Unlike

RSV, however, HIV contains basic amino acid clusters only within the NT and L regions.

Using site directed mutagenesis, the Cys-His boxes were deleted and the mutant NC protein remained nucleolar, indicating that the Cys-His motifs were not necessary for nucleolar localization (Fig. 3.6B). We next examined the localization of each Cys-His flanking region.

The CT was diffuse throughout the cell (Fig. 3.6B). The NT and L regions, when 115 Fig. 3.6. Localization of HIV NC mutants. (A) Schematic diagram of YFP-HIV.NC is shown with the WT amino acid sequences in the amino acids immediately flanking CH1.

Mutant amino acids are depicted in bold. (B) YFP is used as a negative control and is seen localized to the nucleoplasm and cytoplasm, while excluding nucleoli. Wildtype NC is seen localized to nucleoli. Deletion of the Cys-His boxes does not disrupt nucleolar retention.

None of the basic flanking regions (NT, L, or CT) are sufficient for nucleolar localization of

NC. Mutations M1 and M2 show NC in the cytoplasm and nucleoli, with little protein localized to the nucleoplasm. M1/M2 is seen localized to the nucleoplasm and cytoplasm, and excludes nucleoli.

116

117 expressed separately, were within the nucleus and nucleoli (Fig. 6B). This observation suggested that sequences within the NT region and/or the L region contribute to the nucleolar localization of NC.

We next mutated the basic clusters located within the NT and L regions using site- directed mutagenesis (Fig. 3.6A). The M1 or M2 mutants each caused loss of nuclear localization of NC; however, the proteins still accumulated within nucleoli (Fig. 3.6B). The double mutant M1/M2 eliminated nucleolar localization completely (Fig. 3.6B). These results suggest that either basic cluster was sufficient for nucleolar localization. However, both sequences were necessary for proper localization of NC.

Relocalization of HIV Gag to nucleoli

Given that RSV Gag and NC interact within nucleoli, we next asked whether HIV

Gag might be detectable within nucleoli when NC is present. We thus performed FRET experiments using a HIV Gag fused to CFP and HIV NC fused to YFP. Given the lack of a substantiated report of HIV Gag trafficking within nuclei, we did not expect to see an interaction between Gag and NC within nucleoli. Surprisingly, when both constructs were co-transfected into HeLa cells, we observed a slight relocalization of HIV Gag into nucleoli

(Fig. 3.7A). This relocalization was also observed when HIV Gag was co-transfected with

HIV Rev-YFP (Fig. 3.7B, top panel). However no relocalization was observed when HIV

Gag and fibrillarin-YFP were co-transfected, implying a specific interaction between Gag and NC or Rev is occurring (Fig. 3.7B, middle panel). Considering the strong evidence of

Gag-NC and Gag-Gag interactions within the nucleus and nucleoli, we were surprised by the lack of relocalization of RSV Gag by RSV NC under normal, steady state conditions (Fig.

3.7B, bottom panel). The implications of this observation are addressed in the discussion.

However, it appears that the relocalization of HIV Gag to nucleoli is specific when HIV NC or

Rev is present.

To detect whether the interaction between Gag and NC or Rev was a direct or indirect interaction, FRET was performed similar to experiments using RSV constructs. A 118 Fig. 3.7. Interaction of Rev-independent HIV Gag with NC and Rev in nucleoli. (A)

FRET was taken as in Fig 2. HeLa cells were co-transfected with HIV.Gag-CFP and YFP-

HIV.NC. YFP was selectively photobleached by selecting the entire nucleus (dashed white circle). Nucleoli were then measured both before and after bleaching (white solid circles).

(B) Confocal images showing HIV Gag-CFP co-transfected with either Rev-YFP or fibrillarin-

YFP. RSV Gag-CFP and YFP-RSV.NC, expressed in QT6 cells, are shown last. (C) FRET efficiency (percent) of HIV.Gag-CFP with YFP-HIV.NC, Rev-YFP, or fibrillarin-YFP is shown.

The mean with standard error has been plotted. (D) Confocal images of either Rev- dependent Gag-CFP (top) or a HA-tagged Rev-dependent Gag co-transfected with Rev-

YFP. Relocalization of Gag to nucleoli is not seen in either transfection.

119

120 representative FRET experiment between HIV Gag-CFP and HIV NC-YFP is shown (Fig.

3.7A). The entire nucleus was selected for acceptor photobleaching (Fig. 3.7A, dashed circle). Individual nucleoli were then examined for an increase in donor fluorescence between pre- and post-bleaching (Fig. 3.7A, solid circle). FRET was performed on ten separate cells and the average FRET efficiency plotted (Fig. 7C). Gag had a significantly higher FRET efficiency when coexpressed with either NC (7.8%) or Rev (16.4%). However,

FRET efficiency of Gag coexpressed with fibrillarin (3.0%) was similar to the background

FRET between free CFP and YFP (2.6%). These data demonstrate a direct protein-protein interaction between Rev-independent HIV Gag and both HIV NC and HIV Rev within nucleoli.

The HIV Gag-CFP construct used in the above experiments contains several mutations that eliminate the need for the Rev export protein to facilitate translation of the

Gag protein (374). However, the absence of Rev results in a different export pathway for the Gag mRNA. We therefore asked whether this nucleolar relocalization of HIV Gag was also present when the Gag mRNA used the Rev export pathway. Co-transfecting either a

Rev-dependent HIV Gag-CFP or Gag-6HA constructs along with Rev-YFP did not reveal

Gag relocalized to nucleoli (Fig. 3.7D). This observation suggests that the nucleolar relocalization of Gag is dependent upon the mRNA export pathway taken for translation.

3.5 Discussion

Retroviral Gag protein trafficking during assembly is an important, yet poorly understood process. Although we previously reported that RSV Gag transiently traffics through the nucleus (363), we now report that Gag accumulates within nucleoli and at discrete subnuclear foci. Both the formation of Gag nuclear foci and the nucleolar retention of Gag depend on the presence of the NC domain, suggesting that RNA bunding might be required. It is possible that the interaction of Gag with a viral or cellular RNA contributes to nucleolar accumulation of Gag. 121 In HIV and MLV, the NC protein localizes to nucleoli during the early stages of infection (67, 350). When we performed mutational analysis of RSV NC, we found the basic sequences within the regions flanking the Cys-His boxes are necessary for nucleolar localization. This is not surprising given NoRSs are rich in basic residues (104). It is interesting to note the relationship between nucleolar localization and the proximity of basic residues to the Cys-His boxes. Basic amino acids not immediately adjacent to the Cys-His motifs were dispensable for nucleolar localization of YFP-NC. However, mutations immediately flanking either Cys-His motif cause a reduction in nucleolar accumulation of

NC. One explanation for the mutations flanking the Cys-His boxes could be a change in the structure of NC. Yet the basic regions adjoining the Cys-His boxes were found to be unstructured when NC is expressed by itself, making a structure change directly within the basic regions unlikely (456). Therefore, the work presented here may suggest a link between the presence of basic residues, the structure of the Cys-His boxes, and nucleolar localization of NC. Unfortunately, the data presented here does not exclude the possibility of NC being unable to bind RNA. One hypothesis for nucleolar localization of proteins links

RNA binding to nucleolar localization (75). Thus, loss of nucleolar localization of NC may represent a decrease in RNA binding. Previous studies involving HIV NC found that a lower number of basic amino acids correlated to reduced RNA binding (89). Therefore, NC localization may show how well NC or Gag can bind to RNA. In vitro RNA binding assays will be needed to identify a reduction of NC interaction with RNA in our basic residue mutants.

We found a partial link between NC nucleolar localization and L219A.Gag nucleolar accumulation. Neither the M1 mutant nor the M3/M4 double mutant were able to localize to nucleoli in either NC or Gag, whereas the other mutants (M2, M3, M4) were capable of some nucleolar accumulation in both NC and Gag. This correlation suggested that NC is necessary for the nucleolar localization of Gag. However, the localization phenotype between YFP-NC and L219A.Gag-YFP differed when basic residues were altered within the 122 NT region. Neither M5 nor M6, when present within Gag, localized to nucleoli, though both mutations retained some nucleolar localization when present in NC alone. This observation could be due to a change in Gag structure. Further studies will need to be performed to determine what effect the differences between the subnuclear localization of NC and Gag have on virus replication.

From confocal images of the M1 mutant, we found that the PKKRK sequence was necessary for nuclear localization of both NC and Gag. L219A.Gag.M1-YFP localized primarily to the plasma membrane with a diffuse signal throughout the nucleus and cytoplasm. NC has previously been implicated in the plasma membrane association of Gag

(52, 225, 316). Furthermore, inactivating NC-mediated nuclear localization by mutating the

NLS, rather than by deletion of the NC domain altered the trafficking pathway of Gag, resulting in the accumulation of Gag at the plasma membrane. A detailed analysis of the effects of the Gag.M1 mutant in plasma membrane binding, particle release will be needed to understand why this mutant traffics so strongly to the plasma membrane.

Previously, we demonstrated that Gag nuclear trafficking is transient, and very little

Gag protein is detected in the nucleus under steady-state conditions (213). Treatment with the CRM1 inhibitor Leptomycin B causes a redistribution of Gag from the cytoplasm to the nucleus (363). Additionally, the L219A.Gag NES mutant is also concentrated in nuclei

(366). Under these conditions, Gag localizes to nuclear foci and nucleoli in a subset of cells.

The finding that Gag is dynamic, moving between nucleoli and the nucleoplasm, suggests that the protein passes through the nucleolus. However, the observation that wildtype Gag-

CFP is not relocalized to nucleoli when co-expressed with YFP-NC (Fig. 3.7B) suggested that wildtype Gag may not traffic to nucleoli. Thus, it is possible that the LMB treated or

L219A Gag proteins, which cannot be exported from the nucleus, also have aberrant intranuclear transport. It is also possible that a cellular transport protein is needed for proper intranuclear trafficking of Gag, but the high concentration of Gag present within the nucleus saturates the transport factor, allowing Gag to accumulate within nucleoli. In 123 support of this idea, inhibition of CRM1 activity impairs the subnuclear trafficking of U3 snoRNA from Cajal bodies to nucleoli (44). Therefore, because the LMB treated and L219A mutant Gag proteins do not interact with CRM1, its intranuclear trafficking might be impaired, causing nucleolar accumulation.

Another hypothesis for Gag nucleolar localization is to facilitate interaction with a cellular protein or RNA that resides within the nucleolus. For example, both MLV and HIV virions contain an enriched population of small nuclear RNAs (314, 315). RSV also preferentially packages small nuclear and small nucleolar RNAs (158). These packaged small RNAs are all transcripts made by RNA Polymerase III. Newly synthesized Pol III transcripts are often found within or adjacent to nucleoli (269). This link between small RNA packaging and the nucleolus suggests that nucleolar localization of Gag may be necessary for these packaging events to occur. Further studies will determine whether Gag nucleolar localization is important in packaging of nucleolar RNAs.

It is also possible accumulation of the L219A.Gag-YFP mutant within nucleoli is a consequence of the RNA-binding capability of NC, and therefore Gag nucleolar localization is not important for viral replication. However, because the NC protein is observed within nucleoli during early infection (147, 350, 454), it is possible that nucleolar localization of NC is important for an unknown function during viral entry. Further studies are needed to test these proposed hypotheses.

We also asked whether there was a similar role of basic residues in nucleolar localization for HIV NC. In HIV-1, the basic residues within the NT and L regions in NC were responsible for nucleolar localization. Mutants similar to M1 and M2 in NC were found to inhibit gRNA packaging (339). Additionally, it has been demonstrated that the number of basic residues in HIV NC is important for general RNA binding and assembly (72, 89, 339).

It is possible that both gRNA binding and nucleolar localization are inseparable, as for HIV

Rev (37, 44). However, HIV gRNA has been reported to have nucleolar localization properties (285). Furthermore, a combination of HIV Gag, gRNA, and the cellular nucleolar 124 protein nucleolin promoted efficient assembly of HIV virus-like particles (421). These lines of evidence suggest that HIV gRNA packaging may also be linked to nucleoli.

Other than Tat and Rev, no other HIV proteins with nucleolar localization properties have been reported. Surprisingly, we observed the relocalization of HIV Gag to nucleoli when co-expressed with either HIV NC or Rev. To our knowledge, the findings presented in this work are the first observation of a possible nucleolar interaction of HIV Gag within nucleoli. It is interesting to note, however, that both NC and Rev are capable of relocalizing

HIV Gag to nucleoli. Rev has recently been implicated in enhanced gRNA packaging during viral assembly (43). It is possible that Gag and Rev interact by direct protein-protein contacts or through an RNA intermediate. However we only observed nucleolar localization with the Rev-independent Gag. The Rev-dependent Gag did not display the same phenotype. Thus it is possible that the nucleolar localization of Rev-independent Gag is related to its different RNA export pathway.

A possible selection mechanism is through a Rev-RNA-Gag interaction at a site immediately after gRNA nuclear export facilitated by Rev. Gag has been reported to first accumulate at a perinuclear site before assembling at the membrane (329). The perinuclear site was identified as the centrosome, and the ψ-containing RNA was required to induce the localization phenotype (337). However, further studies are needed to examine whether Gag and Rev colocalize within the cell, and whether disruption of the Rev export pathway alters

HIV Gag trafficking.

125

Chapter 4

Effect of NC Nucleolar Localization Signal Mutations on Viral

Genomic RNA Packaging and Infectivity

126 4.1 Abstract

The nucleocapsid (NC) domain of the Gag polyprotein is involved in multiple steps of the retroviral life cycle. During assembly, NC mediates the specific selection of viral genomic RNA (gRNA), and facilitates assembly and budding of new virus particles.

Previously we identified nucleolar localization of the L219A.Gag mutant, which is mediated by NC. Mutation of several specific basic amino acids flanking the Cys-His motifs prevented the NC protein from localizing to nucleoli. Furthermore, mutant Gag proteins containing these mutations were also reduced in nucleolar localization. These observations led us to explore the role of Gag and NC nucleolar localization in retroviral assembly and replication.

Analysis of wildtype NC and mutants containing changes in basic amino acids led to the conclusion that the presence of positively charged residues is an important factor for NC nucleolar localization. In the context of provirus, NC basic amino acid mutants did not affect virus release. Additionally, loss of Gag nucleolar accumulation did not alter gRNA packaging in the virus. However, infectivity was inhibited in viral mutants where the NC protein did not accumulate within nucleoli, indicating nucleoli may be important for viral replication. Furthermore, we found that mutations to the N-terminal region of NC affected proteolytic processing of the Gag protein or delayed infectivity, suggesting a defect in viral maturation.

4.2 Introduction

Viruses commandeer cellular processes to facilitate infection, and the trafficking of viral RNA and proteins through the cell is essential for viral replication. The Rous sarcoma virus (RSV) Gag protein assembles new virus particles at the plasma membrane, after selecting the viral genomic RNA (gRNA) from the milieu of both cellular and viral RNAs.

During the assembly process the Gag protein transiently traffics through the nucleus, a process that is linked to efficient incorporation of gRNA into virus particles (151, 365).

127 The observations reported in the previous two chapters found that nuclear trapped

Gag localizes to multiple subnuclear bodies. Chapter 2 found the presence of Gag nuclear foci, which were anchored within the nucleus, yet Gag protein was able to continually travel through the foci. Chapter 3 identified nucleolar localization of the Gag polyprotein. NC was essential for both subnuclear localization patterns of Gag, and basic amino acid clusters necessary for nucleolar localization were identified. NC basic amino acids have been implicated in multiple functions of viral assembly. There are 16 basic residues within NC, however only 8 are required for both Gag-Gag interactions and virus particle assembly

(232). Although the Cys-His boxes are required for gRNA packaging (51, 119, 282), basic residues play an essential role in specific RNA binding and packaging (231, 232). However, whether basic residues in NC are important for only RNA binding or if they serve multiple functions (i.e. protein localization/trafficking) is not known.

Virus-host interactions within the nucleus have been better studied in other viruses, which have been found to localize to subnuclear bodies during infection. The ICP27 protein from the herpes simplex virus interacts with splicing components within the splicing speckles to disrupt cellular splicing (376). ICP27 may also localize within nucleoli, although the role of nucleolar localization is not known (277, 279). The herpesvirus saimiri ORF57 protein, however, requires nucleolar association for nuclear export of intronless RNA (54). Deletion of the nucleolar localization signal (NoLS) in ORF57 sequesters the intronless RNA in the nucleus. Restoration of nucleolar localization via insertion of the HIV Rev NoLS restores the ability of the virus to export intronless RNA. The HIV Rev protein also localizes to nucleoli before interaction with the Rev-response element and facilitating unspliced RNA export from the nucleus (37, 101, 130). Thus, cellular localization of viral proteins is important for many aspects of viral replication.

