Control of Retroviral Translation and Relationship to Genomic Rna Packaging

Home , Bovine leukemia virus, Human foamy virus, Lentivirus, Murine leukemia virus, Rous sarcoma virus

CONTROL OF RETROVIRAL TRANSLATION AND RELATIONSHIP TO GENOMIC RNA PACKAGING

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Melinda Sue Butsch, A.S., B.S.

* * * * *

The Ohio State University

2002

Dissertation Committee:

Dr. Kathleen Boris-Lawrie Approved by

Dr. James Dewille

Dr. Patrick Green ______Advisor Dr. Daniel Schoenberg Ohio State Biochemistry Program

ABSTRACT

Retroviruses are obligate intracellular parasites that utilize the eukaryotic translational machinery for viral protein synthesis. In the cytoplasm, an unspliced version of the primary retroviral RNA functions as mRNA for synthesis of viral protein and genomic RNA for packaging into infectious virions. Initiation is the rate-limiting step of translation and is a critical determinant of translational efficiency. Central goals of this dissertation were to determine whether or not de novo translation is a prerequisite to package retroviral genomic RNA and to examine the mechanism of retroviral translation initiation. To evaluate the possible requirement for obligate translation, we used three mechanistically distinct translation antagonists to inhibit protein synthesis in

HIV-1 infected T-cells. RNase protection assay (RPA) revealed that RNA packaging efficiency is increased upon inhibition of translation. The results indicate that de novo translation is not required for HIV-1 RNA packaging and that a single functional pool of

HIV-1 unspliced RNA functions interchangeably as mRNA or genomic RNA. To apply these findings to vector RNA and to specifically repress translation of the vector RNA, we developed a unique lentivirus-based luciferase reporter vector that contains the iron response element (IRE), a potent translational repressor.

ii Following deferoxamine or hemin treatment, Luciferase assays demonstrated that IRE confers iron-regulated translational repression on lentivector RNA and does not interfere with lentivector RNA packaging or helper virus production. Future studies will test the hypothesis that targeted translational repression augments lentivector RNA packaging and transduction efficiency.

We characterized HIV-1 reporter RNAs that contain the 5’ LTR of spleen necrosis virus (SNV) and defined a new RNA element that modulates retroviral translation initiation. RNA and protein analysis identified a novel retrovirus post- transcriptional control element located at the 5’ terminus of SNV RNA that facilitates

Rev/RRE-independent expression of unspliced and spliced HIV RNAs. Polysome analysis and RPA indicate that the SNV 5' RU5 enhances cytoplasmic accumulation and polysome association of HIV transcripts. Luciferase assay of SNV gag-luciferase reporter proviruses revealed that SNV RU5 also augments expression of homologous SNV RNA.

Following encephalomyocarditis virus (EMCV) infection, which inhibits cap-dependent translation, Luciferase assay indicates that SNV translation initiation is not cap- independent. The EMCV data and results of bicistronic reporter assays support the model that SNV RU5 is a cap-dependent translational enhancer. These experiments reveal a novel 5’ terminal translational enhancer that facilitate cytoplasmic expression of

HIV reporter RNA and constitute an important step toward defining control of retroviral translation. Future analyses will identify cellular binding partner(s) of SNV RU5 and investigate the role of RU5 in RNA packaging.

iii

Dedicated to my mom, Cecelia Gail Butsch. My biggest fan and one of my greatest supporters. In memory of Ruedog.

ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Kathleen Boris-Lawrie for giving me the opportunity to work in your lab. Many of my successes are directly attributed to your own zest for success and thirst for knowledge. I would also like to thank my committee members, Drs. James Dewille, Patrick Green and Daniel Schoenberg, for your advice and support. A special thanks to Dr. Dewille for serving as my co-advisor in the earlier years of my project.

I would like to thank Dr. Michael Lairimore and Dr. Charles Capen, for their advice, support and letters of recommendation. Thanks to Dr. Lawrence Mathes for the many times I stopped in to ask an immunological question. You never hesitated to stop what you were doing to help me out. To Dr. Kate Hayes, not only for the occasional assistance you provided on my projects but for your personal stories and timely hugs.

I would like to thank the past and present members of the KBL lab - Tiffiney Roberts, Stacey Hull, Drew Dangel, Jennifer Frey, Alper Yilmaz, and Sura Radhakrishna. Thank you for your patience, willingness to teach and willingness to learn, for your friendship and listening ears.

I would like to thank my best friend Uma Sivaprasad - this journey would not have been possible without you! You have taught me what true friendship is.

v I would like to thank my family. My mom and dad (Cecelia and Dan Butsch) have always supported my quest even though they still don't understand exactly what I do. My sister Dawn Bloemer has listened to my frustrations on too many occasions. My brother in-law Mike Bloemer has always been willing to stand in place of Dawn when I needed a good cry or laugh. My sister Danielle Butsch and Nathan Frolo have made me laugh even when I didn't think I could. My in-laws, Al and Bonnie Kovacic, have treated me like family from the start. My brother in-law Tom Kovacic has provided me with some well needed trips to Florida. You all will never know how much I have appreciated your support these last years and particularly these last 6 months. Thank you.

To my wonderful doggies – Scooter Rue, Dustin Buddy and Rude Rudy. You always made me feel loved and provided an outlet for my frustration. You have been with me through it all!

Finally, I would like to thank my husband Al and son Aidan. Al, your support, faith and strength provided for me when my own driving force faltered. My successes are your successes. And to my son Aidan - for putting everything into perspective.

VITA

Birthday...... May 23, 1974

1994...... Associate of Science The University of Cincinnati

1996...... Bachelor of Science The University of Cincinnati

PUBLICATIONS

1. Butsch, M. and Boris-Lawrie, K. 2002. The Destiny of Unspliced Retroviral RNA: Ribosome and/or Virion. (Minireview) Journal of Virology 76(7) 3089-94.

2. Butsch, M. and Boris-Lawrie, K. 2000. Translation is Not Required to Generate Virion Precursor RNA in Human Immunodeficiency Virus Type I Infected T- Cells. Journal of Virology 74(24):11531-7.

3. Butsch, M., Hull, S., Wang, Y., Roberts, T., Boris-Lawrie, K. 1999. The 5’ RNA Terminus of Spleen Necrosis Virus Contains a Novel Post-Transcriptional Control Element that Facilitates Human Immunodeficiency Virus Rev/RRE- Independent Gag Production. Journal of Virology 73(6):4847-55.

FIELDS OF STUDY

Major Field: Ohio State Biochemistry Program

vii

TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication...... iv

Acknowledgments...... v

Vita...... vii

List of Tables ...... x

List of Figures ...... xi

Abbreviations...... xiv

Chapters:

1. Introduction Statement of the Problem...... 1 The Retroviral Genome...... 2 Overview of the Retroviral Lifecycle ...... 5 Retroviral Nucleocytoplasmic Export...... 8 Mechanisms of Translation Initiation...... 12 RNA Packaging...... 23 Lentivirus-based Retroviral Vectors ...... 26

2. Translation is Not Required to Generate Genomic RNA in Human Immunodeficiency Virus Type I Infected T Cells ...... 30

viii Abstract ...... 30 Introduction...... 31 Materials and Methods...... 34 Results...... 38 Discussion...... 49

3. Development of IRE as a Positive Switch for Translational Repression in Novel Lentivector RNAs ...... 52

Abstract ...... 52 Introduction...... 53 Materials and Methods...... 55 Results...... 60 Discussion...... 70

4. The 5’ RNA Terminus of Spleen Necrosis Virus Contains a Novel Post- Transcriptional Control Element that Facilitates Human Immunodeficiency Virus Rev/RRE-independent Gag Production...... 73

Abstract ...... 73 Introduction...... 74 Materials and Methods...... 76 Results...... 80 Discussion...... 98

5. Translation Initiation of Spleen Necrosis Virus RNA is Cap-dependent ...... 102

Abstract ...... 102 Introduction...... 103 Materials and Methods...... 106 Results...... 109 Discussion...... 115

6. Perspectives...... 119

Overview...... 119 Hypothetical Models of Unspliced RNA Cytoplasmic Trafficking .....119 Selection of RNA for Translation or Packaging...... 123 Future Work ...... 126

Bibliography...... 129

LIST OF TABLES

Table Page

1.1 Retroviral Genera...... 4

4.1 HIV Gag Production...... 82

4.2 Comparison of Gag Protein Production and Cytoplasmic Accumulation of HIV RNA ...... 93

4.3 Summary of Gag Production and Subcellular Localization of pYW99 and pYW100 ...... 95

4.4 Gag Production and Subcellular Localization of pYW99 and pYW100 RNA Compared to pSVgagpol-rre RNA...... 98

LIST OF FIGURES

Figure Page

1.1 Retroviral Genome ...... 4

1.2 Retroviral Lifecycle ...... 7

1.3 Retroviral Nucleocytoplasmic Export...... 11

1.4 Mechanisms of Translation...... 14

1.5 Cap-dependent Translation Initiation...... 16

1.6 Iron Responsive Element Mediated Ferritin Translation...... 20

1.7 Encephalomyocarditis Virus Internal Ribosome Entry...... 22

1.8 The 5' Terminus of HIV-1 Genomic RNA ...... 25

1.9 Structure of a Prototypical Retroviral Vector and Propagation in Helper Cells ...... 27

2.1 Incorporation of 35S-Cysteine/Methionine is Inhibited by Incubation With Pactamycin, Cycloheximide and Anisomycin ...... 39

2.2 Ribosomal RNA Profile Analysis in Response to Pactamycin, Cycloheximide and Anisomycin...... 40

2.3 Pulse-chase Analysis of Gag Incorporation into Virions...... 42

2.4 Pactamycin, Cycloheximide and Anisomycin Significantly Inhibits Gag Synthesis ...... 44

2.5 Incorporation of 3H-uridine into Newly Synthesized Virions ...... 45

2.6 HIV-1 Genomic RNA Remains Available for RNA Packaging During Translation Inhibition...... 46

2.7 RNA Packaging Efficiency is Not Reduced Upon Inhibition of De Novo Translation...... 48

3.1 Genomic Structures of Lentivector DNAs...... 62

3.2 Partial Nucleotide Sequence of IRE SIN ...... 63

3.3 IRE Does Not Reduce RNA Packaging Efficiency...... 65

3.4 IRE Does Not Preclude Gag Processing...... 69

3.5 Targeted Translational Repression of Lentivector RNA Requires a Functional IRE...... 70

4.1 Structures of Subgenomic HIV Plasmid pSVgagpol-rreMPMV and Derivatives that Contain the SNV 3' UTR and SNV LTR...... 81

4.2 Structures of Hybrid SNV-HIV Plasmids and HIV Gag Production...... 84

4.3 Western Blot Immunoassay...... 88

4.4 Quantification of Steady-state RNA Levels by RPA...... 91

4.5 Sequence Comparison of the 5' Termini of pYW99 and pYW205 RNA...... 92

4.6 Polysomal RNA Accumulation...... 94

4.7 Comparison of Polysomal and Nonpolysomal RNA Localization...... 97

5.1 Structure of Plasmids Permanently Transfected into D17 Cells...... 110

5.2 Amplification of Luciferase or Cyclophilin in pSNVGagLuc and pSNVDRU5GagLuc Cell Lines ...... 112

5.3 SNV RU5 is Necessary for Luc Activity...... 112

5.4 SNV Translation Initiation is Not Cap-independent...... 113

5.5 SNV Does Not Contain an IRES ...... 115

xii 5.6 Model for Translational Enhancement by SNV RU5 ...... 118

6.1 Models of Retroviral Translation and Packaging ...... 121

6.2 Model of Potential Function of SNV RU5 in Translation and Packaging...... 126

xiii

ABBREVIATIONS

3' ss 3' splice site

5’ cap 5’ methyl-7-G(5’)pppN cap structure

5' ss 5' splice site

ACS autocomplementary sequence actD actinomycin D aniso anisomycin

ATP adenine triphosphate

BLV bovine leukemia virus

CaMV cauliflower mosaic virus

CAT chloramphenicol acetyltransferase chx cycloheximide

CPE cytopathic effect

CPM counts per minute

CTE constitutive transport element def deferoxamine mesylate

DIP defective interfering particle

DIS dimerization initiation site

DNA deoxyribonucleic acid dr direct repeat

xiv E or Y encapsidation/packaging signal eIF eukaryotic initiation factor

ELISA enzyme-linked immunosorbent assay

EMCV encephalomyocarditis virus

Env envelope

GAPDH glyceraldehyde dehydrogenase

GDP guanosine diphosphate

GTP guanosine triphosphate hGH human growth hormone

HIV-1 human immunodeficiency virus type 1

HMSV Harvey murine sarcoma virus hr(s) hour(s)

IRE iron responsive element

IRES internal ribosome entry sequence

IRP iron responsive protein

ITAF IRES transacting factors

Kb kilobase

KLD kissing-loop domain

LTR long terminal repeat

MOI multiplicity of infection

MPMV Mason-Pfizer monkey virus mRNA messenger RNA

MuLV murine leukemia virus

xv NC nucleocapsid domain

NES nuclear export signal

NLS nuclear localization signal nt nucleotide

ORF major open reading frame

PABP1 poly(A)-binding protein pac pactamycin

PAGE polyacrylamide gel electrophoresis

PBS primer binding site

PCE post-transcriptional control element

PEG polyethylene glycol

Pol polymerase

Poly polysomes

PPT polypurine tract

Pro protease

PTB pyrimidine tract-binding protein

R direct repeat

RanGAP Ran-specific GTPase-activating protein

RanGEF Ran-specific guanine nucleotide exchange factor

RevA reticuloendotheliosis virus A

RIPA radioimmunoprecipitation assay

RNA ribonucleic acid rRNA ribosomal RNA

xvi RPA RNase protection assays

RRE Rev responsive element

RSV Rous sarcoma virus

RxRE Rex-responsive element

SD splice donor

SDS sodium dodecyl sulfate

SIN self-inactivating vector

SIV simian immunodeficiency virus

SL stem–loop

SNV spleen necrosis virus

SRV-1 simian retrovirus-1

TAR trans-activating responsive RNA

TCA trichloroacetic acid

TfR transferrin receptor

U3 unique 3’ region

U5 unique 5' region

UTR untranslated region

VSV-G vesicular stomatitis virus G protein

xvii

CHAPTER 1

STATEMENT OF THE PROBLEM

The ability to control gene expression at the level of translation is central to productive retroviral gene expression and sustained production of infectious progeny virus. The rate-limiting step of translation is the initiation phase and is critical for determination of translational efficiency. A central goal of this dissertation was to examine the mechanism used to regulate retroviral translation initiation and to determine the relationship between retroviral translation and ribonucleic acid (RNA) packaging.

One unique characteristic of retroviruses is their ability to subvert typical cellular quality control measures and achieve gene expression of an unspliced, genome-length primary viral transcript. Cytoplasmic expression of the primary unspliced RNA is a mandatory step in the retroviral lifecycle. A portion of the unspliced RNA interacts with the cellular

RNA processing machinery and, similar to a typical cellular pre-mRNA, is spliced and exported to the cytoplasm. Another portion of this retroviral pre-mRNA avoids typical

RNA processing by interacting with viral and/or cellular nucleocytoplasmic shuttle proteins. These shuttle proteins facilitate the export of unspliced RNA to the cytoplasm

(36).

In the cytoplasm, the unspliced RNA transcript has two functions as mRNA template for synthesis of viral structural and enzymatic proteins, and as genomic RNA

1 that is packaged into virions. A longstanding unknown in retroviral biology is the relationship between translation and packaging of unspliced RNA. One of the primary outcomes of this dissertation was the elucidation of the relationship between translation and RNA packaging in human immunodeficiency virus type 1 (HIV-1) chronically infected human T-cells. These findings have important utility for the improvement of retroviral vector systems for gene delivery. Another outcome of this dissertation was a new understanding of the process by which spleen necrosis virus (SNV) facilitates translation of viral RNA. These studies reveal a unique mechanism of retroviral translational enhancement that has broad potential for optimizing diverse gene expression systems and contribute to the basic understanding of retroviral replication and host translational control. The following brief overview of the retroviral replication cycle and the adaptation of retroviruses as vectors for gene transfer will serve as background for these studies and emphasize three post-transcriptional steps of the lifecycle: nucleocytoplasmic export, translation, and RNA packaging, which are the topics of the subsequent chapters.

THE RETROVIRAL GENOME

Retroviruses are a large and diverse family of enveloped RNA viruses. The retroviral virion contains two copies of a single-stranded linear RNA that is 7–12 kb in size, is nonsegmented, and of positive polarity (32). Retroviruses are unique in that they use the virally encoded reverse transcriptase to replicate their genomic RNA into double- stranded DNA, which is subsequently integrated into the cellular genome by retroviral integrase. Reverse transcriptase and integrase are products of the pol gene. In addition to

2 pol, all retroviruses encode gag, pro and env genes (Figure 1.1). Translation of the gag

RNA produces the internal virion proteins that form the matrix, the capsid, and the nucleoprotein structures. The pro gene encodes the virion protease, which is necessary for the cleavage of immature Gag-Pol polyprotein to the mature form. The translation product of env includes the surface and transmembrane portions of the viral envelope protein. Retroviruses are classified as either simple or complex based on their genomic structure. While simple retroviruses contain the above-mentioned genes (Figure 1.1A), complex retroviruses also encode several auxiliary genes involved in virus-host interactions important for viral replication (Figure 1.1B). Retroviruses are further subdivided into seven genera defined by evolutionary relatedness (Table 1.1). Four of the genera contain simple retroviruses and the other three contain complex retroviruses.

3 A. Proviral structure of spleen necrosis virus

PBS PPT Y U3 R U5 gag pro pol env U3 R U5

5’ ss 3’ ss

B. Proviral structure of human immunodeficiency virus type 1

PBS PPT Y vif U3 R U5 U3 R U5 gag pro pol env vpu 5’ss vpr tat rev nef

Figure 1.1: (A) Representative simple retroviral genome. The genetic map of spleen necrosis virus (SNV) contains four major coding regions, gag, pro, pol, and env. The long terminal repeats include the unique 3’ sequences (U3), direct repeat (R), and a unique 5' region (U5). (B) Representative complex retroviral genome. The genetic map of human immunodeficiency virus type 1 (HIV-1) also contains two regulatory genes, tat and rev, and four accessory genes, vif, vpu, vpr, and nef, produced from singly spliced and multiply spliced RNAs. PBS, primer binding site; Y, packaging signal; 5' ss, 5' splice site; 3' ss, 3' splice site; PPT, polypurine tract.

Genus Example Genome Structure 1. Alpharetroviruses Avian leukemia viruses simple 2. Betaretroviruses Mason-Pfizer monkey virus simple 3. Gammaretroviruses Murine leukemia virus, simple Spleen necrosis virus 4. Deltaretroviruses Human T-cell leukemia virus, complex Bovine leukemia virus 5. Epsilonretroviruses Walleye dermal sarcoma virus complex 6. Lentiviruses Human immunodeficiency virus complex 7. Spumaviruses Human foamy virus complex

Table 1.1: Retroviral Genera. Table adapted with modification from reference (100).

RETROVIRAL LIFECYCLE

Retroviruses are RNA viruses that replicate through a DNA intermediate (32)

(Figure 1.2). Entry of virions involves attachment of viral envelope to specific cellular surface receptors and fusion and entry of the core. In the core, the virally encoded enzyme reverse transcriptase generates a double-stranded DNA copy of the RNA genome. The double-stranded DNA copy within the pre-integration complex is transported by host cell proteins into the nucleus and is integrated into chromosomal

DNA by integrase, another virally encoded enzyme. The host RNA polymerase II transcribes the provirus to generate the primary viral RNA or pre-mRNA.

A portion of the primary transcript interacts with the cellular RNA processing machinery and is spliced and exported to the cytoplasm. Another portion of the unspliced pre-mRNAs is transported to the cytoplasm by viral and/or cellular nucleocytoplasmic shuttle proteins. Nucleocytoplasmic export is one obstacle that must be overcome to produce progeny virions. Once this unspliced RNA reaches the cytoplasm, a second post-transcriptional obstacle is the competition between the host translation machinery and viral assembly complexes for the unspliced transcript. The primary role of the transcript is the synthesis of the viral Gag, Polymerase, and Envelope structural and enzymatic proteins. The newly synthesized proteins undergo N-terminal myristylation (62,66,169) and glycosylation (33,166), which subsequently targets and facilitates binding of Gag to the plasma membrane, a function that promotes assembly of viral core particles. To produce an infectious virion, two copies of the unspliced, genome-length RNA are packaged into the newly assembled particles (32). The nascent

5 virions bud from the cell and undergo proteolytic maturation in which the viral protease cleaves the immature Gag-Pol polyprotein to produce a mature, infectious virion. Failure to package two intact genomic RNAs or spontaneous mutation of the genome results in the release of defective interfering particles (DIP). DIP are defective viruses that lack the essential genes required for autonomous replication and interfere with the replication of infectious virions (95).

Defective interfering particle Protein Assembly & RNA Packaging Entry Reverse Transcription

Attachment Integration Translation

Transcription Maturation Splicing RNA Export Parental Virus

Progeny Virion

Figure 1.2: A schematic view of the retrovirus life cycle. The major steps in the replication of a typical retrovirus are indicated. Briefly, the parental virus attaches to a receptor on the cell surface and the viral core enters the cell. The genomic RNA is reverse transcribed to produce double-stranded DNA and integrates into the cell chromosome. The provirus is transcribed by the host transcription machinery. Spliced and unspliced genome-length RNAs undergo nucleocytoplasmic export and, in the cytoplasm, the viral RNAs are translated by host cell ribosomes. Viral proteins traffic to the plasma membrane and assemble. Two unspliced RNAs are packaged into immature Gag core particles. Immature virions (shown with gray round core) that contain or lack RNA (defective interfering particle shown with empty gray core) bud from the plasma membrane. Particles mature to infectious virus upon Gag –Pol polyprotein processing (shown with white icosahedral core).

RETROVIRAL NUCLEOCYTOPLASMIC TRANSPORT

Complex retroviruses

Nuclear export is a mandatory step in post-transcriptional gene expression that is necessary to produce mRNAs for translation to viral proteins. In complex retroviruses, the export of unspliced RNA is mediated by viral regulatory protein that binds to structured viral RNA element. HIV regulatory protein Rev binds to the cis-acting Rev responsive element, RRE [reviewed in (36,97,164)]. The RRE is a complex stem-loop structure present within intronic sequences of a subset of HIV transcripts (130). Rev is a

116 amino nucleocytoplasmic shuttling RNA binding protein (58). The amino terminus of Rev contains an arginine rich nuclear localization signal (NLS) that coordinates nuclear import and subsequent binding to RRE (141,196). Rev transactivation also enhances the stability and translational efficiency of RRE-containing RNAs (4,38,129).

The carboxyl terminus of Rev contains a leucine rich nuclear export signal (NES) that serves as a binding site for Crm1, an essential nuclear export receptor belonging to the

Imp family of nuclear transport receptors (36,130,141,146). Crm1 typically exports 5s rRNA and proteins with leucine-rich nuclear export signals (16,36). Following multimerization of Rev monomers at the RRE, Rev facilitates recruitment of Crm1 to the

RRE-Rev complex (146) (Figure 1.3). Crm1-Ran-GTP interaction mediates delivery of the RRE-Rev complex to the nuclear pore complex and nuclear export to the cytoplasm by binding directly to select nucleoporins, including Nup214/Can (61,146).

8 Release of the RNA into the cytoplasm occurs upon hydrolysis of Ran-GTP to Ran-GDP by cytoplasmic Ran-specific GTPase-activating protein (RanGAP). Both Rev and the

Ran-GDP are recycled back to the nucleus, where Ran-specific guanine nucleotide exchange factor (RanGEF) converts the Ran-GDP to Ran-GTP.

Simple retroviruses

Simple retroviruses face a similar export problem but lack viral regulatory protein analogous to Rev. Recent studies have identified export elements in Mason-Pfizer monkey virus (MPMV) (19), the related simian retrovirus-1 (SRV-1) (217), and avian

Rous sarcoma virus (RSV) (149,188). A 160 nt stem-loop structure in MPMV and SRV-

1 acts as constitutive/cytoplasmic transport element (CTE) and functions to directly recruit cellular export receptor(s) (19,55,56,83,148,188,197,199,217). The highly structured CTE is located in the 3’ untranslated region (UTR) (55,172,197) and was identified by its ability to replace HIV Rev/RRE function in subgenomic HIV plasmids

(19,149,217). While functionally analogous to the HIV-1 RRE, CTE is distinct in that it is not dependent on the Crm1 nuclear export receptor (14,154,160,179). Instead, CTE binds the cellular nuclear export receptor Tap. Tap mediates the sequence nonspecific nuclear export of cellular mRNAs, as well as the sequence-specific export of unspliced retroviral mRNAs bearing the CTE (83). Tap contains a nucleocytoplasmic shuttling domain at the carboxyl terminus that functions as both a NLS and a NES (6,106). The Tap nucleocytoplasmic shuttling domain directly binds specific nucleoporins (6,83,106)

(Figure 1.3). Tap also contains an additional NLS that binds to transportin, a nuclear import factor belonging to the Imp family (204). This second NLS is not essential for Tap function and is proposed to promote the rapid recycling of Tap to the cell nucleus.

9 Like MPMV, RSV and avian sarcoma virus contains a redundant stem-loop structure that is approximately 160 nt in length located near the 3’ UTR (149). These structured direct repeat (dr) elements have been shown to mediate stability and cytoplasmic accumulation of intron-containing RNA and RNA packaging

(5,148,149,188,193) (Figure 1.3). The cellular nuclear export receptor protein used by dr- containing transcripts has not yet been identified, but dr is known to function independently of Tap and Crm1 pathways (155).

dr modulates RNA stability, RRE/Rev modulates RNA export, cytoplasmic accumulation and stability, and RNA packaging translation CTE modulates RNA export

NUCLEUS ? CYTOPLASM Tap

RSV dr-containing mRNAs Crm1 MPMV CTE-containing mRNAs Rev

HIV RRE-containing mRNAs

Figure 1.3: Retroviral RNA elements involved in nucleocytoplasmic export. HIV Rev responsive element (RRE)-containing RNAs interact with viral Rev protein, which is an adapter protein to nuclear export receptor Crm1 and activates export by a pathway generally used for export of 5s rRNA and proteins with leucine-rich nuclear export signal. RNAs that contain the simian retrovirus type 1 or Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) achieve nuclear export by interaction with the nuclear export receptor Tap. The nuclear export receptor used by direct-repeat (dr)- containing transcripts has not yet been identified. Adapted with modification from reference (16).