We therefore asked what effects do our basic residue mutants have on the release of virus particles, gRNA packaging, and replication of RSV? In context of a proviral construct, none of the NC basic amino acid mutants were significantly reduced for virus particle 128 release. However, several NC basic amino acid mutants were deficient in gRNA packaging.

Although no relationship was found between gRNA packaging and nucleolar localization, infectivity was inhibited in a viral mutant that contained an NC protein that was unable to localize to nucleoli. Furthermore, we identified two basic amino acid mutants within the N- terminal region of the NC protein that appear to affect protease processing or maturation of virus particles.

4.3 Materials and Methods

Expression vectors, plasmids, and cells

Prague C RSV Gag and NES mutant (L219A and NES-A) expression vectors containing a CFP or YFP fluorophore were previously described (213). RSV NC was expressed from a pEYFP-N1 containing vector (Clontech) (67). pKoz.Gag-YFP was created through PCR amplification of the gag coding region of pL219A.Gag-YFP, which included the first twelve nucleotides upstream of the ATG start site of Gag. The PCR product was then inserted into a pEYFP-N2 vector (Clontech) using NheI-ApaI restriction sites and screened via size analysis using NheI-ApaI. p5’UTR.L219A.Gag-YFP was created by PCR amplification of the entire 5’UTR and gag coding region from a pRC.V8 vector containing the

L219A NES mutation (366). The 5’UTR and a portion of the N-terminal coding region of gag were ligated into pEYFP-N2 using NheI-ApaI and screened using PstI. The NC and Gag mutants M1, M2, M3, M4, M5, M6, SKL, and M3/M4 were described within the Materials and

Methods section of Chapter 3. The mutants RAA, RAA/M4, and M3/RKR were introduced into pEYFP-NC and pGag-YFP using site-directed mutagenesis (QuikChange, Stratagene).

Proviral constructs were created by site-directed mutagenesis in pCMV-GagPol (from

Rebecca Craven, Penn State College of Medicine). The NC region was then exchanged between the GagPol and RS.V8 plasmids using SbfI-HpaI restriction sites. Endonuclease digestion was used to identify clones containing the mutations and all cloning was confirmed with DNA sequencing. Primers used for RAA and M3/RKR are listed (Table 4.1). All 129 experiments were performed using the quail fibroblast QT6 cell line or the chicken fibroblast

DF1 cell line (183, 326). Transfections were performed using the calcium phosphate method (QT6) (144).

Confocal imaging

Approximately 2x105 cells were seeded onto 35-mm glass-bottomed dishes (MatTek

Corporation) and imaged using a Leica AOBS SP2 confocal microscope at 14 to 24 h post- transfection. Sequential scanning settings were used to differentiate CFP (excitation at 458 nm, emission at 465-490 nm, and 50% laser power) and YFP (excitation at 514 nm, emission at 530-600 nm, and 10% laser power) emission spectra. Light images were obtained using the transmitted light channel during fluorescence acquisition.

Profiling

Cells were co-transfected with wildtype or mutant pYFP-NC plasmid DNA and pfibrillarin-CFP plasmid DNA. Images of cells were acquired and the profiling application within the Leica LCS software was used. Briefly, a line was drawn through the center of a nucleolus, indicated by fibrillarin-CFP, and fluorescence intensity was measured for both the

CFP and YFP channels across the line. The peaks of both the CFP and YFP channels were compared to determine the nucleolar localization of NC or the NC mutants. If both CFP and

YFP intensity peaked at the same location within the cell, a score of 3 was given and the localization was considered “nucleolar”. When the YFP intensity remained static through the entire nucleus, a score of 2 was assigned and the localization was considered “nuclear diffuse”. If the YFP-NC intensity decreased when the fibrillarin-CFP peaked, a score of 1 was given and the NC protein was considered to “exclude nucleoli”.

Budding analysis

Budding assays were performed as previously described (71). Immunoprecipitated

RSV proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and quantified using a PhosphorImager (Bio-Rad). Budding efficiency was

130 Table 4.1. Primers for additional NC basic amino acid mutations. Primers are listed for

NC basic acid mutations in NC, Gag and Gag-Pol. Altered residues are marked in bold.

Screen site indicates the restriction enzyme used for identifying positive clones.

131

132 calculated as a ratio of the CA present within the media divided by the amount of Gag expressed in the lysate. The release of RS.V8 was set at 100% and all mutants were expressed as a percentage of the wildtype RS.V8.

Western blotting

Media from transfected QT6 cells expressing RS.V8, RS.V8 M5, Gag-YFP, or

Gag.M5-YFP was collected for 24-hours. Collected media samples were concentrated by ultracentrifugation at 126,000 X g through a 25% sucrose cushion and resuspended in sample buffer. Cell lysates were harvested using sample buffer as previously described

(151) and the media and lysates were resolved by SDS-PAGE. Samples were transferred to PVDF, blocked in 5% NFDM, probed with a rabbit antibody against RSV (1:15,000), a secondary anti-rabbit antibody conjugated to HRP (1:15,000, Sigma), and viewed by chemiluminescence using a Super Signal West Pico substrate solution kit

(ThermoScientific).

Ribonuclease protection assays

QT6 cells were transfected with either wildtype or mutant proviral DNA constructs.

Culture media was collected after 48 hours and filtered (0.2µ). Virus was pelleted by spinning at 126,000 X g through a 25% sucrose cushion. After resuspension of the pellet, aliquots were removed for reverse transcriptase (RT) assays. The mean RT values were used to normalize the amount of virus particles for each sample as previously described

(94). Viral RNA was extracted using a QIAamp viral RNA mini kit (QIAGEN). Ribonuclease protection assays were performed as previously described (150). A 318-nucleotide antisense probe was transcribed with T7 RNA polymerase with [32P]CTP (spanning the splice acceptor site of the env gene) and used to detect both unspliced (263-nucleotide fragment) and spliced (183-nucleotide fragment) viral RNA. Samples were separated by

7.5% polyacrylamide/12 M Urea SDS-PAGE and then quantified using a PhosphorImager

(BioRad).

Viral infectivity assay 133 Infectivity assays were performed as previously described (397). Media samples were collected from QT6 cells expressing either wildtype or mutant provirus after 48 hours.

Virus was concentrated by ultracentrifugation at 126,000 X g through a 25% sucrose cushion. RT assays were performed as described above to normalize the amount of virus particles between wildtype and mutant virus. Equal amounts of virus were added to uninfected DF1 cells. Cells were then assayed for the presence of GFP, which is expressed from the RS.V8 provirus, by flow cytometry (FACSCanto, BD Biosciences). The percent of cells expressing GFP were examined every three days until all infectious virus reached approximately 95% green cells, or after 21 days for less infectious virus. A minimum of three infection assays was performed from two separate transfections.

4.4 Results

Subcellular localization of NC basic mutants

Chapter 3 previously characterized the importance of basic residues in Gag nucleolar localization. This chapter expands on that work and explores the role that these mutations have on viral replication. Several new basic residue mutations have been added to help identify the role of Gag nucleolar localization (Fig 4.1A). The new mutants, in the context of YFP-NC, are shown (Fig. 4.1B). Previous studies found that the first arginine of the RKR cluster immediately flanking the second Cys-His motif was crucial for gRNA packaging (231, 232). However, mutation of RKR to RTL did not drastically decrease the amount of viral RNA incorporated into virus particles. The YFP-NC.RAA mutant, similar to the RTL mutant, was diffuse throughout the nucleoli and nucleoplasm (Fig. 4.1B). Mutation of further downstream basic residues, YFP-NC.M4/RAA, caused the fluorescent signal to exclude nucleoli (Fig. 4.1B). The mutant YFP-NC.M3/RKR maintained an overall net charge identical to the wildtype NC protein, but shifted the RKR basic cluster downstream 3 amino acids. The YFP-NC.M3/RKR mutant localized to nucleoli (Fig. 4.1B).

Profiling localization phenotypes of wildtype and mutant NC proteins 134 Fig. 4.1. Schematic diagrams of YFP-RSV.NC and basic residue mutants. (A) Wildtype residues are shown for the N-terminal, Linker, and C-terminal basic regions. Altered residues are depicted in bold; unaltered residues are shown as dots. (B) Cells expressing fibrillarin-CFP (middle column), and YFP-NC.RAA, M4/RAA, or M3/RKR (left column) were imaged using confocal microscopy. A single representative image is shown for each protein combination.

135

136 With confocal imaging, many of the NC basic residue mutants displayed multiple nuclear localization phenotypes. Both YFP-NC.M3 and YFP-NC.SKL are given as examples of this difference in appearance (Fig. 4.2A). The fluorescent signal was observed as localized to nucleoli, diffuse through both the nucleus and nucleoli, and within the nucleus excluding nucleoli. To determine the overall localization trend of each mutant, we used a method called profiling, which is commonly used to determine the co-localization of two proteins (307, 333).

Cells co-expressing fibrillarin-CFP and either wildtype or mutant YFP-NC were imaged using confocal microscopy. Using the Leica software, a line was drawn through the nucleolus, marked by fibrillarin-CFP, and both the CFP and YFP intensities were measured across the line. If the CFP and YFP channels peak at the same point in the cell, as shown with YFP-NC (Fig. 4.2B, panel a), the NC protein was considered localized to nucleoli, and a score of 3 was given. When the YFP intensity remained the same across the nucleus, represented by YFP-NC.SKL (Fig. 4.2B, panel b), the localization was categorized as diffuse through both the nucleus and nucleoli. This profile was given a score of 2. When the YFP signal decreased as CFP increased, shown by YFP-NC.M1 (Fig. 4.2B, panel c), the NC protein was excluding nucleoli, and a score of 1 was assigned.

A minimum of 15 cells from each of the NC basic mutants was analyzed by profiling and plotted on a scattergram (Fig. 4.2C). The line represents the average score with standard error bars, and individual cell scores were plotted. The results can be separated into three groups. The wildtype (score = 2.8), M4 (2.9), M5 (2.6), and M3/RKR (2.9) mutant

NC proteins scored the highest. Middle range scores were assigned to the M2 (2.2), M3

(1.9), M6 (1.7), RAA (2.0), and SKL (2.0) NC mutants. The lowest scores were assigned to the M1 (1.0), M3/M4 (1.1), and M4/RAA (1.2) mutant NC proteins. Although the NC protein is not Gag, we have previously demonstrated that the Gag protein localizes to nucleoli.

137 Fig. 4.2. Localization profile of NC basic residue mutants. (A) QT6 cells expressing basic residue mutants show three distinct localizations: nucleolar, nuclear diffuse, and nucleolar exclusion. Examples of all three localization phenotypes for YFP-NC.M3 and

YFP-NC.SKL are shown. (B) Profiling was used to determine the average localization of the various NC basic residue mutants. QT6 cells co-transfected with fibrillarin-CFP (red) and

YFP-NC or mutant NC (green) were imaged using confocal microscopy. After image acquisition, a line was drawn through the center of the nucleoli, as determined by the localization of fibrillarin-CFP. At each pixel across the line, fluorescence intensity was measured in both the CFP (fibrillarin, red) and YFP (NC, green) channels. If the NC protein was localized to nucleoli, the red and green channels peaked at the same location (a, YFP-

NC), and a localization score of 3 was given. If the NC protein showed a nuclear diffuse phenotype, the green channel intensity remained flat throughout the nucleus (b, YFP-

NC.SKL) and a score of 2 was assigned. If the NC protein excluded nucleoli, the green fluorescence decreased as the red channel increased (c, YFP.NC.M1), and the cell was scored a 1. (C) An average score for localization phenotypes of wildtype NC protein and basic residue mutants are plotted within the scatterplot. The bars represent the standard error of the mean. Each dot surrounding the mean and error bars represents a single cell scored for the NC or mutant protein. A minimum of 20 cells was counted for each construct plotted.

138

139 Effect of basic residue mutations on particle assembly

RNA binding properties of proteins frequently depend upon basic amino acids (44,

433). Furthermore, the formation and stability of immature virus particles requires the presence of RNA, and the ability of the Gag protein to bind RNA (52, 72, 302, 432). In RSV, the requirement for Gag-Gag interactions is reliant upon the number of basic amino acids within the NC domain (232). Using particle release assays, we therefore asked whether any of our basic residue mutations eliminated the ability of Gag to form virus particles. We expected that if we affected general NC RNA binding, we would observe a reduction in virus particle assembly.

Wildtype or mutant RS.V8 proviral constructs were transfected into two identical plates of cells. One plate was metabolically labeled for 2.5 hours, while the second plate was starved for 30 minutes, followed by a 5-minute metabolic labeling. Following labeling, lysates (5 minute) or media (2.5 hour) was collected and immunoprecipitated with an αRSV antibody. Lysates and media were then resolved on a 12% SDS-PAGE gel and visualized by a PhosphorImager. A single band of Gag was detected within the lysate (Fig. 4.3A). A triplet representing the three CA species was observed in the media (Fig. 4.3B).

Quantitation of particle release for each mutant revealed no significant change from wildtype

(Fig. 4.3C). These results indicated that the basic residue mutations within the NC domain of Gag do not affect the level of particle release. It is also suggestive that general RNA binding of the Gag proteins containing these mutations was not affected.

Effect of M5 mutant on mature virus particle stability

Through the course of expression studies of these NC basic mutants, we made an unexpected observation. Media was collected for 24 hours from cells expressing either

RS.V8 or RS.V8 M5 and virus particles were concentrated through a 25% sucrose cushion using ultracentrifugation. Both media and lysates were separated using SDS-PAGE, and viral proteins were identified using an αRSV antibody for western blotting. Although the wildtype virus produced Gag within the lysate and produced mature particles (Fig. 4.3D, 140 Fig. 4.3. Viral budding of NC basic amino acid mutants. (A) Cells expressing wildtype or mutant RS.V8 provirus were starved for 30 minutes, followed by metabolic labeling with

35S for 5 minutes. After collection of cell lysates and immunoprecpitation using an αRSV antibody, the proteins were separated by SDS-PAGE and visualized by phosphorimaging.

The result of a representative lysate gel is presented. The gap between the gels represents two separate experiments; the line separating RS.V8 from M3/RKR, RAA, and RAA/M4 is a crop from the same gel. The arrow indicates the size of Gag. (B) Cells expressing wildtype or mutant RS.V8 provirus were metabolically labeled with 35S for 2.5 hours. After labeling, media was collected and immunoprecipitated using an αRSV antibody. Proteins were then separated by SDS-PAGE and visualized by phosphorimaging. This gel is coupled with the lysates presented in A. The arrow indicates the size of CA. (C) The average of a minimum of three 35S budding assays is shown on the bar graph. Calculations were performed by dividing the CA from the media by the Gag from the lysate. Mutants were then set as a percent of wildtype. The bars represent the standard error of the mean. The graph is separated into L (light gray), CT (dark gray), and NT (black) mutants. Statistics were performed by student’s t test; no statistical difference was found between wildtype and any of the mutants. (D) Immunoblot analysis of both lysates and media collected from cells expressing Mock transfected (lane 1), RS.V8 (lane 2) or RS.V8 M5 (lane 3), or Gag-YFP

(lane 4) or two different clones of Gag.M5-YFP (lanes 5 and 6). Proteins were detected using an αRSV antibody. Molecular weight markers and indicated to the left. Arrows on the right identify Gag-YFP, Gag, and CA.

141

142 lane 2), no CA was detectable in the media for RS.V8 M5 (Fig. 4.3D, lane 3). Additionally, several degradation products were detected in the M5 lysates, indicating a possible processing defect or degradation of the protein might have occurred. Given that the M5 mutant was measured at wildtype levels during the metabolic labeling (Fig. 4.3C), this result suggested that the M5 virus particles were unstable after release from the plasma membrane.

To test the hypothesis that the M5 virus particles were unstable, we performed the same western blot assay on two separate clones of Gag.M5-YFP, which lack the viral protease and only form immature VLPs. Both wildtype Gag-YFP (Fig. 4.3D, lane 4), and

Gag.M5-YFP c1 and c3 (Fig. 4.3D, lanes 5 and 6, respectively), were capable of forming

VLPs at detectable levels. Quantitation of virus or VLP release was performed on both proviral and Gag-YFP constructs using densitometry. RS.V8 M5 was extremely reduced from wildtype (1%). Gag.M5-YFP produced much higher levels for clones c1 (30%), and c3

(90%) when compared to wildtype Gag-YFP. Furthermore, no degradation products were detected with the Gag.M5-YFP constructs, supporting the idea that a processing defect is occurring with RS.V8 M5. Together, these findings indicate a maturation or stability defect with the M5 mutant in the context of full virus.

Incorporation of genomic RNA into virus particles

The particle release assays suggested that general RNA binding was unaffected in any of the NC basic residue mutants. We next asked whether the specific binding of gRNA was affected. To determine the efficiency of gRNA packaging, sensitive and quantitative ribonuclease protection assays were performed. The amount of RNA used from the wildtype virus, and each mutant, was normalized via RT activity. The RT activity was measured prior to RNA extraction as described in the materials and methods. A representative blot for each mutant is shown (Fig. 4.4A). Using a probe for the 3’ splice acceptor site in env, we are able to detect both the spliced vRNA and the unspliced gRNA.

143 Fig. 4.4. Effect of NC basic residue mutations on gRNA packaging. (A) RS.V8 or

RS.V8 NC mutant DNA was transfected into QT6 cells. Virus was collected for 48 hours and normalized via an RT assay. RNA was extracted from equal numbers of virus particles and gRNA concentration was measured by RPA. After digestion of the 318-nt 32P- radiolabled antisense riboprobe and digestion of the unprotected fragment with RNase treatment, the RNA was separated by gel electrophoresis and visualized by

PhosphorImager. Spaces between the gels represent results from independent experiments. Arrows on the left identify nucleotide sizes, and arrows on the right denote the species of RNA (free probe, unspliced gRNA, and spliced vRNA). (B) The results of four

RPAs were graphed, with the bars representing standard error of the mean. The bars were colored based on infectivity data presented in Fig. 4.5. Black bars represent wildtype infectivity, gray bars denote an intermediate phenotype, and white bars were noninfectious.

144

145 The averages of at least 3 independent RPA experiments for each mutant were plotted in a bar graph (Fig. 4.4B). The amount of gRNA packaging of the wildtype was set to 100%, and all mutants were calculated as a fraction of that amount. RS.V8 M5 was the only mutant that was not significantly lower than wildtype (117%). M4 packaged a high amount of gRNA (70% of wildtype). M2, M6, and RAA/M4 packaged levels slightly lower than wildtype (66%, 62%, and 45% of the wildtype level, respectively). The RAA, M4/RAA,

M3/RKR, SKL, M3, M3/M4, and M1 mutants all were severely reduced for gRNA packaging

(39%, 29%, 26%, 17%, 12%, 11%, and 10% of wildtype levels, respectively).

These results show that most mutations immediately flanking the C-terminus of the

Cys-His boxes reduce gRNA packaging dramatically (M1, M3, M3, SKL, and M3/M4), but mutations further away from the Cys-His motifs are unchanged or only modestly affected

(M4 and M5). M2 and M6, which flank the N-terminus of the Cys-His motifs, are also only slightly reduced for gRNA packaging. RAA is also reduced for gRNA packaging, but it is interesting to note that the loss of two more basic amino acids in NC, RAA/M4, does not significantly change the amount of gRNA incorporated into virus particles. Finally, the

M3/RKR mutant is strongly reduced in gRNA packaging, suggesting that the location of the basic amino acids rather than the number of residues is important for gRNA packaging.

Infectivity of proviruses containing NC basic amino acid mutations

We next asked whether the basic amino acid mutants were able to replicate in culture. Many of the viral vectors containing NC basic amino acid mutations incorporated less than 30% of wildtype levels of gRNA (M1, M3, M3/RKR, SKL, and M3/M4). If the virus required a certain amount of gRNA to propagate an infection, these mutants would be noninfectious. Intermediate mutants (M2, M6, RAA, and RAA/M4), which packaged between

30% and 70% of wildtype gRNA levels were predicted to be weakly infectious or noninfectious. Mutants that incorporated 70% of wildtype gRNA levels or higher (M4 and

M5) were expected to be fully infectious. However, because the M5 mutant did not produce mature virus (Fig. 4.3D), we expected M5 to be noninfectious. 146 To assess the effect of the NC mutations on viral propagation, a spreading assay was performed. Wildtype or mutant proviral vectors were transfected into QT6 cells, and media was collected for 24 to 48 hours. After the collection period, a small portion of the media was measured for RT activity, and equal amounts of virus were added to uninfected

DF1 cells. The newly infected cells were examined for the spread of GFP, which is expressed from the RS.V8 genome, using flow cytometry. Measurement of GFP spread was performed every three days after the initial infection. The percentage of green cells was plotted per time-point, and representative infection experiments are shown (Fig. 4.5).

These assays were performed at least three times. Wildtype virus peaked at day 6 post- infection for each experiment performed (Fig. 4.5A, B, and C, solid square). As predicted,

M4 was fully infectious, increasing in GFP-positive cells at a rate similar to wildtype (Fig.

4.5A, open circle). Additionally, the M1, M3, M3/M4, and SKL mutants were noninfectious, as expected (Fig. 4.5A, solid triangle, solid circle, open square, solid diamond, respectively).

The M5 mutant, which we were able to detect only a small amount of mature virus from via the RT assay, was also noninfectious (Fig. 4.5B, open circle).

The infectivity of the M6 mutant was unexpected (Fig. 4.5B, solid diamond). GFP positive cells would remain extremely low for 6 to 9 days post-infection, until the percentage of green cells would increase nearly as rapidly as wildtype. Sequencing analysis confirmed that the M6 mutation was present within the cells at day 18 post-infection, and no other mutations were found within the gag coding region. This result suggested that M6 establishes infection slowly, until enough cells within the population were infected that the slow phenotype was no longer detectable. It is also possible that a second-site suppression mutation existed outside of the gag coding region.

147 Fig. 4.5. Infectivity of virus containing NC basic amino acid mutations. Virus collected from transfected QT6 cells was placed onto uninfected DF1 cells and examined every 3 days post infection for spread of infection. Cells were counted using flow cytometry, and the percentage of green cells per time-point is plotted. Each graph is a representative assay of at least three independent infection assays. (A) RS.V8.M4 was as infectious as wildtype, but RS.V8.M1, M3, M3/M4 and SKL were noninfectious. (B) RS.V8.M2 increased in GFP- positive cells at a rate similar to wildtype. RS.V8.M6 was delayed for 6 days post-infection before spreading at a rate similar to wildtype. (C) RS.V8.RAA was slightly slower in spreading than wildtype. RS.V8.M4/RAA was severely inhibited for infectivity, showing only a small percentage of infected cells 21 days after infection. RS.V8.M3/RKR was noninfectious.

148

149 The arginine immediately flanking the second Cys-His motif was necessary for gRNA packaging (231), however the role of charge and infectivity has not previously been explored. The mutant M3/RKR was unable to restore infectivity to the M3 mutant (Fig 4.5C, solid triangle), indicating that the location of charge within NC is important, or that it is below the necessary amount of gRNA to allow infection. The RAA mutant, which still possessed the essential arginine residue, was infectious and reached wildtype levels within 9 to 12 days post infection (Fig. 4.5C, solid circle). Further mutations to basic residues within the N- terminal region of NC (M4/RAA) severely reduced viral infectivity (Fig. 4.5C, open circle).

Together, the results from this section suggest a link between the loss of specific basic amino acid clusters (M2, M6, RAA, M4/RAA) and a reduction in infectivity.

Viral infectivity was scored based on the time-point of peak infectivity of the wildtype virus (day 6 post-infection, ++++). Mutants that were slightly delayed (reaching maximum

GFP-positive cells on day 9 or day 12 post-infection) were considered intermediate phenotypes (+++ and ++, respectively). The M4/RAA mutant was slightly infectious, and never reached wildtype levels during the period of these experiments; it was scored “+”. All other mutants that were noninfectious were scored “-“. The summary of these results is presented in Table 4.2.

4.5 Discussion

In this report, we set out to understand the role of NC and Gag nucleolar localization with respect to virus replication. To this end we examined the effect of NC basic residue mutants on virus budding, gRNA packaging, and viral infectivity. The summary of the results from these functional studies is presented in Table 4.2. Several interesting mutants are discussed here, followed by a general discussion of overall trends and observations.

We previously identified a cluster of basic amino acids (RKRR in the L region) that was necessary for nuclear localization of both the NC and Gag protein. The M1 mutant virus (RKRR->AAAA) released virus particles at a rate similar to wildtype. Because RNA is 150 Table 4.2. Summary of NC basic amino acid mutations and effects on virus replication. The table presented summarizes the quantitative data described in the text.

The M5 mutant is highlighted in bold due to the inability of this mutant to produce collectable mature virus particles. Gag localizations were determined from Fig. 3.5: No, nucleolar; N, nuclear; C, cytoplasmic; M, membrane. N/D indicates experiments were not done.

151

NC Basic Localization L219A.Gag % % gRNA Mutant AA # Score Localization Budding Packaging Infectivity

Wildtype 16 2.8 No 100 100 ++++ L M1 12 1.0 M,C,N 99 10 - M2 14 2.2 No 103 66 +++ CT M3 13 1.9 No 113 12 - M4 14 2.9 No 64 71 ++++ SKL 14 2.0 N/D 78 17 - RAA 14 2.0 N/D 77 39 +++ M3/M4 11 1.1 N 93 11 - M4/RAA 12 1.2 N/D 62 45 + M3/RKR 16 2.9 N/D 96 29 - NT M5 14 2.7 N 94 117 - M6 14 1.7 N 96 62 ++

152 required as a structural element for particle assembly and infectivity (52, 72, 302, 359, 431), this result indicated that general RNA binding of Gag was not adversely affected. However, the M1 mutant was extremely reduced for gRNA packaging and is noninfectious. The strong plasma membrane localization of this mutation in context of L219A.Gag.M1-YFP presented in the previous chapter (Fig. 3.5) was reminiscent of the Myr1E mutant (151,

363). Myr1E localizes strongly to the plasma membrane of infected cells and is largely insensitive to the CRM-1 export-blocking drug, leptomycin B. The similarity between the

Myr1E and M1 mutants suggested that M1 was directed to the plasma membrane to a larger extent than wildtype. It is possible that the loss of nuclear localization was responsible for the inefficient incorporation of gRNA into virus particles. However, the studies presented here do not differentiate loss of nuclear localization and loss of Gag-ψ RNA interaction.

Interestingly, although Gag localized strongly to the plasma membrane, metabolic labeling studies show that the M1 mutant was not increased for particle production like the Myr1E mutant (363). Thus, additional studies are needed to identify what effect the M1 mutant has on Gag RNA binding, intracellular trafficking, and particle assembly.

In addition to identifying a sequence important for nuclear localization within NC, we found several basic amino acids involved in nucleolar localization of the NC and Gag proteins, located within the C-terminal basic region of NC (M3/M4). We examined the effects of basic amino acid mutations immediately flanking the N-terminal and C-terminal ends of the second Cys-His motif within NC. Changing the basic residues on the N-terminal end of the second Cys-His motif to alanines (M2) had little effect on viral budding, packaging, or replication. However, altered basic residues immediately flanking the C- terminus of the Cys-His box (M3) severely inhibited incorporation of gRNA into virions and was noninfectious. This result was similar to the previously reported SKL mutation, which also reduces gRNA packaging (231, 232). Although mutations further downstream of the

RKR basic cluster did not drastically effect viral replication (M4), the M3/M4 double mutant packaged gRNA at levels similar to the M3 mutant, suggesting that the M3 mutation is 153 dominant. Although the M3 and M3/M4 results suggest that nucleolar localization is necessary for gRNA packaging, prior studies demonstrated the importance of a single basic amino acid immediately flanking the second Cys-His motif (the first arginine of RKR) (231).

From this previous report it was proposed that, for gRNA packaging, the location of the basic amino acids is as important as the net charge of the NC domain within Gag. Thus it is possible that the M3 mutant is incapable of binding the ψ packaging sequence, and localization studies directed at this mutant will be difficult to interpret. Thus more studies are needed to understand why the first arginine of the RKR basic cluster is essential for the interaction of Gag with ψ-RNA.

To further test whether the location of the basic amino acids is important for packaging, we made the M3/RKR mutant, which maintains the same number of basic residues as wildtype NC, but shifts the basic amino acids three positions toward the C- terminus. When expressed alone, the YFP-NC.M3/RKR mutant had a nucleolar localization score (2.9) similar to wildtype. However, the M3/RKR mutant provirus packaged relatively little gRNA (29%) and was noninfectious. These results demonstrate that a high NC nucleolar localization score is not sufficient for gRNA packaging or viral infectivity. These data also support the previous hypothesis that the location of the basic amino acids within

NC is important for incorporation of gRNA into virus particles (232).

The RAA and M4/RAA mutants revealed different NC localization scores (2 and 1.2, respectively), yet both proviral mutants released virus particles at a rate similar to wildtype and incorporated equivalent amounts of gRNA into released particles (39% and 45% of wildtype, respectively). This observation indicates that a high NC nucleolar localization score is not necessary for gRNA packaging. However, the ability to spread during an infection drastically differed between the two mutants: the RAA mutant was nearly as infectious as wildtype (+++), but the M4/RAA mutant was unable to efficiently spread to uninfected cells (+). We found a correlation between the number of basic residues in NC and the nucleolar localization of YFP-NC (Pearson’s coefficient = 0.87), suggesting that a 154 high NC localization score is necessary for efficient replication, probably during early infection. It is possible that Gag is unable to properly acquire a necessary cellular factor during early assembly. However, it is more likely that the loss of basic residues inhibits the

RNA chaperone activity of NC during early infection because NC is essential for tRNA priming, and efficient reverse transcription during entry of the virus (412). Further experiments are needed to examine how these basic residue mutations affect the chaperone capabilities of NC.

In addition to the basic amino acid mutations within the L and CT regions of NC, we examined the effect of altered basic amino acids within the NT region. The M6 mutant had an intermediate NC localization score (1.7), similar to the other basic amino acid mutants that were immediately flanking either Cys-His motif (M2, M3). However, unlike the

L219A.Gag.M2-YFP and L219A.Gag.M3-YFP mutants, L219A.Gag.M6-YFP was not observed within nucleoli. In context of the provirus, the RS.V8.M6 mutant packaged an intermediate amount of gRNA (62%). This mutant, however, consistently lagged for a 3-day period before spreading at a rate similar to wildtype during the infectivity assays. A similar delay in infectivity is seen in mutants with maturation defects in both RSV and HIV (53, 221).

In RSV, a mutation causing maturation defects was found within the spacer peptide (SP), which is located between CA and NC domains of the Gag polyprotein (53). This SP mutant showed rapid CA processing compared to wildtype, and was delayed for 3 days before reaching reverse transcription levels similar to wildtype. A similar mutation was found within the HIV-1 Gag SP1 region, which intervenes the CA and NC domains (221). In the HIV-1

SP1 mutant, CA processing appeared inhibited, although a similar 3-day delay in infectivity was seen. Together, these data suggest that a processing or maturation defect is responsible for delayed infectivity of the M6 mutant presented here. Although RSV NC is part of a CA-NC intermediate during maturation of virus particles, interplay between NC and

SP has not been reported. It will be necessary to examine the effect of the M6 mutant on

155 CA processing and capsid maturation to understand why the lag-time exists for the previously reported SP mutants and the RS.V8.M6 mutant reported here.

The second N-terminal mutant of NC (M5) presented an interesting phenotype in context of the provirus. Under metabolic labeling, the M5 mutant released virus particles at an efficiency similar to wildtype virus. However, we were unable to concentrate virus particles from media of RS.V8 M5 transfected cells using ultracentrifugation, as shown by

Western blot analysis. Examination of the lysate revealed multiple processing and/or degradation products, suggesting that the viral protease was activated before release of assembling virus particles. Further support for this possibility stems from cells transiently transfected with Gag.M5-YFP, which lack the viral protease. No processing or degradation products were detected within the lysates of Gag.M5-YFP expressing cells, and we were able to collect VLPs from the media of these cells. This defect in budding is similar to that seen in RSV mutants where the viral protease is mutated to increase catalytic activity (443,

444). Although NC has been shown to be vital for activation and the first proteolytic steps of the viral protease in both RSV and HIV (443, 444, 461), to the best of our knowledge the M5 mutant reported here is the first known identification of a mutation in NC that increases the proteolytic cleavage rate during viral assembly. Further examination of the M5 mutant is needed to understand the mechanisms of NC and PR interplay during Gag processing.