MECHANISMS OF TRANSLATION INITIATION

Translation is the step in post-transcriptional gene expression that involves synthesis of proteins encoded by an mRNA template. The rate-limiting step of translation is initiation. Translational efficiency is nearly always determined by the rate of initiation and is directly influenced by the accessibility of the 5’ methyl-7-G(5’)pppN cap structure (5’cap), the context of the initiator AUG codon, the length and structure of the 5’ UTR and the absence of upstream AUGs or upstream open reading frames (ORFs)

(132). Translation initiation is tightly regulated at the level of RNA sequence and structure and often involves interaction(s) with one or many regulatory proteins. The majority of mRNAs contain 5’ cap that interact with translation initiation factors to facilitate cap-dependent ribosome scanning and initiation at the first AUG start codon.

Instead of scanning, specific eukaryotic cellular and viral mRNAs use an alternative mechanism to initiate protein synthesis by internal initiation. These mRNAs contain highly structured internal ribosome entry sites (IRESs) by which ribosomes bind in a cap- independent fashion and initiate translation at the next downstream AUG codon. Stable secondary structures inhibit ribosomal scanning when placed between the 5' terminus of the RNA and the initiation codon (114). IRESs are hypothesized to allow ribosomes to overcome the inefficient scanning of long and/or structured 5' UTRs.

The 5' UTR of retroviral unspliced RNA contains a collection of complex structures required for several steps of viral replication, including reverse transcription,

RNA packaging, and dimerization (8,143,202). Efficient cap-dependent translation of the unspliced RNA is expected to require localized melting of RNA structure, which would

12 distort presentation of the RNA packaging signal. Further, interaction between the retroviral nucleocapsid protein and the RNA packaging signal is expected to arrest ribosome scanning and inhibit efficient cap-dependent translation of the viral RNA

(11,156,156). This scenario implies that the cellular translation machinery and viral assembly complexes compete for cytoplasmic unspliced RNA or that translation is initiated by a cap-independent mechanism. Other viruses encounter similar obstacles.

Plant caulimoviruses use cap-dependent ribosome shunts to avoid the long and complex

RNA secondary structure that precedes their major ORFs (180) (Figure 1.4).

Alternatively, picornaviruses are uncapped and use a cap-independent mechanism to initiate translation (104) (Figure 1.4). An outcome of this dissertation is that retroviruses contain structured RNA elements that recruit cellular or viral RNA binding proteins that modulate the translation of their viral RNAs. Below is a brief overview of cap-dependent and cap-independent mechanisms used by retroviruses to initiate translation.

Additionally, specific translational control mechanisms are highlighted that are directly applied in this dissertation.

Mechanisms of Translation Initiation Example

Cap-dependent

5’ UTR A. Ribosomal scanning 7 m G AUG An Cellular and viral mRNAs

B. Ribosomal shunt 7 m G AUG A Cauliflower mosaic virus n

Cap-independent

C. Internal ribosome entry AUG An Encephalomyocarditis virus

7 m G AUG An Reticuloendotheliosis virus

Figure 1.5: Mechanisms of translation initiation of typical cellular and viral RNAs compared to particular viral RNAs that contain highly structured 5’ UTRs. A) Illustration of a representative 5' capped mRNA that utilizes cap-dependent ribosome scanning to initiate translation. Black arrows indicate translation initiation at the first open reading frame (denoted by a rectangle). Specific viral RNAs use B) ribosomal shunts, C) internal ribosome entry sequences (IRESs) to initiate translation. UTR, untranslated region. Figure adapted with modification from reference (64).

The cap-dependent ribosomal scanning model

Cap-dependent translation initiation occurs following recognition of 5’ cap of the mRNA by cap-associated eukaryotic initiation factors (eIFs) and ribosome scanning (78)

(Figure 1.5). Required factors include eIF4F and eIF4B (reviewed in (163)). eIF4F is a heterotrimeric cap-binding complex composed of eIF4E, eIF4G and eIF4A (21,80,81). eIF4E is the rate-limiting translation initiation factor (42,121). eIF4E binds the 5' cap

14 structure and facilitates the recruitment of the trimeric eIF4F complex to the 5' cap (26). eIF4E interacts with the amino terminus of eIF4G (127). eIF4G acts as a bridge between eIF4E, eIF4A and eIF3. eIF3 is the ribosomal dissociation factor and interacts with the central region of eIF4G (117). eIF4A is an ATPase-dependent RNA helicase and melts highly structured regions of mRNA (80). eIF4A binds eIF4G at two sites, one in the central domain and one in the carboxyl-terminal domain (112). The helicase activity of eIF4A is enhanced by the RNA binding protein eIF4B (175). Interaction between eIF4G and the poly(A)-binding protein (PABP) increases the affinity of PABP for poly(A), which subsequently increases the affinity of the eIF4F for the 5' cap structure (termed the closed-loop model) (209). Formation of the 48S preinitiation complex occurs upon assembly of the eIF4F cap-binding complex, 43S preinitiation complex and eIF4B at the

5' cap structure (171) (Figure 1.5). The 43S preinitiation complex contains a 40S subunit, a ternary complex (eIF2/GTP/Met-tRNAi) and eIF3. Following scanning of the RNA and recognition of an AUG start codon by the 48S preinitiation complex, the initiation codon base pairs to the anticodon of initiator tRNA and eIF5 triggers hydrolysis of the

GTP in the ternary complex (40). The initiation factors are released and the large 60S ribosomal subunit joins the 48S complex to form an 80S ribosome (Figure 1.5). The elongation phase of initiation ensues. The factors that promote the recognition of the

AUG start codon are discussed below.

Figure 1.5: Cap-dependent translation initiation. Step (1a): A ternary complex is formed between eIF-2, GTP, and the initiator tRNA. Step (1b): Dissociation of ribosomal subunits is aided by initiation factors including eIF-3. Step (2): The 40S ribosomal subunit recruits the ternary complex to form the 43S pre-initiation complex. Step (3): eIF4F facilitates binding of the 43S pre-initiation complex to the 5' of the mRNA. Step (4): The 43S pre-initiation complex, aided by associated factors, migrates to the initiator AUG to form the 48S pre-initiation complex. Step (5a): eIF5 hydrolyzes the GTP in the ternary complex, the initiation factors are released, and in Step (5b), the 60S ribosomal subunit joins. (Adapted from reference (78)).

16 Selection of the translation initiation site

Productive translation initiation site selection is determined by the context of the nucleotide sequence surrounding the first AUG codon encountered by the scanning preinitiation complex. The Kozak consensus sequence flanks the initiation codon and facilitates recognition of a particular AUG translation start codon (115). The recognition sequence GCC(A/G)CCATGG is the most efficient context for translation initiation.

Departure from this canonical consensus sequence, particularly by to mutations at the –3 or +4 positions, can result in reduced AUG recognition frequency and encourages initiation of translation from a stronger downstream Kozak consensus (41,82).

Initiation at an AUG that has a poor consensus sequence can result in leaky scanning. Ribosomes use leaky scanning to bypass a weak HIV Vpu AUG codon in favor of the downstream Env AUG codon to initiate synthesis of HIV Env proteins (185).

Similarly, viruses can bypass an in-frame termination codon in a process called functional recoding (69). In murine leukemia virus (MuLV) RNAs, insertion of a glutamine at the gag stop codon allows the ribosomes to read through the gag-pol junction (170) and synthesize the Gag-Pol polyprotein, an event that is essential for viral replication (60,96).

The presence of small upstream ORFs can also control the translation of the major downstream ORF by impeding ribosome scanning and suppressing the translation of downstream ORF. When this occurs, ribosomes scan the first open reading frame and reinitiate at the downstream ORF. The 380-nucleotide RSV RNA leader contains three

ORFs upstream of the AUG initiator of the gag gene (84) that are postulated to control

RSV Gag translation by reinitiation (89) and subsequently facilitate RNA packaging

(47,48,192). However, recent studies question whether or not RSV uses reinitiation and,

17 instead, suggests a cap-independent mechanism of translation initiation (43,44). A mechanism not yet evaluated in retroviruses involves the use of small ORFs to shunt ribosomes over highly structured leaders (reviewed by Hohn (94)) (Figure 1.5). While there are no identified retroviral ribosome shunts, RNA viruses such as cauliflower mosaic virus (CaMV) use ribosome shunts to promote cap-dependent translation of pregenomic 35S RNA (180). Despite a 612-nucleotide leader that contains several highly structured motifs and small ORFs, downstream translation of the 35S RNA is possible due to non-linear migration of ribosomes from a take-off site within a small ORF near the

5' cap to a landing site within a small ORF near the 3' end of the leader (165). Following nonlinear migration, the ribosomes resume scanning and initiate translation of the first long ORF (46,178).

Translation control by the iron responsive element

Translation can be potently regulated by binding of proteins to sequences within the 5' or 3' UTR. The best-characterized regulatory interaction occurs between the iron regulatory protein (IRP) and the non-coding iron responsive element (IRE) of ferritin mRNA. Interaction of IRP with the ferritin IRE represses formation of 80S ribosomes

(144). While retroviruses have not been found to contain IREs, the presence of an IRE near the retroviral 5' transcription start site is postulated to mediate translation repression in a manner similar to that of ferritin mRNA (to be discussed in Chapter 3). The ability to regulate the translation of retroviral RNAs, particularly retroviral vector RNAs, has the potential utility for increasing vector RNA packaging and the improvement of transduction efficiency in gene transfer applications.

18 The IRE of ferritin mRNA is located in the 5' UTR of the mRNA and controls ribosome binding. Other IREs, such as the IRE of transferrin receptor (TfR) mRNA, are located in the 3' UTR and regulate RNA degradation. IRPs are members of the aconitase family and bind the IRE of ferritin mRNAs when iron is limited (Figure 1.6), This interaction represses the recruitment of the 43S translation preinitiation complex (77,144)

(Figure 1.6). When iron is in excess, IRP binds iron instead of IRE and protein synthesis is derepressed by IRP (Figure 1.6). IRPs are regulated either by assembly or by disassembly of an iron-sulfur cluster (IRP1) or by rapid degradation in the presence of iron (IRP2). For IRP binding to efficiently repress translation, the IRE must be located within 67 nucleotides of the 5’ cap of the mRNA, a location that is phylogenetically conserved in mammalian IRE-containing RNAs (74,75,91). In Chapter 3, translation of retroviral vectors that have the ferritin IRE near their 5' transcription start site is postulated to be repressed by limiting iron using the iron chelator deferoxamine mesylate and derepressed in iron-rich hemin-treated media.

19 A. Fe IRP

IRE

7 ferritin mRNA m G An mRNA translated FERRITIN MADE

B. IRP

7 ferritin mRNA m G An mRNA translation is inhibited

NO FERRITIN MADE

Figure 1.6: IRE/IRP-mediated translation of ferritin. A) In iron-rich cells, IRP is bound to iron instead of IRE and ferritin is synthesized in order to bind the extra iron. B) In iron-poor cells, IRP binds IRE and synthesis of ferritin is repressed. IRE, iron responsive 7 element; IRP, iron responsive protein; m G, methyl-7-G(5’)pppN cap structure; An, poly (A) tail.

Cap-independent translation initiation

A cap-independent mechanism to initiate translation involves recognition of an internal ribosomal entry sequence (IRES) rather than a 5’ cap. Picornaviruses are the hallmark family of viruses that utilize IRESs to initiate protein synthesis (103).

Encephalomyocarditis virus (EMCV) is a classic example of a picornavirus that utilizes

IRES-mediated initiation. EMCV initiation does not involve scanning and does not 20 require eIF4E. EMCV IRES-mediated translation is sustained when eIF4E is unavailable and cap-dependent initiation is inefficient. EMCV IRES function is ATP-dependent and requires only eIFs 2, 3, eIF4A and the carboxyl-terminus of the scaffold protein, eIF4G, to which eIF4A binds (162). It is for this reason that cleavage of eIF4G by many picornavirus protease 2A proteins specifically reduces cap-dependent translation but not

IRES-mediated translation (35,76). Instead of the 5' cap structure, the eIF4A and eIF4G bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes (125) (Figure 1.7). Similar to the role of eIF4E on capped mRNA, the interaction of eIF4G with the IRES recruits the preinitiation complex to the RNA and promotes ribosomal attachment (131).

Initiation on some EMCV and EMCV-like IRESs requires additional noncanonical IRES transacting factors (ITAFs). Binding of ITAFs such as pyrimidine tract-binding protein (PTB) stabilizes IRES conformation and promotes binding of essential factors like eIF4A/4G. The diversity of IRES sequences and structures leads to the requirement for a variety of ITAFs. Initiation at several picornavirus IRESs including

EMCV require binding of PTB and a 45kDa ITAF (ITAF45) (Figure 1.7).

Figure 1.7: EMCV IRES-driven translation initiation. ITAF45 and PTB bind the IRES and facilitate binding of essential initiation factors, eIF4A and eIF4G, immediately upstream of the EMCV initiation codon. Interaction of eIF4G and eIF4A with the IRES promotes the formation of the 48S preinitiation complex by recruiting 43S complexes to the RNA. EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; PTB, pyrimidine tract-binding protein; ITAF45, 45kDa IRES transacting factor. Figure from reference (163).

22 Experiments with bicistronic reporter plasmids have identified IRES activity in the 5’ UTRs of Harvey murine sarcoma virus (HMSV), MuLV, Rous sarcoma virus

(RSV), avian reticuloendotheliosis virus A (RevA) and simian immunodeficiency virus

(SIV) and in the gag ORF of HIV-1 (12,13,20,43,44,126,150,205). The data suggest that these IRESs function to used to overcome inefficient ribosome scanning in retroviral

RNAs (102,167) and promote synthesis of Gag and/or glyco-Gag proteins, although a functional role for internal ribosome entry during retroviral replication has not been demonstrated.

RETROVIRAL RNA PACKAGING

Retroviral RNA packaging is the process by which a homodimer of the full-length retroviral genome is incorporated into assembling virions. RNA packaging is a critical step in the retrovirus lifecycle because viral particles that lack or contain a defective viral genome function as DIPs (11). The selection of full-length viral RNA for packaging and exclusion of spliced viral RNAs and cellular mRNAs is a result of recognition by the Gag precursor protein of the cis-acting structured RNA element in the 5’ UTR designated the

RNA packaging signal (E or Y). Y is located in the 5’-noncoding region of the RNA between the primer-binding site (PBS) and into the 5’ terminus of the gag gene. Two copies of the unspliced genome-length RNA are specifically packaged into the assembling virion via interaction between the Cys-X2-Cys-X4-His-X4-Cys motifs of the nucleocapsid domain of Gag precursor protein and the highly structured Y (reviewed in

(11,62,169)). The presence of the Y increases packaging of retroviral genomic RNAs up to 200-fold over cellular and viral RNAs that lack Y (169). Additional Gag domains are 23 involved in particle assembly. Basic residues of the Gag matrix domain and an amino- terminal myristylation signal target nascent Gag structural protein to the plasma membrane (92,151,214,215). N-terminal myristylation then facilitates binding of Gag to the plasma membrane and promotes assembly of viral cores, which subsequently package

RNA.

Y is one of many complex structures within the 5' UTR of retroviral unspliced

RNAs (8,143,202). Localized melting of the RNA by the translation machinery may distort Y and inhibit RNA packaging. The interaction between Y and nucleocapsid is expected to arrest ribosome scanning and inhibit efficient translation of the viral RNA

(11,156,156). The dual function of the unspliced RNA implies that the cellular translation machinery and viral assembly complexes compete for cytoplasmic unspliced

RNA.

HIV-1 RNA packaging

The primary HIV-1 Y has been extensively characterized and consists of a stable

RNA secondary structure that embodies four hairpin loops referred to as stem–loops 1–4

(SL1, SL2, SL3 and SL4) (136). Deletion of SL1, SL3 and SL4 has been associated with a defect in RNA packaging (136). Because deletion of SL3 has the greatest effect on packaging, SL3 is often termed the major packaging signal of HIV-1 (136) (Figure 1.8).

SL1 is also important for genomic RNA dimerization and has been termed the kissing- loop domain (KLD) (118). The KLD contains an almost invariant hexameric autocomplementary sequence (ACS) called a palindrome (119) (Figure 1.8).

24 The GCGCGC palindrome is the dimerization initiation site (DIS) of genomic RNA (189) and stimulates genomic RNA dimerization (28,85), genomic RNA packaging

(10,28,119,135,156), and proviral DNA synthesis (156). A dimeric genome is required for HIV-1 replication (156). SL2 contains the major splice donor. Inclusion of the splice donor within the packaging signal may provide a mechanism for preferential selection of the full-length genomic RNAs for packaging into progeny virions (136).

Figure 1.8: The 5' terminus of HIV-1 genomic RNA contains structured motifs necessary for viral replication, including the RNA packaging signal. Highlighted are the trans- activating responsive RNA element (TAR), the R-U5 stem loop which contains sequences in R and U5, the primer binding site (PBS), the GCGCGC palindrome, the kissing loop domain (KLD), the splice donor (SD), the stem loop 3 (SL3) and the AUG initiation codon of the gag gene. Figure adapted with modification from reference (186).

LENTIVIRUS-BASED RETROVIRAL VECTORS

One of the most promising applications of retroviruses is their modification for use in gene expression studies and as delivery vehicles for gene therapy. The ability of retroviral genomes to function as vectors was established in the early 1980’s when mutated murine and avian retroviral RNAs that lack viral protein coding regions were shown to be eligible for packaging if the missing viral proteins were provided in trans

(70,187,198). The term vector refers to a modified virus that contains one or more transgenes in place of viral ORFs and the cis-acting elements required for gene expression and replication (99). Most vectors contain an intact retroviral packaging signal (Y+), but lack some or all of the viral protein coding sequences. Vectors are therefore replication-incompetent. To support a single cycle of replication of the vector

RNA, viral genes lacking in the vectors are expressed in a helper cell (Figure 1.9). The helper cell may contain either a helper virus or cotransfected plasmids that express the missing proteins in trans, but do not contain the retroviral packaging signal (Y-). Upon co-transfection of a helper plasmid, the Y+ vector transcripts function as the genomic

RNA that are packaged into the helper virus, while the Y- helper RNAs function exclusively as mRNA template for translation of viral Gag, Polymerase, and Envelope proteins. The transduction of target cells with the progeny helper virus results in a single cycle of vector RNA replication. Subsequently, the integrated vector provirus functions exclusively as mRNA for synthesis of vector-encoded protein by the host cell machinery.

Importantly, the vector does not express any viral proteins and infectious virions are not produced and do not spread to other target cells.

Y Y+

Transduce

(Y-)

Figure 1.9: Structure of a prototypical retroviral vector, propagation in a helper cell, and a single cycle of replication in a transduced target cell. A retroviral vector containing a desired transgene (diagonally stripe square) is transfected into a helper cell. The helper cell produces the viral proteins (shown as black ellipses and white circles) that are necessary to assemble viral particles and package the vector RNA. Transduction of progeny particles into target cells results in reverse transcription and integration of the vector genome into the target cell genome by the helper proteins. Transcription of the vector provirus occurs and the resulting RNA is translated to produce transgene protein. The target cells, however, do not express viral proteins and therefore do not produce a second round of progeny vector virus. Figure adapted from reference (99).

Retroviral vector technology is the most commonly used delivery system for gene transfer in gene therapy applications (177). An ideal retroviral vector for gene delivery is efficient, regulatable and safe. While the use of retroviral vectors in biomedical research is commonplace, the success of retroviral vectors in clinical trials has been restricted due to limited gene expression, low viral titer and questionable safety. However, their promise has made overcoming these deficiencies the focus of study for molecular retro- biologists and gene therapists alike. Recently, lentivectors have been used in place of traditional retroviral vectors to increase host range. Lentivectors are vectors based on

27 lentiviruses that have the unique ability to transduce both nondividing and terminally differentiated cells in addition to dividing cells, thus expanding the host cell range of the vector virus (124). The ability to transduce nondividing cells would be particularly useful for gene transfer to somatic tissues of humans for gene therapy. Replacement of retroviral Envelope proteins with the G protein of vesicular stomatitis virus (VSV-G) also increases host range and improves virion stability, which subsequently allows concentration of the vectors to high titer (22,211,212). Maximal viral titer is one measure critical for efficient transduction. HIV-based lentivectors pseudotyped with VSV-G have proven to be relatively high-titer lentivectors (3,145). Additionally, ultracentrifugation

(57), hollow fiber filtration (161), tangential flow filtration polyethylene glycol (PEG)- precipitation (113), and column chromatography (133) promotes concentration of retroviral vectors to titers of 106 to >107.

Also critical for retroviral vector design and maximization of transgene expression is promoter choice. Certain promoters located within the vector can also provide the ability to express more than one transgene from a single vector. However, transcript interference by the retroviral promoter can occur (52,53). Use of a self- inactivating (SIN) vector in conjunction with an internal promoter prevents promoter interference in transduced cells (70). SINs are designed so that the viral promoter in the

U3 region of the LTR is inactivated following reverse transcription in transduced cells

(50,213). Additionally, inactivation of the 5’ LTR reduces the possibility of generating a replication-competent virus through recombination in vivo.

A new, yet to be developed method to increase virus titer is maximization of RNA packaging. Increased packaging efficiency is expected to decrease defective interfering

28 particles (DIP). DIPs are void of genomic RNA or contain partial or defective genomic

RNA and reduce infectious virus titer by interfering with viral replication (111). Analysis of the ratio of DIP versus infectious units of MLV and HIV-based vectors using electron microscopy and a functional transduction assay has revealed approximately 100-350

DIPs per infectious particle (93). Increased RNA packaging efficiency is proposed to reduce the proportion of DIP and increase vector titer. An inverse relationship between translation and RNA packaging implies that targeted repression of vector RNA translation will increase the vector RNA available for packaging into vector virus.

Subsequently, the ratio of DIP to infectious particles will decline and augment vector virus titer.

CHAPTER 2

TRANSLATION IS NOT REQUIRED TO GENERATE GENOMIC RNA IN HUMAN IMMUNODEFICIENCY VIRUS TYPE I INFECTED T CELLS

ABSTRACT

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as genomic RNA for packaging remains an important open question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate genomic

RNA. HIV-1 infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady state

Gag is competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 hours after transcription, newly synthesized RNA is detectable in cell-free virions and packaging is sustained upon inhibition of host cell translation. 3H-uridine incorporation assays and HIV-1 specific

RNase protection assays (RPAs) agree that translation inhibition reduces the absolute amount of both cytoplasmic and virion-associated RNA.

30 Evaluation of packaging efficiency by RPA revealed that the cytoplasmic availability of genomic RNA is increased indicating that HIV-1 unspliced mRNA can be rerouted to function as genomic RNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as genomic RNA.

INTRODUCTION

The genomes of RNA viruses are multifunctional molecules. In retroviruses, including human immunodeficiency virus type 1 (HIV-1), the primary RNA transcript functions as pre-mRNA for splicing, mRNA for synthesis of viral protein, and genomic

RNA for packaging into infectious virions. The unspliced HIV-1 mRNA and genomic

RNA are physically indistinguishable and are defined experimentally by their association with ribosomes or virions, respectively. The relationship between mRNA and genomic

RNA remains poorly understood, and its characterization may yield a new strategy to inhibit production of infectious HIV-1 and to improve lentiviral vector systems for gene transfer applications.

Initial investigation of the relationship between retroviral unspliced mRNA and genomic RNA focused on cells productively infected with the genetically simple murine leukemia virus (MuLV) (123,139,159). Levin and colleagues (122,123) analyzed cells treated with the transcription inhibitor actinomycin D (actD) and showed that viral mRNA remains available to direct viral protein synthesis, but the particles do not contain genomic RNA. These data implied that MuLV transcripts segregate into two functionally distinct populations of mRNA for translation or genomic RNA for packaging (123).

31 Stoltzfus et al. (194) applied isotopic equilibrium assay to cells infected with avian sarcoma virus (ASV) and observed not two but rather a single RNA population that functions as both ASV mRNA and genomic RNA. Sonstegard and Hackett (192) came to similar conclusions in their studies of Rous sarcoma virus (RSV) vector RNAs.

Transfection studies with vectors that contain or lack most of the RSV packaging signal, y, indicate that interaction of Gag with y autogenously modulates competition between the translational machinery and assembling viral proteins. The data indicate that equilibrium exists between vector RNA destined for translation or RNA packaging, which is determined by the cytoplasmic availability of Gag protein and ribosomes (192).

Investigation of the fate of genomic RNA from genetically complex retroviruses has been largely limited to genetic studies with HIV vectors and has not been pursued for

RNA expressed from HIV-1 provirus in human T-cells. Studies with HIV-1 based vectors have shown that the RNA structure inherent in the HIV-1 RNA packaging signal inhibits efficient translation (67,142). These results imply that HIV-1 RNA packaging and translation are competing processes. McBride et al. (136) evaluated a subgenomic HIV-1 vector that contains a premature gag stop codon and found that RNA packaging remained efficient. These data are consistent with the successful use of HIV-1 as a gene transfer vector (109,145) and eliminate a requirement for ongoing Gag protein synthesis.

However, the question remains open whether or not it is necessary for genomic RNA to serve as mRNA template prior to packaging. Contrasting results were obtained in a study of HIV-2 based vectors that contain deletions at the 3' end of the gag open reading frame.

These results indicated that Gag protein translation from vector template was necessary to generate HIV-2 genomic RNA (107).

32 HIV-1 differs from HIV-2 in that the complete packaging signal exists only on unspliced viral RNA and not on spliced RNAs as in HIV-2 (137). The requirement for prior translation of HIV-2 gag mRNA is a potential mechanism for selective packaging of

HIV-2 unspliced RNA into progeny virions (107).

The primary goal of this project was to evaluate the hypothesis that translation is a prerequisite to generate HIV-1 genomic RNA in chronically infected human T-cells.

Definition of the relationship between HIV-1 unspliced mRNA and genomic RNA will shed light on whether the transcripts constitute a single RNA pool or two functionally independent pools of RNA that are dedicated as either mRNA or as genomic RNA. If

HIV-1 unspliced transcripts function as a single pool of RNA, inhibition of protein synthesis is expected to increase the availability of genomic RNA and augment packaging efficiency. However, in the case that prior utilization as mRNA is necessary to generate genomic RNA, packaging efficiency would be decreased upon inhibition of protein synthesis. If HIV-1 unspliced transcripts represent two independent pools of

RNA that are committed to either translation or packaging, translation inhibition would not alter generation of genomic RNA.

To examine these possibilities, HIV-1 infected human T cells were treated with translation inhibitors under conditions that maintain virus assembly. Comparison of ribosomal RNA profiles of HIV-1 infected and mock-infected T cells verifies that HIV-1 infection does not mediate shut-off of host cell translation or disrupt the mechanistic effects of pactamycin (pac), cycloheximide (chx), or anisomycin (aniso). Analysis of newly synthesized HIV-1 protein and genomic RNA after short-term treatment with the inhibitors established that de novo translation is not necessary to maintain assembly,

33 release, and processing of Gag precursor protein, or packaging of genomic RNA. RNase protection assays (RPAs) demonstrate that HIV-1 packaging efficiency is increased upon

80%-90% inhibition of de novo translation. The data indicate that prior translation of

HIV-1 unspliced RNA is not a prerequisite to generate genomic RNA. HIV-1 unspliced transcripts constitute a single population of RNA that can be selected interchangeably as genomic RNA and as mRNA.

MATERIALS AND METHODS

Cells and translation inhibitors

CEM(A) T-cells infected with HIV-1NL4-3 (CEM(A)/HIV-1) were cultured in

RPMI 1640 medium supplemented with 10% fetal calf serum and 1% antibiotic- antimycotic (Gibco-BRL). Cell viability in response to chx, pac, or aniso was assayed by propidium iodide and flow cytometry at 4 hours after treatment (31). Maximal concentrations having minimal cytopathic effect during 4 hour incubation were used in subsequent experiments: 5 ´ 10-8 M pac (Pharmacia & UpJohn, Kalamazoo, MI), 0.5 mg/ml chx (Sigma, St. Louis, MO), and 0.1 mg/ml aniso (Sigma).

Protein analysis

CEM(A)/HIV-1 T cells were lysed in RIPA buffer (0.05M Tris-HCL, pH 8, 0.1%

SDS, 1% Triton-X-100, 2mM PMSF, 0.15M NaCl, 2mM) containing 1% deoxycholic acid and the nuclei were removed after centrifugation at 13,400 ´ g for 10 minutes. Total protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad

Laboratories, Hercules, CA). Virion-containing medium was clarified by centrifugation at

2000 × g for 10 min and virions were collected by centrifugation at 156,000 × g for 1.5

34 hours at 4o C in a Beckman SW41 rotor. Gag ELISA was performed by the manufacturer’s protocol (Beckman-Coulter, Brea, CA). 35S-labeling experiments were performed by incubating CEM(A)/HIV-1 T cells in cysteine/methionine - free RPMI media with 5% dialyzed fetal bovine serum for 30 minutes followed by addition of pac,

35 chx, or aniso coincident with 10 mCi /ml S - cysteine/methionine (1175 Ci/mmol, 43.5

MBq/ml) (ICN Biohemicals, Irvine, CA). The cells were lysed in RIPA buffer containing 1% deoxycholic acid. Fifty nanogram of cytoplasmic lysate and virion lysates equivalent to 30 ng of Gag were precipitated by trichloroacetic acid (TCA) (20% TCA,

0.1mg/ml bovine serum albumin) onto 25 mm glass fiber filters (type A/C) (Pall Corp.,

Ann Arbor, MI), washed three times in 10% TCA and in 100% ethanol, and subjected to scintillation counting. For pulse-chase experiments, CEM(A)/HIV-1 T cells were incubated for 30 minutes in cysteine/methionine - free RPMI media with 5% dialyzed fetal bovine serum followed by incubation for 1 hour in 35S - cysteine/methionine- supplemented media with or without pac, chx or aniso. The cells were washed and complete RPMI was added. After 1, 2, 4, 6 or 8 hours post-chase, cells were harvested as above. For immunoprecipitation, the 35S-labeled lysates were incubated for 16 hours with protein A sepharose beads (Pharmacia) and polyclonal rabbit sera against HIV Gag (gift of N. Panganiban; (134). The beads were washed once in high salt RIPA (1M NaCl) and once in low salt RIPA (0.15M NaCl), and boiled to elute the proteins. The 35S-labeled precipitated proteins were subjected to SDS-PAGE, visualized and quantified by

PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA) with ImageQuaNT

Software version 4.2 (Molecular Dynamics).

35 RNA analysis

CEM(A)/HIV-1 cells were plated in T150 flasks, cultured overnight to 80%

3 confluence, and incubated for 4 hours in medium with 30 mCi /ml H-uridine (Amersham,

Piscataway, NJ) and with or without pac, chx, or aniso. Cytoplasmic extracts were prepared in 0.9 ml cold cell lysis buffer (10 mM Tris pH 8.3, 150 mM NaCl, 1.5 mM

MgCl2) and 0.1 ml NP40. Following centrifugation to pellet the nuclei, the supernatant was mixed with TriReagent LS and cytoplasmic RNA was isolated by the manufacturer’s protocol (Molecular Research, Cincinnati, OH). One microgram of cytoplasmic 3H-RNA and virions equivalent to 30 ng of extracellular Gag were applied to glass fiber filters, which were washed 4 times with 5% TCA containing 20 mM sodium pyrophosphate, once with 100% ethanol, dried, and subjected to scintillation counting. For polyribosome profiles, clarified cytoplasmic extract from three 80% confluent T150 flasks was layered onto a 10 ml linear gradient of 15% to 45% sucrose. The gradient was centrifuged

0 225,000 × g for 2.25 hours at 4 C in a Beckman SW41 rotor. Gradients were fractionated and monitored for A254 on an ISCO model 160 gradient fractionator

(Lincoln, NE). To prepare virion RNA, cell medium was clarified by centrifugation at

2000 × g for 10 min, virions were pelleted by centrifugation at 156,000 × g for 2.5 hours at 4o C in a Beckman SW28 rotor, and lysed in 1 ml Trizol ReagentTM and isolated by the manufacturer’s protocol (Gibco BRL, Gaithersburg, MD).

32P-labeled antisense RNA probes were generated by in vitro transcription of pGEM(600-900), which contains the 5’ UTR of HIV-1NL4-3 (134), and pGAPDH, which contains human glyceraldehyde dehydrogenase (gapdh) gene (25). Following digestion of pGEM(600-900) with NotI and pGAPDH with NcoI, antisense run-off RNA transcripts

36 were synthesized with MAXIscriptTM T7 RNA polymerase (Ambion, Austin, TX) and the probes were isolated by gel elution. RPA was performed using RPA III (Ambion) according to the instruction manual with some modifications (25). Typically, 10 mg of cytoplasmic RNA or viral RNA from virions equivalent to 250 ng of Gag was precipitated by ethanol with 2 × 105 CPM HIV-1 probe and 2 × 104 CPM gapdh probe.

Samples were resuspended in 10 ml of hybridization buffer, denatured at 94o C for 3 min, and hybridized at 42o C overnight. RNAse A/T1 was diluted 1:100 in Ambion RNAse digestion buffer and 150 ml was added to each sample and incubated at 37o C for 30 minutes. SDS and proteinase K were added to final concentrations of 1% and 0.5 mg/ml, respectively, and samples were incubated at 37o C for 30 minutes. A 100 base pair 32P- labeled DNA fragment was added to virion RNA samples followed by phenol-chloroform and chloroform extraction and precipitation with ethanol in the presence of 10 mg of yeast

RNA. Following centrifugation, the pellets were dissolved in 6 ml of loading buffer, denatured at 94o C for 3 min, and subjected to 5% denaturing polyacrylamide gel electrophoresis. RNase protection products were visualized and quantified by

PhosphorImager analysis.

37 RESULTS

HIV-1 infection does not alter host cell response to pac, chx, or aniso.

Our goal was to evaluate trafficking of HIV-1 unspliced RNA expressed from provirus in T-cells. A genetic approach using conventional transfection methods was of limited utility for this purpose because over-expression of RNA from transfected DNA may saturate the assembly process and obscure the natural relationship between HIV-1 unspliced mRNA and genomic RNA that is exhibited by authentic provirus in an infected

T cell. Therefore, a biochemical approach was developed that limits de novo translation of HIV-1 RNA under conditions that maintain virus production.

CEM(A) T-cells productively infected with HIV-1NL4-3 were subjected to short- term incubation with three mechanistically distinct biochemical antagonists of translation: pac, chx, and aniso. To determine the magnitude and onset of inhibition of protein synthesis, 35S-cysteine/methionine incorporation into whole cell protein was evaluated after 4-hour incubation with pac, chx or aniso. Comparison with mock-treated cells indicated that incorporation of 35S-cysteine/methionine into whole cell protein was inhibited 80-90%; the reduction in protein synthesis commenced by 30 minutes post- treatment and continued up to 4 hours post-treatment (Figure 2.1). Propidium iodide staining and flow cytometry detected no overt cytopathic effects on the cells during the 4- hour incubation period. Relative to the mock control, percent cell viability remained

100% in response to pac, 96% in response to chx, but was reduced to 86% in response to aniso.

Figure 2.1: Incorporation of 35S-cysteine/methionine is inhibited by incubation with pac, chx and aniso. Inhibition of 35S-cysteine/methionine incorporation occurs 0.5 hours post- treatment and is sustained over a 4-hour period. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine - free RPMI media for 0.5 hours followed by the addition of 35S-cysteine/methionine with pac, chx, or aniso. Total cell lysates were collected at 0.5, 2 or 4 hours post-treatment and 35S-cysteine/methionine incorporation were quantified by TCA precipitation assay. Average results of at least four experiments are shown. Error bars indicate standard deviation.

Ribosomal RNA profile analysis of the cells indicated that the treatments exert the expected mechanistic effects on the translational machinery (Figure 2.2). Pac produced an accumulation of 80S monosomes, which is attributable to interference with translation initiation (63,73,206). Chx resulted in the accumulation of polyribosomes in response to a block in EF-2-dependent peptide translocation (59,147,206). Aniso reduced polyribosome abundance associated with defective peptide bond formation during elongation of the polypeptide (206). 39 Comparison with the ribosomal RNA profiles of mock-infected CEM(A) T-cells (Figure

2.2) indicated that HIV-1 infection did not change the ribosomal RNA profile of CEM(A)

T-cells (2), nor alter the response to pac, chx, or aniso. These results indicate that HIV-1

infection does not disrupt the host translation machinery.

HIV-1-infected

Mock PactamycinPactamycin CycloheximideCycloheximide AnisomycinAnisomycin Mock

80S

80S 80S A254 80S poly 40/60S poly poly 40/60S poly 40/60S 40/60S

Mock-infected

Mock PactamycinPactamycin CycloheximideCycloheximide AnisomycinAnisomycin

80S 80S 80S

A 254 80S poly poly 40/60S poly 40/60S poly 40/60S 40/60S

Figure 2.2: Ribosomal RNA profile analysis in response to pac, chx, and aniso. HIV-1 infected or uninfected CEM(A) T cells were treated for 4 hour with or without pac, chx, or aniso and cytoplasmic extracts were placed on 10 ml linear gradients of 15% to 45% sucrose. After ultracentrifugation, the gradients were fractionated and monitored at A254 using an ISCO gradient fractionation system.

40 De novo translation is not necessary for virion production or Gag processing.

Pulse-chase labeling was used to define the onset and duration of accumulation of newly synthesized Gag in virions. Cells were incubated for one hour in 3H-uridine- supplemented media, and then washed and incubated with media without 3H-uridine.

One to 8 hours later, virions were isolated by ultracentrifugation and quantified by Gag

ELISA. One microgram of whole cell lysate or virions equivalent to 30 ng Gag were subjected to precipitation assay with TCA to determine 35S-cysteine/methionine incorporation into newly synthesized proteins. Nonspecific accumulation of 35S- cysteine/methionine was quantified in control cultures that were treated with pac to inhibit protein synthesis. Background incorporation of 35S cysteine/methionine into the pac-treated whole cell lysates and virion samples was similar at each time point; 1000

CPM or less for whole cell lysate and 30 CPM or less for virion samples (representative figure, Figure 2.3). Incorporation of 35S-labeled protein into virions was maximal at 1 hour post-chase indicating that newly synthesized Gag is readily incorporated into virions

(Figure 2.3). Production of 35S-virions continued for 6 hours post-chase indicating that newly synthesized Gag is not required for continued virion production. Over time, 35S- cysteine/methionine incorporation into virions diminished and this trend matched the decline in intracellular 35S-labeled protein. The results indicate a concentration- dependent relationship between intracellular and extracellular Gag.

Figure 2.3: Pulse-chase analysis of Gag incorporation into virions. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine – free media for 0.5 hours followed by a 1 35 -8 hour incubation with of 10 mCi /ml S - cysteine/methionine and 5 ´ 10 M pac in control plates. Cells were washed and incubated in complete media with or without pac. Cell lysates and virion lysates were collected at intervals between 1 and 8 hour post- chase. TCA precipitation assay was performed with 50 ng of cellular protein or virion lysate equivalent to 30 ng of Gag. Representative results of three experiments are shown.

Gag radioimmunoprecipitation assay (RIPA) was used to evaluate the effect of pac, chx, and aniso on Gag protein synthesis and processing. We also evaluated the effect of actD, an RNA synthesis inhibitor that is known to disrupt shuttling of HIV-1

Rev between the nucleus and the cytoplasm (140). The cells were incubated for 4 hours in media supplemented with 35S-cysteine/methionine. Subsequent Gag ELISA of cell- free supernatant indicated that Gag production was reduced, but not abrogated in response to the translation inhibitors.

42 Compared to the mock-treated cells, Gag production from pac, chx, aniso, and actD- treated cells was 68 ± 11%, 69 ± 16%, 71 ± 19% and 93 ± 15%, respectively. These results indicate that the inhibitor treatments do not prevent previously synthesized Gag from being released from the cell.

RIPA detected similar levels of 35S-labeled Gag in mock-treated cells and actD- treated cells (100% and 120%, respectively) (Figure 2.4). Similar levels of 35S- incorporation were also observed in virion samples indicating that de novo RNA synthesis is not necessary for synthesis and processing of HIV-1 Gag. Treatment with pac, chx, or aniso reduced intracellular 35S-labeled Gag levels to 28%, 40%, and 28%, respectively. Extracellular 35S-labeled Gag levels were similarly reduced to 20%, 20%, and 26%, respectively, indicating that extracellular 35S-Gag levels are proportional to the cytoplasmic abundance of 35S-labeled Gag. Each virion sample displayed fully processed

Gag p24 indicating that de novo translation is not required for Gag protein processing.

Minor differences were observed in the intracellular ratio of unprocessed Gag p55 to Gag p24 and may be attributable to variation among the cells in the intracellular concentration of Gag p55. The observation that the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag validates a concentration-dependent relationship between extracellular and intracellular Gag.

Figure 2.4: Pac, chx and aniso significantly inhibit Gag synthesis. CEM(A)/HIV-1 T cells were incubated with cysteine/methionine - free RPMI media for 0.5 hours followed by 35 the addition of 10 mCi /ml S - cysteine/methionine with or without pac, chx, or aniso. Cell lysates and cell-free supernatants were collected 4 hours post-treatment and virions were isolated by centrifugation. Fifty nanogram samples of 35S-labeled cell lysate and virions equivalent to 100 ng Gag were subjected to radio-immunoprecipitation assay with Gag p24 antibody followed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis.

Newly synthesized RNA accumulates in virions within 2 hours.

3H-uridine labeling was performed to evaluate the time course in which newly synthesized genomic RNA becomes available for RNA packaging. CEM(A)/HIV-1 cells were incubated with 3H-uridine over a 6 hour period and cytoplasmic and virion- associated RNAs were isolated and subjected to the TCA precipitation assay. One microgram of cytoplasmic RNA and virion RNA equivalent to 30 ng Gag were analyzed.

By 2 hours post-labeling, 3H-labeled RNA was present in the cytoplasm of the mock- treated cells and was incorporated into virions (representative figure, Figure 2.5). These

44 results indicate that as early as 2 hours post-labeling, changes in cytoplasmic RNA are manifested in virions. As a negative control for nonspecific incorporation of 3H-uridine,

RNA synthesis was inhibited by treatment with actD. The actD treated samples exhibited low level 3H-uridine incorporation at each time point; 1000 CPM in cytoplasmic RNA and 100 CPM virion RNA (Figure 2.5).

Figure 2.5: Incorporation of 3H-uridine into newly synthesized virions. CEM(A)/HIV-1 3 cells were incubated for 2, 4 and 6 hours in media containing 30 mCi /ml H-uridine with or without actD [0.5 mg/ml]. 3H-uridine in cytoplasmic RNA and in virion RNA were quantified by TCA precipitation analysis. Representative results of at least three experiments are shown.

To evaluate the possibility that de novo translation is a prerequisite for generation of genomic RNA, 3H-uridine incorporation into virion RNA was evaluated with or without treatment with the pac, chx or aniso. Again treatment with actD was used to determine the value of background incorporation of 3H-uridine into virion preparations.

45 Compared to mock-treated cells, 3H-incorporation into cellular RNA of the actD-treated cells was reduced to 15%. As expected, minimal background 3H-uridine incorporation was observed into virions. The level was 8% or less at each time point (Figure 2.6). 3H- uridine incorporation into cytoplasmic RNA of pac, chx, or aniso-treated cells was 53 ±

8%, 70 ± 21%, and 52 ± 21% of the mock-treated cells, respectively. These reductions in

3H-uridine incorporation are in part attributable to turnover of short-lived proteins that facilitate the stability of steady state cellular RNA. 3H-uridine incorporation into virion

RNA displayed coincident reduction to 48 ± 8%, 30 ± 5%, and 31 ± 9% of the mock- treated control, respectively. These results indicate that inhibition of de novo translation decreases, but does not abrogate the supply of genomic RNA.

Figure 2.6: HIV-1 genomic RNA remains available for packaging during translation inhibition. CEM(A)/HIV-1 cells were incubated for 4 hours in medium containing 30 3 3 mCi /ml H-uridine with or without pac, chx, or aniso. H-uridine in cytoplasmic RNA and in virion RNA were quantified by TCA precipitation analysis. Average results of three experiments are shown. Error bars indicate standard deviations. 46 RNA packaging efficiency is sustained upon translation inhibition.

To evaluate the effect of translation inhibition on packaging efficiency of HIV-1 genomic RNA, RPAs were performed with an RNA probe complimentary to the HIV-1

5’ untranslated region. To control for variation in cytoplasmic RNA loading, cytoplasmic

RNA samples were also hybridized to a probe complimentary to cellular gapdh RNA. To monitor for possible variation in virion RNA processing, the virion samples were supplemented with a 100 base pair 32P-labeled DNA following probe hybridization and digestion and before phenol extraction and ethanol precipitation. Four independent RPAs were performed using 10 mg of cytoplasmic RNA and virion RNA equivalent to 250 ng

Gag p24. A representative RPA is shown in Figure 2.7A and the data from the 4 RPAs are summarized in Figure 2.7B. RNA packaging efficiency was calculated as the level of

HIV-1 virion RNA relative to the level of cytoplasmic HIV-1 unspliced RNA.

Consistent with the 3H-uridine results, the absolute abundance of cytoplasmic

HIV-1 unspliced RNA was reduced upon treatment with pac, chx, or aniso. The absolute amount of HIV-1 RNA in virions was also reduced. Compared to the mock-treated control, the genomic RNA packaging efficiency was 165 ± 22%, 147 ± 52%, and 97 ±

21% in response to pac, chx, and aniso, respectively (Figure 2.7B). Packaging efficiency was also increased by treatment with actD (200%, Figure 2.7B). These data indicate that genomic RNA remains available for packaging during inhibition of de novo translation and that prior translation of the HIV-1 unspliced transcripts is not necessary for generation of genomic RNA. The actD data indicate that de novo RNA synthesis and

Rev shuttling is not necessary for packaging of genomic RNA.

Figure 2.7: RNA packaging efficiency is not reduced upon inhibition of de novo translation. A) Representative RNase protection assay of cytoplasmic and virion RNA that was harvested after 4 hour incubation with or without pac, chx, or aniso. Labels indicate the sizes of the protected RNAs and control 100 base pair DNA fragment used to control for viral RNA processing (virus control), cell treatment, and undigested probes. B) Summary of four RPAs. Average results are shown and error bars indicate standard deviations. RNA packaging efficiency was determined by dividing virion RNA level by the corresponding cytoplasmic RNA level.

48 DISCUSSION

We developed a biochemical assay to examine the relationship between HIV-1 unspliced mRNA and genomic RNA. The assay uses three mechanistically distinct translation antagonists to inhibit protein synthesis in HIV-1 infected T cells under conditions that maintain virion production. Our ribosomal RNA analysis comparing

HIV-1 infected and mock-infected T cells agrees with the ribosomal profile of Agy et al.

(2) with the exception that we do not observe a significant reduction in the overall abundance of ribosomal RNA in response to HIV-1 infection. Our experiments show that uninfected and HIV-1 infected cells exhibit the expected distinct profiles in response to pac, chx, or aniso, which selectively inhibit either the initiation or elongation step of translation. These data confirm that HIV-1 infection does not disrupt the translation machinery.

Pulse-chase experiments and immunoprecipitation assays established that de novo translation is not necessary for HIV-1 particle assembly and release, and that a concentration-dependent relationship exists between cell-associated Gag and virion- associated Gag. The newly synthesized Gag can be readily assembled into virions, but steady-state Gag is also sufficient to produce virions. Immunoprecipitation results also indicate that inhibition of protein synthesis does not interfere with processing of Gag precursor protein. Examination of cytoplasmic and virion RNA by RPA and 3H-uridine labeling demonstrated that de novo translation is not required for RNA packaging of genomic RNA. The absolute level of virion RNA is reduced upon translation inhibition.

49 The magnitude of this reduction was greater when measured by the 3H-uridine labeling approach, which detects both host and viral transcripts, than by the HIV-1 specific RPA.

One possible explanation for this difference is that the 3H-labeling technique is detecting changes in packaging of host RNAs.

Treatment with the inhibitors increased the cytoplasmic availability of genomic

RNA and yielded increased packaging efficiencies indicating that HIV-1 mRNA can also be utilized as genomic RNA. This ability to increase genomic RNA availability indicates that generation of genomic RNA does not require prior utilization of the HIV-1 unspliced

RNA as mRNA template for protein synthesis. We speculate that disruption of the protein synthesis machinery reduces competition by ribosomes and the HIV-1 mRNA is rerouted to function as genomic RNA. Our data imply that HIV-1 genomic RNA and mRNA do not follow a separate intracellular RNA pathway. Instead HIV-1 unspliced

RNA constitutes a single functional pool that can function interchangeably as mRNA and as genomic RNA. Our results are similar to results with avian simple retroviruses in which Stoltzfus et al. (194) and Sonstegard and Hackett (192) concluded that a single metabolic pool of viral RNA exists that functions as both mRNA and genomic RNA.

Contrasting results in the MuLV system suggested that there are two nonequilibrating pools of MuLV RNA, each functioning as either mRNA or as genomic RNA (123). In the MuLV system, actD-treated cells produced virions without genomic RNA. In our

HIV-1 system, actD-treated cells sustain production of virions with genomic RNA and exhibit increased packaging efficiency.

Our biochemical results from HIV-1 infected human T-cells are in agreement with genetic analysis of HIV-1 based vectors (136) and indicate that translation of HIV-1

50 vector mRNA is not a rate-limiting step in production of vector virus. Our data contrast with HIV-2 experiments in which continued protein synthesis was required for packaging of genomic RNA (107). This feature of the HIV-2 system is presumed to be necessary for sorting HIV-2 genomic RNA because the RNA packaging signal is present on both the HIV-2 unspliced genomic RNA and spliced mRNA (107,137). We speculate that for

HIV-1, interaction of HIV-1 Gag protein with the RNA packaging signal modulates the competition between host translational machinery and virus assembly complexes, similar to the mechanism originally proposed from study of RSV (192).

This chapter was previously published in reference (23).

Journal of Virology 74:11531-11537.

CHAPTER 3

DEVELOPMENT OF IRE AS A SWITCH FOR TRANSLATIONAL REPRESSION IN NOVEL LENTIVECTOR RNAS

ABSTRACT

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. We have previously used metabolic inhibitors to inhibit translation in HIV-1 T-cells and observed sustained virus production accompanied by an increase in the efficiency of RNA packaging. Our long-term goal is to apply our findings to lentivector RNA and use translational repression to augment vector

RNA packaging efficiency. Increased RNA packaging efficiency has the potential to augment viral titer and provide a new strategy to optimize lentivector gene transfer for medical applications.

Here we seek to develop a unique lentivirus-based vector that contains the iron response element (IRE). We hypothesize that interaction between IRE and IRP in lentivector RNA will be a productive strategy to target translational repression specifically to vector RNA in helper cells. As a negative control for iron responsiveness and unexpected effects of IRE sequence insertion on vector replication, IRE SIN was compared to a derivative, DC IRE SIN. DC IRE SIN contains a deletion of a single critical cytidine residue located in the loop of IRE. 293 or HeLa cells transfected with

52 IRE SIN or DC IRE SIN were treated with the iron chelator deferoxamine mesylate (def) or the iron-rich hemin. RNase protection assay (RPA) of lentivector RNA reveals that the introduction of IRE or DC IRE does not reduce RNA packaging efficiency compared to

HIV SIN. Radioimmunoprecipitation assay indicates that treatment with def or hemin does not preclude Gag protein processing, particle assembly or release. Luciferase reporter assay determined that Luc translation of IRE SIN, but not DC IRE SIN, was reduced to 60% following def treatment. As expected, hemin increased Luc activity of

IRE SIN, but not DC IRE SIN, to 150%. These data indicate that addition of def and hemin modulates translational repression of IRE-containing lentivector RNAs. Further, the data reveals that introduction of IRE is an effective and novel approach to target translational repression of retroviral vector RNA and will be used to investigate the effect of targeted translational repression on lentivector RNA packaging efficiency.

INTRODUCTION

Previously, we used metabolic inhibitors (cycloheximide [chx], pactamycin, and anisomycin) to inhibit translation of primary HIV-1 RNA and demonstrated that translation repression in HIV-1 infected T-cells increases the efficiency of genomic RNA packaging (23). We predict that translational repression will likewise augment lentivector packaging efficiency. An increase in lentivector RNA packaging efficiency is a potential method to augment vector virus titer by converting particles that lack or contain defective genomic RNA to dimmer-containing infectious virions (111). Analysis of MLV and

HIV-based vectors using electron microscopy and a functional transduction assay has revealed approximately 100-350 defective particles per infectious virion (93). An 53 inverse relationship between translation and RNA packaging efficiency implies that targeted repression of vector RNA translation will increase the vector RNA available for packaging into vector virus and, subsequently augment vector virus titer. To increase lentivector RNA packaging efficiency, we have proposed to specifically repress translation with novel IRE-containing lentivector RNAs. Here, we test the hypothesis that introduction of IRE at the 5’ terminus of lentivector RNA will provide a potent switch for translational repression without disruption of helper virus replication.