In addition to specific mutants discussed above, we noticed several general trends with the NC basic amino acid mutants presented in this report. None of the mutants presented here released virus particles at a rate significantly different from wildtype, as seen from the metabolic labeling assays. This observation agrees with previous studies that suggest a minimum of eight basic amino acids are needed to maintain Gag-Gag interactions and virus particle assembly (232). Because RNA is an essential structural element for the formation and stability of retroviral particles (302), it is unlikely that any of the basic amino acid mutants presented in this report are unable to bind RNA. Thus, any mutant that is unable to package gRNA is not due to a loss of general RNA binding, but loss of specific 156 (i.e. ψ-containing) RNA interactions. This inability to bind ψ-RNA may be due to a loss of ψ sequence recognition by NC or an inability of Gag to travel to the site of Gag-gRNA interactions. In vitro studies examining the ψ-binding ability of these basic residue mutants in context of Gag will be necessary to separate the two possibilities.

There also appears to be a threshold or correlation relating gRNA packaging to infectivity. In our experiments, any mutant that packages gRNA at levels from 70% to 100% of wildtype was indistinguishable from wildtype for infectivity. Mutants within the packaging range of 30% to 70% of wildtype gRNA levels were reduced in infectivity. Any mutant that packaged gRNA at less than 30% of wildtype was found to be noninfectious. This result suggests that a minimum amount of incorporated gRNA is necessary to facilitate virus infection. It is possible that viral RNA contaminated the samples, as no RNase treatment was performed to eliminate RNA that may be on the outside of virus particles. However, the possibility of RNA contamination would not change the interpretations of these results, only the amount of RNA necessary for infection to occur. The possibility of a minimal amount of gRNA required for infectivity has not been identified or discussed prior to this report, yet similar interpretations can be inferred from a study using HIV: several basic amino acid mutations were made on HIV NC, all of which caused a reduction in gRNA packaging (89).

One mutant, packaging gRNA at 45% of wildtype was infectious, but a second mutant packaging 22% of wildtype gRNA levels was noninfectious. Thus, there may be some minimal amount of RNA required for a virus to enable an ongoing infection.

There are also several caveats to the studies in this report. Although we make conclusions relating nucleolar localization and viral function, it should be noted that the localization is based on the NC protein and not the Gag polyprotein. Chapter 3 demonstrated several differences between the localization of NC and Gag (Fig 3.4 and 3.5).

Both the YFP-NC.M5 and YFP-NC.M6 mutants localized to nucleoli (2.9 and 1.7, respectively), yet neither mutant was observed within nuclear foci or nucleoli when expressed as a Gag mutant. It is likely that the RNA binding properties of NC are different 157 from that of Gag, possibly due to structural differences or varying interacting partners of NC and Gag. A difference between NC- and Gag-RNA interaction has been observed in HIV studies, where minor effects on NC binding caused a more drastic decrease of gRNA packaging in context of the virus (89). To separate the differences in RNA-binding of the NC and Gag proteins, in vitro binding assays will be performed.

It has been proposed that, because ribosomal RNA is present within nucleoli, any protein within the nucleus that binds RNA will localize to nucleoli (75). If this hypothesis were correct, loss of nucleolar localization of NC would potentially correlate with a loss of

RNA binding ability. Indeed, we found that loss of basic amino acids in NC correlated with a lower NC nucleolar localization score. Previous studies involving HIV NC found that the number of basic amino acids correlated to RNA binding ability (89). Taken together, our NC localization may identify the ability of NC to bind RNA, but a reduction in RNA binding seems unlikely given that none of the mutants presented in this report were deficient for virus particle production. Thus, further studies are needed to identify potential differences in

Gag and NC interactions with RNA.

Although no link between nucleolar localization and gRNA packaging was found, we discovered a possible relationship between nucleolar localization and infectivity. Future studies will use a gain-of-function approach, restoring nucleolar localization of NC and Gag through the insertion of a heterologous nucleolar localization signal. With localization restored, we will be able to examine the effect on virus particle production, gRNA packaging, and infectivity. These studies will provide a better understanding of the role of NC and Gag subnuclear localization during retroviral assembly.

158

Chapter 5

Insertion of Heterologous Nucleolar Localization Signals Restores

Viral gRNA Packaging to a Nucleolar Deficient Gag Protein

159 5.1 Abstract

Subcellular trafficking and localization of proteins to the proper compartment are often essential for their function. The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus that leads to efficient incorporation of the viral genomic RNA

(gRNA) into virus particles. Selection of gRNA is mediated through the nucleocapsid (NC) domain. When expressed alone, NC localizes to nucleoli. We recently discovered that NC facilitates nucleolar localization of Gag, and although nucleolar localization of NC is neither necessary nor sufficient for gRNA packaging, it is necessary for efficient infectivity. To further explore the role of Gag nucleolar localization in viral replication, we used a gain-of- function approach, inserting a heterologous nucleolar localization signal (NoLS) from either the HIV-1 Rev protein or the HSV ICP27 protein into a viral mutant deficient in nucleolar localization of NC and Gag. Each sequence was sufficient to restore nucleolar localization of NC and Gag. Surprisingly, although the Gag protein from either viral mutant appeared nuclear under steady state condition, the NoLS ICP27 did not hinder viral assembly, but the

NoLS from Rev decreased viral budding. However, both NoLSs were capable of rescuing gRNA packaging in the context of a proviral vector, but neither could restore infectivity.

Although these results support the hypothesis that gRNA packaging is linked to nucleolar localization of the Gag protein, because both NoLSs are also RNA-binding domains, an increase of RNA binding may explain the restoration of gRNA packaging.

5.2 Introduction

The eukaryotic cell nucleus is an organized and complex environment containing many discrete and dynamic compartments (290). The nucleolus is the largest of these subnuclear components and was originally thought to be responsible for only ribosomal biogenesis. However, recent studies have identified many new roles for nucleoli, including cell cycle regulation, mRNA processing, tumorigenesis, and cell stress responses (75, 313,

380). Continuing studies have identified a nucleolar protein population that exceeds 700 160 (13, 14, 241, 367). Given that nucleoli are involved in a variety of different cellular functions, it is likely that the ability to localize to and interact with nucleoli is important in the regulation of the non-ribosomal functions of the nucleolus.

In addition to cellular proteins, retroviruses also use nucleoli during infection. HIV-1

Rev, a protein responsible for nuclear export of unspliced viral RNAs. Although Rev normally shuttles between the nucleus and the cytoplasm, it is found localized to nucleoli under steady-state conditions (90, 284, 328). The nucleolar localization signal (NoLS) of

Rev overlap with the NLS, and it is also an RNA binding domain, suggesting a link between nuclear export of viral RNA and nucleolar localization (37, 43, 64, 266, 267, 328). Support for this hypothesis comes from multiple studies. Multimerization of Rev is required for function and occurs within nucleoli (101). The nucleolar protein B23 is bound to Rev until an

RNA containing the Rev response element (RRE) displaces B23, suggesting that the site of interaction between unspliced viral RNA and Rev occurs within nucleoli (130). Rev recruits the nuclear export protein CRM1 to nucleoli, presumably for export of the Rev-RNA complex

(459). Rev is also capable of interacting with the U16 small nucleolar RNA fused to the

RRE and exporting it to the cytoplasm, further supporting the functional significance of Rev nucleolar localization (64). Experiments using a ribozyme, specific for cleavage of HIV-1

RNA, fused to a small nucleolar RNA examined the possibility of viral RNA accumulation within nucleoli (285). The presence of the nucleolar ribozyme inhibited HIV-1 replication, suggesting that retroviral RNA associates with nucleoli. Furthermore, the nucleolar protein nucleolin has been implicated in enhancing HIV assembly. Together, HIV Gag, nucleolin, and an RNA containing the psi packaging sequence greatly increase the rate of budding for virus-like particles (421). Taken together, these studies with suggest that nucleoli serve as an RNA-viral protein interaction site for HIV.

In addition to retroviruses, many DNA and RNA viruses also express proteins with nucleolar localization properties. These include proteins from coronaviruses, influenza viruses, herpesviruses, and adenoviruses (185). Although nucleolar localization properties 161 were first thought to occur due to the RNA binding capability of the viral proteins found in nucleoli, recent studies demonstrates a vital role for nucleolar localization in viral life cycles including viral RNA transport, disruption of the cell cycle, and replication processes. One specific example is the herpesvirus saimiri ORF57 (54). ORF57 mediates the nuclear export of intronless viral mRNA. When the NoLS is deleted from ORF57, the intronless mRNA was trapped within the nucleus. Export of the viral mRNA is restored through the insertion of the

Rev NoLS. These experiments indicate that nucleolar association of the ORF57 protein is necessary for viral intronless RNA export.

Chapter 4 explored the relationship between loss of NC nucleolar localization and virus particle formation, gRNA packaging, and infectivity. Mutational analysis revealed that basic residues in the C-terminal region of NC (residues 61-73) were necessary for nucleolar localization of NC. Loss of these basic amino acids did not alter budding, but reduced gRNA packaging and made the virus noninfectious. In this chapter, we further test the relationship between nucleolar localization and viral replication using a gain-of-function approach: inserting a heterologous NoLS within the C-terminus of NC. The HIV Rev or

HSV ICP27 NoLS was inserted into the deletion mutant, and was able to restore NC and

Gag nucleolar localization. Additionally, the NoLSs restored incorporation of gRNA into mutant virus particles.

5.3 Materials and Methods

Expression vectors, plasmids, and cells

pYFP-NC, pGag-YFP and pL219A.Gag-YFP have been described previously (67)

(213). The mutants Δ61-73, ICP27, and Rev were introduced into the NC domain of pCMV.GagPol using site-directed mutagenesis (QuikChange, Stratagene). NC or Gag was then PCR amplified from the mutant GagPol and inserted into the pEYFP-C1 vector or pL219A.Gag-YFP vector, respectively. To make pRS.V8.Gag. Δ61-73, pRS.V8.Gag.ICP27, and pRS.V8.Gag.Rev proviral mutants, the NC region was then swapped between the 162 GagPol and RS.V8 plasmids using SbfI-HpaI restriction sites. Endonuclease digestion was used to identify clones containing the mutations and all positive clones were confirmed using sequence analysis. Primers and restriction digest screen sites are described (Table 5.1). All experiments were performed using the quail fibroblast QT6 cell line or the chicken fibroblast

DF1 cell line (183, 326). Transfections were performed using the calcium phosphate method (QT6) (144).

Confocal imaging

0.2 x 106 cells were seeded onto 35-mm glass-bottomed dishes (MatTek

Corporation) and imaged using a Leica AOBS SP2 confocal microscope at 14 to 24 h post- transfection. Sequential scanning settings were used to differentiate CFP (excitation at 458 nm, emission at 465-490 nm, and 50% laser power) and YFP (excitation at 514 nm, emission at 530-600 nm, and 10% laser power) emission spectra. Light images were obtained using the transmitted light channel during fluorescence acquisition.

Immunofluorescence

QT6 cells, seeded onto a 1.5 glass coverslip, were transfected with a wildtype or mutant provirus plasmid overnight and fixed using 2% paraformaldehyde. Cells were permeablilized using 100% Methanol at RT for 5 minutes, and subsequently blocked with

5% goat serum (Rockland). After one hour, the cells were washed using 0.1% Tween-20 in

PBS. The cells were incubated using a rabbit αRSV antibody (1:300) and a Cy3-conjugated

αrabbit secondary antibody (1:100, Abcam). DAPI was used to stain the heterochromatin within the nucleus. Coverslips were mounted to slides using SlowFade reagent (Invitrogen) and imaged using confocal microscopy (Cy3 was excited at 543nm, DAPI was excited at

405nm).

Budding analysis

Budding assays were performed as previously described (71). Immunoprecipitated

RSV proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and quantified using a PhosphorImager (Bio-Rad). Budding efficiency was 163 Table 5.1. Primers for insertion of heterologous NoLS into RSV NC. Altered residues are marked in bold; flanking nucleotides of deletion mutants are indicated with underlined residues. Screen site indicates the restriction enzyme used for identifying positive clones.

Size comparisons were performed by performing a restriction digest identical to the insertion digest (see text for restriction enzymes used).

164

165 calculated as a ratio of the CA present within the media divided by the amount of Gag expressed in the lysate. The release of RS.V8 was set at 100% and all mutants were expressed as a percentage of the wildtype RS.V8.

Ribonuclease protection assays

QT6 cells were transfected with either wildtype or mutant proviral DNA constructs.

Culture media was collected after 48 hours and cells were removed by passing the media through a 0.2µ filter. Virus was pelleted by ultracentrifugation at 126,000 X g through a 25% sucrose cushion. After resuspension of the pellet, aliquots were removed for reverse transcriptase (RT) assays. The mean RT values were used to normalize the amount of virus particles for each sample as previously described (94). Viral RNA was extracted using a

QiaAMP viral RNA mini kit (Qiagen). Ribonuclease protection assays were performed as previously described (95). A 318-nucleotide antisense probe transcribed with T7 RNA polymerase with [32P]CTP, spanning the splice acceptor site of the env gene, was used to detect both unspliced (263-nucleotide fragment) and spliced (183-nucleotide fragment) viral

RNA as previously described (150). Samples were separated by SDS-PAGE and quantified using a PhosphorImager (BioRad).

Viral infectivity assay

Infection assays were performed as previously described (397). Media was collected from QT6 cells expressing either wildtype or mutant provirus after 48 hours. Virus was concentrated by ultracentrifugation at 126,000 X g through a 25% sucrose cushion. RT assays were performed, as described above, to normalize the amount of virus used for infection. Equal numbers of virus were added to uninfected DF1 cells. Cells were then assayed for the presence of GFP, which is expressed from the RS.V8 provirus, by flow cytometry (FACSCanto, BD Biosciences). The percent of cells expressing GFP were examined every three days until all infectious virus reached approximately 95% green cells, or after 21 days for weakly infectious virus. A minimum of three infection assays was performed from two separate transfections. 166 5.4 Results

Introduction of heterologous nucleolar localization signals restores NC nucleolar localization

Chapters 3 and 4 investigated the consequences for the virus when nucleolar localization of Gag and NC was prevented. We next asked whether we could replace a sequence, necessary for nucleolar localization of NC, with a heterologous NoLS. Two different sequences were selected. The first sequence chosen was from the HSV-1 ICP27 protein, which contains a NoLS separate from the NLS (277). The second heterologous sequence chosen was from the Rev protein of HIV-1, which consists of overlapping nuclear and nucleolar localization signals (90). Both the Rev and ICP27 NoLSs also function as

RNA binding domains (44, 279). A segment containing 13 amino acids of the NC protein was deleted (Δ61-73), and the ICP27 or Rev localization sequence was subsequently used to replace the deleted amino acids, forming the chimeric proteins YFP-NC.ICP27 and YFP-

NC.Rev.

To examine the subcellular localization of each NC mutant, cells were co-transfected with wildtype or mutant YFP-NC and the nucleolar protein fibrillarin-CFP, and imaged using confocal microscopy. Wildtype NC localized to nucleoli (Fig. 5.1B, panel a), and the

NC.Δ61-73-deletion mutant excluded nucleoli (Fig. 5.1B, panel b). Insertion of the ICP27 or

Rev NoLS between residues 60 and 74 restored nucleolar localization of the NC protein

(Fig. 5.1B, panels c and d, respectively). However, no fluorescent cells were found after transfection of either of these chimeric proteins from 1µg of DNA or greater. Titration of the protein down to 100ng of DNA per transfection allowed visualization of the NC mutant proteins within live cells. Although the amount of protein expressed was lower than wildtype, both restoration mutants appeared to have less fluorescence within the cytoplasm, suggesting that their localization to nucleoli was stronger than YFP-NC. These data show that replacement of amino acids 61-73 with either the ICP27 or Rev NoLS is sufficient to direct NC nucleolar localization. 167 Fig. 5.1. Localization of NC restoration mutants using confocal microscopy. (A)

Schematic diagram of RSV NC and nucleolar restoration mutants. Wildtype residues are shown for the C-terminal basic region. Dashed lines represent deleted residues (Δ61-73).

Bold residues depict sequences used to replace the deleted amino acids (ICP27 and Rev).

(B) QT6 cells were co-transfected with wildtype or mutant YFP-NC, and fibrillarin-CFP as a marker for nucleoli. YFP-NC localized to nucleoli, with some fluorescent signal present within the nucleus and cytoplasm (panel a). YFP-NC.Δ61-73 was observed excluding nucleoli (panel b). Both YFP-NC.ICP27 and YFP-NC.Rev were found co-localized with fibrillarin-CFP in nucleoli (panels c and d, respectively). 1µg of DNA was used for both wildtype NC and NC.Δ61-73, 100ng was used for NC.ICP27 and NC.Rev.

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169 Localization of restoration mutant Gag proteins

In addition to facilitating nucleolar localization, the peptide sequences from both

ICP27 and Rev are capable of general RNA binding (44, 278). Therefore, the addition of a heterologous NoLS/RNA binding domain may alter the normal trafficking patterns of Gag during retroviral assembly. To test this possibility, we examined cells expressing wildtype or mutant proviral constructs by immunofluorescence staining using a polyclonal αRSV antibody. Wildtype provirus exhibited fluorescence in the cytoplasm, forming punctate foci at the plasma membrane (Fig. 5.2A, top). The Δ61-73 mutant showed similar localization, with fluorescent signal primarily within the cytoplasm (Fig. 5.2A, bottom). Unexpectedly, although both RS.V8 ICP27 and Rev mutants contain a functional NES, they both formed punctate foci in the nucleoplasm and accumulated within nucleoli (Fig. 5.2B, top and middle, respectively). The RS.V8 M3/Rev mutant also localized to nuclear foci and nucleoli (Fig

5.2B, bottom). The formation of nuclear foci was observed within all cells expressing the proviral constructs containing heterologous NoLSs. These observations suggested that Gag proteins are trapped or retained within the nucleus.

Insertion of a heterologous NoLS alters the rate of virus particle budding

If the mutant Gag proteins are unable to leave the nucleus, they would be unable to reach the plasma membrane, and we would expect a decrease in the rate of virus particle release similar to that seen in leptomycin B drug-treated cells and with Gag p10 NES mutants (363, 366). To test the possibility that the heterologous NoLSs alter virus particle production, a radioimmunoprecipitation assay was performed as described in materials and methods. A representative experiment showing Gag expression within the cell lysates after a 5-minute labeling period is presented (Fig. 5.3A). The differences in apparent molecular weight of the different Gag mutants are visible within the lysates. Additionally, CA is detectable within the media after a 2.5-hour metabolic labeling (Fig. 5.3B).

Quantitation of virus release (% release) was performed by dividing the amount of

CA in the media by the amount of Gag in the cell lysates (71). The budding efficiency for 170 Fig. 5.2. Localization of RS.V8 Gag restoration mutants. QT6 cells expressing wildtype or mutant proviral constructs were processed for immunofluorescence using a rabbit αRSV primary antibody, followed by a Cy3 conjugated αrabbit secondary antibody. Cells were imaged using confocal microscopy and the Cy3 channel and an overlay showing the nucleus (DAPI) are presented. The wildtype virus was seen primarily in the cytoplasm and as punctate foci at the membrane (panel a). Both RS.V8 constructs containing either the

ICP27 or Rev localization sequence were observed forming punctate foci within the nucleus

(panels c and d, respectively).

171

172

Fig. 5.3. Budding analysis of proviral nucleolar restoration mutants. (A) Cells expressing wildtype or mutant RS.V8 provirus were starved in Met-/Cys- DMEM for 30 minutes, followed by metabolic labeling with 35S-Met/Cys for 5 minutes. After collection of cell lysates and immunoprecpitation using an αRSV antibody, the proteins were separated by SDS-PAGE and visualized by phosphorimaging. A representative cell lysate gel is presented. Differences in molecular weight are noticeable, with Δ61-73 mutant Gag running raster than wildtype, and both Rev and ICP27 running slower. The arrow indicates the position of the Gag band. (B) Cells expressing wildtype or mutant RS.V8 provirus were metabolically labeled with 35S-Met/Cys for 2.5 hours. After labeling, media was collected and immunoprecipitated using an αRSV antibody. Proteins were then separated by SDS-

PAGE and visualized by phosphorimaging. The gel presented is matched with the lysates presented in A. The arrow indicates CA. (C) The average of four independent budding assays is presented within the bar graph, with the bars representing the standard error.

173

174 each mutant was compared to wildtype (100%) (Fig. 5.3C). RS.V8.Δ61-73 was not significantly different from wildtype (147%). A provirus containing the Rev NoLS was severely reduced in budding (12%). Surprisingly, although the RS.V8.ICP27 Gag protein, expressed from the proviral construct, was nuclear when observed by immunofluorescence, viral particle release was unchanged from wildtype (75%). These results indicate that the

Rev NoLS prevents virus particle formation or release, but the ICP27 NoLS does not noticeably affect virion production.

Heterologous NoLS in NC restores gRNA packaging

In addition to changing the localization and kinetics of virus release, introduction of a heterologous NoLS into the NC domain could prevent the incorporation of gRNA into newly formed virions due to a change in structure, RNA binding, or mislocalization. However, the

Cys-His boxes specifically select and interact with the psi-packaging sequence (233, 282,

456). Therefore, if RNA binding has not been eliminated, packaging efficiency will likely remain unchanged, because no mutations have been made to the zinc fingers. This prediction was tested through RPA analysis. The amount of RNA used from the wildtype and mutant virus was normalized via RT activity (as described in materials and methods).

Using a probe for the 3’ splice acceptor site in env we are able to detect both the spliced vRNA, and the unspliced gRNA. Representative blots for each mutant are shown (Fig.

5.4A).

The averages from at least 3 independent RPA experiments for each mutant were plotted in a bar graph (Fig. 5.4B). The amount of gRNA packaging of the wildtype was set to 100%, and all mutants were compared to wildtype. The Δ61.73 mutant virus was unable to package gRNA efficiently (15%). Both RS.V8.Rev and RS.V8.ICP27 were able to rescue the Δ61.73 packaging deficiency (73% and 78%, respectively). These results indicate that insertion of a heterologous NoLS/RNA binding domain restores incorporation of gRNA into virus particles.

175 Fig. 5.4. Effect of NC nucleolar restoration mutations on gRNA packaging. (A) A wildtype RS.V8 vector or RS.V8 vector containing the deletion of heterologous NoLS was transfected into QT6 cells. Virus was collected for 48 hours and virus particle numbers were normalized via an RT assay. Equivalent numbers of virus particles were used to extract

RNA, and RPA was used to measure the levels of gRNA present. After digestion of the

318-nt 32P-radiolabled antisense riboprobe, spanning the 3’-splice acceptor site within the env coding region, and digestion of the unprotected fragment with RNase treatment, the

RNA was separated by gel electrophoresis and visualized using a phosphorimager. Gaps between the gels represent results from independent experiments. Arrows on the left identify nucleotide sizes, and arrows on the right denote the species of RNA (Free probe, unspliced gRNA, and spliced vRNA). (B) The results of four independent RPAs are shown on the graph, with the bars representing standard error of the mean.

176

177 Chapter 3 found that shifting the charge away from the Cys-His motif (M3/RKR) retained nucleolar localization of NC, while reducing the incorporation of gRNA in context of a proviral vector (11%). However, the M3/Rev was able to rescue the M3 gRNA packaging deficiency (69%). This result suggests that the loss of basic residues immediately flanking the second Cys-His motif can be overcome by introduction of a heterologous NoLS/RNA binding domain.

Virus containing heterologous NoLS insertions are noninfectious

Although the insertion of a heterologous NoLS/RNA-binding domain is capable of restoring gRNA packaging, the virus may still be unable to reestablish infectivity. Virus particles were collected from QT6 cells expressing either wildtype or mutant proviral constructs, the particles were normalized via RT activity, and equal numbers of virus were placed on uninfected DF1 cells. Because the RS.V8 provirus contains a GFP gene, the ability of mutant viruses to spread through the cell culture was measured by FACS every 3 days for 3 weeks (Fig 5.5).

Wildtype virus (RS.V8) was able to efficiently infect and replicate within cells, reaching approximately 95% green cells by day 6 post infection. The Δ61-73 mutant was noninfectious, which was expected given the low incorporation of gRNA. However, gRNA restoration by insertion of an exogenous NoLS was unable to restore infectivity in ICP27,

Rev, or M3/Rev. The failure of the heterologous NoLSs/RNA-binding domains to rescue infectivity are probably due to additional blocks along the replication pathway.

5.5 Discussion

Previous studies from this laboratory reported that transient nuclear trafficking of the RSV

Gag protein is linked to efficient gRNA packaging (151, 363). In chapter 3 of this dissertation, I reported that a mutant of Gag that is unable to leave the nucleus accumulates within nucleoli. Chapter 4 found that nucleolar localization of Gag is not involved in incorporating gRNA into virus particles. The work reported here further explores the 178 Fig. 5.5. Infectivity of virus containing nucleolar restoration mutations. Virus was collected from transfected QT6 cells transfected with an RS.V8 or RS.V8 mutant provirus and placed onto uninfected DF1 cells. Cells were examined every 3 days post infection for spread of GFP using flow cytometry, and the percentage of green cells per time-point is plotted. Neither the deletion mutant (Δ61-73) nor any restoration mutant (ICP27, Rev, or

M3/Rev) was infectious. The graph shown is a representative assay of at least three independent infection assays.

179

180 potential for nucleolar localization in virus replication using a gain-of-function approach.

Deletion of 12 amino acids immediately after the second Cys-His motif (Δ61-73) eliminated nucleolar localization of the YFP-NC.Δ61-73 mutant. Furthermore, deletion of these residues reduced the amount of gRNA incorporated into virus particles. Restoration of nucleolar localization of NC and Gag, via a heterologous NoLS, resulted in an increase of packaged gRNA compared to a nucleolar deficient NC/Gag protein.

To restore nucleolar localization to the NC.Δ61-73 deletion mutant, I selected two nucleolar localization signals that are known to be sufficient to drive nucleolar accumulation.

The NoLS of HIV-1 Rev was selected as a heterologous signal because is well studied and has been used to restore nucleolar localization and function to the herpesvirus saimiri

ORF57 protein (54). However, the NoLS of Rev also contains a NLS (44). The ICP27 RGG domain is also capable of directing nucleolar localization of the green fluorescent protein, but is separate from the NLS within ICP27 (277). When inserted into the YFP-NC.Δ61-73 mutant, either of these signals was sufficient to direct NC to nucleoli. Surprisingly, the insertion of either the Rev or ICP27 NoRS into Gag redirected the Gag protein to nuclear foci and nucleoli. This nucleolar relocalization occurred in the context of a proviral construct, without drug treatment or mutation of the nuclear export signal. Because the Gag was primarily localized to nuclei, these results suggested that both heterologous NoLSs would inhibit viral assembly, similar to NES mutants (366).

Because the Gag proteins were found localized to the nucleus, we used metabolic labeling assays to determine whether virus release had been affected. RS.V8.Rev virus particle release was reduced as expected from its nuclear localization phenotype.

Surprisingly, although RS.V8 ICP27 was also predominantly nuclear in localization, budding efficiency was not significantly changed from wildtype. Although both NoLSs are also RNA binding domains (44, 279), it is unlikely that this function is preventing Rev but not ICP27 from releasing new virus particles. However, the relative RNA binding affinities of both signals are unknown, therefore it remains a possibility. Furthermore, it is possible that the 181 trafficking kinetics are different between Rev and ICP27, and Rev is imported into the nucleus faster than it can be exported, accumulating within the nucleus.

We asked whether the insertion of the heterologous NoLSs could rescue gRNA packaging of the RS.V8.Δ61-73 mutant provirus. Insertion of either NoLS restored both nucleolar localization to NC/Gag and gRNA packaging to the virus. This restoration of packaging could be due to the restoration of Gag localization to the nuclear foci and nucleoli. We have previously observed that the L219A.Gag nuclear foci are colocalizing with or adjacent to sites of viral RNA (86). Although we do not know whether the nuclear foci formed by the Gag.Rev and Gag.ICP27 mutants are similar to a Gag protein with a wildtype NC, it is possible that the nuclear foci are the site of gRNA packaging.

A second possibility is that RNA binding has been restored to the NC domain of the

RS.V8.Rev and RS.V8.ICP27 mutants. Both the ICP27 and Rev NoLSs are RNA binding domains (279, 328). It has been proposed that the presence of multiple nonspecific RNA- binding domains connote specific RNA binding to a protein, although the mechanism for specific RNA recognition is not understood (260). It is possible that the Δ61-73 mutant eliminates specific binding of the ψ-sequence, and restoration via the ICP27 and Rev heterologous RNA-binding domains restores that specific interaction. Given that the basic residue immediately after the second Cys-His motif is important for incorporating gRNA into virus particles (231), the replacement of that basic residue may have been responsible for the restoration of gRNA packaging. But the M3/Rev mutant restored gRNA packaging and the M3/RKR mutant (Chapter 4) does not. Thus, the addition of a large number of basic residues from the ICP27 and Rev NoLSs may be responsible for the restoration (3 and 5 more basic residues than wildtype, respectively). It has been proposed that specific RNA binding of a protein is facilitated through multiple weak RNA binding domains (260).

Therefore whether the selective RNA packaging is due to the addition of these RNA binding domains or simply the addition of basic amino acids is not known. Further mutants inserting a random sequence of basic residue sequences and RNA binding domains that do not 182 contain basic amino acids (KH domain or additional zinc fingers) will be needed to test these possibilities.

Although insertion of a heterologous nucleolar localization signal into NC rescued incorporation of gRNA into virus particles, these mutants were noninfectious. These data indicate that nucleolar localization of Gag is not sufficient for infectivity and there are several possibilities why the RS.V8.Rev and RS.V8.ICP27 mutants are noninfectious. It is possible that the insertion of an exogenous amino acid sequence alters proteolytic cleavage of the virus during maturation. An increase in proteolytic cleavage during virus assembly was observed in chapter 4 of this dissertation (RS.V8.M5). Western blotting is needed to examine whether there are cleavage products in either the RS.V8 Rev or RS.V8.ICP27 mutants.

A second possibility for the lack of infectivity is a decrease of Gag-Pol incorporation into virus particles. Gag-Pol, which contains both the reverse transcriptase (RT) and integrase (IN) proteins, is essential for reverse transcription of the gRNA into DNA and integration of the viral DNA into the host genome (93, 199, 381). CA:RT ratios are needed to determine whether wildtype levels of Gag-Pol are packaged into virus particles (151,

398). Finally, the envelope protein (Env) is essential for viral entry (30, 31). Whether Env is incorporated into RS.V8.Rev or RS.V8.ICP27 virus particles is unknown. Western blotting for Env will determine the amount of Env present within each mutant virus.

A final hypothesis for the noninfectious nature of the RS.V8.Rev and RS.V8.ICP27 mutants is during virus entry. The NC protein is involved during many aspects of virus replication, including gRNA dimerization (23, 51, 341), reverse transcription (8, 27, 107, 117,

173), and import of the preintegration complex (412). Insertion of these heterologous

NoLSs may alter the chaperone activities of NC during virus entry, leading to a noninfectious virus. Further examination into where the block during infection occurs is needed to better understand what effect the heterologous NC mutants have on viral infectivity.

183

Chapter 6

Dissertation Discussion

184 6.1 Introduction

Although several retroviral proteins are reported to have nucleolar localization properties, the work presented here is the first known study to identify subnuclear localization of the RSV Gag protein. This chapter is separated into two main discussion sections. The first will examine the possibilities of why the L219A.Gag-YFP protein accumulates within nuclear foci and nucleoli. The second section will discuss the implications of Gag subnuclear localization on biological processes (e.g. assembly and replication).

6.2 Discussion of Gag subnuclear localization

Ideas presented in the next several sections provide a detailed discussion of what

L219A.Gag-YFP nuclear foci might be and how they may form. Additionally, the potential mechanisms of Gag nucleolar localization are considered.

6.2.1 Gag nuclear foci

Chapter 2 demonstrated that RSV Gag proteins deficient in nuclear export accumulated within discrete nuclear foci (Fig. 2.1). No electron dense structures were visible under TEM, making aberrant virus-like particle formation in the nucleus unlikely (Fig.

2.4). Additionally, the average size of the L219A.Gag-YFP nuclear foci was significantly larger than the average size of Gag-YFP VLPs at the plasma membrane (Fig. 2.3). The nuclear foci were anchored within the nucleus, but Gag proteins moved between the punctate foci and the nucleoplasm (Fig. 2.5 and Fig. 2.6). These observations suggest that

Gag is retained at a subnuclear compartment, raising several questions about L219A.Gag-

YFP intranuclear trafficking: what are these nuclear foci and how do they accumulate within these discrete punctate locales?