IRE facilitates translation of ferritin and heterologous mRNAs by interaction with

IRE binding protein (IRP) (145). IRP competitively binds iron. In iron poor cells, binding of IRP to IRE prevents recruitment of 43S translation preinitiation complexes to the RNA and represses protein synthesis (77,144,157). In iron-rich cells, IRP is sequestered from IRE and translation is derepressed. The advantage of IRE-regulated translation over inhibition by metabolic inhibitors is the ability to specifically target translational repression to the vector RNA and eliminate secondary effects on cell metabolism. Iron responsiveness is determined by treating transiently transfected cells with the iron chelator deferoxamine mesylate (def) or the iron source, hemin and determining Luc activity. The hemin to def (H/D) ratio indicates the range of iron regulation that is observed. Previous studies commonly examined the range of iron regulation of heterologous RNAs in murine fibroblast cells.

54 Chloramphenicol acetyltransferase (CAT) assay of IRE-containing CAT reporter RNA and radioimmunoassay of human growth hormone (hGH) reporter RNA in murine fibroblasts exhibit H/D ratios of 6.4 and 11.5, respectively (90). Since HIV Rev does not efficiently function in murine cells and is required for lentivector RNA export, we employed permissive 293 kidney cells and HeLa cells for our experiments (216).

RNase protection assay (RPA) of cytoplasmic and vector virus RNA isolated from 293 cells transiently transfected with IRE SIN or DC IRE SIN reveals that the introduction of IRE or DC IRE at the 5' RNA terminus does not reduce RNA packaging efficiency compared to HIV SIN. Results of radioimmunoprecipitation assays and

Luciferase reporter assays indicate that treatment with def or hemin does not preclude

Gag protein processing, particle assembly or release and that modulation of translation is specific to lentivectors that contain a functional IRE. These results establish that introduction of IRE to lentivector RNA is an effective approach to target lentivector translational repression.

MATERIAL AND METHODS

Plasmid Construction

IRE SIN and DC IRE SIN were constructed in two phases. First, IRE sequences were amplified by polymerase chain reaction (PCR) from pCATIRE (29,174) with primers containing HindIII sites. The HindIII fragment was inserted into the HindIII site of pGL3 (Promega, Madison, WI) to produce pGL3IRE. To produce pMBIREHIVLuc, pGL3IRE was digested with NcoI and a PCR fragment containing HIV-1NL4-3 5' RU5 and

55 UTR sequences (492-793) with NcoI ends was inserted. A linker containing a central

PvuII site was introduced into the XbaI site of pMBIREHIVLuc to make pMBIREHIVLuc+. HIV RRE was amplified by PCR and inserted into pPCRScriptCam, digested with PvuII and inserted into the linker PvuII site of pMBIREHIVLuc+ to make pMBIREHIVLucRRE.

The second phase of construction of IRE SIN involved the modification of parental HIV SIN to produce SNV/HIV SIN. To make the hybrid SNV/HIV SIN, HIVBru sequences from +1 to 1586 were amplified by PCR with primers containing ClaI and

ApaI sites and subcloned into pPCRScriptCam to make pSH500. pKB504 (25) was digested with HindIII and AflIII and a linker containing HindIII, NotI, NcoI and AflIII sites was inserted to produce pSH501. A ClaI site immediately 3’ of the 5' U3 region of pSH501 was introduced by PCR-based site directed mutagenesis to produce pSH502. pSH502 was digested with ClaI and EcoRI and HIVBru 5' RU5, UTR and gag-containing

ClaI/EcoRI fragment of pSH500 was inserted to produce pSH503, which contains a single 5' SNV/HIV hybrid LTR. The new hybrid 5’ LTR was amplified by PCR with primers containing NotI and NcoI sites and inserted into the NotI/NcoI sites of pSH503 to produce pSH504. HIV SIN (145,213) sequences which include the HIV RRE, CMV promoter and gfp gene (1066 to 3389) were amplified by PCR and subcloned into pPCRScriptCam to produce pSH701. pSH504 was digested with SalI and SmaI and the

SalI/SmaI fragment (containing the HIV RRE, CMV promoter and gfp gene) of pSH701 was inserted to produce pSH702. To replace the intact 3’LTR with a SIN LTR, the 3’

LTR of HIV SIN was amplified by PCR and subcloned into pPCRScriptCam to produce pSH705. pSH702 was digested with SalI and AflIII and the SalI/AflIII fragment of

56 pSH705 was inserted to produce SNV/HIV SIN. A linker removing the original ClaI near U3 site and introducing another ClaI site 17 nts downstream was inserted into the

ClaI site of SNV/HIV SIN to produce SNV/HIV SIN+. SNV/HIV SIN+ was digested with ClaI and the backbone gel-eluted. PCR-based site directed mutagenesis was used to add a ClaI site to pMBIREHIVLucRRE upstream of the IRE to produce pMBIREHIVRREClaI. pMBIREHIVRREClaI was digested with ClaI and the fragment ligated into the gel-eluted backbone of SNV/HIV SIN+ to make IRE SIN. PCR-based site directed mutagenesis to remove a single cytidine residue of the IRE SIN yielded DC

IRE SIN. All plasmids and lentivectors were verified by restriction digestion and sequence analysis.

Cells and drug treatment

293 cells or HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Gibco-BRL). 293 cells were transfected by a CaCl2 protocol (110). To transfect HeLa cells, the cells were plated at

60% confluency and incubated for 6 hrs with a 3:1 ratio of Fugene 6 to plasmid DNA

(Roche Biochemicals) in DMEM medium supplemented with 2% fetal bovine serum.

After 6 hrs, the media was removed and replaced with DMEM medium supplemented with 10% fetal bovine serum. Cells were co-transfected with 7 mg of IRE SIN or DC IRE

SIN plasmid, 3 mg pCMV259D21 helper plasmid (136) and 0.5 mg pRL-CMV for transfection efficiency. Transfected 293 cells or HeLa cells were treated with def (100 mM in water) (Sigma) or hemin (100 mM in 1.4M NH4OH) (Sigma) 24 hrs following transfection and incubated at 37°C for 16 hrs. After 16 hrs, fresh media containing def or hemin was added and the cells were incubated at 37°C for another 6 hrs. The cells were 57 harvested in phosphate buffered saline (PBS), centrifuged at 2,000 × g for 3 minutes, and resuspended in 0.1 ml or 0.05 ml of ice-cold lysis buffer (20mM Tris-HCl, pH 7.4,

150mM NaCl, 2mM EDTA, 1% NP40). Particle abundance was quantified by Gag

ELISA (Coulter Corp., Miami, FL). Luc activity (relative light units [RLU]) was normalized for transfection efficiency by Renilla Luc activity. Luc assays were performed with 10 ml lysate and 100 ml Luciferase Substrate (Promega, Madison, WI) and quantified in a Lumicount Luminometer (Packard, Meriden, CT). Dual measurement of Luciferase expressed from firefly (Photinus pyralis) luc and sea pansy (Renilla reniformis) ren was performed by the Dual-Luciferaseâ Reporter Assay System

(Promega, Madison, WI).

RNA analysis

Cytoplasmic extracts were prepared in 0.9 ml cold cell lysis buffer (10 mM Tris pH 8.3, 150 mM NaCl, 1.5 mM MgCl2) and 0.1 ml 5% NP40. Following centrifugation to pellet the nuclei, the supernatant was mixed with TriReagent LS and cytoplasmic RNA was isolated by the manufacturer’s protocol (Molecular Research, Cincinnati, OH). Each cytoplasmic RNA sample was treated with DNase (Gibco-BRL, Rockville, MD), twice, extracted with phenol:chloroform and chloroform:isoamyl, and the RNA was precipitated with ethanol. To prepare virion RNA, cell medium was clarified by centrifugation at

2000 × g for 10 min, virions were pelleted by centrifugation at 156,000 × g for 2.5 hours at 4o C in a Beckman SW41 rotor, and lysed in 1 ml Trizol ReagentTM and isolated by the manufacturer’s protocol (Gibco BRL, Gaithersburg, MD).

32P-labeled antisense RNA probes were generated by in vitro transcription of pGEM(400-600) (25) which contains a portion of the HIVNL4-3 5’ UTR, and pGAPDH,

58 which contains human glyceraldehyde dehydrogenase (gapdh) gene (25). Following digestion of pGEM(400-600) with NotI and pGAPDH with NcoI, antisense run-off RNA transcripts were synthesized with MAXIscriptTM T7 RNA polymerase (Ambion, Austin,

TX) and the probes were isolated by gel elution. RPA was performed using RPA III

(Ambion) according to the instruction manual with some modifications. Typically, 20 mg of cytoplasmic RNA or viral RNA from virions equivalent to 200 ng of Gag was precipitated by ethanol in the presence of 10 mg of yeast RNA with 2 × 105 CPM Luc probe and 2 × 104 CPM gapdh probe. Samples were resuspended in 10 ml of hybridization buffer, denatured at 90o C for 3 min, and hybridized at 42o C overnight.

RNAse A/T1 was diluted 1:100 in Ambion RNAse digestion buffer and 150 ml was added to each sample and incubated at 37o C for 30 minutes. SDS and proteinase K were added to final concentrations of 1% and 0.5 mg/ml, respectively, and samples were incubated at 37o C for 30 minutes. Samples were phenol-chloroform and chloroform extracted and precipitated with ethanol. Following centrifugation, the pellets were dissolved in 6 ml of loading buffer, denatured at 94o C for 3 min, and subjected to 5% denaturing polyacrylamide gel electrophoresis. RNase protection products were visualized and quantified by PhosphorImager analysis.

Radioimmunoprecipitation assay

Transfected cells were lysed in RIPA buffer (0.05M Tris-HCL, pH 8, 0.1% SDS,

1% Triton-X-100, 2mM PMSF, 0.15M NaCl, 2mM) containing 1% deoxycholic acid and the nuclei were removed after centrifugation at 13,400 ´ g for 10 minutes. Total protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad Laboratories,

Hercules, CA). Virion-containing medium was clarified by centrifugation at 2000 × g for 59 3 minutes and virions were collected by centrifugation at 156,000 × g for 1.5 hours at 4o

C in a Beckman SW41 rotor. Gag ELISA was performed by the manufacturer’s protocol

(Beckman-Coulter, Brea, CA). 35S-labeling experiments were performed by incubating the transfected cells in cysteine/methionine - free RPMI media with 5% dialyzed fetal

35 bovine serum and 10 mCi /ml S - cysteine/methionine (1175 Ci/mmol, 43.5 MBq/ml)

(ICN Biohemicals, Irvine, CA) for 30 minutes. The cells were lysed in RIPA buffer containing 1% deoxycholic acid. Fifty nanograms of cytoplasmic lysate and virion lysates equivalent to 30 ng of Gag were immunoprecipitated. The 35S-labeled lysates were incubated for 16 hours with protein A Sepharose beads (Pharmacia) and polyclonal rabbit sera against HIV Gag (gift of A. Panganiban). The beads were washed once in high salt RIPA (1M NaCl) and once in low salt RIPA (0.15M NaCl), and boiled for 3 minutes to elute the proteins. The 35S-labeled precipitated proteins were subjected to

SDS-PAGE, visualized and quantified by PhosphorImager analysis (Molecular

Dynamics, Inc., Sunnyvale, CA) with ImageQuaNT Software version 4.2 (Molecular

Dynamics).

RESULTS

IRE SIN development

To specifically target translational repression, we have developed and characterized a unique lentivirus-based vector, IRE SIN, which contains the ferritin iron response element (IRE) (Figure 3.1). IRE translational repression requires that the IRE be located less than 67 nucleotides from the RNA start site (68,75). Using the cytidine residue in the IRE bulge as the reference point, the IRE was introduced 28 nucleotides

60 downstream of the transcription start site of a hybrid spleen necrosis virus (SNV)/HIV-1

SIN (Figure 3.2) (75). The hybrid SNV/HIV SIN is Tat-independent and contains a self- inactivating deletion in the 3' HIV U3 region that, after reverse transcription, is copied into the 5’ LTR and inactivates the 5’ LTR-driven transcription (17). Inactivation of the

5’ LTR in transduced cells reduces the possibility of generating a replication-competent virus through recombination in vivo and, when used in conjunction with an internal promoter, prevents promoter interference (70). The novel IRE SIN contains a luciferase

(luc) reporter gene expressed from Rev-responsive vector genomic RNA and Luc expression indicates the level of translational repression. A gfp transgene is expressed from an internal cytomegalovirus immediate early (CMV IE) promoter and is used to indicate transduction efficiency of the lentivector RNA (Figure 3.1). Controls include the functionally inactive DC IRE SIN which contains a single deletion of the cytidine residue in the loop of IRE (Figure 3.2) (174) and the well-characterized parental HIV SIN

(Figure 3.1).

Y HIV SIN HIV U3 R U5 gag RRE CMV gfp SIN R U5 5’ ss 3’ ss

IRE

Y IRE SIN SNV U3 R U5 luc RRE CMV gfp SIN R U5 5’ ss 3’ ss DCIRE

Y DCIRE SIN SNV U3 R U5 luc RRE CMV gfp SIN R U5 5’ ss 3’ ss

Figure 3.1: Genomic structures of lentivector DNA. Parental HIV SIN, IRE SIN and DCIRE SIN are shown. IRE SIN and DCIRE SIN are derivatives of HIV SIN but contain the 5’ SNV U3 region, R, U5 and UTR sequences from HIVNL4-3 and the luciferase gene. IRE SIN contains the ferritin iron response element (IRE) at +28 (with the C residue in the IRE bulge as reference), and DC IRE SIN contains a deletion of the C residue at +34 of the IRE loop. HIV and SNV U3 regions are labeled. Y, HIV packaging signal, 5’ ss and 3’ ss, 5’ and 3’ splice sites, respectively; RRE, Rev responsive element; luc, luciferase gene; CMV, cytomegalovirus immediate early promoter; gfp, green fluorescent protein gene.

TATA Box +1 RNA start TATATAAGCC GGGTACATCG CTTGCTC GGG GTCATCGATG +28 GTACCCGGGG ATCCTGCTT C AACAGTGCTT GGACGGATCC

TCTAGAGTAA GCTTGGCATT CCGGTACTGT TGGTAAAGCC HIV R ACCATGGACT GGGTCTCTCT GGTTAGACCA GATCTGAGCC

TGGGAGCTCT CTGGCTAACT AGGGAACCCA CTGCTTAAGC

CTCAATAAAG CTTGCCTTGA GTGCTCAAAG TAGTGTGTGC

CCGTCTGTTG TGTGACTCTG GTAACTAGAG ATCCCTCAGA

CCCTTTTAGT CAGTGTGGAA AATCTCTAGC AGTGGCGCCC

GAACAGGGAC TTGAAAGCGA AAGTAAAGCC AGAGGAGATC

TCTCGACGCA GGACTCGGCT TGCTGAAGCG CGCACGGCAA

GAGGCGAGGG GCGGCGACTG GTGAGTACGC CAAAAATTTT

GACTAGCGGA GGCTAGAAGG AGAACCATGG GAAGACGCCA

luciferase start site®

Figure 3.2: Partial nucleotide sequence of IRE SIN. The proposed transcription start site is numbered +1 and marked by an arrow. Boxed sequences indicate the IRE. The gray- circled “C” residue, +28, serves as a reference point for distance from the transcription start site. The clear-circled “C” residue, +34, is the residue shown to be critical for IRE function and is deleted in DIRE SIN. The TATA box, the start of HIV transcription and the luciferase translation start site are indicated as labeled.

IRE does not reduce lentivector RNA packaging efficiency

To evaluate the possibility that introduction of IRE disrupts the vector RNA packaging signal, 293 cells were cotransfected with IRE SIN, DC IRE SIN or HIV SIN plasmids (10 mg) and HIV helper virus expressed from pCMV259D21 (3 mg). Twenty- four hrs later, cytoplasmic and vector virus was harvested, and RNA isolated for RPA.

Briefly, fifteen micrograms of cytoplasmic RNA and RNA from equivalent helper virions

(250,000 pg of Gag p24 measured by ELISA) were subjected to the RPA using an RNA probe complementary to the HIV-1 5’ untranslated region (Figure 3.3A). To control for variation in cytoplasmic RNA loading, cytoplasmic RNA samples were also hybridized to a probe complementary to cellular gapdh RNA. Duplicate RPAs are shown in Figure

3.3B and the quantified data are in Figure 3.3C. RNA packaging efficiency is expressed as the ratio of virion-associated to cytoplasmic genomic RNA in equivalent virions. The data reveal that the absolute abundance of cytoplasmic IRE SIN RNA was reduced following introduction of IRE. The reduction in cytoplasmic RNA may be attributable to interference of the IRE with host transcription machinery or RNA stability. Nevertheless, the absolute amount of lentivector RNA packaged in vector virus was not diminished by addition of the IRE or the DC IRE stem loop. In fact, the IRE SIN lentivector packaging efficiencies were 2.5 and 1.6 relative to the HIV SIN control, (Figure 3.3C). Similarly, the packaging efficiencies of DC IRE SIN were 1.5 and 1.8 (Figure 3.3C). These data indicate that introduction of IRE to the HIV SIN does not disrupt the HIV RNA packaging signal and therefore does not preclude lentivector RNA packaging.

64 A. r u5 5’ ss gag HIV SIN RNA

antisense RNA probe 344 nt unspliced gag RNA 303 nt

spliced RNA 146 nt

IRE luc r u5 5’ ss IRE SIN RNA

antisense RNA probe 344 nt

unspliced gag RNA 180 nt

spliced RNA 146 nt

continued

Figure 3.3: IRE does not reduce RNA packaging efficiency. A) Regions of complementarity between the SINs and the antisense HIV RNA probe, and the protected unspliced and spliced transcripts. HIV U5 and 5’ untranslated region (narrow line) gag, and luciferase (luc), r, u5 (white boxes), 5’ splice site (5’ ss) and IRE are indicated. B) RNase protection assays. 293 cells were transiently transfected by CaCl2 and 2 days post- transfection cytoplasmic and virion RNA was harvested and subjected to DNase treatment. Twenty mg aliquots of cytoplasmic RNA or helper virions equivalent to 250,000 pg Gag p24 (as measured by ELISA) were subjected to RPA with uniformly- labeled antisense HIV RNA and gapdh RNA probes, PAGE, and PhosphorImager analysis. Duplicate samples of each RNA are shown. The protected RNAs are labeled. C) Two replicate experiments are shown. Values are shown relative to the first HIV SIN RNA sample (rep 1). Packaging efficiency was determined by dividing virion RNA level by the corresponding cytoplasmic RNA level. Rep, replicate.

65 Figure 3.3 continued

Cytoplasmic RNA

IRE SIN DC IRE SIN HIV SIN replicate experiments marker GAPDH probe HIV probe 1 2 1 2 1 2

gapdh RNA

HIV SIN unspliced RNA

IRE & DC IRE SIN unspliced RNA

IRE, DC IRE & HIV SIN spliced RNA

Vector virus RNA

IRE SIN DC IRE SIN HIV SIN replicate experiments 1 2 1 2 1 2

HIV SIN unspliced RNA

IRE & DC IRE SIN unspliced RNA

IRE & HIV SINs spliced RNA

continued

Figure 3.3 continued

3.0 Cytoplasmic RNA

2.5 Vector virus RNA Packaging Efficiency 2.0

1.5

1.0

0.5

Level Relative to HIV SIN Rep 1 0.0

rep 1 rep 2 rep 1 rep 2 rep 1 rep 2

HIV SIN IRE SIN DC IRE SIN

Def or hemin treatment does not disrupt Gag helper protein synthesis or processing

To evaluate the effect of def and hemin on helper virus Gag protein synthesis and processing, 293 cells were cotransfected with IRE SIN (10 mg) and the helper plasmid pCMV259D21 (3 mg) (136). 24 hrs later, the cells were treated with def or hemin. Sixteen hrs later, the cells were washed and fresh def or hemin-containing media supplemented with 35S-cysteine/methionine was added. 6-hrs post def or hemin treatment, total cell protein and vector virus was harvested. 75 ng of cytoplasmic lysate and virion lysate from equivalent helper virions (50 ng of Gag p24 measured by ELISA) were subjected to

67 RIPA using polyclonal rabbit sera against HIV Gag. RIPA of cytoplasmic 35S-labeled

Gag p24 from def-treated and hemin-treated cells (98% and 65% of the untreated control, respectively) indicate that treatment with def or hemin does not abrogate accumulation of

35S-labeled Gag (Figure 3.4). Similarly, extracellular 35S-labeled Gag p24 levels were

125% and 72% compared to the untreated control, following def or hemin treatment, respectively. These data and the observation that vector virus contains fully processed

Gag p24 indicates that treatment does not significantly alter release of Gag from the helper cells or inhibit Gag protein processing (Figure 3.4). Consistent with the RIPA results, ELISA reveal comparable levels of Gag from the untreated IRE SIN transfected cells (2.5 ´ 105 pg/ml), and def or hemin-treated cells (2.7 ´ 105 pg/ml and 2.6 ´ 105 pg/ml, respectively). These data indicate that the neither def nor hemin prevent the synthesis, release or processing of vector virus from helper cells.

Cytoplasmic Gag Vector virus Gag

def hemin def hemin mock mock

p55

p24

Figure 3.4: IRE does not preclude Gag processing. 35S-labeled total cell proteins from 293 cells cotransfected with IRE SIN and pCMV259D21 were immunoprecipitated with polyclonal rabbit sera against HIV Gag and visualized by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. The sizes of Gag p55 and Gag p24 are indicated based on non-isotopic molecular weight markers.

Def specifically inhibits translation of the IRE SIN

To quantify the effects of the def and hemin on lentivector RNA translation, Luc activity of triplicate lysates from transfected HeLa was determined. Following def- treatment, Luc activity from IRE SIN is reduced to 60% of the untreated control.

Conversely, hemin treatment increased Luc activity to 150% (Figure 3.5). As expected,

Luc activity from DC IRE SIN was unaltered by addition of either def or hemin (Figure

3.5). These data indicate that translation of the IRE SIN lentivector RNA can be regulated by iron. Further, the H/D ratios of the SINs are 2.4 and 0.9, respectively.

69 These data reveal that addition of a functional IRE is necessary for regulation of translational repression. Further, the results establish that introduction of IRE to lentivector RNA confers iron responsiveness to lentivector RNA.

200% mock

160% def hemin 120%

80%

40% Percent Luc Activity

DC IRE SIN IRE SIN

Figure 3.5: Translational repression of lentivector RNA requires a functional IRE. HeLa cells were co-transfected with 10 mg lentivector DNA, 3 mg pCMV259D21 and 1 mg pCMV-RL by Fugene 6 protocol. Following 24 hrs, cells were incubated in deferoxamine (100 mM), or hemin (100 mM)-containing medium. After 22 hrs, cytoplasmic lysates were harvested and dual Luceriferase assay performed. Luc levels are normalized to Renilla Luc for transfection efficiency. Data is shown as Luciferase activity relative to mock/untreated. Assays were performed in triplicate and average results are shown. Error bars indicate standard deviations.

DISCUSSION

We have developed a novel lentivirus-based vector that contains IRE at its 5' terminus (Figure 3.1) 28 nucleotides from the RNA start site (Figures 3.2). RPA indicates that addition of the IRE does not preclude RNA packaging (Figure 3.3). RIPA reveals

70 that neither def nor hemin treatment interferes with processing of Gag structural protein, assembly or release of progeny vector virions (Figures 3.4). Luc activity of IRE SIN compared to DC IRE SIN following def or hemin treatment indicates that a functional

IRE is necessary to confer iron-regulated translational repression on lentivector RNA

(Figure 3.5). Further, the range of iron regulation or H/D ratio of IRE SIN is 2.5-fold greater than that of DC IRE SIN.

The range of iron regulation that is observed for the IRE SIN, however, is lower than reported ratios for IRE-containing chloramphenicol acetyltransferase (CAT) and human growth hormone (hGH) reporter RNAs, which were 6.4 and 11.5, respectively

(90). Our observed H/D ratio may be attributable to cell type specific differences in IRE function. Previous studies examining the effect of iron regulation on heterologous reporter RNAs were performed in permanently transfected B6 murine fibroblast cells.

Since HIV Rev does not efficiently function in murine cells (203,216) but is necessary for HIV vector propagation, we employed 293 kidney cells and HeLa cells for our experiments. Earlier experiments with transfected HeLa cells have shown that a fraction of IRE activity is observed compared to results with murine fibroblasts. Specifically, only one-fourth to one-fifth of the 20-30-fold iron-dependent change in ferritin synthesis is due to presence of IRE (75). These data imply that various cells use diverse mechanisms to regulate translation of IRE-containing RNA (34). Other studies have reported H/D ratios of HeLa cells transfected with hGH reporter plasmids was between

6.0 – 6.5 when the IRE was placed 17 to 44 nts from the RNA start site (75). Our inability to observe the larger range of iron regulation attained in these HeLa studies may be a result of the context of the sequences that are adjacent to the IRE. Previous reports

71 indicate that the regions flanking the ferritin IRE influence translational repression (45).

Flanking regions may affect binding of IRP and/or binding of translation initiation factors or ribosome scanning (34). The highly structured HIV 5' UTR has been shown to inhibit translation (67,142,158). The juxtaposition of IRE to HIV 5' UTR sequences in our IRE

SIN may interfere with IRP binding to IRE and diminish the ability of def to modulate translational repression.

Future studies will test IRE SIN in human rhabdomyosarcoma cells, which have been demonstrated to allow both IRP and Rev regulation (216), and examine the effect of alternate flanking sequences on responsiveness of our IRE-containing lentivector RNA.

Ultimately, we will utilize an optimized IRE SIN to test the hypothesis that translational repression increases lentivector packaging efficiency. We predict that an increase in lentivector RNA packaging will augment viral titer by reducing the accumulation of defective interfering particles. Additionally, we will compare translational repression of

IRE SIN by def to translational repression by the metabolic inhibitor, chx to examine the effect of targeted translational repression compared to unilateral translational repression on lentivector RNA packaging efficiency.

CHAPTER 4

THE 5’ RNA TERMINUS OF SPLEEN NECROSIS VIRUS CONTAINS A NOVEL POST-TRANSCRIPTIONAL CONTROL ELEMENT THAT FACILITATES HUMAN IMMUNODEFICIENCY VIRUS REV/RRE- INDEPENDENT GAG PRODUCTION

ABSTRACT

Previous work has shown that spleen necrosis virus (SNV) long terminal repeats

(LTRs) are associated with Rex/Rex-responsive element-independent expression of bovine leukemia virus RNA and supports the hypothesis that SNV RNA contains a cis- acting element that interacts with cellular Rex-like proteins. To test this hypothesis, the human immunodeficiency virus type 1 (HIV) Rev/RRE-dependent gag gene was used as a reporter to analyze various SNV sequences. Gag enzyme-linked immunosorbent assay and Western blot analyses reveal that HIV Gag production is enhanced at least 20,000- fold by the 5' SNV LTR in COS, D17, and 293 cells. Furthermore, SNV RU5 in the sense but not the antisense orientation is sufficient to confer Rev/RRE-independent expression onto a cytomegalovirus-gag plasmid. In contrast, the SNV 3' LTR and 3' untranslated sequence between env and the LTR did not support Rev-independent gag expression.