Because Gag is an RNA-binding protein, L219A.Gag-YFP may accumulate where

RNA is present within the nucleoplasm. The idea that Gag accumulates at sites rich in RNA 185 is supported by the observation that nuclear foci do not form upon deletion of the RNA- binding NC domain (Fig. 2.1). The nucleus is the cellular site for RNA transcription, which occurs within multiple discrete transcription factories (405). Furthermore, the nucleus is compartmentalized but highly dynamic (458), encompassing discrete subnuclear structures including speckles (222), PML bodies (272), and transcription factories (405). Active transcription sites may consist of all three subnuclear structures, which are found colocalized and/or juxtaposed (216). Our laboratory has recently inserted MS2 loops within a proviral vector (86). A MS2 protein fused to YFP binds to retroviral RNA, allowing for visualization of RNA localization (40). These experiments have led to the observation that retroviral RNA form nuclear foci within a subset of cells. When L219A.Gag-YFP is expressed within these cells, it is found colocalized with or juxtaposed to the MS2-tagged viral RNA. These data suggest that Gag may associate with a nuclear body involved in the transcriptional process (i.e. speckles, PML bodies). Because the Gag protein is juxtaposed to the viral RNA, it is possible that the L219.Gag-YFP mutant localizes to one of these transcription/processing bodies.

To determine whether Gag is accumulating at transcriptional sites, colocalization experiments will be performed with markers/proteins of subnuclear bodies via overexpression and immunofluorescence. Immunofluorescence will be used where antibodies are available. When antibodies are unavailable, overexpression experiments using YFP/CFP tagged cellular proteins will be used. Experiments examining colocalization between SC35 (splicing speckles) and Pol II (transcription factories) are currently ongoing within our laboratory. Although previous experiments, which compared Gag nuclear and

PML bodies found no colocalization between L219A.Gag and PML (unpublished data), these experiments involved human PML proteins in chicken cells. The PML protein is approximately 44% conserved between humans and chickens. Thus it is possible that the human PML protein is mislocalized or nonfunctional when expressed in chicken cells.

Therefore, a chicken PML protein or antibody will be obtained to determine whether Gag 186 associates with avian PML bodies. Ideally, these localization experiments will also be performed using antibodies against endogenous subnuclear compartment proteins. If Gag is not associating with nuclear bodies involved with transcription, further experiments with other nuclear body markers will determine what L219A.Gag-YFP associates with.

To determine whether RNA is needed to form the Gag nuclear foci, RNase treatment will be used to look at the effects on Gag nuclear foci formation. Literature on cellular nuclear bodies indicate that the presence of RNA is not required for proteins associated with transcriptional complexes, and RNase treatment did not noticeably alter splicing speckles or

PML bodies (396, 436). Additionally, transcription factories remain intact in the absence of active transcription (291). Thus treatment of L219A.Gag-YFP nuclear foci with RNase will determine whether RNA is required for their existence. The presence of Gag nuclear foci after RNase treatment would indicate that protein-protein interactions are necessary, whereas disappearance of the nuclear foci would demonstrate an RNA requirement. If RNA is a requirement of Gag nuclear foci, examination into why the localization of L219A.Gag-

YFP forms nuclear foci whereas YFP-NC does not will be performed. Both the Gag protein and the NC protein interact with RNA, implicating the role of another domain within the Gag protein (120, 456). This hypothesis can be tested via Gag domain deletions. The MA domain, which possesses nucleic acid binding activity (400), is a good candidate for the formation of an RNA-dependent nuclear focus. Otherwise, stepwise deletions of the p2, p10, CA, and SP domains will be performed to determine whether these domains are involved in Gag nuclear puncta formation.

Should L219A.Gag-YFP nuclear foci form via a protein-mediated pathway, the host or viral proteins involved will be determined. We have adapted a tandem affinity purification

(TAP) vector for use with the Gag protein, which allows for stringent purification of proteins

(343). After isolating nuclei of cells expressing either TAP-L219A.Gag or TAP-NC, this TAP system will be used to collect proteins interacting with L219A.Gag and NC. Mass spectrometry will be used to identify the isolated proteins. Through this method, we may 187 identify proteins interacting with Gag but not NC. With putative cellular proteins identified, we can use knockdown or overexpression experiments to determine whether the identified proteins affect Gag localization or trafficking.

A problem with the idea that L219A.Gag-YFP accumulates at nuclear foci and nucleoli due to RNA binding is that these localization phenotypes are observed within a subset of transfected cells. If only RNA binding were responsible, every cell expressing

L219A.Gag-YFP should form Gag nuclear foci. However, there are several possible reasons why L219A.Gag forms nuclear foci within some cells whereas Gag is found diffuse in other cells. One possibility is that Gag is oversaturating or unable to interact with cellular transport proteins and is therefore trapped within nuclear foci. A similar result is seen with the cellular U3 snoRNA (48). Although U3 is normally found within nucleoli, if the export protein CRM1 is blocked via LMB treatment the U3 snoRNA becomes relocalized to Cajal bodies. This result suggests that CRM1 is not only involved in nuclear export, but also intranuclear trafficking. Because we are using LMB treatment or a Gag mutant that should be unable to interact with CRM1, Gag may become trapped within a nuclear body similar to the U3 snoRNA. These reasons would also explain why the formation of Gag nuclear foci is not observed within infected cells. The amount of Gag expressed from a proviral vector or an infected cell is likely lower than that expressed from a CMV-Gag-YFP vector. Therefore, even though Gag in infected cells becomes concentrated within the nucleus upon LMB treatment, nuclear foci are unable to form.

The formation of Gag nuclear foci may also be dependent upon cell cycle. Nuclear bodies are dynamic structures that dissipate as cells enter mitosis and reassemble during interphase. During mitosis, PML proteins remain within distinct foci in the cytoplasm until after reformation of the nuclear envelope (113). PML is then imported into the nucleus, where it reforms PML bodies during G1, and the number of PML bodies doubles during S- phase (112, 410). Although most of the PML bodies are associated with active transcription sites during G1, very few are observed near transcription sites during G2 (216). This result 188 demonstrates the dynamic nature of subnuclear structures, and shows that function and localization may change during the cell cycle. Therefore the subnuclear localization of

L219A.Gag-YFP will be looked at in synchronized cells to determine whether Gag nuclear foci form at a specific phase (i.e. G1 or G2).

6.2.2 Gag nucleolar localization

Chapter 3 reported the nucleolar localization of LMB treated cells expressing Gag-

YFP or of cells expressing the L219A.Gag-YFP mutant (Fig. 3.3A). The Gag protein required basic residues within the NC domain for nucleolar localization (Fig. 3.5). This result suggests that nucleolar localization of Gag is mediated via the NC domain, similar to the formation of Gag nuclear foci. Additionally, L219A.Gag-YFP proteins were shown to be exchanging between nucleoli and the nucleoplasm (Fig. 3.3B and C). Thus the interaction between Gag and nucleoli appears to be a dynamic process. However, it is still unknown whether nucleolar localization of the Gag protein is RNA- or protein-mediated.

Although both the YFP-NC protein and L219A.Gag-YFP protein localize to nucleoli, there are differences in where they accumulate. Cells expressing YFP-NC colocalized with fibrillarin-CFP, indicating that NC accumulates within the dense fibrillar component (DFC) of nucleoli where newly transcribed rRNAs bind to proteins. The L219A.Gag-YFP protein concentrated around fibrillarin-CFP, indicating that Gag localized to the outer granular component (GC) of nucleoli where late steps of rRNA processing occur.

There were also differences between the localizations of YFP-NC basic amino acid mutants and L219A.Gag-YFP basic amino acid mutants. Mutations that affected the NT region of NC either did not affect YFP-NC localization (M5), or partially reduced nucleolar accumulation (M6). However, neither the M5 nor the M6 L219A.Gag-YFP mutant proteins localized to nucleoli. These data indicate that the basic amino acids within the NT region are required for Gag nucleolar localization, but not for the nucleolar accumulation of YFP-

NC. Given that both RS.V8.M5 and RS.V8.M6 mutants release virus particles as efficiently 189 as wildtype, and both mutants package gRNA, it is unlikely that RNA binding has been abolished. It is possible that conformation of Gag is altered and thus cannot localize to nucleoli, or that we have eliminated an interaction site for Gag with a nucleolar protein.

Several nucleolar proteins localize to nucleoli via either direct trafficking or retention at nucleoli via protein-protein interactions. For example, nucleolin and B23 encode stretches of acidic amino acids that bind to basic residues in target proteins to mediate nucleolar localization (250, 370). The hepatitis delta virus antigen (HDAg) and the hepatitis C NS5B protein directly bind to nucleolin to facilitate nucleolar localization (184, 229). In a similar fashion, B23 appears to mediate nucleolar localization of HIV1 Rev and Tat (130). CRM1 has been implicated as a nucleolar localization protein and is involved in transporting cellular proteins, including the PHAX-U3 snoRNA complex to nucleoli (48).

Co-immunoprecipitation and TAP assays will be performed to examine nucleolar binding partners of NC and Gag. Nucleoli can be specifically isolated from nuclei (14), facilitating an examination of proteins involved primarily of the nucleolar localization step of

Gag. If nucleolar proteins do interact with Gag, altering Gag-nucleolar protein interaction sites may give further insight into the role of nucleoli during viral replication. Testing the mechanisms of nucleolar localization may be more complicated than with the nuclear foci, as disruption of Pol I transcription or nucleolin function disperses nucleoli (422, 453). These observations indicate that nucleoli are a delicate subnuclear structure, which are disrupted by changes in RNA or protein content.

Previously, a C-terminal fragment of nucleolin was identified that binds to RSV Gag, presumably at the NC domain (24). This fragment does not noticeably affect cell function, but when expressed with MLV Gag, the C-terminal fragment of nucleolin inhibits assembly of virus particles. Although it is unknown whether this protein fragment is nucleolar, it does contain the previously defined nucleolar targeting signals located within the C-terminal half of nucleolin (370). It is possible that a nucleolin truncation mutant would sequester RSV

Gag in the nucleolus, demonstrating a potential nucleolar partner of Gag. Thus, these and 190 other studies will be required to understand the mechanism behind Gag nucleolar localization.

6.2.3 Interplay between Gag nuclear foci and nucleolar Gag

The L219A.Gag-YFP protein localized to both discrete nuclear foci and nucleoli.

Although the previous sections discuss each subnuclear location individually, no discussion has yet been offered as to whether Gag localization to nuclear foci and nucleoli are interrelated. In this section, two different models of Gag subnuclear trafficking are presented and discussed.

The first model presented is a general diffusion model (Fig. 6.1A). In this scenario,

Gag is imported into the nucleus through the importin α and importin 11 pathways (67, 172).

Following nuclear import, Gag proteins passively diffuse between the nucleoplasm, nuclear foci (NF), and nucleoli (No). Interactions with nuclear foci or nucleoli, through protein or

RNA interactions, cause Gag to be temporarily retained at these compartments. Eventually

Gag interacts with the CRM1 export factor and is exported from the nucleus. Evidence for this model comes from the Gag nuclear foci-nucleoplasmic and nucleolar-nucleoplasmic shuttling observed during FRAP assays (Fig. 2.6 and Fig. 3.3, respectively). Furthermore, several cellular proteins have been proposed to follow a general diffusion model between subnuclear bodies and nucleoli, including the small nucleolar ribonucleoprotein associated

Sm protein (389), and the similarly associated NHPX protein (240). Both of these proteins are found to freely move between Cajal bodies and nucleoli.

The second model of Gag subnuclear trafficking involves directed transportation (Fig.

6.1B). In this model, Gag is first trafficked to the nuclear subcompartment (e.g. nuclear foci).

Following interaction with proteins contained in that subnuclear compartment, Gag is trafficked to the next nuclear subcompartment (e.g. nucleoli). Finally, interaction with the

CRM1 export protein facilitates nuclear export of Gag. Although the model presented depicts directionality from nucleoplasm to nuclear foci to nucleoli, the direction can be 191 Fig. 6.1. Two models of Gag subnuclear trafficking. (A) Diffusional model of Gag subnuclear localization. After nuclear import of Gag, diffusion across the nucleoplasm allows Gag to be retained within nucleoli (No) or nuclear foci (NF). The Gag proteins transiently localized within the subnuclear compartments eventually return to the nucleoplasm and are free to associate with other nuclear components. After interaction with the CRM1 export protein, Gag is exported from the nucleus. (B) Directional model of Gag subnuclear localization. After nuclear import of Gag, retention within nuclear foci occurs first. After association with nuclear foci, Gag then localizes to nucleoli, which initiates CRM1 mediated export. For this model, the opposite pattern is also possible: nucleolar localization, followed by NF localization, culminating in nuclear export of Gag.

192 193 reversed (nucleoli localization followed by nuclear foci trafficking). Support for this model is not evident from the data in this dissertation, but there is evidence for step-wise and directional subnuclear trafficking. The cellular protein PHAX binds to the U3 snoRNA in

Cajal bodies (48). Following binding to U3, CRM1 interacts with PHAX and transports the

RNA-protein complex to nucleoli, where maturation and nuclear export of the snoRNA occurs.

Fluorescence loss in photobleaching (FLIP) using a multi-photon confocal microscope will be used to determine whether Gag undergoes unidirectional trafficking (83,

331). In FLIP, a single region of interest is constantly photobleached during the assay, and a separate region of interest is monitored during the bleaching. Multi-photon lasers allow bleaching in a single z-section, unlike single-photon lasers, which photobleach fluorophores through the entire z-axis of a cell (114). Thus we can specifically photobleach a nucleolus and observe nuclear foci without affecting cytoplasmic proteins. A decrease in nuclear foci fluorescence after bleaching nucleoli would indicate nucleolar Gag proteins subsequently trafficking to nuclear foci. Conversely, we can bleach nuclear foci and watch for fluorescence decrease in nucleoli. If fluorescence intensity decreases, this result would indicate that Gag traffics from nuclear foci to nucleoli. If FLIP data indicates that proteins travel both ways (nucleoli to nuclear foci and nuclear foci to nucleoli), this result would support the passive diffusion model, and a positive FLIP in a single direction would support the directional model. These models are not mutually exclusive, as there may be scenarios where both diffusion and directed trafficking may occur. However, these initial studies will begin to unravel the directionality of Gag subnuclear trafficking.

6.2.4 Nucleolar restoration mutants

In Chapter 5, the Gag mutants containing heterologous NoRSs from HSV-1 ICP27 and HIV-1 Rev presented an interesting localization phenotype: both mutants localized to nucleoli and nuclear foci without LMB treatment or mutation of the NES (Fig. 5.2). These 194 mutants may serve as tools to help determine the mechanisms and steps of Gag subnuclear trafficking. Under LMB treatment or with a nuclear export mutant, Gag no longer interacts with CRM1, which eliminates a step of nuclear trafficking that may confound interpretation of the assays proposed in the previous sections because the Gag protein is stuck in a trafficking loop within the nucleus. However, the Gag.ICP27 mutant was presumably capable of interacting with CRM1 because it maintained an intact NES. Additionally, the

RS.V8.ICP27 mutant released virus particles as efficiently as wildtype (Fig. 5.3), suggesting that the Gag proteins undergo similar trafficking patterns to the wildtype. Furthermore,

ICP27 and wildtype RS.V8 proviral constructs packaged gRNA with similar efficiencies (Fig.

5.4). Because the subnuclear accumulations are observable under “wildtype” export conditions, the Gag.ICP27 mutant may be a good candidate protein to study the mechanisms of nuclear foci and nucleolar trafficking in the studies proposed above.

Insertion of the HIV-1 Rev NoLS into Gag produced the same localization as the

ICP27 NoLS. In contrast to the ICP27 NoLS, RS.V8 Rev failed to efficiently assemble virus particles (Fig. 5.3). This result, along with the nuclear localization phenotype, suggests that the Gag.Rev may be trapped within the nucleus even though it maintains a wildtype NES. A possible reason for these results is the presence of a strong NLS within the Rev NoLS.

Additionally, importin α binds directly to the NLC in NC (67, 172), and importin β directly binds to the NLS present within the Rev NoLS (419). It is possible that two strong NLSs in the same protein cause a change in the nuclear import kinetics, and sequester Gag in the nucleus. This seems unlikely, as the NES should allow the Gag protein to shuttle between the nucleoplasm and ultimately form virus particles at the plasma membrane. It is also possible that the extra NLS prevents a conformational change within Gag, which would normally expose the NES within p10. FRAP experiments will determine whether Gag.Rev can shuttle between the nuclear foci, nucleoli, and nucleoplasm. FLIP experiments will be used to determine whether nuclear export of the Gag.Rev mutant occurs. In vitro assays are 195

6.3 Biological implications of Gag subnuclear trafficking

This dissertation reports the nuclear foci and nucleolar localization of the RSV Gag polyprotein, but a clear function of subnuclear trafficking has yet to be uncovered. The next several sections discuss why the Gag protein might use nuclear foci and nucleoli during virus replication.