Quantitative RNase protection assays indicate that the SNV 5' RNA terminus enhances cytoplasmic accumulation and polysome association of HIV unspliced and spliced transcripts.

73 However, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for the significant increase in Gag production by the SNV sequences. Our analysis reveals that the SNV 5' RNA terminus contains a unique cis-acting posttranscriptional control element that interacts with hypothetical cellular Rev-like proteins to facilitate HIV RNA transport and efficient translation

INTRODUCTION

Retroviruses require cytoplasmic expression of unspliced RNA to produce infectious progeny. Complex retroviruses including human immunodeficiency virus

(HIV) and bovine leukemia virus (BLV) exert similar post-transcriptional control by their regulatory protein and cis-acting responsive element, Rev/Rex and RRE/RxRE, respectively. Rev/RRE are necessary for efficient transport, stability, and translation of unspliced HIV RNAs (1,4,9,37,38,54,58,86,87,120,129,130,183). Simple retroviruses lack analogous regulatory protein. However, recent studies have dentifiedi cis-acting elements in some simple retroviruses that function in conjunction with Rev-like cellular factor(s) to modulate cytoplasmic expression of their unspliced RNA

(19,83,149,188,197,199,217). These elements are designated constitutive/cytoplasmic transport elements or cis-acting trans-activation elements (CTEs), and have been identified in Mason-Pfizer monkey virus (MPMV) (19), the related simian retrovirus-1

(SRV-1) (217), and the avian Rous sarcoma virus (RSV) (149,188). The CTEs are structured RNA elements positioned in the 3’ untranslated region (3’ UTR) (55,172,197) and were identified by their ability to replace HIV Rev/RRE function in subgenomic HIV

74 plasmids (19,149,217). They function to increase stability and nucleocytoplasmic transport of unspliced transcripts. The RSV CTE is proposed also to facilitate efficient processing of Gag precursor protein (188).

Recent characterization of BLV retroviral vector genomes that contain spleen necrosis virus (SNV) long terminal repeats (LTRs) revealed Rex/RxRE-independent expression of BLV structural gene vectors (15,18). This observation indicates that SNV

RNA may contain a cis-acting element that interacts with cellular Rex-like factors. SNV is an avian simple retrovirus that is unrelated to MPMV/SRV-1 or RSV, and is instead related to murine leukemia virus (210). The goal of this study was to test the hypothesis that SNV RNA contains a CTE that would interact with cellular Rev-like factors. Our analysis focused on two SNV regions: the 3’ UTR that corresponds to the position of the

MPMV, SRV-1, and RSV CTEs; and the LTRs, because BLV structural gene vectors that contain the SNV LTRs are Rex/RxRE-independent. Our data eliminate the possibility that the SNV 3’ UTR and 3’ LTR facilitate Rev-independent gene expression, and establish that the SNV 5’ LTR functions in a position-dependent manner to facilitate

Rev/RRE-independent expression of HIV gag RNA. The SNV 5’ LTR facilitates cytoplasmic accumulation and polysome association of HIV unspliced and spliced RNAs.

These data identify a novel retrovirus post-transcriptional control element located at the 5’ terminus of a simple retrovirus RNA that facilitates Rev/RRE-independent expression of unspliced and spliced HIV RNAs.

75 MATERIALS AND METHODS

Plasmid construction

HIV-based plasmids pSVgagpol-rre, pSVgagpol, pBBgagpol encode HIV Gag and either contain RRE, lack RRE but contain b–globin intron, or lack RRE and b–globin intron, respectively (190). To construct derivatives of these plasmids, the SNV 3’UTR was excised from pKB477 on BamHI/BglII fragment and subcloned into the BamHI site of pSVgagpol-rre, pSVgagpol, or pBBgagpol to construct pKB634, pKB636, or pKB637, respectively. The SNV LTR was excised from pKB404 on a BamHI fragment and subcloned into the BamHI site of pSVgagpol-rre, pSVgagpol, or pBBgagpol to construct pKB624, pKB628, or pKB632, respectively.

pKB504gagpol was constructed in 5 steps beginning with pKB404, which contains 2 copies of the SNV LTR ligated at opposite ends of the multiple cloning site in pUC19 (18). pKB404 modified by insertion of the HIV polypurine tract (ppt) (HIVBRU coordinates 8662-8699 (208)) on an oligonucleotide at the SphI/HindIII sites adjacent to the 3’ SNV LTR to create pKB504. HIVBRU sequences from U5 through gag (100 to

2040) were amplified by 8 cycles of PCR by Taq polymerase with primers having

EcoRI/XbaI termini, and ligated into pUC19. Subsequently, the EcoRI fragment that encompasses HIV sequences 100-2040 was subcloned at the EcoRI site of pKB504. HIV sequences 1521 to 4655 (HIV gag-pol) were amplified with primers having XbaI termini, ligated into pUC19, and then subcloned into the preceding plasmid at ApaI (1552) and

XbaI (4655) sites to construct pKB504gagpol.

76 MPMV CTE was PCR amplified from MPMV provirus pSHRM-15 (coordinates 8022-

8193, gift of Eric Hunter (197)) with primers having XhoI termini. MPMV CTE PCR product was inserted into the SalI site of pKB504gagpol to create pKB504gagpolCTE.

To construct pYW100, the region between HIV ppt and the 3’ SNV LTR of pKB504gagpol was replaced with a heterologous p(A). Briefly, pKB504gagpol was digested with AflIII, treated with Klenow, and digested with XbaI. pCMVglobinSPA

(gift of Dan Schoenberg), which contains an optimized 47 base synthetic p(A), was digested with HindIII, Klenow treated, digested with XbaI, and the fragment containing p(A) was ligated with the vector backbone to make pYW100. An intermediate plasmid, pYW201, was constructed by ligation into pUC19 of the BamHI fragment of pKB504gagpol that contains the sequence from HIV U5 through the 3’ SNV LTR. Then the CMV IE promoter of pCMVglobinSPA was excised using SalI, treated with Klenow, and ligated at the SmaI site to make pYW202. To construct pYW203, a deleted SNV

LTR that lacks the 3’ 29 bases of R and 60 bases of U5 (D486-575) was amplified by

PCR, treated with Klenow, and ligated at the SmaI site of pYW201. To construct pYW209, a deleted SNV LTR that lacks the U5 (D512-575) was amplified by PCR, treated with Klenow, and ligated at the SmaI site of pYW201. To construct pYW99, the

SphI fragment of pYW202 that contains the CMV promoter and 5’ gag gene were ligated to the SphI fragment of pYW100 that contains the 3’ region of gag and p(A). pYW204 was constructed by ligation of SphI fragments of pYW203 and pYW100. pYW205 was constructed by deletion of RU5 sequences starting at +2 (D436-575) by AvaI/BamHI digestion followed by Klenow treatment and blunt end ligation. To construct pYW207 and pYW208, SNV RU5 was excised with AvaI from pKB402 (sequences SNV 435-599

77 and pUC19 396-412), Klenow treated and ligated at the Klenow-treated BamHI site of pYW99. The plasmid with the antisense RU5 orientation is pYW207, and the plasmid with the sense orientation is pYW208. The 5’ transcriptional control region between gag and the 3’ UTR through the LTR or heterologous p(A) sequence of each plasmid was verified by DNA sequencing. pGEM(140-440) was derived from pGEM(400-600) of

McBride and Panganiban (134) by replacement of the HIVNL4-3 5’ UTR with the HIVBRU

5’UTR. Plasmid pMBSVT7 was constructed by PCR amplification of the 5’ UTR regions of pSVgagpolrre and ligation into the SrfI site of pPCRScriptCam (Stratagene).

ELISA and western blot

3 ´ 105 293 cells were cotransfected with 2 µg of test plasmid and 0.2 µg of pEGFPN1 (Clonetech) or pGL3 (Promega) reporter plasmid by the calcium phosphate protocol and maintained in DMEM with 10% fetal calf serum. Three days post- transfection cell-free media and total cell protein was harvested in NP40 lysis buffer

(20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA, 1% NP40) and the nuclei removed by centrifugation at 13,400 ´ g for 10 minutes. Gag levels were quantified by Gag ELISA from cell free media and normalized to transfection efficiency according to the manufacturer’s protocol (Beckman-Coulter, Brea, CA). For western blot immunoassay, total cell proteins were harvested at 3 days post-transfection and total protein concentration determined by Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules,

CA). Equivalent total cell protein was separated by SDS-PAGE, transferred to nitrocellulose, and reacted with polyclonal rabbit sera against HIV Gag (gift from

Antonito Panganiban, University of Wisconsin). HIV Gag proteins were detected by enhanced chemiluminescence (ECL kit; Amersham).

78 RNA preparation

Total, nuclear or cytoplasmic RNA was prepared with Tri-ReagentTM or Tri-

ReagentTM LS, respectively, by the manufacturer’s instructions (Molecular Dynamics,

Inc.). Transfected COS or 293 cells from two 10 cm plates or T150 flasks were harvested into PBS, centrifuged at 2000 × g for 5 min, and resuspended in cold 0.9 ml cell lysis buffer (10 mM Tris pH 8.3, 150 mM NaCl, 1.5 mM MgCl2) and 0.1 ml 5% NP40. After thorough mixing, incubation on ice for 10 min, and centrifugation twice at 2000 × g for

10 min at 40 C, the nuclei were treated with 1 ml Tri-Reagent and frozen for future extraction of nuclear RNA. Following a second centrifugation step, the cytoplasmic supernatant was mixed with 3 volumes of Tri-Reagent LS, and RNA was extracted. To prepare polysomal RNA, the clarified cytoplasmic extract was supplemented with cycloheximide (50mg/ml), RNasin (100U/ml) and dithiothreitol (2 mM) and layered onto a 9 ml linear gradient of 15% to 40% sucrose in 30 mM Tris [pH 7.4], 2 mM DDT, 10 mM EGTA, 5 mM MgCl2 that is underlayed with 2 ml of 60% sucrose in 30 mM Tris pH

7.4, 2 mM DTT, 10 mM EDTA, 5 mM MgCl2, (182). The gradient was centrifuged

0 225,000 × gmax for 3.5 hr at 4 C in a Beckman SW41 rotor. The EDTA in the 60% sucrose pad causes free polysomes to dissociate and sediment with membrane-bound polysomes at the 60% boundary (188). Polysomal RNA was extracted from the 60% boundary (1 ml) and nonpolysomal RNA was extracted from the upper fraction (8 ml) with Tri-Reagent. All RNA preparations were treated extensively with RQ DNase

(Promega), phenol extracted and ethanol precipitated.

79 RNA analysis

Antisense run-off a32P-labeled RNA transcripts were synthesized with

MAXsciptTM T7 RNA polymerase (Ambion) by the manufacturer’s instructions.

Template pGEM(140-440) was digested with NotI and pGAPDH was digested with NcoI.

Template from pMBSVT7 was prepared by PCR-amplification. The in vitro transcribed

RNAs were isolated by gel elution and the RNAse protection assays were performed using RPAIII (Ambion) according to the instruction manual with some modifications.

Typically, fifteen mg of RNA was ethanol precipitated with 3 × 105 CPM HIV probe and

3 × 103 CPM GAPDH probe. Samples were resuspended in 10 ml of hybridization buffer, heated at 900 C for 3 min, and hybridized at 420 C for 16 hr. An RNAse digestion mixture (1:100) was added to each sample (150 ml) and incubated at 370 C for 30 min.

SDS and proteinase K were added to final concentrations of 1% and 0.5 mg/ml, respectively, the samples were incubated at 370 C for 30 min, followed by extraction with phenol-chloroform and chloroform, and precipitation with ethanol in the presence of 10 mg of yeast RNA. Pellets were dissolved in 6 ml of loading buffer, heated at 900 C for 3 min, and subjected to denaturing polyacrylamide gel electrophoresis on 5% gels. RNAse protection products were visualized by PhosphorImager analysis (Molecular Dynamics,

ImageQuaNT Software version 4.2, Sunnyvale, CA).

RESULTS

SNV LTR facilitates Rev/RRE-independent production of HIV Gag

The MPMV CTE was identified originally by its ability to modulate Rev/RRE- independent expression of Gag from subgenomic HIV plasmids (19). These HIV 80 plasmids encode gag and either contain RRE, lack RRE and contain a 3’ b–globin intron, or lack both RRE and b–globin intron (pSVgagpol-rre, pSVgagpol, and pBBgagpol, respectively) (207). Two SNV regions were evaluated for Rev/RRE-independent Gag production in comparison to MPMV CTE: 1) the SNV 3’ UTR between env and the 3’

LTR, which is analogous to the position of the previously defined CTEs (19,172,181); and 2) the SNV LTR, which is associated with Rex/RxRE-independent expression of

BLV structural genes (15,18). pSVgagpol-rre-MPMV and derivatives containing SNV 3’

UTR (pSVgagpol-rre-3’UTR) or SNV LTR (pSVgagpol-rre-LTR) are shown in Figure

4.1.

Figure 4.1: Structures of subgenomic HIV plasmid pSVgagpol-rreMPMV (5) and derivatives that contain the SNV 3' UTR and SNV LTR. SV40, SV40 late promoter; black rectangle, HIV gag-pol-vif. The SNV 3' UTR extends from the 3' 200 base pairs of env through the PPT.

The plasmids were transfected into COS cells in the presence or absence of

Rev expression plasmid, pRev1 (gift of David Rekosh, University of Virginia) (190).

Cell-associated Gag protein was quantified by ELISA as an endpoint for gag RNA transcription, cytoplasmic accumulation, and translation. The Gag antigen capture

ELISA uses antibodies specific for the capsid domain of Gag to detect precursor Gag p55 and processed Gag p24 (Coulter Corp). The minimum detectable by the assay is 15 pg.

As expected (19), Gag production from pSVgagpol-rre was Rev-dependent, while Gag

81 production from pSVgagpol-rre-MPMV was Rev-independent (Table 4.1). Gag production from the plasmids containing SNV sequences (pSVgagpol-rre-3’UTR, pSVgagpol-rre-LTR) remained Rev-dependent (Table 4.1). The Rev-responsiveness of the plasmids indicates that they are competent for Gag production.

PLASMID COS D17

pSVgagpol-rre

Table 4.1: HIV Gag Production (pg/ml). Three days post-transfection cell-free supernatant media from 3 × 105 cells was harvested and Gag levels were quantified by Gag ELISA and normalized to transfection efficiency. COS cells were transfected with a mixture of LipofectamineTM (Gibco-BRL) and test DNA (1.5 mg) and 0.3 mg pRev1 plus 0.2 mg pEGFPN1 reporter plasmid. After 5 hr, the cells were washed and cultured in 2 ml DMEM with 10% fetal calf serum. Transfection efficiency was determined as percent of green fluorescent cells in 1000 cells. D17 cells were transfected with a mixture of polybrene (30 mg/ml), test DNA, and pEGFPN1 or pCMVluc, and cultured in DMEM supplemented with 5% fetal calf serum. Representative data from at least 3 independent transfections. < MD, less than the minimum detectable.

We evaluated potential cell type specificity of the SNV sequences by transfecting the various plasmids into D17 cells, a dog osteosarcoma cell line that supports SNV replication and Rex/RxRE-independent replication of hybrid SNV/BLV structural gene

82 vector (15,18). As expected, Gag production from pSVgagpol-rre was Rev-dependent

(Table 4.1). The D17 cells also supported Rev-independent Gag production from pSVgagpol-rre-MPMV, consistent with the presence of appropriate MPMV CTE- interacting factors. However, Gag production from the derivatives containing the SNV sequences remained Rev-dependent. In summary, in the context of the 3’ UTR of pSVgagpol-rre and derivative plasmids, neither the SNV 3’ UTR nor the SNV LTR facilitate Rev-independent Gag production in COS and D17 cells.

In our previous characterization of the Rex/RxRE-independent hybrid

SNV/BLV structural gene vectors, the SNV LTRs sequences corresponded to the 5’ and

3’ termini of the RNA (5, 6). Therefore, the position-dependence of the putative SNV cis-acting element was considered by analyzing hybrid SNV/HIV structural gene vectors in which the SNV sequences comprise the 5’ and 3’ termini of the RNA (pKB504gagpol and pKB504gagpol-MPMV, Figure 4.2).

Figure 4.2: Structures of hybrid SNV-HIV plasmids and HIV Gag production. Black lines and rectangle, HIV 5' UTR beginning at HIV U5 and extending through gag-pol, and the HIV PPT through the attL site, respectively (HIVBRU coordinates 100 to 4655 and 8662 to 8699, respectively); *, major HIV splice donor (left) and vif splice acceptor (right); arrows, sense and antisense orientation of SNV RU5. Shown at the right are representative data from 10 independent Gag ELISAs (Coulter Corp.), in which 105 293 cells were cotransfected with 2 µg of test plasmid and 0.2 µg of pEGFPN1 (Clonetech) or pGL3 (Promega) reporter plasmid by the calcium phosphate protocol and maintained in DMEM with 10% fetal calf serum. Total cell proteins were harvested at 3 days post- transfection. Gag production was quantified by Gag ELISA (Coulter Corp.), and transfection efficiency was quantified as percentage of green fluorescent cells in 2,000 cells by UV microscopy or relative luciferase activity. Gag levels are normalized to transfection efficiency.

Upon transfection into COS and D17 cells, Rev/RRE-independent HIV Gag production was detected from pKB504gagpol (Table 4.1). The MPMV CTE in pKB504gagpol-MPMV provided a stimulatory effect on Gag production in COS cells.

The plasmids were also transfected into 293 human embryonic kidney cells, which consistently exhibited a higher transfection efficiency than the COS or D17 cells. pKB504gagpol and pKB504gagpol-MPMV also exhibited Rev/RRE-independent HIV

Gag production in 293 cells, although a stimulatory effect of MPMV CTE is not detected

(Figure 4.2). Possible reasons for the increased level of Gag in 293 cells include higher transfection efficiency and/or increased availability of pertinent cellular factors. These results indicate that the SNV LTRs facilitate Rev/RRE-independent Gag expression in

COS, D17, and 293 cells. Furthermore, the SNV sequences function in a position- dependent manner that corresponds to the termini of the RNA.

SNV RU5 RNA facilitates Rev/RRE-independent Gag production

To evaluate the contribution of the individual SNV LTR sequences, a panel of hybrid SNV/HIV replacement plasmids was analyzed (Figure 4.2). In retrovirus DNA, the LTRs are present in two copies that are segregated into three regions: U3, R, and U5.

The 5’ U3 region corresponds to the promoter/enhancer, and the 3’ RU5 region contains the 3’ RNA processing signals. In the retrovirus RNA, R sequences are repeated at both ends of the RNA transcript, U5 is unique to the 5’ RNA terminus, and U3 is unique to the

3’ RNA terminus. pKB504gagpol was modified by replacement of both SNV LTRs with heterologous transcriptional control sequences. The 5’ SNV LTR was replaced with the cytomegalovirus immediate early (CMV IE) promoter and the 3’ LTR was replaced with

85 a synthetic polyadenylation (p(A)) signal to generate pYW99. Less than the minimum detectable level of Gag protein is exhibited in cells transfected with pYW99 (Figure 4.2).

When the 5’ LTR is replaced and the 3’ LTR is maintained (pYW202), low levels of Gag are observed. In contrast, when the 5’ LTR is maintained and the 3’ LTR is replaced

(pYW100), Gag is produced at a level similar to pKB504gagpol. These results indicate that sequences within the 5’ SNV LTR modulate the Rev/RRE-independent Gag production.

To determine the region of the 5’ SNV LTR necessary for the Rev/RRE- independent Gag expression, LTR deletion mutants were analyzed (Figure 4.2).

Complete deletion of SNV RU5 (pYW205), or partial deletion of R and all of U5

(pYW204) yields low, but detectable levels of Gag (Figure 4.2). This defect is not complemented by concurrent addition of the 3’ SNV LTR (pYW203). These results suggest that the SNV RU5 RNA encoded by the 5’ LTR is necessary for maximal levels of Gag production. To test directly the contribution of SNV RU5 to Gag expression,

SNV RU5 was inserted adjacent to the CMV IE promoter in pYW99 to make pYW207 and pYW208. The presence of RU5 in the sense orientation (pYW208), but not the antisense orientation (pYW207) correlates with Gag production (Figure 4.2). These results indicate that SNV RU5 RNA is sufficient for Rev/RRE-independent expression of

HIV Gag.

Western blot assay with Gag antibody was used to confirm that the differences observed by ELISA are not attributable to differential specificity of the Gag ELISA antibodies for precursor Gag p55 or processed Gag p24. This was important to evaluate directly because the RSV CTE has been proposed to facilitate Gag protein processing

86 (19), and because simian immunodeficiency virus constructs containing the SRV-1 CTE exhibit impaired Gag processing in 293 cells (207). As expected, Western blot analysis does not detect Gag proteins in cells transfected with mock DNA or with pYW99 (Figure

4.3). A low level of Gag p55 is observed in cells transfected with pYW203, which contains a deletion of 5’ RU5 sequence, whereas high levels of Gag p55 are observed in cells transfected with pKB504gagpol, which maintains the 5’ RU5. Thus, the Western blot data are consistent with the ELISA results. Consistent results were also observed for control cells transfected with HIV provirus (pMSMDenv2 (134)); high levels of Gag are detected by ELISA (200,000 pg) and by Western blot analysis (Figure 4.3). For the HIV control, the ratio between precursor Gag p55 and processed Gag p24 is low, consistent with high-level Gag production and efficient Gag processing. The analysis of a similar amount of Gag protein expressed from pKB504gagpol also detects both precursor Gag p55 and processed Gag p24, but the ratio between precursor Gag p55 and processed Gag p24 is high (150,000 pg/Figure 4.3). These results indicate that either the subcellular concentration of precursor Gag p55 is inadequate to drive Gag processing, or that Gag processing is inefficient for pKB504gagpol. Future experiments will address the relationship between the SNV element and inefficient Gag precursor processing.

In summary, ELISA results and Western blot analysis are in agreement that the

5’ SNV RU5 RNA facilitates maximal levels of Rev-independent Gag production.

Comparison of Gag levels produced from pYW205, pYW100, and pYW208 indicates that maximal Gag levels are observed with the combination of SNV RU5 and the SNV

U3 promoter/enhancer, rather than the combination of SNV RU5 and the CMV IE promoter/enhancer.

87 The apparent synergy between SNV U3 and RU5 may reflect cooperative interaction between cellular factors mediated by U3 and RU5 that together stimulate high level Rev- independent Gag production. Consistent with this model, the R region of murine leukemia virus and other related simple retroviruses has been shown to be important for stimulation of gene expression (37).

Figure 4.3: Western blot immunoassay. Total cell proteins from transfected 293 cells were separated by SDS-PAGE, transferred to nitrocellulose, and reacted with polyclonal rabbit sera against HIV Gag (gift from Antonito Panganiban, University of Wisconsin). HIV Gag proteins were detected by enhanced chemiluminescence (ECL kit; Amersham). The sizes of Gag p55 and Gag p24 indicated are based on comparison with molecular weight markers (not shown).

Unexpectedly pYW205, which encodes the SNV promoter/enhancer alone, exhibits low level Rev/RRE-independent Gag production, whereas, pYW99, which encodes the CMV promoter/enhancer alone, exhibits the expected undetectable level of

88 Gag. One possible explanation for this difference is that the RNAs expressed from pYW205 and pYW99 have different 5’ ends and exhibit different splicing patterns.

Low level Rev/RRE-independent Gag production from pYW205 may be attributable to expression of gag transcripts that either lack a 5’ splice donor (27) or contain an excisable intron upstream of gag (88)). The following experiments use RNA protection analyses (RPAs) to evaluate role of SNV LTR sequences in HIV RNA expression, steady state level, cytoplasmic accumulation, and polysome loading.

Rev/RRE-independent Gag levels are not attributable to differences in steady state RNA

Steady state RNAs from pYW99, pYW100, pYW205, and HIV provirus were subjected to quantitative RPAs with antisense RNA probe that extends across the HIV major splice donor, and distinguishes unspliced and spliced HIV transcripts (Figure 4.4A)

(134). A gapdh probe was used to normalize differences in RNA loading (gift of Ing-

Ming Chiu, Ohio State University). Control RNA expressed from HIVNL4-3 exhibited the

HIV unspliced RNA and spliced RNAs previously characterized by McBride and

Panganiban (134) (Figure 4.4B). These HIV unspliced and spliced RNAs were also expressed from pYW100, pYW99, and pYW205. Interestingly, pYW100 exhibits an increased amount of spliced RNA. The size of spliced transcripts corresponds to pre- mRNAs spliced at the HIV major 5’ splice donor upstream of gag and the vif splice acceptor. Results from four independent experiments indicate that steady state gag RNA levels are 3.5 ± 1.6-fold higher for pYW99 than pYW100, and indicate that the

Rev/RRE-independent Gag production is not attributable to increased promoter activity or RNA stability. While a difference in splicing pattern is not observed among the

RNAs, experiments were performed to more completely evaluate the possibility that the

89 low level Gag production from pYW205 is attributable to altered RNA splicing. The 5’

RNA terminus was characterized by primer extension analysis with antisense primer in

HIV 5’ UTR (Figure 4.5A). Compared to pYW99 RNA, pYW205 RNA was 1 nt longer and differed in sequence at the 5’ terminal 9 nts (Figure 4.5B). Comparison of the protected RNAs against DNA sequence ladders confirmed that, as expected, the protected pYW99 RNAs are 10 nt shorter than the pYW205 RNAs. These data eliminate the possibility that low level of Gag production from pYW205 is attributable to altered RNA splicing that yields new gag transcripts that either lack a 5’ splice site (134), or contain an excisable intron positioned upstream of gag (88). Further experiments are necessary to explain the low-level Gag production from pYW205.

90 A.

Hybrid SNV/HIV RNA gag r u5 u5 5’ ss

antisense RNA probe 344 nt

unspliced gag RNA 303 nt spliced RNA 146 nt pSVgagpol-rre RNA 5’ ss SV40 promoter gag

antisense RNA probe 346 nt unspliced gag RNA 244 nt

spliced RNA 82 nt

Figure 4.4: Quantification of steady-state RNA levels by RPA. A) Regions of complementarity between hybrid SNV-HIV sequence and the antisense HIV RNA probes, and the protected unspliced and spliced transcripts. SNV R and U5 RNA regions are shown in white. HIV U5 and 5' UTR (narrow line) and gag are shown in black. 5' ss, 5' splice site; nt, nucleotide. B) Two days post-transfection, total cellular RNA was harvested and subjected to DNase treatment. Aliquots of 15 µg were subjected to RPA with uniformly labeled antisense HIV RNA and gapdh RNA probes, PAGE, and PhosphorImager analysis. The protected RNAs are labeled.