6.3.1 Nuclear foci in viral replication

Although chapter 2 of this dissertation observed the formation of Gag nuclear foci when Gag is concentrated in the nucleus, whether there is a functional role for these punctate spots is unknown. As discussed in section 6.2.1, it is possible that the RNA- binding properties of the Gag polyprotein cause it to localize to sites of RNA within the nucleoplasm. However, when the RNA-binding protein NC was expressed alone, nuclear foci were not observed.

One possibility for the function of Gag nuclear foci is to affect cellular splicing. For example, the HSV-1 ICP27 protein forms nuclear foci and is implicated in downregulating cellular splicing via interaction with SR splicing proteins (174, 254, 376). RSV maintains a high level of unspliced RNA via a negative regulator of splicing (NRS) found within the gag- coding region and the NRS directly interacts with SR proteins involved in RNA splicing (20,

274). However, the NC domain of the Gag protein is dispensable for the cellular accumulation of unspliced viral RNA (28, 283). Therefore, it appears unlikely that the Gag protein is involved in the regulation of unspliced RNA during infection, however it is unknown whether other domains within Gag may affect viral RNA splicing.

A second possible function for Gag accumulating at these nuclear foci is to facilitate gRNA packaging. Using a MS2 system to label full-length and spliced viral RNAs, our laboratory has observed L219A.Gag-CFP foci immediately adjacent to or colocalized with foci formed by viral RNA (86). Other viruses use subnuclear bodies for replication and 196 packaging, including HSV-1, which forms replication complexes within PML bodies (392).

Additionally, the ICP27 protein localizes to PML bodies and facilitates the export of unspliced viral mRNA transcripts (175, 254). However, the results with the M5 and M6 mutants presented within this dissertation makes nuclear foci related to gRNA packaging unlikely. Nuclear foci were not observed for the L219A.Gag.M5-YFP or the L219A.Gag.M6-

YFP mutants, yet both the RS.V8.M5 and RS.V8.M6 mutants incorporated gRNA into virus particles (Fig. 4.4). Furthermore, the M6 mutant virus was able to replicate, albeit delayed when compared to wildtype. Though nuclear foci do not readily form when these mutants are expressed, there is no noticeable exclusion of nuclear foci within the nucleoplasm. Thus it is possible that the Gag proteins are still interacting with nuclear foci, but at a lesser extent. Fortunately, many of the discrete nuclear bodies of cells can be isolated from the nucleoplasm (288, 356). If the Gag nuclear foci are found to colocalize with transcriptional complexes, then the subnuclear bodies of cells expressing the Gag.M5 and Gag.M6 mutants will be isolated. These experiments will determine wither the Gag.M5 and Gag.M6 mutant proteins still associate with the isolated nuclear bodies. Currently, however, the possibility of Gag nuclear foci involved in gRNA packaging appears unlikely.

6.3.2 Nucleoli in viral replication

In Chapter 3 I discovered that Gag, when concentrated within the nucleus, also accumulated at nucleoli. Given that the Gag.SKL mutant is reduced for gRNA packaging

(231), and that YFP-NC.SKL was diffuse through the nucleus and nucleolus, the original hypothesis was that nucleolar localization was necessary for incorporation of gRNA into virus particles. However, chapter 4 in this dissertation demonstrates that nucleolar localization of Gag is not required for gRNA packaging, and other possible explanations will be discussed here.

One possibility is that Gag may traffic to nucleoli to interact with or package a host protein or RNA. One such cellular protein candidate is nucleolin. Using a yeast two-hybrid 197 assay, a C-terminal fragment of nucleolin was found to bind RSV Gag, presumably via an interaction with the NC domain (24). Interestingly, the C-terminal nucleolin fragment also inhibited MLV particle assembly. It is likely that this nucleolin fragment is nucleolar, as the

NoLS is found in the C-terminal half of nucleolin (370). Experiments have also revealed that nucleolin promotes the budding efficiency and infectivity of HIV-1 virus particles (421).

Together, these findings suggest nucleolin is involved in the assembly pathway, potentially in nucleoli, though nucleolin is known to traffic throughout the entire cell. Experiments examining virus assembly and infectivity with overexpression or knockdown of nucleolin with

RSV are needed to test this hypothesis.

A second possible host protein is the CRM1 export protein, which is known to directly interact with RSV Gag (172). The HIV Rev protein relocalizes the CRM1 protein and other nuclear export factors to nucleoli under steady-state conditions, implicating nucleoli as a site for nuclear export (459). CRM1 is also involved in transporting the U3 snoRNA from Cajal bodies to nucleoli to facilitate processing and nuclear export of U3 (48). Furthermore,

CRM1 is necessary for the nuclear export of ribosomal subunits, although whether CRM1 initiates this function within nucleoli or the nucleoplasm remains unclear (411). Together, these studies suggest that CRM1-mediated export may occur through nucleoli. Further studies involving the interaction of CRM1 with Gag will be needed to examine the involvement of nucleoli.

It is also feasible that Gag localizes to nucleoli to mediate incorporation of small cellular RNAs into virus particles. Several cellular RNAs are specifically incorporated into

RSV, MLV, and HIV particles (84, 158, 314, 315, 361). Although U6 and 7SL are specifically packaged into RSV particles (84, 158), the studies discussed here examine the relationship between virus particles and small cellular RNAs have been performed in MLV

(152, 314). In these studies multiple pol III transcribed small RNAs were found preferentially packaged into MLV particles (314). It was reported that Pol III transcription generally occurs in or adjacent to nucleoli, which produces a localization phenotype strikingly similar to the 198 nucleolar accumulation of the L219A.Gag-YFP mutant (269). Additionally, at least one of the preferentially packaged small RNAs, mY, occurs during early Y RNA biogenesis (152), which happens to be a perinucleolar compartment (269). It is feasible that the small RNAs packaged into virions are selected in the nucleus, possibly at the nucleolus. However, the importance of these packaged small RNAs for viral replication is currently unknown.

To test the hypothesis that Gag is selecting small RNAs at nucleoli, we will determine whether the RNAs packaged in MLV are also packaged in RSV. If the small RNAs are preferentially packaged into RSV virions, we can then determine whether our nucleolar trafficking mutants are unable to incorporate the small RNAs. A decrease in small RNA packaging would suggest that Gag nucleolar trafficking is responsible for the selection of these RNA species.

Lastly, the role of nucleoli in viral replication may be important during viral entry. The results from Chapter 4 revealed a complex picture for the infectivity of NC basic amino acid mutants in context of virus. For example, YFP-NC.RAA localized to the nucleoplasm and nucleoli, but YFP-NC.M4/RAA excluded nucleoli. The RS.V8.RAA and RS.V8.M4/RAA mutants both packaged similar levels of gRNA. However, the RS.V8.RAA mutant was nearly as infectious as wildtype, whereas the RS.V8.M4/RAA mutant was deficient for viral replication. Together, these results suggest that nucleolar localization of NC may be important during viral entry. In support of this hypothesis, the NC protein of HIV and MLV are localized to nucleoli in early infection (147, 350, 454). Disruption of nucleoli during early infection (i.e. integration) will be performed to determine if the presence of nucleoli is important for viral replication. Additionally, experiments will be performed using the viral basic amino acid mutants to determine whether there is a block in early replication events

(i.e. reverse transcription or integration).

The reduction in infectivity with the RAA and M4/RAA basic amino acid mutants may be due to alterations in NC’s chaperone ability. Basic amino acids are important for many steps during virus replication, including tRNA annealing, gRNA maturation, and reverse 199 transcription (27, 176, 190, 423). Future studies will determine where NC basic residue mutants are blocked during infection. However, considering how many steps of the life cycle NC is involved in (see section 1.7 for the role of NC in infection), it is difficult to predict what step or steps these NC mutants affect or whether these mutants influence multiple processes during viral entry.

6.4 Summary of conclusions

The work presented in this dissertation found two subnuclear localizations of nuclear trapped RSV Gag polyprotein: an as-yet-unidentified nuclear body, and nucleoli (Chapters 2 and 3). The effect of reducing or eliminating nucleolar localization of NC on viral replication was therefore examined, and was found to show a reduction in both gRNA packaging and infectivity (Chapter 4). Using a gain-of-function approach, heterologous NoLSs were inserted into the nucleolar deficient NC mutants and used to further explore the role of nucleolar localization on gRNA packaging and viral infection (Chapter 5). The following section briefly highlights the main conclusions from each of these chapters.

Data presented within Chapter 2 revealed that the Gag polyprotein, when overexpressed within nuclei, localized to discrete nuclear foci. Although particle tracking revealed that these nuclear foci were anchored within the nucleoplasm, Gag proteins were able to travel between the puncta and the nucleoplasm. This result suggested that the Gag protein is retained at the nuclear foci for an unknown purpose. Furthermore, the NC domain of Gag was necessary for the formation of these nuclear foci.

The experiments in Chapter 3 identified nucleolar localization of Gag proteins deficient for nuclear export (e.g. L219, NES-A, or LMB treatment). Using site-directed mutagenesis, Gag nucleolar localization was found to require basic amino acids within the

NC domain. Specifically, mutations to basic amino acid clusters immediately flanking the

Cys-His motifs within NC caused a reduction in NC nucleolar localization. Mutations to basic amino acids distal to the Cys-His boxes did not appreciably affect nucleolar 200 accumulation of the NC protein. Additionally, we found that the basic amino acids within the

PKKRK motif in NC were necessary for nuclear localization of both the NC and Gag proteins, and likely represents a NLS. These results led us to ask the question: what biological role does Gag or NC nucleolar localization have in retroviral replication?

The results in Chapter 4 addressed this question by comparing nucleolar localization profiling scores of the NC protein to virus particle release, gRNA packaging, and viral infectivity. Using the profiling assay, nucleolar localization of NC was found to correlate to the number of basic amino acids within NC, which suggested a loss of RNA-binding capability by NC. However, virus particle release was not affected by any of the NC basic amino acid mutants. Because RNA is an essential structural element for the formation of virus particles, this result indicated that general RNA binding (cellular and/or viral RNA) of

Gag was not affected. Although several NC basic residue mutations reduced incorporation of gRNA into virus particles, Gag nucleolar localization was neither necessary nor sufficient for gRNA packaging. However, nucleolar localization of NC was found to be necessary for efficient virus spreading in the infectivity assays. In addition to the general conclusions of nucleolar localization and viral replication, several interesting mutants were also found within the N-terminal region of NC, which may potentially increase catalytic cleavage by the viral protease or affect maturation of newly formed virus particles.

Lastly, in Chapter 5 we used heterologous NoLSs from the HIV Rev protein and the

HSV ICP27 protein, to restore nucleolar localization of a NoLS deficient NC mutant (Δ61-

73). Surprisingly, the addition of either heterologous NoLS was sufficient to localize Gag to both nuclear foci and nucleoli without LMB treatment or a mutation of the NES. Either NoLS was sufficient to restore gRNA packaging to the Δ61-73 NC mutant. However, because the

Rev and ICP27 NoLSs are also RNA binding domains, it is unknown whether this restoration is due to restoration of Gag nucleolar localization, or the addition of an RNA-binding region.

This dissertation contributes to understanding the role of RSV Gag nuclear trafficking through the discovery of novel subnuclear localizations. However, due to the pleiotropic 201 nature of the NC and Gag proteins, we are left with more questions than answers. The work presented here lays the groundwork to better understand the complex processes of viral replication.

202

Appendix

Effect of ψ-Containing RNA on RSV Gag Trafficking

203 A.1 Abstract

Formation of a new Rous sarcoma virus (RSV) particle requires thousands of the

Gag structural protein subunits to assemble in concert at the plasma membrane. During this assembly process, cellular and viral RNAs are incorporated into the nascent virions.

Interestingly, only two copies of the viral genomic RNA (gRNA) are selected by the Gag protein and packaged into each virus particle. Where Gag selects gRNA and what occurs to regulate the specific packaging of only two copies of viral RNA per virion are currently unknown. The work presented here examines what effect the sequence required for gRNA incorporation, the ψ-packaging sequence, has on the trafficking kinetics of the Gag polyprotein within the cytoplasm. To facilitate these studies, we used fluorescently tagged

Gag proteins and used fluorescence recovery after photobleaching to determine whether the

ψ-sequence affected the trafficking kinetics of Gag. Results show that the presence of the

ψ-sequence, in cis or in trans, causes Gag to slow significantly within the cytoplasm, suggesting that this specific RNA sequence alters the trafficking properties of Gag.

Previously, we had demonstrated nucleolar accumulation of a Gag nuclear export mutant.

Presence of the ψ-sequence decreased the frequency of nucleolar accumulation, suggesting the packaging sequence alters Gag subnuclear trafficking. Together, these results suggest that although Gag is responsible for incorporating the gRNA into virus particles, the viral RNA may alter how Gag traffics through the cell.

A.2 Introduction

The Rous sarcoma virus Gag polyprotein directs assembly and release of retrovirus particles from the plasma membrane of infected cells. Expression of Gag alone is both necessary and sufficient for the formation of virus-like particles (VLPs) that are similar to complete virions (224, 440). The RSV Gag polyprotein consists of the matrix (MA), capsid

(CA), nucleocapsid (NC), and protease (PR) domains, which are cleaved into individual proteins during viral maturation. Additional cleavage products consist of p2a, p2b, p10, and 204 spacer (SP). During the virus assembly, Gag must also selectively package two copies of the viral genomic RNA (gRNA) (19, 38). The NC domain within the Gag protein mediates the interaction between Gag and gRNA and the ψ-packaging sequence located at the 5’ untranslated region of the viral RNA (1, 26, 39, 120, 230).

The incorporation of gRNA into virus particles requires export of the viral RNA from the nucleus. The unspliced RNA also serves as the template for translation of the Gag polyprotein. Normally, unspliced transcripts are retained and degraded in the nucleoplasm by cellular proofreading machinery, unless splicing occurs. For HIV-1, the Rev protein interacts with RNAs encoding the Rev-response element (RRE) to facilitate nuclear export of unspliced HIV RNA (21, 130). Although RSV has no accessory proteins, two direct repeat (DR) elements within the viral RNA facilitate nuclear export through the cellular

Tap/Dbp5 export pathway (227, 322). Although these mechanisms mediate export of viral mRNA for translation, whether they are involved in gRNA packaging is unknown.

HIV appears to require Rev-RRE interactions for appropriate Gag trafficking and efficient virus particle assembly (202, 406). Although Rev is primarily known to facilitate translation of the Gag protein, recent studies have identified Rev involvement in gRNA packaging (43). The presence of ψ-containing RNA localizes HIV Gag to a perinuclear/centrosomal compartment (337). Without the ψ-sequence, Gag does not localize to the centrosome, suggesting that viral RNAs may affect trafficking of Gag.

RSV does not have an accessory protein to facilitate the export of unspliced viral

RNA. Studies from our laboratory have identified transient nuclear trafficking of the Gag polyprotein (363). Nuclear entry is mediated by nuclear localization signals (NLSs) within the NC and MA domains of Gag (67, 172). Nuclear export of Gag is mediated by a CRM1 dependent nuclear export signal (NES) within the p10 domain (366). Gag nuclear trafficking is essential for efficient gRNA packaging, and bypassing the nuclear step greatly reduces the amount of viral RNA incorporated into virus particles (151, 363). Chapters 3 through 5 of this dissertation established the nucleolar trafficking of the Gag protein, and implicated 205 this localization event as necessary for gRNA packaging and infectivity. From these data, we concluded that trafficking of Gag is important for incorporation of gRNA into virus particles. We next asked whether the viral RNA was influencing trafficking of Gag after binding.

Previous studies into HIV used fluorescence recovery after photobleaching (FRAP) to show that Gag is highly mobile within the cytoplasm, and significantly decreases in mobility as it assembles at the membrane (160). Additionally, truncations of the Gag protein, deletion of the NC and p6 domains, changed the kinetics of Gag within the cytoplasm, suggesting that cellular binding partners of wildtype and mutant Gag proteins are different. FRAP is often used to identify mobile proteins, and their binding kinetics within specific subcellular compartments (331) . Mobility studies have provided many insights into biochemical processes within living cells (346).

This report characterizes the effect of the ψ-sequence on the mobility of Gag protein in the cell. We found that the ψ-sequence, either in cis or in trans, is capable of altering the kinetics of Gag in the cytoplasm. Additionally, we found that ψ-containing RNAs influence the nucleolar accumulation of Gag nuclear export mutants, but do not apparently alter Gag nuclear foci.