91 A. pYW99 pYW205 C T A G PE C T A G PE

B. pYW99 RNA CUCGUCGAG GGAUCCGGAC UGAAUCCGUA GUACGAAUUC pYW205 RNA UCUCUUGCUC GGAUCCGGAC UGAAUCCGUA GUACGAAUUC

Figure 4.5: Sequence comparison of the 5' termini of pYW99 and pYW205 RNA. A) Primer extension analysis (PE) was performed on total cell RNA from transfected cells with murine leukemia virus reverse transcriptase and primer complementary to the HIV 5' UTR. The extension products were approximately 100 bases in length and were analyzed by electrophoresis in parallel with homologous DNA sequencing reactions and PhosphorImager analysis. Arrows indicate the proposed RNA start site based on the primer extension product. B) RNA sequences of pYW99 and pYW205. Differences are indicated in boldface.

SNV sequences facilitate cytoplasmic accumulation of HIV RNA

To begin to address the role of SNV RU5 in the cytoplasmic accumulation of HIV

RNAs, RPAs were used to analyze total and cytoplasmic RNAs from cells transfected with pYW99, pYW100, pYW205, or pYW208. The presence of the SNV RU5 correlates

92 with a 2 to 4-fold increase in nucleo-cytoplasmic transport of both unspliced and spliced

RNA (compare pYW99 with pYW208 and pYW205 with pYW100, Table 4.2). Also,

the presence of the SNV LTR in pYW100 also increased the relative amount of spliced

RNA significantly. The modest increase in nucleo-cytoplasmic transport by SNV RU5 is

not sufficient to account for the significant increase in Gag production in the presence of

the element. Therefore, experiments were pursued to test the hypothesis that SNV RU5

enhances the polysome association of the HIV RNAs. Previous research has shown that

Rev increases polysome association of Rev-dependent mRNAs (4,38).

GAG PRODUCTION AND CYTOPLASMIC ACCUMULATION OF HIV RNA

DNA TOTAL CYTOPLASMIC FOLD Gag (pg) Unspliced Spliced Unspliced Spliced Unspliced Spliced pYW99

pYW208 8,000 6.4 (0.4) 23.0 (1.4) 3.0 (1.3) 23.4 (10.2) 3.3 4.3

pYW205 12,000 10.4 (0.6) 43.8 (2.6) 1.8 (0.8) 14.6 (6.3) 1.3 1.1

pYW100 81,000 9.1 (0.5) 56.0 (3.4) 2.4 (1.0) 35.6 (15.5) 2.0 2.3

Table 4.2: Comparison of Gag protein production and cytoplasmic accumulation of HIV RNA. Total cellular or cytoplasmic RNAs were harvested from duplicate cultures of 1.0 x 106 293 cells at two days post-transfection and subjected to DNase treatment. Fifteen mg aliquots were subjected to RPA with uniformly labeled antisense HIV and gapdh RNA probes, PAGE, and PhosphorImager analysis. Cell-associated Gag protein measured by Gag ELISA. Fold indicates cytoplasmic RNA/total RNA level relative to cytoplasmic RNA/total RNA level of pYW99. RNA level presented relative to level of pYW99 unspliced RNA are indicated within parenthases. Values are presented as PhosphorImager units × 105 normalized to gapdh RNA signal. .

93 RPAs were performed on total RNA, nuclear and polysomal RNA from duplicate cell cultures transfected with pYW99 or pYW100. Data from three replicate experiments are summarized in Table 4.3 and a representative RPA is shown in Figure 4.6. Consistent with our previous results, total steady state gag RNA levels were lower for pYW100 than pYW99 by a factor of 3, and the amount of spliced RNA from pYW100 was increased.

Comparison of gag RNA levels in polysomal and nuclear RNA indicates that pYW100

RNA exhibits an average 3.8-fold increase in polysome association compared to pYW99

RNA (Table 4.3). Spliced HIV transcripts from pYW100 increased by an average 2.4- fold for pYW100.

Figure 4.6: Polysomal RNA accumulation. RNase protection assay of nuclear, polysomal and total RNA. RNAs were harvested two days post-transfection, subjected to DNase treatment, and 5-10 mg aliquots were subjected to RPA with uniformly labeled antisense HIV and GAPDH RNA probes, PAGE, and PhosphorImager analysis. Labels indicate the protected RNAs.

SUMMARY OF GAG PRODUCTION AND SUBCELLULAR LOCALIZATION OF HIV RNA DNA TOTAL NUCLEAR POLYSOMAL

Gag Unspliced Spliced Unspliced Spliced Unspliced Fold Spliced Fold pYW99

Table 4.3. Summary of Gag production and subcellular localization of HIV RNA. Total RNA, and nuclear or polysomal RNA was harvested 2 days post-transfection of duplicate cultures of 2.5 x 106 293 cells, subjected to DNase treatment and RPA with uniformly- labeled antisense HIV and gapdh RNA probes, PAGE, and PhosphorImager analysis. Cell-associated Gag protein measured by Gag ELISA in picograms per milliliter. Fold indicates polysomal RNA/nuclear RNA level relative to polysomal RNA/nuclear RNA level of pYW99. Values are presented as PhosphorImager units × 105 normalized to gapdh RNA level. RNA level presented relative to level of pYW99 unspliced RNA is indicated in parentheses. ND, not done;

To further evaluate the cytoplasmic localization of the HIV transcripts, RPAs were also performed on nuclear, polysomal and nonpolysomal RNAs from transfected cells. As a control, expression of Rev-dependent HIV RNAs was evaluated from pSVgagpol-rre in the absence and presence of Rev, and from pSVgagpol-rre-MPMV.

Similar antisense RNA probes were used for the SNV plasmids and pSVgagpol-rre-based plasmids (Figure 4.4A). In the absence of Rev, gag transcripts from pSVgagpol-rre are readily observed in nuclear RNA, while levels in polysomal and nonpolysomal cytoplasmic RNA are significantly lower (Figure 4.7A, Table 4.4). In the presence of

95 Rev in trans or MPMV CTE in cis (pSVgagpol-rre-MPMV), polysomal gag RNA levels increase 3.4-fold and 6.1-fold, respectively. Nonpolysomal gag RNA levels increase 3.4- fold and 2.7-fold, respectively. For spliced HIV transcripts, the level in polysomal increased 1.5-fold in the presence of Rev and was not increased by CTE. Consistent with the previous RPAs, polysomal gag RNA levels expressed by pYW100 were increased compared to pYW99; polysomal gag RNA was increased 2.4-fold, while nonpolysomal gag RNA levels differed by 1.4-fold compared to pYW99 (Figure 4.6, Table 4.4). In addition, polysomal and nonpolysomal levels of spliced HIV transcripts increased 2-fold.

The results summarized in Figure 4.3 and Figure 4.4 results confirm that the SNV LTR facilitates cytoplasmic accumulation and polysome association of both HIV unspliced

RNA (3.3 ± 0.8-fold) and spliced RNAs (2.3 ± 0.9-fold). By comparison, MPMV CTE selectively facilitates cytoplasmic accumulation and polysome association of the HIV unspliced transcripts (6-fold). Still, comparison of the absolute amounts of polysomal gag RNA indicates that enhanced polysome association of gag RNA is not sufficient to account for the large increases in Gag production by the SNV LTR. These results imply that enhanced translation efficiency may account for the increase in Gag production by

SNV sequences.

Figure 4.7. Comparison of polysomal and nonpolysomal RNA localization. RNase protection assay of nuclear, polysomal and free RNA. RNAs were harvested two days post-transfection, subjected to DNase treatment, and 5-15 mg aliquots were subjected to RPA with uniformly labeled antisense HIV and GAPDH RNA probes, PAGE, and PhosphorImager analysis. Labels indicate the protected RNAs. A) RNase protection assay of RNA from cells transfected with pSVgagpol-rre minus and plus pCMVRev and pSVgagpol-rre-MPMV. B) RNase protection assay of RNA from cells transfected with pYW99 and pYW100.

SUMMARY OF GAG PRODUCTION AND SUBCELLULAR LOCALIZATION OF HIV RNA

DNA NUCLEAR POLYSOMAL NONPOLYSOMAL Gag Unspliced Spliced Unspliced Fold Spliced Fold Unspliced Fold Spliced Fold

-Rev

+Rev 347,000 34.2 (1.4) 137.7 (5.8) 12.6 (4.8) 3.4 243.0 (93.5) 1.5 5.2 (4.7) 3.4 357.1 (324.6) 1.2

+MPMV 3,000 29.4 (1.2) 212.7 (9.0) 18.9 (7.3) 6.1 115.1 (44.3) 0.4 3.5 (3.2) 2.7 91.6 (83.3) 0.2

pYW99

Table 4.4. Summary of Gag production and subcellular localization of HIV RNA. Nuclear RNA and polysomal or nonpolysomal RNA was harvested two days post- transfection of 2.5 x 106 293 cells, subjected to DNase treatment and RPA with uniformly-labeled antisense HIV and gapdh RNA probes, PAGE, and PhosphorImager analysis. Values are presented as PhosphorImager units × 105 normalized to gapdh RNA level. Cell-associated Gag protein (pg/ml) was normalized to transfection efficiency, which was determined by co-transfection of pGL3 and luceriferase assay. Parentheses indicate RNA level relative to unspliced RNA from pSVgagpol-rre minus Rev or pYW99. Fold indicates either polysomal RNA/nuclear RNA level relative to polysomal RNA/nuclear RNA level of pSVgagpol-rre without pRev1 or pYW99, or nonpolysomal RNA/nuclear RNA level relative to nonpolysomal RNA/nuclear RNA level of pSVgagpol-rre without pRev1 or pYW99. pSVgagpol-rre minus pRev1, plus pRev1, or pSVgagpol-rre-MPMV.

DISCUSSION

The goal of this study was to test the hypothesis that SNV sequences contain a cis-acting element that facilitates Rev/RRE-independent expression of HIV gag RNA.

SNV 3’ UTR and LTR regions were analyzed in the context of HIV-based plasmids that were used previously for identification of the MPMV CTE (19). Exchange of MPMV

CTE with SNV 3’ UTR or SNV LTR indicated that these SNV regions do not function in 98 the 3’ UTR of pSVgagpol-rre to replace the function of MPMV CTE, even in the absence of RRE or a b-globin intron (Table 4.1). Previous observation of BLV Rex/RxRE- independent gene expression in the context of hybrid SNV/BLV retrovirus vectors implicated the SNV LTRs to possess a position-dependent CTE-like function (15,18).

Consistent with this prediction, the SNV LTRs facilitated HIV Rev/RRE-independent

Gag expression in the context of a hybrid SNV/HIV retrovirus vector (Table 4.1, Figure

4.2). The observation of Rev/RRE-independent gene expression in COS, D17, and 293 cells indicates that putative cellular factors necessary for function of the SNV element are expressed in each of these cell lines (Table 4.1, Figure 4.2). Analysis of Gag production from a panel of LTR deletion mutants indicates that the SNV element functions in a position- and orientation-dependent manner that corresponds to the 5’ LTR (Figure 4.2,

Figure 4.3). Specifically, the SNV RU5 RNA is necessary and sufficient for efficient

Rev/RRE-independent Gag production. Quantitative RPAs were used to evaluate the contributions of transcription, RNA stability, nucleo-cytoplasmic transport, and translation efficiency to Rev/RRE-independent Gag production. Analysis of steady state

RNA indicates that the effect of SNV RU5 is not attributable to increased steady state gag RNA level nor changes in splicing pattern, although SNV sequences do increase the amount of spliced HIV transcripts (Figure 4.4B, Figure 4.6, Figure 4.7B). RPAs of nuclear and cytoplasmic gag RNA indicate that SNV RU5 RNA facilitates cytoplasmic accumulation of both unspliced and spliced HIV transcripts (Table 4.3). Because the 2 to

4-fold increase in transport is insufficient to account for the significant increase in Gag production, a translational effect of the SNV sequence was investigated.

99 Analysis of cytoplasmic localization of the RNAs indicates that the SNV 5’ LTR enhances polysome association of HIV unspliced and spliced RNAs (Figure 4.6, Figure

4.7; Table 4.3, Table 4.4). Control experiments with the Rev-dependent RNAs indicate that MPMV CTE selectively facilitates polysome association of HIV unspliced transcripts (Figure 4.6, Table 4.4).

Unexpectedly, low level Rev/RRE-independent Gag production is detected from pYW205, which encodes the SNV U3 promoter/enhancer region alone. As expected, Gag production is not detectable from pYW99, which encodes the CMV IE promoter/enhancer alone. Comparative analysis of pYW205 and pYW99 by RPA and primer extension eliminated the possibilities that pYW205 RNA lacks a 5’ splice site

(27), or contains an excisable intron positioned upstream of gag (88). Further experiments are necessary to understand the low-level Gag production from pYW205.

Comparison of gag RNA and protein levels from pYW205, pYW100, pYW99, and pYW208 indicates that the combination of SNV RU5 with SNV U3 promoter/enhancer, rather than combination of SNV RU5 with CMV promoter/enhancer, produces maximal levels of Gag. A possible explanation for the apparent synergy between SNV U3 promoter/enhancer and RU5 is cooperative interaction between cellular factors mediated by U3 and RU5 that stimulates high level Rev-independent Gag production. The R regions of related simple retroviruses (i.e. murine leukemia virus and chicken syncytial virus) have been shown to be important for stimulation of gene expression and the mechanisms involved in RNA processing (37).

Trans-activation of Gag production by Rev/RRE involves derepression of cis- acting translational repressive sequences in HIV RNA (30,128,181,184) that bind

100 cytoplasmic poly(A)-binding protein (PABP1) (1). The release of PABP1 is proposed to enhance polysome association and efficient translation of gag RNA by facilitating interaction between the 3’ poly(A) tail and 5’ 7-methylguanosine cap (1,200). Future experiments will consider whether the SNV sequences are neutralizing these translational repressive sequences in the HIV RNA or supplying stimulatory sequences.

In summary, SNV encodes a position- and orientation-dependent post- transcriptional control element that is distinct in location and function from the MPMV

CTE. The possibility remains of a CTE elsewhere in the SNV genome. It is also possible that the MPMV LTR contains a post-transcriptional control element similar to

SNV. The SNV element corresponds to the 5’ terminus of the SNV RNA and increases cytoplasmic accumulation and polysome association of both unspliced and spliced HIV transcripts. In contrast, MPMV CTE selectively stimulates cytoplasmic accumulation and polysome association of unspliced viral transcripts. Importantly, comparison of the absolute amounts of polysomal RNA indicates that polysome association is not sufficient to account for Rev/RRE-independent Gag production by SNV sequences. The data imply that a significant effect of the SNV element is enhancement of the translation efficiency of HIV gag RNA. Elucidation of SNV primary sequence and RNA structure that are necessary and sufficient for Rev/RRE-independent transport and translation will be important for identification of cellular Rev-like factors that modulate the function of this unique post-transcriptional control element.

This chapter was previously published in reference (25).

Journal of Virology 73:4847-4855.

101

CHAPTER 5

TRANSLATION INITIATION OF SPLEEN NECROSIS VIRUS RNA IS CAP-DEPENDENT

ABSTRACT

Our previous work has shown that the spleen necrosis virus (SNV) RU5 facilitates

Rev/RRE-independent expression of intron-containing HIV-1 gag RNA and augments expression of intronless luc RNA. However, the potential positive effect of SNV RU5 on expression of the homologous SNV RNA has not been determined. SNV exhibits 90% sequence homology with reticuloendotheliosis virus type A (RevA) and the RevA distal

5’ untranslated region has been shown to contain an internal ribosome entry sequence

(IRES). Here we sought to determine the effect of SNV RU5 on expression of homologous SNV Gag and to determine whether or not SNV translation initiation is cap- independent. D17 cells were permanently transfected with SNV gag-luc reporter proviruses that contain firefly luc fused in-frame with the SNV gag gene. To examine the role of RU5 on SNV Gag expression, we compared Luc activity of proviruses that either contain or lack the 5' RU5. The analysis revealed that SNV RU5 increases Gag-

Luc expression by 5-12 fold and indicates a functional role for RU5 in the homologous

RNA. Infection with encephalomyocarditis virus (EMCV) was used to inhibit cap- dependent translation initiation. Following EMCV infection, Luc activity of SNV Gag-

Luc RNA and a cap-dependent control RNA were inhibited, while the cap-independent

102 EMCV IRES control RNA was resistant. The results indicate that translation of SNV

RNA initiates by a cap-dependent mechanism. Luciferase assay of transiently transfected bicistronic reporter plasmids verify that neither SNV RU5 nor distal 5' UTR sequences function as an IRES. The data supports the model that SNV RU5 is a cap-dependent 5' terminal translational enhancer.

INTRODUCTION

Retroviruses are obligate intracellular parasites that depend exclusively on the host translational machinery for the synthesis of viral proteins. Translational initiation is the rate-limiting step in protein synthesis and the primary target for translational control

(72). Retroviruses contain 5' methyl-7-G(5')pppN cap structures, which facilitate cap- dependent ribosomal scanning in conjunction with host translation initiation factors including cap binding protein, eIF4E. Structured replication motifs between the 5' cap and distal translation start codon of retroviral RNAs have been shown to inhibit efficient cap-dependent ribosome scanning and translation initiation of the downstream open reading frame (ORF) (67,142,158). Selected viral and cellular RNAs overcome structured barriers to ribosome scanning by internal ribosome entry (51). The picornavirus encephalomyocarditis virus (EMCV) is a well-studied example of IRES- mediated initiation (162). EMCV initiation does not involve cap-dependent ribosomal scanning and does not require eIF4E. IRES-mediated translation is sustained when eIF4E, the rate limiting initiation factor, is unavailable and cap-dependent initiation is inefficient. Generally, picornaviruses modulate specific shut-off of cap-dependent host translation by modifying eIF4F and therefore limiting eIF4E (reviewed in reference

103 (201)). eIF4F is a multicomponent cap-binding complex which is comprised of the cap- binding protein, eIF4E, eIF4G and eIF4A (138). eIF4G acts as a scaffold that connects eIF4E to eIF4A and eIF3 (112,117,127). Circularization of the mRNA upon simultaneous binding of eIF4E and poly (A)-binding protein (PABP) to eIF4G is thought to facilitate translation initiation (167). Cleavage of the amino terminus of eIF4G, which interacts with the cap-binding factor eIF4E (35,76), dephosphorylation and activation of 4E-BP1, a repressor of eIF4E function (71) and cleavage of PABP, which subsequently prevents communication of the PABP with eIF4E (105,108), have been shown to directly inhibit cap-dependent translation.

Unlike picornaviruses, retroviral mRNAs are capped, polyadenylated, and have appropriate start site context (Kozak consensus) (116). These features imply that translation is initiated by a cap-dependent mechanism. Experiments with bicistronic reporter plasmids, however, have identified IRES activity in the 5' untranslated regions

(UTRs) of avian reticuloendotheliosis virus A (RevA), Harvey murine sarcoma virus

(HMSV), murine leukemia virus (MuLV), simian immunodeficiency virus (SIV), Rous sarcoma virus (RSV) and in the gag ORF of HIV-1 (12,13,43,44,126,150,205). The implication of these results is cap-dependent internal ribosome entry is used to overcome inefficient ribosome scanning in retroviral RNA (103,167). However, authentic cap- independent translation has not been characterized in genomic RNAs.

RevA and spleen necrosis virus (SNV) are closely related members of the amphotropic bird retrovirus family called the reticuloendotheliosis viruses (REVs) (168).

RevA and SNV share 90% sequence homology and exchange of cis- and trans-acting

104 sequences sustains replication of the reciprocal virus (49). The mechanism by which these viruses initiate translation of their genomic RNA is not known. However, IRES activity in RevA was identified in bicistronic reporter RNAs analyzed by in vitro translation assay in rabbit reticulocyte lysate (126). The RevA IRES was mapped to within a 129 nt fragment (nucleotides 452-580) of the distal 5' UTR between the RevA packaging signal and gag AUG codon (126). IRES-activity in the distal SNV 5' UTR has not been previously examined. However, the highly structured 5' terminal RU5 sequences of SNV have been shown to function in an orientation and position-dependent manner to enhance the translation of intron-containing HIV-1 gag RNA and nonviral luc

RNA (25,173). Ribosome analysis further revealed that SNV RU5 enhances polysome loading on the heterologous HIV gag and luc reporter RNAs. Analysis of the SNV 5’

RU5 region in bicistronic reporter plasmids transiently transfected in D17 and 293 cells excluded the possibility that RU5 is a 5' terminal IRES (173). The experiments did not, however, address whether or not RU5 and distal 5’ UTR function synergistically in internal ribosome entry, if the distal 5’ UTR contains an IRES, or if SNV translation is cap-independent.

Here, we developed SNV gag-luciferase (luc) reporter proviruses that contain firefly luc fused in-frame with SNV gag. The proviruses were first used to evaluate SNV

RU5 activity in the homologous viral RNA and second, to determine whether or not SNV contains an IRES that facilitates cap-independent translation of the gag ORF. Our analyses revealed that SNV RU5 augments expression of the homologous SNV gag gene.

To examine the mechanism of translation initiation, the effect of EMCV infection on proviral Luc expression was examined. The data revealed that SNV does not utilize cap- 105 independent translation. Furthermore, analysis of SNV RU5 and extended 5' UTR sequences in bicistronic reporter plasmids indicate that unlike RevA, the SNV 5' UTR does not contain an IRES. Instead, the data indicates that translation of SNV genomic

RNA is cap-dependent.

MATERIALS AND METHODS Plasmid Construction

Three steps were performed to construct the bicistronic reporter plasmids. First,

SNV 5' UTR sequences and polio IRES sequences were amplified by polymerase chain reaction (PCR) from the SNV proviral clone pPB101 (7) and pP2'-5' (a gift from N.

Sonenberg), respectively, with primers containing EcoRI, NcoI and XbaI sites. Second, the resulting PCR product was inserted adjacent to luciferase at EcoRI and NcoI sites in pTR250 (173). Third, the SNV UTR-luc and polio IRES-luc cassettes were excised with

XbaI and ligated at the XbaI site in pRL-CMV (Promega, Madison, WI) to create pRenRU5UTRLuc, pRenUTRLuc, pRenDUTRLuc and pRenPolioIRESLuc. pRenRU5UTRLuc contains the entire SNV 5’ RU5 and 5’ UTR (sequences 1 - 615). pRenUTRLuc contains the 5’ UTR only (sequences 274 - 615). pRenDUTRLuc contains the UTR sequences proximal to the Gag AUG start codon (sequences 401 - 615). pRenIRESLuc, pRenRU5Luc (sequences 1 - 165), and pRenLuc were constructed as described by Roberts and Boris-Lawrie (173).

To construct pSNVGagLuc, pPB101 was initially modified by SmaI deletion of polymerase (pol) sequences (2964 to 5632). PCR-based site-directed mutagenesis was used to disrupt the pol stop codon by replacement with a NcoI site to make pMB109.

106 Luciferase was amplified by PCR from pGL3 (Promega, Madison, WI) and subcloned into pPCRScriptCam to make pCAMLuc. pMB109 was digested with NcoI and SmaI and the NcoI/SmaI fragment of pCAMLuc was inserted to create pSNVGagLuc. To create pSNVDRU5Luc (which lacks 5’ RU5), the SNV LTR and 5' SNV gag sequences from 395 to 2487 were amplified by PCR, and subcloned into pPCRScriptCam (Promega,

Madison, WI). The SNV sequences were excised on a AsnI/SmaI fragment and ligated into the AsnI/SmaI sites of pGFP-N1 to make pSNVGFP. To provide a SpeI site at the 5' end of U5 region for the easy exchange of the SNV LTR for a cassette that contains the

U3 region alone, PCR-based site-directed mutagenesis of pSNVGFP was performed.

The U3 region was amplified by PCR with primers that introduced a 5' AsnI site and a 3'

SpeI site and subcloned into pPCRScriptCam. The U3 region was excised from the intermediate plasmid on a AsnI/SpeI fragment and ligated into pSNVGFP at the AsnI and new SpeI site to make pSNV205GFP. pSNV205GFP was digested with AsnI and NcoI and inserted into pSNVGagLuc digested with EcoRI/Klenow and NcoI to create pSNVDRU5GagLuc.

DNA transfection, clone selection and protein analysis

Bicistronic reporter gene assays were performed following transient transfection of D17 cells by a polybrene protocol (18) or transient transfection of 293 cells by calcium phosphate protocol in three replicate 33 mm plates (110). The cells were harvested 48 hours post-transfection in phosphate buffered saline (PBS), centrifuged at 2,000 × g for 3 minutes, and resuspended in 0.1 ml or 0.05 ml of ice-cold NP40 lysis buffer (20mM Tris-

HCl, pH 7.4, 150mM NaCl, 2mM EDTA, 1% NP40). Luc levels were quantified and normalized to Renilla Luc activity (relative light units [RLU]). Luc assays were

107 performed with 10 µl lysate and 100 µl Luc Substrate (Promega, Madison, WI) and quantified in a Lumicount Luminometer (Packard, Meriden, CT). Dual measurement of

Luc expressed from firefly (Photinus pyralis) luc and sea pansy (Renilla reniformis) ren was performed by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

Reporter gene assays of pSNVGagLuc and pSNVDRU5GagLuc plasmids were performed with cultures of D17 cells permanently transfected by a polybrene protocol

(18). Cells were co-transfected with pSV2Neo and G418 resistant clones were selected with 0.4 mg/ml of G418 within 2 weeks. To measure plasmid copy number, approximately 1-2 months after selection with G418, cells were harvested in PBS, centrifuged at 3,000 × g for 3 minutes, and resuspended in 0.5 ml of genomic DNA lysis buffer (10mM Tris-HCl, pH 8, 100mM NaCl, 25mM EDTA, pH 8, 0.5% SDS).

Following addition of proteinase K (100 mg/ml) the lysates were incubated overnight at

37°C. An equal volume of phenol: chloroform:isoamyl alcohol (25:24:1) was added and the samples were passed through a 22 gauge needle/10 ml syringe twice. The samples were then subjected to chloroform:isoamyl alcohol (24:1) extraction and ethanol precipitation at -80°C. Following resuspension in distilled water, PCR was performed using 1 mg of DNA. Luc was amplified by 28 cycles of PCR to produce a 400 base pair band. Cyclophilin was amplified by 21 cycles of PCR to produce a 250 base pair band.

The amplification products were visualized by agarose gel electrophoresis and quantified by Alpha ImagerTM analysis (Alpha Innotech Corporation, San Leandro, CA).

EMCV infection

For EMCV experiments, EMCV was propagated by repeated passage on HeLa cells. Typically, a fraction (e.g., 10%) of the supernatant containing free virus was 108 transferred to a new population of uninfected HeLa cells, to the experimental permanently transfected cells, or stored at –80°C for future use, approximately 5–6 days post-infection or when 80% of the infected cells showed considerable cytopathic effect

(CPE). To determine multiplicity of infection (MOI), D17 cells in triplicate wells of 24 well plates were infected with 10-fold serial dilutions of stock EMCV. After 5 days, the cytopathic effect of the virus was examined. MOI was calculated by dividing the lowest dilution of EMCV that produced CPE by the number of cells plated. To examine cap- dependence of SNV, the permanently transfected D17 cells were plated at 60-80% confluency and 2 mls of stock EMCV (MOI>0.5) added. Cells were incubated at 37°C and harvested over a 6-hr period in NP40 lysis buffer. Total protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Luc assays were performed with 25 ng lysate and 100 µl Luc Substrate (Promega, Madison,

WI) and Luc activity was quantified in a Lumicount Luminometer (Packard, Meriden,

CT).

RESULTS

SNV RU5 enhances expression of a SNV-Luciferase reporter proviral clone

To examine SNV gene expression in the context of the homologous virus, SNV gag-luc reporter proviruses were constructed that contain luc fused in-frame with SNV gag, and either lack (pSNVDRU5GagLuc) or contain the 5' RU5 (pSNVGagLuc) (Figure

5.1). D17 cells were cotransfected with pSV2neo and either pSNVGagLuc, pSNVDRU5GagLuc, the pRenIRESLuc cap-independent control or the pGL3 cap- dependent control (Figure 5.1), and G418 resistant cells were selected with 0.4 mg/ml of

109 G418 within a 2-week period. Permanently transfected cells are designated

D17/SNVGagLuc, D17/SNVDRU5GagLuc, D17/RenIRESLuc and D17/GL3, respectively. SmaI SmaI

2964 5632 SmaI

3795 6068 PBS 2484 PPT Y pPB101 SNV gag pol U3 R U5 U3 R U5 env clone 5’ ss 3’ ss

EcoRI NcoI SmaI pol

U3 R U5 gag luc env U3 R U5 pSNVGagLuc

AsnI NcoI pol

U3 gag luc env U3 R U5 pSNVDRU5GagLuc

CMV ren IRES luc p(A) pRenIRESLuc

SV luc p(A) pGL3 V

Figure 5.1: Structure of plasmids permanently transfected into D17 cells. U3RU5, spleen necrosis virus long terminal repeat; black line, SNV 5' untranslated region; PBS, primer binding site; E, packaging signal; 5' ss, 5' splice site; 3' ss, 3' splice site; PPT, polypurine tract; luc, luciferase gene fused in-frame with gag; env, SNV envelope gene; ren, renilla luciferase gene; IRES, encephalomyocarditis virus IRES; CMV, CMV immediate early promoter; SV, simian virus promoter 40; p(A), poly(A) tail; 3' post-transcriptional control sequence.

To examine the relative copy number of pSNVGagLuc and pSNVDRU5GagLuc, luc was amplified by PCR from genomic DNA isolated from D17/SNVGagLuc and

D17/SNVDRU5Luc. The expected 400 base pair product was quantified by spot

110 densitometry (Figure 5.2). Parallel analyses of the cellular cyclophilin gene (250 base pairs) was used to standardize equivalent levels of input genomic DNA. The analysis revealed that the relative copy number of pSNVDRU5GagLuc (1.65 integrated density value, IDV) was similar in magnitude to pSNVGagLuc (1.0 IDV). However, Luc reporter assay on replicate populations of each cell line determined that the magnitude of Luc activity was 5-12 fold higher for D17/SNVGagLuc (Figure 5.3). The data indicate that

RU5 is necessary for robust expression of SNV Gag-Luc and that expression of SNV

Gag-Luc is reduced in the absence of SNV RU5.

111

1 2 3 4 5 6 7 8 A Luciferase

B Cyclophilin

Figure 5.2: Amplification of luciferase (A) or cyclophilin (B) in pSNVGagLuc and pSNVDRU5GagLuc cell lines. Genomic DNA was isolated from D17 cells (Lane 1), D17 cells permanently transfected with either pSNVGagLuc (Lane 2) or pSNVDRU5GagLuc (Lane 3) and compared to a negative control (Lane 4) and standard curve (Lanes 5-8). 1.0, 0.01, 0.001, and 0.0001 ug of DNA were use to construct the luc standard curve. 5.0, 0.5, 0.25 and 0.1 ug of DNA were use to construct the cyclophilin standard curve. The 400 base pair luc amplification product and the 250 base pair cyclophilin products were visualized by agarose gel electrophoresis and quantified with by Alpha ImagerTM analysis (Alpha Innotech Corporation, San Leandro, CA).

10,000 D17/SNVGagLuc 8,000 D17/SNVDRU5GagLuc 6,000

4,000

Relative Light Units 2,000

Experiment 1 Experiment 2 Experiment 3

Figure 5.3: SNV RU5 is necessary for robust Luc activity. Cell lysates of D17/SNVGagLuc or D17/SNVDRU5GagLuc cells were subjected to Luciferase assay. Three separate experiments were performed in triplicate (labeled Experiment 1, 2 and 3) and bars indicate standard deviations. D17/SNVGagLuc, D17 cells permanently transfected with pSNVGagLuc; D17/SNVDRU5GagLuc, D17 cells permanently transfected with pSNVGagLuc.

112 SNV translation initiation is not cap-independent

To determine whether or not SNV uses cap-independent translation,

D17/SNVGagLuc or D17/RenIRESLuc and D17/GL3 control cell lines were infected with EMCV (MOI>0.5) and Luc activity at 4 time points post-infection was quantified by

Luc assay. As expected, EMCV infection increased cap-independent translation from

D17/RenIRESLuc to 200% of the 0-hr time point (Figure 5.4). Luc activity from

D17/GL3 (student t-test compared to D17/RenIRESLuc at 5.75 hrs, p = 0.05) and

D17/SNVGagLuc (p = 0.007) was not increased. These data indicate that SNV does not utilize cap-independent internal ribosome entry to initiate translation.

2.5

2 D17/RenIRESLuc 1.5 D17/GL3 1 D17/SNVGagLuc

0.5 Relative Light Units

Compared to Untreated 0

0 1 2 3 4 5 6 Hour Post-EMCV Infection

Figure 5.4: SNV translation initiation is not cap-independent. Total cellular proteins were harvested at 4 time points after infection with EMCV in NP40 lysis buffer and equal protein was subjected to Luc assay. Duplicate experiments were performed and bars indicate standard deviations. D17/RenIRESLuc, cap-independent control; D17/Luc, cap- dependent control; D17/SNVGagLuc, D17 cells permanently transfected with pSNVGagLuc.

113

SNV 5' UTR Does Not Contain an IRES

We used bicistronic reporter RNAs to evaluate the entire SNV 5' UTR for IRES activity. Previous studies using bicistronic reporter RNAs have determined that the 5’ terminal 165 nt SNV RU5 sequence does not have IRES activity (173) and that the highly related distal 5' UTR of RevA exhibits IRES activity in in vitro translation assays (126).

We also considered that RU5 may function in conjunction with the distal 5' UTR to support internal ribosome entry. We transiently transfected the bicistronic reporter plasmids that contain various segments of the SNV 5’ UTR into D17 and 293 cells in three replicate experiments (Figure 5.5). The bicistronic reporters encode renilla luc (ren) in the first cistron and firefly luc in the second cistron, which is IRES-dependent. Firefly

Luc levels were standardized to Renilla Luc activity, a measure of transfection efficiency.

As expected, cap-independent translation of Luc is observed in response to the polio

IRES and EMCV cap-independent IRES controls (Figure 5.5). Baseline activity was exhibited in response to pRenLuc. The data reveal that SNV RU5 RNA and 5' UTR sequences exhibit Luc activity near or below baseline. The results indicate SNV RU5 and extended 5' UTR sequences do not support cap-independent translation initiation in bicistronic reporter plasmids and do not contain an IRES.

114

ren Polio luc

ren EMCV luc

ren luc

185-615 ren RU5 luc

ren RU5 UTR luc D17 Cells 1-273 ren UTR luc 293 Cells 1-400 ren DUTR luc

0 20 40 60 80 100 120 140 Relative Light Units

Figure 5.5: SNV does not contain an IRES. Reporter gene assays were performed with bicistronic plasmids that express the indicated reporter RNA in transiently transfected D17 or 293 cells. At 48 hours post-transfection dual measurement of Luc and Renilla Luc was measured by Dual-Luciferase Reporter Assay System (Promega). Bars indicate standard deviations. Dashes indicate deleted 5’ UTR sequences. Labeled rectangles, coding region of renilla luciferase, ren and luciferase, luc; Polio, internal ribosome entry sequence of polio virus; EMCV, internal ribosome entry sequence of encephalomyocarditis virus; RU5, SNV 5’ R and U5 regions (+1 - 184); R-UTR, SNV sequences including RU5 to the gag open reading frame (+1 - 615); UTR, SNV 5’ UTR sequences (274 - 615); DUTR, 3' distal sequences of the 5' UTR (401 - 615).

DISCUSSION

To characterize the role of SNV 5' RU5 on the expression of the homologous

SNV RNA, Luc activity was compared between SNV gag-luc proviruses that either contain or lack RU5. The analysis revealed that, similar to our findings with HIV gag and nonviral luc reporter RNAs, the SNV 5' RU5 augments gene expression of the homologous SNV gag (Figure 5.3). To evaluate the mechanism of SNV translation 115 initiation, we inhibited cap-dependent translation by EMCV infection (Figure 5.4).

Results of Luc assays following EMCV infection indicate similar trends for

D17/SNVGagLuc and the GL3 cap-dependent/D17 control and indicate that SNV does not contain sequences that sustain cap-independent translation. The results indicate that

SNV utilizes a cap-dependent mechanism to initiate translation. The bicistronic data also supports the conclusion that the SNV 5' UTR does not function as an IRES (Figure 5.5).

Our data do not, however, address the possibility that the SNV RU5 or distal 5' UTR sequences function as a cap-dependent ribosomal shunt. Presently, there are no identified retroviral ribosome shunts. The RNA virus cauliflower mosaic virus (CaMV) uses a ribosome shunt to promote translation of their pregenomic RNAs despite a highly structured leader (180). The non-linear migration of ribosomes occurs from a take-off site within a small ORF near the 5' cap to a landing site within a small ORF proximal to the start of the first coding ORF (46,165,178). There are no upstream ORFs in SNV, however, future study is necessary to directly determine whether SNV contains a noncanonical ribosomal shunt.

Our results support the model that SNV RU5 functions as a cap-dependent translational enhancer (Figure 5.6). We speculate that the RU5 provides a cap-dependent mechanism for RNAs to overcome translation inhibition imposed by the structured leader. Recent identification of a 5' terminal translational enhancer in Mason-Pfizer monkey virus (MPMV) (101) and possibly human foamy virus (HFV) (176) indicate that a 5' terminal translational enhancer is a shared feature among divergent retroviruses that confers interaction with cellular proteins that facilitate productive cytoplasmic expression. Our long-term goal is to characterize virus-host interactions that mediate

116 translational enhancement by SNV RU5. These proteins may include RNA helicase, elF4A, which melts the long and highly structured 5' UTR to promote ribosome scanning.

The requirement for elF4A has been shown to be in direct proportion to the degree of mRNA 5' secondary structure by in vitro translation assays (195). Poor translational efficiency of mRNAs with structured 5' UTRs is attributed to inadequate levels of eIF4A. eIF4E stimulates delivery of eIF4A to the 5' RNA terminus. An attractive model is that the SNV 5' translational enhancer recruits eIF4E and stimulates delivery of eIF4A helicase activity, which subsequently neutralizes the structured RNA and promotes polysome loading. Our previous results indicate that SNV 5’ RU5 facilitates cytoplasmic accumulation and polysome association of HIV-1 gag and nonviral luc reporter RNAs

(25,173). An important future experiment will be to perform ribosomal RNA profile analyses on RNA isolated from D17/SNVGagLuc. These experiments constitute an important step toward defining cellular control of SNV translation and virus-host interactions that define RU5 activity.

117

AUG RU5 Y AAAAAAAA

eIF4F AUG

PABP RU5

AAAAAAA

Figure 5.6: Model for translational enhancement by SNV RU5. SNV RU5 interacts with an unknown cellular factor (X) and augments recruitment of translation initiation complexes, which contains the trimeric cap-binding complex, eIF4F, and poly(A) binding protein (PABP) to the RNA (black lines). Presence of eIF4E, a component of eIF4F, facilitates ribosome scanning and polysome loading onto the RNA.

118

PERSPECTIVES

OVERVIEW

A longstanding question in retroviral RNA biology is the relationship between translation and packaging of genomic RNA. For retroviruses, an extensive body of work has characterized nuclear export of the unspliced genome-length transcript

(16,36,36,97,97), but the cytoplasmic trafficking of the RNA has remained relatively undefined. Comparison of our results to those of others has revealed a consensus on the relationship between translation and packaging of retroviral RNA. An unexpected finding is that retroviruses have adapted two divergent approaches to manage the cytoplasmic fate of genomic RNA (see review (24)). The following paragraphs discuss the interdependent relationship between translation and packaging of retroviral RNA and postulate models of retroviral RNA trafficking in the cytoplasm. Subsequently, we speculate on the role(s) of translational enhancers in the decision to translate or package unspliced RNA and present future experiments to test our hypotheses.

HYPOTHETICAL MODELS OF UNSPLICED RNA TRAFFICKING IN THE CYTOPLSAM

In the cytoplasm of an infected cell, the unspliced genome-length RNA serves two essential roles. The immediate function is to serve as mRNA template for translation of viral proteins. Another function is to serve as genomic RNA that is packaged in

119 assembling virions. We have postulated three models to describe the trafficking of the unspliced RNA in the cytoplasm. Model 1 postulates that unspliced RNA segregates into functionally independent RNA populations for packaging or translation (Figure 6.1).

Model 2 postulates that a single population of unspliced RNA functions interchangeably as mRNA template and virion RNA (Figure 6.1). A related possible model (model 2B) postulates that translation is an obligate step in RNA packaging (Figure 6.1). In this model, recruitment of the mRNA template protein to the viral assembly complexes by the newly synthesized Gag would improve genomic RNA packaging specificity.

Historically, studies with retroviral vectors have provided clues to the cytoplasmic trafficking of genomic RNA of their parental replication-competent retrovirus.

The ability of retroviral genomes to function as vectors was established in the early 1980’s when mutated murine and avian retroviral RNAs lacking viral protein coding regions were shown to be eligible for packaging if the missing viral proteins were provided in trans (70,70,187,198,198). The ability to segregate the translation and RNA packaging functions to helper and vector RNAs gives retroviruses their utility as gene transfer vectors and indicates that in murine and avian vectors, coordinate translation of

Gag structural protein is not necessary to target RNA for packaging. These conclusions are in agreement with recent studies with HIV-1 and HIV-2 vector RNAs that indicate that coordinate Gag protein synthesis is not absolutely necessary for packaging of lentivector RNA (79,107,136).

120

Model 1. mRNA

Nuclear Export

nucleus genomic RNA cytoplasm

Model 2A. mRNA

Nuclear

Export genomic RNA nucleus cytoplasm

Model 2B.

mRNA Nuclear

Export genomic RNA nucleus

cytoplasm

Figure 6.1: Models of retroviral translation and packaging. Model 1. The unspliced genome-length RNA (gray lines with intronic sequences denoted in black) achieves nuclear export, and segregate into functionally distinct population of either mRNA template for translation of viral proteins on host cell ribosomes or as genomic RNA that is packaged into assembling virions. Model 2A. The unspliced RNA can function interchangeably as both mRNA and genomic RNA with no requirement for prior translation. Model 2B. Prior translation is an obligate requirement to generate genomic RNA.

121

In 1974, chemical inhibitors were first applied to study the interdependent relationship between the processes of translation and packaging of unspliced RNA. These early experiments were recently validated by quantitative RPA and extended to human retroviruses. Evaluation of the analyses with the genetically simple MLV and ASV or complex HIV-1 and HIV-2 indicates that retroviruses have adapted at least two approaches to manage competition for unspliced RNA by the translational machinery and viral assembly components. Cytoplasmic trafficking of MLV unspliced RNA is distinct from that of ASV, HIV-1 and HIV-2. Levin et al. showed that the half-life of MLV genomic RNA is shorter than that of MLV cytoplasmic unspliced RNA (122), which supports model 1 (Figure 6.1). Model 1 posits that MLV RNA segregates into two distinct populations that function independently as genomic RNA for packaging into progeny virions or mRNA template for protein synthesis (122,123). The possibility remains that packaged MLV RNA is inherently less stable than cytoplasmic transcripts. An approach to either corroborate or disprove this hypothesis would be to apply the translation inhibitor approach developed in Chapter 2 to the MLV system.

In contrast to MLV, unspliced RNA of ASV, HIV-1, and HIV-2 exhibit dual functionality within the cytoplasm of an infected cell (23,107,192,194) (Figure 6.1, model 2A). Experiments with HIV-1 infected T-cells and translation inhibitors indicate that prior utilization as template for translation is not an obligate requirement to generate an HIV-1 genomic RNA (23) and eliminate model 2B. The observation that HIV-1 packaging efficiency is not only sustained, but can be increased upon translation inhibition indicates that cytoplasmic HIV-1 mRNA transcripts can be rerouted from

122 translation machinery to assembly complexes. Although analogous experiments have not been performed with ASV or HIV-2 proviruses, studies with ASV or HIV-2 vector RNAs are in agreement with the replication-competent HIV-1 data. Results with HIV-1 are consistent with model 2A and reveal that coordinate translation is not absolutely required for vector RNA packaging (107,192).

Why is the pattern of cytoplasmic trafficking of unspliced RNA different for

MLV? Complex retroviruses, like HIV-1, utilize the well-characterized RNA export element, RRE, and cognate viral Rev protein to recruit host Crm1 nuclear export protein to activate nuclear export of intron-containing RNA (97). A RRE-like RNA element has been observed in the 3’ UTR of ASV that is believed to be important for recruitment of cellular Rev-like proteins (155). MLV has not been shown to contain a RRE-like element in the 3’ UTR and recent studies have focused on the possibility that nuclear export of MLV unspliced RNA is mediated by sequences in proximity of a gag splice site

(98,152,153). We hypothesize that MLV unspliced RNA accesses a specialized nucleocytoplasmic trafficking pathway that dictates dedicated cytoplasmic utilization of

MLV unspliced RNA as either mRNA or genomic RNA.

SELECTION OF RNA FOR TRANSLATION OR PACKAGING

The Gag polyprotein facilitates the specific packaging of two copies of the retroviral unspliced RNA. The nucleocapsid domain (NC) of Gag contains redundant

Cys-X2-Cys-X4-His-X4-Cys motifs that interact with the highly structured packaging signal (y or E), which is located in the 5’ untranslated region (UTR) (62). Interaction of

NC with Y implies that the availability of NC modulates selection of the unspliced RNA

123 for packaging. This hypothesis is consistent with our conclusion that the cytoplasmic availability of Gag protein may determine whether or not the RNA is packaged or translated (Chapter 2). To effectively select unspliced RNA for packaging, NC may function as a chaperone that localizes the viral genomic RNA to the plasma membrane.

Alternatively, NC may function to sequester the unspliced RNA away from the ribosomes and interact with host or other viral proteins that consequently localize the

RNA to the plasma membrane. Abundant NC would facilitate the interaction of the RNA with the assembling viral proteins and limit translation by hindering the scanning ribosomes. Conversely, limited NC would promote the synthesis of viral proteins.

Distal RNA segments of retroviruses that have been shown to have IRES activity

(12,13,13,13,20,20,43,44,44,126,126,150,205,205) may allow constitutive synthesis of

Gag and/or glyco-Gag polyprotein and diminish the competition between the scanning ribosomes and assembling viral proteins. Subsequently, constitutive NC production would facilitate interaction with Y and increase production of progeny virions that contain genomic RNA

Studies in our laboratory have identified 5' terminal translational enhancers in

SNV (Chapter 4) and Mason-Pfizer monkey virus (MPMV) (101) that facilitate productive cytoplasmic expression of viral RNA. An additional hypothetical role for a 5' terminal translational enhancer is to modulate selection of unspliced RNA for packaging

(Figure 6.2). In this model, RU5 interacts with an unknown cellular factor (factor X) such as a translation initiation factor or a transport factor that localizes the RNA to the translation machinery, sequesters the RNA to the host translation machinery, and/or prohibits NC from trafficking the RNA to assembling viral proteins.

124 We have not yet determined the cellular factor that binds SNV RU5, however, one possible protein partner is the cap-binding initiation factor, eIF4E. eIF4E is the rate- limiting translation initiation factor. Over-expression of eIF4E has been shown to increase translation of myc, vegf, fgf-2 and odc mRNAs, which also contain highly structured 5' UTRs (191). This stimulation is attributed to increased available eIF4F, which delivers helicase (eIF4A) to the 5’ RNA terminus. Ribosomes utilize helicase to overcome the inhibitory effect of a highly structured 5’ UTR (67,142,158). An attractive model is that SNV 5’ translation enhancer facilitates recruitment of eIF4E to enhance delivery of eIF4A helicase activity. Interaction between eIF4E and the SNV RU5 would facilitate melting of the RNA secondary structure. This would increase ribosome scanning and translational efficiency. Localized melting of the RNA packaging signal would coordinately decrease productive interaction with NC and reduce RNA packaging efficiency.

Alternatively, factor X may interact with the 5’ terminal translational enhancer in a manner similar to the interaction between HIV-1 Rev and RRE, which exports unspliced transcripts from the nucleus to the cytoskeletal polysomes and activates their translational efficiency (16). Rev acts as an adapter protein that connects RRE- containing RNA to the Crm1 nuclear export receptor (61,146). Ribosomal profile analysis revealed that in the absence of Rev, polysome association of gag mRNA is 4%.

In the presence of Rev, 20% of gag mRNA is associated with polysomes (38). Parallel in situ hybridization indicated that the Rev/RRE localizes the viral RNA to distinct cytoplasmic compartments, which were speculated to be important for the increased translational efficiency of the unspliced RNA. The nuclear export pathway of SNV RU5-

125 containing RNAs remains to be dissected. To date our laboratory has shown that the export pathway of SNV is distinct from the Crm1-dependent pathway accessed by

Rev/RRE (39). The possibility that interaction of RU5 with an export protein that facilitates localization of the RNA to the ribosomes is consistent with model 2 (Figure

6.1).

Packaging

X X does not bind to RU5

eIF4F AUG X PABP RU5 Y AAAAAAAA X binds RU5 Translation

Figure 6.2: Model of potential function of SNV 5' RU5 in translation and packaging. An unknown cellular factor (X) binds PCE and augments the binding of the cap-binding complex (CBC) and poly A binding protein (PABP) to the RNA (black lines) thus enhancing binding of ribosomes (connected circles) and translation of the downstream Gag open reading frame. Absence of the unknown cellular factor, however, allows the RNA to be selected by Gag structural proteins for packaging into progeny virions.

FUTURE WORK

A long-term goal of this work is to characterize the structure-function relationship of SNV RU5 and to determine primary sequences necessary for the 5' terminal translational enhancer activity. Presently, members of the lab are examining the effect of

RNA context on translational enhancement by SNV RU5 and determining the required

126 RU5 structural motifs that interact with cellular proteins using mutagenesis. Other important experiments include UV-cross linking and gel shift assays with wild type and a nonfunctional RU5 mutant RNA. These experiments will identify RU5 protein partners which we expect will be eIF4E or a Rev-like export factor.

To determine the role of translational enhancement by SNV RU5 on selection of

RNA for packaging, novel vectors that contain both SNV RU5 and IRE could be developed. Introduction of IRE would facilitate translational repression or derepression of the vector RNA by def or hemin, respectively, and modulate association of factor X or

NC with the RNA. To identify the level of RNA associated with either factor X or NC, immunoprecipitation assay could be performed followed by RT-PCR (65). If either protein is involved in selection of RNA packaging, upon translational repression, RNA levels bound to NC will increase and RNA levels bound to factor X will diminish.

Following derepression of translation by hemin, inverse RNA levels are expected. In situ hybridization would provide further clues about the localization of the protein/RNA complexes and their involvement in selection of RNA for packaging or translation.

Localization of factor X to the polyribosomes would imply a role in translational efficiency. Similarly, localization of factor X to the plasma membrane would imply a role in RNA packaging.

Our RNA packaging and RU5 studies also have application for optimization of retroviral vectors for gene transfer. Continued optimization of our IRE-containing lentivectors presented in Chapter 3 will allow us to utilize translational repression as a positive switch for transduction efficiency. Introduction of SNV RU5 into vector RNA proximal to a therapeutic transgene and downstream of an internal promoter will augment

127 translational efficiency of the transgene RNA in transduced target cells. Our novel lentivector RNAs may provide a new strategy to optimize lentivector gene transfer for medical applications.

Portions of this chapter were previously published in reference (24).

Journal of Virology 76:3089-3094.

128

BIBLIOGRAPHY

1. Afonina, E., M. Neumann, and G. N. Pavlakis. 1997. Preferential binding of poly(A)-binding protein 1 to an inhibitory RNA element in the human immunodeficiency virus type 1 gag mRNA. J.Biol.Chem. 272:2307-2311.

2. Agy, M. B., M. Wambach, K. Foy, and M. G. Katze. 1990. Expression of cellular genes in CD4 positive lymphoid cells infected by the human immunodeficiency virus, HIV-1: evidence for a host protein synthesis shut-off induced by cellular mRNA degradation. Virology 177:251-258.

3. Akkina, R. K., R. M. Walton, M. L. Chen, Q. X. Li, V. Planelles, and I. S. Chen. 1996. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J.Virol. 70:2581-2585.

4. Arrigo, S. J. and I. S. Chen. 1991. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 5:808-819.

5. Aschoff, J. M., D. Foster, and J. M. Coffin. 1999. Point mutations in the avian sarcoma/leukosis virus 3' untranslated region result in a packaging defect. J.Virol. 73:7421-7429.

6. Bachi, A., I. C. Braun, J. P. Rodrigues, N. Pante, K. Ribbeck, C. von Kobbe, U. Kutay, M. Wilm, D. Gorlich, M. Carmo-Fonseca, and E. Izaurralde. 2000. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA. 6:136-158.

7. Bandyopadhyay, P. K. and H. M. Temin. 1984. Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector. Mol.Cell Biol. 4:743-748.

8. Baudin, F., R. Marquet, C. Isel, J. L. Darlix, B. Ehresmann, and C. Ehresmann. 1993. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J.Mol.Biol. 229:382-397.

9. Benko, D. M., R. Robinson, L. Solomin, M. Mellini, B. K. Felber, and G. N. Pavlakis. 1990. Binding of trans-dominant mutant Rev protein of human

129 immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA. New Biol. 2:1111-1122.