A.3 Materials and Methods

Expression vectors, plasmids, and cells

Prague C RSV Gag and NES mutant (L219A) expression vectors containing YFP and GFP fluorophores have previously been described (213). Untranslated region constructs are depicted (Fig. A.1), and described in the text. The Koz or RU5 upstream sequences were made using primers engineered to PCR amplify the entire RU5 region and gag coding sequence, or only 12 nt immediately upstream of Gag (Koz). PCR products were digested with NheI-ScaI, and inserted into the wildtype or L219A pGag-GFP or YFP vectors. pRU5.mCherry was created by PCR amplification of the 5’UTR of the RSV 206 provirus. The PCR product was inserted into pmCherry-n2 vector using HindIII-ApaI restriction sites. Mutants were screened by endonuclease digestion and positive clones were confirmed through automated sequencing. All experiments were performed using the

QT6 cell line (183). Transfections were performed using the calcium phosphate method

(144).

Laser scanning confocal microscopy

Live cells were seeded onto 35-mm glass-bottomed dishes (MatTek Corporation) and imaged using a Leica AOBS SP2 confocal microscope at 14 to 24 h post-transfection.

Sequential scanning settings were used to differentiate CFP (excitation at 458nm, emission at 465-490nm, and 50% laser power), YFP (excitation at 514 nm, emission at 530-600nm, and 10% laser power), and mCherry (excitation at 543 nm, emission at 575-700 nm) emission spectra.

FRAP analysis

Fluorescence recovery after photobleaching (FRAP) was performed at 38.5°C on live cells transfected with the Gag-XFP constructs (Fig. A.1). All assays were performed at a scan speed of 400 Hz with acquisition time of 1.6 seconds, or 800 Hz with an acquisition time of 0.8 seconds. Five pre-bleach images were acquired using the YFP channel settings at 10% laser power. YFP in nucleoli (L219A.Gag-YFP) or at the membrane (Gag-YFP) was specifically photobleached using the 514nm laser at 100% power over four time-points.

Recovery was monitored for approximately 200 seconds at 10% laser power. As a control for bleaching and acquisition fluorescence loss, fluorescence of the entire cell was monitored during the experiment. The background from both the region of interest and the total cell fluorescence was subtracted. Corrections for photobleaching and normalization of the data was performed as previously described (331). Briefly, the relative fluorescence

intensity was calculated by , where T0 is the total cell intensity before bleaching,

Tt is the total cell intensity at time-point t, I0 is the intensity of the region of interest before

207 bleaching, and It is the intensity of the region of interest at time-point t. Kinetic analysis was performed using a nonlinear, one-phase association curve fit (Prism 5, GraphPad). Half- times were automatically calculated. The mobile fraction of each region was determined

I E - I0 using Fm = , where IE is the fluorescence in the bleached region after full recovery, I0 I I - I0 is the fluorescence immediately after bleaching, and II is the pre-bleach fluorescence. A minimum of 3 assays was performed on Gag within the cytoplasm.

Frequency of nucleolar localization of Gag

QT6 cells co-transfected with a plasmid encoding a nucleolar marker pfibrillarin-CFP and either pKoz.L219A.Gag-YFP or p5’UTR.L219A.Gag-YFP were imaged through a single optical slice. Cells displaying nuclear foci were imaged using both the CFP and YFP channels. Captured images were analyzed for the presence of Gag nucleolar localization.

A positive score was given if Gag was found within nucleoli or within a ring pattern around the nucleolus. The percentage of cells with Gag localized to nucleoli was calculated for both

Koz.L219A.Gag-YFP and 5’UTR.L219A.Gag-YFP. A minimum of 25 cells was counted for each condition.

A.4 Results

Gag and RNA constructs

We asked whether the viral ψ-packaging sequence affected the trafficking pathways of the Gag protein. To facilitate these studies, we created two Gag constructs, differing only in their 5’ untranslated region (Fig. A.1). RU5.Gag-XFP encoded the entire upstream- untranslated region, starting from the repeat R region of the 5’ end (nucleotides 1-380). This construct contains the entire ψ-sequence (Aψ) (210, 402). The pKoz.Gag-XFP vector contains only the 12 nucleotides immediately upstream of the gag start codon. Both constructs express the Gag protein fused to either GFP or YFP (Fig. A.1, XFP). To express the ψ sequence in trans, we fused the mCherry fluorescent protein to the 5’UTR of RSV

208 Fig. A.1. DNA representation of Gag and mCherry constructs. RU5.Gag-XFP encodes the entire 5’UTR of RSV (nt 1-380). pKoz.Gag-XFP encodes only the last 12 nucleotides of the 5’UTR (nt 368-380). The RU5.mCherry construct encodes the entire 5’UTR (nt 1-380).

Thin black lines represent untranslated RNA. Thick colored lines represent translated proteins: Gag (blue), XFP (GFP or YFP, green), or mCherry (red). The ψ- sequence is represented by the dashed line (nt 126-380). Nucleotides numbers are listed above the

DNA schematics (derived from GenBank accession number J02342; beginning of R region in LTR is nucleotide 1).

209

210 (RU5.mCherry). This construct contains most of the Aψ sequence (1-380), and stops immediately prior to the ATG start site of Gag. RU5.mCherry allows us to express a ψ-RNA, and use the expression of mCherry to identify cells transfected with the construct. With these three DNA constructs, we were able to express Gag without the ψ-packaging sequence. We could also express ψ-containing RNA in cis and in trans, allowing us to study the effects of the viral packaging sequence on Gag mobility.

FRAP analysis of RSV Gag in the cytoplasm

To examine the effect the ψ packaging sequence has on Gag trafficking, we used florescence recovery after photobleaching (FRAP) (331). Gag constructs were transfected in singly (Koz.Gag-XFP or RU5.Gag-XFP), or as a cotransfection with the ψ-RNA (Koz.Gag-

XFP + RU5.mCherry). The FRAP assays involved photobleaching a region of interest (ROI) within the cytoplasm (dimensions 3 µm x 5 µm) using the 514 nm laser. After irreversibly photobleaching the YFP fluorophores within the ROI, fluorescence recovery was monitored for approximately 200 seconds. Presence of the ψ-RNA was confirmed by expression of mCherry (Fig. A.2A). A representative assay involving the Koz.Gag-YFP cotransfected with

RU5.mCherry is presented (Fig. A.2B). First, pre-bleach images were collected (Fig. A.2B, pre-bleach). Next, the fluorescent protein was specifically photobleached (GFP, 458 nm;

YFP, 514 nm) within a specific region of interest (Fig. A.2B, red box). After photobleaching the region of interest was reduced in fluorescence intensity (Fig. A.2B, t=0s). Recovery of the fluorescence signal was then tracked for 200 seconds (Fig. A.2B, t=200s). The acquired data is normalized as described in materials and methods to produce a graph of the relative fluorescence intensity over time (Fig. A.2C-E). The half-time of Gag mobility was determined as described in materials and methods.

Koz.Gag-XFP had a recovery half-time of 4.95 seconds. The presence of the viral

5’UTR, either in cis or trans, caused a significant increase in the recovery half-time of Gag.

RU5.Gag-XFP slowed the half-time of recovery to 9.99s (P = 0.023). Similarly, the cotransfection of Koz.Gag-XFP and RU5.mCherry decreased the half-time to 11.02s (P = 211 Fig. A.2. Cytoplasmic Gag FRAP analysis. (A) For Koz.Gag-YFP + RU5.mCherry FRAP assays, the presence of ψ-RNA was established via the expression of the mCherry fluorescent protein. (B) A representative example of FRAP images. A region of interest

(ROI) within the cytoplasm of Koz.Gag-YFP + RU5-mCherry expressing cells was selected for photobleaching (Pre-bleach, red box). Immediately after photobleaching, fluorescence within the ROI was decreased (t=0s). Fluorescence recovery within the ROI was then tracked over time (t=200s). (C-E) Representative normalized FRAP data are plotted for

Koz.Gag-YFP (C), RU5.Gag-YFP (D), and Koz.Gag-YFP + RU5.mCherry (E). Half-times were calculated as described in materials and methods. (F) The average half-time from a minimum of 3 separate FRAP experiments are plotted. Koz.Gag-XFP recovered rapidly

(4.95 s). Both RU5.Gag-XFP and Koz.Gag-YFP + RU5.mCherry half-times were longer

(9.99 s and 11.02 s, respectively). Bars represent the standard error of the mean. Statistics were performed using student’s t-test. (*, P=0.023; **, P=0.014). (G) Mobile fraction of cytoplasmic Gag. Mobile fraction was determined as in materials and methods. Averages of FRAP experiments are plotted with the standard error of the mean. No statistical difference was found between ψ+ and ψ- Gag proteins.

212

213 0.014). These results demonstrate a significant change in trafficking kinetics between Gag alone, and Gag bound to the ψ-packaging sequence.

The amount of Gag proteins mobile within the cytoplasm was not changed by the ψ sequence. Mobile fractions of each FRAP graphs were calculated as described in materials and methods. Koz.Gag-XFP (0.83), RU5.Gag-XFP (0.93), and Koz.Gag-XFP +

RU5.mCherry (0.82) were equally mobile within the cytoplasm (Fig. A.2G). The data indicate that although Gag is slowing down in the presence of ψ, most of the Gag protein remains mobile.

Effect of viral packaging sequence on Gag nucleolar accumulation

Previous studies in this dissertation explored the role of Gag nucleolar trafficking in gRNA packaging (Chapters 3-5). In these chapters we found that Gag nuclear export mutants accumulate within nucleoli. Additionally, we found a correlation between the nucleolar localization of NC and the amount of gRNA incorporated into virus particles (see

Fig. 4.7B). Given that ψ has previously been known to localize HIV Gag to centrosomes

(337), we asked whether the presence of ψ would alter the ability of RSV Gag to localize to nucleoli.

To test this hypothesis we cotransfected the nucleolar protein fibrillarin-

CFP with either Koz.L219A.Gag-YFP or RU5.L219A.Gag-YFP, and acquired confocal microscopy images (Fig A.3). Cells that displayed the Gag nuclear foci phenotype were selectively imaged and analyzed for nucleolar accumulation. If Gag was localized within or immediately around fibrillarin, it was considered a nucleolar phenotype. At least 25 individual cells were analyzed for nucleolar localization. Koz.L219A.Gag-YFP was found localized to nucleoli in 70% of the cells counted (Fig. A.3A). RU5.L219A.Gag-YFP presented a nucleolar phenotype in 46% of cells (Fig. A.3A). These observations indicate that the 5’UTR of RSV is altering the nuclear localization of Gag, presumably through the ψ sequence.

214 Fig. A.3. Frequency of Gag nucleolar accumulation. (A) Cells coexpressing

Koz.L219A.Gag-YFP and fibrillarin-CFP were imaged using confocal microscopy. Cells displaying Gag nuclear foci were analyzed for nucleolar accumulation. Both nucleolar (top) and nuclear foci (bottom) phenotypes are presented. Gag accumulated in or around nucleoli in 70% of cells counted. (B) Cells coexpressing RU5.L219A.Gag-YFP and fibrillarin-CFP were analyzed as in part A. Gag was found within or around nucleoli in 46% of the cells counted.

215

216 A.5 Discussion

The RSV Gag protein must coordinate to form an immature virus particle during a complex assembly. Although virions bud from the plasma membrane, multiple lines of evidence have demonstrated retroviral Gag-Gag interactions and assembly intermediates within the cytoplasm (225, 237, 255, 418). Therefore, the Gag proteins visualized within the cytoplasm likely represent a heterogeneous mix of assembly intermediates. To gain insights into the assembly process, we used FRAP analysis to examine whether changes in Gag binding affect its ability to traffic through the cell.

Viral RNA is not required for assembly of virions, but is necessary for an infectious virus particle. Curiously, the virus is able to package only two copies of the gRNA, however the mechanism of how this is accomplished is currently unknown. We therefore asked whether the ψ-packaging sequence of RSV affects Gag trafficking within the cell. This appendix chapter presents preliminary findings of how the mobility and kinetics of Gag are affected by the ψ-packaging sequence.

Our FRAP data revealed that the presence of the viral 5’UTR, which contains the ψ sequence, slows the protein down in the cytoplasm. However, there was no change in the overall mobility of Gag, as the mobile fractions were statistically similar with and without the

5’UTR. These findings occurred whether the 5’UTR was expressed in cis or in trans. The possibility of accelerated assembly of larger complexes of Gag within the cytoplasm is unlikely. Experiments have shown that protein size has little effect on FRAP half-times

(399). Therefore, these experiments suggest that the ψ sequence affects the binding kinetics or binding partners of Gag within the cytoplasm. However, controls and further experiments are needed to confirm that the ψ sequence is affecting Gag mobility within the cytoplasm.

We first need to confirm that the ψ sequence alone is affecting Gag mobility. A 160 nt fragment of the ψ sequence is packaged as efficiently as wildtype (26). Mψ can be used in place of the 5’UTR to determine whether the ψ sequence is affecting trafficking, or if a 217 sequence outside of Mψ alters Gag mobility. Gag mutants deficient in gRNA packaging

(ΔNC, M3/M4, Myr1E) can be used to determine whether the change in Gag kinetics is NC mediated. These controls are necessary to confirm the effect of the ψ sequence on Gag mobility within the cytoplasm.

Chapters 2 and 3 revealed that Gag VLPs at the membrane are highly immobile (Fig.

2.6 and Fig. 3.3). These data agree with previous FRAP experiments in HIV Gag, which identified Gag VLPs as immobile (160). Chapter 2 VLP analysis combined mobility data from both Gag proteins with and without the ψ sequence present. The results were the same: Gag proteins in VLPs are highly immobile. Thus, it appears unlikely that viral RNA will have much effect on Gag after it assembles at the plasma membrane.

No analysis of nuclear Gag has been successfully performed thus far. We are extremely interested in whether the ψ sequence alters Gag kinetics in the nucleoplasm. A change in kinetics would indicate that Gag is capable of binding the ψ-packaging sequence in the nucleus. However, these studies will need to be performed on wildtype Gag proteins, as the L219A nuclear export mutant would not differentiate between Gag-ψ complexes formed in the cytoplasm of nucleus. It is feasible to predict an alteration of Gag mobility in the presence of ψ. Nucleolar accumulation of Gag was decreased when the ψ-packaging sequence was present. There are two likely possibilities for this result: either Gag is interacting with ψ in nucleoli and is released from the nucleolus, or Gag is binding to ψ prior to interaction with nucleoli and cannot be retained. However, it will be difficult to differentiate between these two scenarios.

HIV Gag localizes to centrosomes in the presence of ψ, suggesting viral RNA affects trafficking of Gag during infection (337). The FRAP analysis presented here offers new insight into the effect that viral RNA has on Gag proteins. Future experiments will look into the binding partners of Gag and Gag-ψ complexes. These experiments will begin to define the mechanisms retroviruses use to selectively package gRNA into virus particles.

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254 Vita Timothy Lewis Lochmann

Academic Degrees

Ph.D. in Genetics 2005-2011 The Pennsylvania State University, College of Medicine (Hershey, PA)

B.S. in Biochemistry and Cell Biology 2001-2003 University of California, San Diego (San Diego, CA)

Selected Abstracts

5th Annual NIH National Graduate Student Research Festival (October 25-26, 2010) Abstract/Poster Title: Nucleolar Trafficking of Retroviral Gag Proteins Linked to Viral RNA Packaging Authors: Timothy Lochmann and Leslie Parent

American Society for Microbiology 110th General Meeting (May 23-27, 2010) Abstract/Poster Title: Nucleolar Trafficking of RSV Gag Linked to Viral RNA Packaging Authors: Timothy Lochmann and Leslie Parent

7th International Retroviral NC Symposium (September 14-16, 2009) Abstract/Talk Title: Nucleolar Trafficking of RSV NC/Gag Linked to Viral RNA Packaging Authors: Timothy Lochmann and Leslie Parent

2008 Dynamic Organization of the Cell Nucleus (September 17-21, 2008) Abstract/Poster Title: The nucleolus: Matchmaker for retroviral protein-RNA complexes Authors: Timothy Lochmann and Leslie Parent

Publications

Nucleolar trafficking of retroviral Gag proteins. Lochmann TL, Ryan EP, Beyer AR, Parent LJ. In preparation. Nucleolar trafficking of RSV Gag and NC is linked to packaging and infectivity. Lochmann TL and Parent LJ. In preparation. Viral RNA and PI(4,5)P2 drive polyhexameric oligomerization of RSV Gag. Gudleski N, Nadariaia-Hoke SN, Lochmann TL, Flanagan JM, Bewely M, Parent LJ. In preparation.

Intermolecular interactions between retroviral Gag proteins in the nucleus. Kenney SP, Lochmann TL, Schmid CL, Parent LJ. J Virol. 2008 Jan;82(2):683-91. Awards

Ruth L. Kirschstein National Research Service Award 2007-2010 Provost’s Honors, University of California, San Diego, CA. 2002, 2003