10. Berkhout, B. and J. L. van Wamel. 1996. Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J.Virol. 70:6723-6732.

11. Berkowitz, R., J. Fisher, and S. P. Goff. 1996. RNA packaging. Curr.Top.Microbiol.Immunol. 214:177-218.

12. Berlioz, C. and J. L. Darlix. 1995. An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors. J.Virol. 69:2214-2222.

13. Berlioz, C., C. Torrent, and J. L. Darlix. 1995. An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors. J.Virol. 69:6400-6407.

14. Bogerd, H. P., A. Echarri, T. M. Ross, and B. R. Cullen. 1998. Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J.Virol. 72:8627-8635.

15. Boris-Lawrie, K., V. Altanerova, C. Altaner, L. Kucerova, and H. M. Temin. 1997. In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex. J.Virol. 71:1514-1520.

16. Boris-Lawrie, K., T. M. Roberts, and S. Hull. 2001. Retroviral RNA elements integrate components of post-transcriptional gene expression. Life Sci. 69:2697- 2709.

17. Boris-Lawrie, K. and H. M. Temin. 1994. The retroviral vector. Replication cycle and safety considerations for retrovirus-mediated gene therapy. Ann.N.Y.Acad.Sci. 716:59-70.

18. Boris-Lawrie, K. and H. M. Temin. 1995. Genetically simpler bovine leukemia virus derivatives can replicate independently of Tax and Rex. J.Virol. 69:1920- 1924.

19. Bray, M., S. Prasad, J. W. Dubay, E. Hunter, K. T. Jeang, D. Rekosh, and M. L. Hammarskjold. 1994. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev- independent. Proc.Natl.Acad.Sci.U.S.A 91:1256-1260.

20. Buck, C. B., X. Shen, M. A. Egan, T. C. Pierson, C. M. Walker, and R. F. Siliciano. 2001. The human immunodeficiency virus type 1 gag gene encodes an internal ribosome entry site. J.Virol. 75:181-191.

130 21. Buckley, B. and E. Ehrenfeld. 1987. The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells. J.Biol.Chem. 262:13599-13606.

22. Burns, J. C., T. Friedmann, W. Driever, M. Burrascano, and J. K. Yee. 1993. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc.Natl.Acad.Sci.U.S.A 90:8033-8037.

23. Butsch, M. and K. Boris-Lawrie. 2000. Translation is not required To generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells. J.Virol. 74:11531-11537.

24. Butsch, M. and K. Boris-Lawrie. 2002. Destiny of unspliced retroviral RNA: ribosome and/or virion? J.Virol. 76:3089-3094.

25. Butsch, M., S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie. 1999. The 5' RNA terminus of spleen necrosis virus contains a novel posttranscriptional control element that facilitates human immunodeficiency virus Rev/RRE- independent Gag production. J.Virol. 73:4847-4855.

26. Carberry, S. E., D. E. Friedland, R. E. Rhoads, and D. J. Goss. 1992. Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure. Biochemistry 31:1427-1432.

27. Chang, D. D. and P. A. Sharp. 1989. Regulation by HIV Rev depends upon recognition of splice sites. Cell 59:789-795.

28. Clever, J. L. and T. G. Parslow. 1997. Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation. J.Virol. 71:3407-3414.

29. Coccia, E. M., E. Perrotti, E. Stellacci, R. Orsatti, N. Del Russo, G. Marziali, U. Testa, and A. Battistini. 1997. Regulation of expression of ferritin H-chain and transferrin receptor by protoporphyrin IX. Eur.J.Biochem. 250:764-772.

30. Cochrane, A. W., K. S. Jones, S. Beidas, P. J. Dillon, A. M. Skalka, and C. A. Rosen. 1991. Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J.Virol. 65:5305-5313.

31. Coder, D. M. and . 1997. Assesment of Cell Viability In J.Paul Robinson (man.ed.) (ed.), Current Protocols in Cytometry. John Wiley & Sons, Inc, New York.

32. Coffin, J. M., S. H. Hughes, and H. E. Varmus. 1997. Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY.

131 33. Corbin, A., A. C. Prats, J. L. Darlix, and M. Sitbon. 1994. A nonstructural gag- encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses. J.Virol. 68:3857-3867.

34. Coulson, R. M. and D. W. Cleveland. 1993. Ferritin synthesis is controlled by iron-dependent translational derepression and by changes in synthesis/transport of nuclear ferritin RNAs. Proc.Natl.Acad.Sci.U.S.A 90:7613-7617.

35. Cuesta, R., G. Laroia, and R. J. Schneider. 2000. Chaperone hsp27 inhibits translation during heat shock by binding eIF4G and facilitating dissociation of cap-initiation complexes. Genes Dev. 14:1460-1470.

36. Cullen, B. R. 2000. Nuclear RNA export pathways. Mol.Cell Biol. 20:4181-4187.

37. Cupelli, L., S. A. Okenquist, A. Trubetskoy, and J. Lenz. 1998. The secondary structure of the R region of a murine leukemia virus is important for stimulation of long terminal repeat-driven gene expression. J.Virol. 72:7807-7814.

38. D'Agostino, D. M., B. K. Felber, J. E. Harrison, and G. N. Pavlakis. 1992. The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Mol.Cell Biol. 12:1375-1386.

39. Dangel, A. W., S. Hull, T. M. Roberts, and K. Boris-Lawrie. 2002. Nuclear interactions are necessary for translational enhancement by spleen necrosis virus RU5. J.Virol. 76:3292-3300.

40. Das, S. and U. Maitra. 2001. Functional significance and mechanism of eIF5- promoted GTP hydrolysis in eukaryotic translation initiation. Prog.Nucleic Acid Res.Mol.Biol. 70:207-231.

41. Dasso, M. C., S. C. Milburn, J. W. Hershey, and R. J. Jackson. 1990. Selection of the 5'-proximal translation initiation site is influenced by mRNA and eIF-2 concentrations. Eur.J.Biochem. 187:361-371.

42. De Benedetti, A. and R. E. Rhoads. 1990. Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. Proc.Natl.Acad.Sci.U.S.A 87:8212-8216.

43. Deffaud, C. and J. L. Darlix. 2000. Characterization of an internal ribosomal entry segment in the 5' leader of murine leukemia virus env RNA. J.Virol. 74:846-850.

44. Deffaud, C. and J. L. Darlix. 2000. Rous sarcoma virus translation revisited: characterization of an internal ribosome entry segment in the 5' leader of the genomic RNA. J.Virol. 74:11581-11588.

132 45. Dix, D. J., P. N. Lin, Y. Kimata, and E. C. Theil. 1992. The iron regulatory region of ferritin mRNA is also a positive control element for iron-independent translation. Biochemistry 31:2818-2822.

46. Dominguez, D. I., L. A. Ryabova, M. M. Pooggin, W. Schmidt-Puchta, J. Futterer, and T. Hohn. 1998. Ribosome shunting in cauliflower mosaic virus. Identification of an essential and sufficient structural element. J.Biol.Chem. 273:3669-3678.

47. Donze, O., P. Damay, and P. F. Spahr. 1995. The first and third uORFs in RSV leader RNA are efficiently translated: implications for translational regulation and viral RNA packaging. Nucleic Acids Res. 23:861-868.

48. Donze, O. and P. F. Spahr. 1992. Role of the open reading frames of Rous sarcoma virus leader RNA in translation and genome packaging. EMBO J. 11:3747-3757.

49. Dornburg, R. 1995. Reticuloendotheliosis viruses and derived vectors. Gene Ther. 2:301-310.

50. Dougherty, J. P. and H. M. Temin. 1987. A promoterless retroviral vector indicates that there are sequences in U3 required for 3' RNA processing. Proc.Natl.Acad.Sci.U.S.A 84:1197-1201.

51. Ehrenfeld, E. 1996. Initiation of Translation by Picornavirus RNAs, p. 549-573. In J. W. B. Hershey, D. H. Mathews, and N. Sonenberg (ed.), Translational Control. Cold Spring Harbor Laboratory Press, Plainview.

52. Emerman, M. and H. M. Temin. 1986. Comparison of promoter suppression in avian and murine retrovirus vectors. Nucleic Acids Res. 14:9381-9396.

53. Emerman, M. and H. M. Temin. 1986. Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol.Cell Biol. 6:792-800.

54. Emerman, M., R. Vazeux, and K. Peden. 1989. The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell 57:1155-1165.

55. Ernst, R. K., M. Bray, D. Rekosh, and M. L. Hammarskjold. 1997. A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol.Cell Biol. 17:135-144.

56. Ernst, R. K., M. Bray, D. Rekosh, and M. L. Hammarskjold. 1997. Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element. RNA. 3:210-222.

133 57. Fekete, D. M. and C. L. Cepko. 1993. Retroviral infection coupled with tissue transplantation limits gene transfer in the chicken embryo. Proc.Natl.Acad.Sci.U.S.A 90:2350-2354.

58. Felber, B. K., M. Hadzopoulou-Cladaras, C. Cladaras, T. Copeland, and G. N. Pavlakis. 1989. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc.Natl.Acad.Sci.U.S.A 86:1495- 1499.

59. Felicetti, L., B. Colombo, and C. Baglioni. 1966. Inhibition of protein synthesis in reticulocytes by antibiotics. II. The site of action of cycloheximide, streptovitacin A and pactamycin. Biochim.Biophys.Acta 119:120-129.

60. Feng, Y. X., T. D. Copeland, S. Oroszlan, A. Rein, and J. G. Levin. 1990. Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus. Proc.Natl.Acad.Sci.U.S.A 87:8860-8863.

61. Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90:1051-1060.

62. Freed, E. O. 1998. HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251:1-15.

63. Fresno, M., L. Carrasco, and D. Vazquez. 1976. Initiation of the polypeptide chain by reticulocyte cell-free systems. Survey of different inhibitors of translation. Eur.J.Biochem. 68:355-364.

64. Gale, M., Jr., S. L. Tan, and M. G. Katze. 2000. Translational control of viral gene expression in eukaryotes. Microbiol.Mol.Biol.Rev. 64:239-280.

65. Gallouzi, I. E. and J. A. Steitz. 2001. Delineation of mRNA export pathways by the use of cell-permeable peptides. Science 294:1895-1901.

66. Garnier, L., J. B. Bowzard, and J. W. Wills. 1998. Recent advances and remaining problems in HIV assembly. AIDS 12 Suppl A:S5-16.

67. Geballe, A. P. and M. K. Gray. 1992. Variable inhibition of cell-free translation by HIV-1 transcript leader sequences. Nucleic Acids Res. 20:4291-4297.

68. Gebauer, F., L. Merendino, M. W. Hentze, and J. Valcarcel. 1998. The Drosophila splicing regulator sex-lethal directly inhibits translation of male- specific-lethal 2 mRNA. RNA. 4:142-150.

69. Gesteland, R. F. and J. F. Atkins. 1996. Recoding: dynamic reprogramming of translation. Annu.Rev.Biochem. 65:741-768.

134 70. Gilboa, E., M. Kolbe, K. Noonan, and R. Kucherlapati. 1982. Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus. J.Virol. 44:845-851.

71. Gingras, A. C., Y. Svitkin, G. J. Belsham, A. Pause, and N. Sonenberg. 1996. Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus. Proc.Natl.Acad.Sci.U.S.A 93:5578- 5583.

72. Gingras, A.-C., B. Raught, and N. Sonenberg. 1999. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu.Rev.Biochem. 68:913-963.

73. Goldberg, I. H. 1974. Mechanisms of action of antitumor antibiotic inhibitors of protein synthesis. Cancer Chemother.Rep. 58:479-489.

74. Goossen, B., S. W. Caughman, J. B. Harford, R. D. Klausner, and M. W. Hentze. 1990. Translational repression by a complex between the iron-responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position- dependent in vivo. EMBO J. 9:4127-4133.

75. Goossen, B. and M. W. Hentze. 1992. Position is the critical determinant for function of iron-responsive elements as translational regulators. Mol.Cell Biol. 12:1959-1966.

76. Gradi, A., Y. V. Svitkin, H. Imataka, and N. Sonenberg. 1998. Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc.Natl.Acad.Sci.U.S.A 95:11089-11094.

77. Gray, N. K. and M. W. Hentze. 1994. Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J. 13:3882-3891.

78. Gray, N. K. and M. Wickens. 1998. Control of Translation in Animals. Annu.Rev.Cell Dev.Biol. 14:399-458.

79. Griffin, S. D., J. F. Allen, and A. M. Lever. 2001. The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity. J.Virol. 75:12058-12069.

80. Grifo, J. A., R. D. Abramson, C. A. Satler, and W. C. Merrick. 1984. RNA- stimulated ATPase activity of eukaryotic initiation factors. J.Biol.Chem. 259:8648-8654.

135 81. Grifo, J. A., S. M. Tahara, M. A. Morgan, A. J. Shatkin, and W. C. Merrick. 1983. New initiation factor activity required for globin mRNA translation. J.Biol.Chem. 258:5804-5810.

82. Grunert, S. and R. J. Jackson. 1994. The immediate downstream codon strongly influences the efficiency of utilization of eukaryotic translation initiation codons. EMBO J. 13:3618-3630.

83. Gruter, P., C. Tabernero, C. von Kobbe, C. Schmitt, C. Saavedra, A. Bachi, M. Wilm, B. K. Felber, and E. Izaurralde. 1998. TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol.Cell 1:649-659.

84. Hackett, P. B., R. Swanstrom, H. E. Varmus, and J. M. Bishop. 1982. The leader sequence of the subgenomic mRNA's of Rous sarcoma virus is approximately 390 nucleotides. J.Virol. 41:527-534.

85. Haddrick, M., A. L. Lear, A. J. Cann, and S. Heaphy. 1996. Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1. J.Mol.Biol. 259:58-68.

86. Hadzopoulou-Cladaras, M., B. K. Felber, C. Cladaras, A. Athanassopoulos, A. Tse, and G. N. Pavlakis. 1989. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J.Virol. 63:1265-1274.

87. Hammarskjold, M. L., J. Heimer, B. Hammarskjold, I. Sangwan, L. Albert, and D. Rekosh. 1989. Regulation of human immunodeficiency virus env expression by the rev gene product. J.Virol. 63:1959-1966.

88. Hammarskjold, M. L., H. Li, D. Rekosh, and S. Prasad. 1994. Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron. J.Virol. 68:951-958.

89. Hensel, C. H., R. B. Petersen, and P. B. Hackett. 1989. Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation. J.Virol. 63:4986-4990.

90. Hentze, M. W., S. W. Caughman, T. A. Rouault, J. G. Barriocanal, A. Dancis, J. B. Harford, and R. D. Klausner. 1987. Identification of the iron- responsive element for the translational regulation of human ferritin mRNA. Science 238:1570-1573.

91. Hentze, M. W., B. Goossen, and S. W. Caughman. 1990. Complex formation between a cytoplasmic protein and the ferritin mRNA regulates translation initiation. Mol.Biol.Rep. 14:61.

136 92. Hermida-Matsumoto, L. and M. D. Resh. 2000. Localization of human immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. J.Virol. 74:8670-8679.

93. Higashikawa, F. and L. Chang. 2001. Kinetic analyses of stability of simple and complex retroviral vectors. Virology 280:124-131.

94. Hohn, T., S. Corsten, D. Dominguez, J. Futterer, D. Kirk, M. Hemmings- Mieszczak, M. Pooggin, N. Scharer-Hernandez, and L. Ryabova. 2001. Shunting is a translation strategy used by plant pararetroviruses (Caulimoviridae). Micron. 32:51-57.

95. Holland, J. 1990. Defective Viral Genomes, p. 151-165. In B. N. Fields and D. M. Knipe (ed.), Virology. Raven Press., New York.

96. Honigman, A., D. Wolf, S. Yaish, H. Falk, and A. Panet. 1991. cis Acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus. Virology 183:313-319.

97. Hope, T. J. 1999. The ins and outs of HIV Rev. Arch.Biochem.Biophys. 365:186-191.

98. Hoshi, S., T. Odawara, M. Oshima, Y. Kitamura, H. Takizawa, and H. Yoshikura. 2002. cis-Elements involved in expression of unspliced RNA in Moloney murine leukemia virus. Biochem.Biophys.Res.Commun. 290:1139- 1144.

99. Hu, W. S. and V. K. Pathak. 2000. Design of retroviral vectors and helper cells for gene therapy. Pharmacol.Rev. 52:493-511.

100. Hughes, S. 2002. Retroviridae Fields Virology, vol. 1. Lipponcott, Williams and Wilkins.

101. Hull, S. and K. Boris-Lawrie. 2001. The RU5 region of the Mason-Pfizer monkey virus 5' long terminal repeat enhances translational efficiency of retroviral and nonviral RNA. submitted.

102. Jackson, R. J., S. L. Hunt, J. E. Reynolds, and A. Kaminski. 1995. Cap- dependent and cap-independent translation: operational distinctions and mechanistic interpretations. Curr.Top.Microbiol.Immunol. 203:1-29.

103. Jackson, R. J. and A. Kaminski. 1995. Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. RNA. 1:985-1000.

104. Jang, S. K. and E. Wimmer. 1990. Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal

137 entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 4:1560-1572.

105. Joachims, M., P. C. Van Breugel, and R. E. Lloyd. 1999. Cleavage of poly(A)- binding protein by enterovirus proteases concurrent with inhibition of translation in vitro. J.Virol. 73:718-727.

106. Kang, Y. and B. R. Cullen. 1999. The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. Genes Dev. 13:1126-1139.

107. Kaye, J. F. and A. M. Lever. 1999. Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J.Virol. 73:3023-3031.

108. Kerekatte, V., B. D. Keiper, C. Badorff, A. Cai, K. U. Knowlton, and R. E. Rhoads. 1999. Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff? J.Virol. 73:709-717.

109. Kim, V. N., K. Mitrophanous, S. M. Kingsman, and A. J. Kingsman. 1998. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. J.Virol. 72:811-816.

110. Kingston, R. E. 1994. Introduction of DNA into mammalian cells, p. 9.1.4-9.1.6. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York.

111. Kirkwood, T. B. and C. R. Bangham. 1994. Cycles, chaos, and evolution in virus cultures: a model of defective interfering particles. Proc.Natl.Acad.Sci.U.S.A 91:8685-8689.

112. Korneeva, N. L., B. J. Lamphear, F. L. Hennigan, W. C. Merrick, and R. E. Rhoads. 2001. Characterization of the two eIF4A-binding sites on human eIF4G- 1. J.Biol.Chem. 276:2872-2879.

113. Kotani, H., P. B. Newton, III, S. Zhang, Y. L. Chiang, E. Otto, L. Weaver, R. M. Blaese, W. F. Anderson, and G. J. McGarrity. 1994. Improved methods of retroviral vector transduction and production for gene therapy. Hum.Gene Ther. 5:19-28.

114. Kozak, M. 1986. Influences of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc.Natl.Acad.Sci.U.S.A 83:2850-2854.

115. Kozak, M. 1987. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J.Mol.Biol. 196:947-950. 138 116. Kozak, M. 1989. The scanning model for translation: an update. J.Cell Biol. 108:229-241.

117. Lamphear, B. J., R. Kirchweger, T. Skern, and R. E. Rhoads. 1995. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation. J.Biol.Chem. 270:21975-21983.

118. Laughrea, M. and L. Jette. 1996. Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation. Biochemistry 35:1589-1598.

119. Laughrea, M., L. Jette, J. Mak, L. Kleiman, C. Liang, and M. A. Wainberg. 1997. Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization. J.Virol. 71:3397-3406.

120. Lawrence, J. B., A. W. Cochrane, C. V. Johnson, A. Perkins, and C. A. Rosen. 1991. The HIV-1 Rev protein: a model system for coupled RNA transport and translation. New Biol. 3:1220-1232.

121. Lazaris-Karatzas, A., K. S. Montine, and N. Sonenberg. 1990. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5' cap. Nature 345:544-547.

122. Levin, J. G., P. M. Grimley, J. M. Ramseur, and I. K. Berezesky. 1974. Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. J.Virol. 14:152-161.

123. Levin, J. G. and M. J. Rosenak. 1976. Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA. Proc.Natl.Acad.Sci.U.S.A 73:1154-1158.

124. Lewis, P. F. and M. Emerman. 1994. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J.Virol. 68:510- 516.

125. Lomakin, I. B., C. U. Hellen, and T. V. Pestova. 2000. Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol.Cell Biol. 20:6019-6029.

126. Lopez-Lastra, M., C. Gabus, and J. L. Darlix. 1997. Characterization of an internal ribosomal entry segment within the 5' leader of avian

139 reticuloendotheliosis virus type A RNA and development of novel MLV-REV- based retroviral vectors. Hum.Gene Ther. 8:1855-1865.

127. Mader, S., H. Lee, A. Pause, and N. Sonenberg. 1995. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol.Cell Biol. 15:4990-4997.

128. Maldarelli, F., M. A. Martin, and K. Strebel. 1991. Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. J.Virol. 65:5732-5743.

129. Malim, M. H. and B. R. Cullen. 1993. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol.Cell Biol. 13:6180-6189.

130. Malim, M. H., J. Hauber, S. Y. Le, J. V. Maizel, and B. R. Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338:254-257.

131. Marcotrigiano, J., I. B. Lomakin, N. Sonenberg, T. V. Pestova, C. U. Hellen, and S. K. Burley. 2001. A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery. Mol.Cell 7:193-203.

132. Mathews, M., N. Sonenberg, and J. Hershey. 1996. Origins and Targets of Translation Control, p. 1-29. In J. Hershey, M. Mathews, and N. Sonenberg (ed.), Translational Control. Cold Spring Harbor Press, Plainview.

133. Matsuoka, H., K. Miyake, and T. Shimada. 1998. Improved methods of HIV vector mediated gene transfer. Int.J.Hematol. 67:267-273.

134. McBride, M. S. and A. T. Panganiban. 1996. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures [published erratum appears in J Virol 1997 Jan;71(1):858]. J.Virol. 70:2963-2973.

135. McBride, M. S. and A. T. Panganiban. 1997. Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo. J.Virol. 71:2050-2058.

136. McBride, M. S., M. D. Schwartz, and A. T. Panganiban. 1997. Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J.Virol. 71:4544-4554.

137. McCann, E. M. and A. M. Lever. 1997. Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2. J.Virol. 71:4133-4137. 140 138. Merrick, W. C. and J. W. B. Hershey. 1996. The Pathway and Mechanism of Eukaryotic Protein Synthesis, p. 31-69. In J. W. B. Hershey, M. B. Mathews, and N. Sonenberg (ed.), Translational control. Cold Spring Harbor Laboratory Press, Plainview.

139. Messer, L. I., J. G. Levin, and S. K. Chattopadhyay. 1981. Metabolism of viral RNA in murine leukemia virus-infected cells; evidence for differential stability of viral message and virion precursor RNA. J.Virol. 40:683-690.

140. Meyer, B. E. and M. H. Malim. 1994. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes Dev. 8:1538-1547.

141. Meyer, B. E., J. L. Meinkoth, and M. H. Malim. 1996. Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals. J.Virol. 70:2350-2359.

142. Miele, G., A. Mouland, G. P. Harrison, E. Cohen, and A. M. Lever. 1996. The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure. J.Virol. 70:944-951.

143. Mougel, M., N. Tounekti, J. L. Darlix, J. Paoletti, B. Ehresmann, and C. Ehresmann. 1993. Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization. Nucleic Acids Res. 21:4677-4684.

144. Muckenthaler, M., N. K. Gray, and M. W. Hentze. 1998. IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap- binding complex eIF4F. Mol.Cell 2:383-388.

145. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono. 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263-267.

146. Neville, M., F. Stutz, L. Lee, L. I. Davis, and M. Rosbash. 1997. The importin- beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr.Biol. 7:767-775.

147. Obrig, T. G., W. J. Culp, W. L. McKeehan, and B. Hardesty. 1971. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J.Biol.Chem. 246:174-181.

148. Ogert, R. A. and K. L. Beemon. 1998. Mutational analysis of the rous sarcoma virus DR posttranscriptional control element. J.Virol. 72:3407-3411.

141 149. Ogert, R. A., L. H. Lee, and K. L. Beemon. 1996. Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm. J.Virol. 70:3834-3843.

150. Ohlmann, T., M. Lopez-Lastra, and J. L. Darlix. 2000. An internal ribosome entry segment promotes translation of the simian immunodeficiency virus genomic RNA. J.Biol.Chem. 275:11899-11906.

151. Ono, A., J. M. Orenstein, and E. O. Freed. 2000. Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly. J.Virol. 74:2855-2866.

152. Oshima, M., T. Odawara, K. Hanaki, H. Igarashi, and H. Yoshikura. 1998. cis Elements required for high-level expression of unspliced Gag-containing message in Moloney murine leukemia virus. J.Virol. 72:6414-6420.

153. Oshima, M., T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura. 1996. Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus. J.Virol. 70:2286-2295.

154. Otero, G. C., M. E. Harris, J. E. Donello, and T. J. Hope. 1998. Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element. J.Virol. 72:7593-7597.

155. Paca, R. E., R. A. Ogert, C. S. Hibbert, E. Izaurralde, and K. L. Beemon. 2000. Rous sarcoma virus DR posttranscriptional elements use a novel RNA export pathway. J.Virol. 74:9507-9514.

156. Paillart, J. C., L. Berthoux, M. Ottmann, J. L. Darlix, R. Marquet, B. Ehresmann, and C. Ehresmann. 1996. A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis. J.Virol. 70:8348-8354.

157. Paraskeva, E., N. K. Gray, B. Schlager, K. Wehr, and M. W. Hentze. 1999. Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements. Mol.Cell Biol. 19:807-816.

158. Parkin, N. T., E. A. Cohen, A. Darveau, C. Rosen, W. Haseltine, and N. Sonenberg. 1988. Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation. EMBO J. 7:2831-2837.

159. Paskind, M. P., R. A. Weinberg, and D. Baltimore. 1975. Dependence of Moloney murine leukemia virus production on cell growth. Virology 67:242-248.

142 160. Pasquinelli, A. E., R. K. Ernst, E. Lund, C. Grimm, M. L. Zapp, D. Rekosh, M. L. Hammarskjold, and J. E. Dahlberg. 1997. The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16:7500-7510.

161. Paul, R. W., D. Morris, B. W. Hess, J. Dunn, and R. W. Overell. 1993. Increased viral titer through concentration of viral harvests from retroviral packaging lines. Hum.Gene Ther. 4:609-615.

162. Pestova, T. V., C. U. Hellen, and I. N. Shatsky. 1996. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol.Cell Biol. 16:6859-6869.

163. Pestova, T. V., V. G. Kolupaeva, I. B. Lomakin, E. V. Pilipenko, I. N. Shatsky, V. I. Agol, and C. U. Hellen. 2001. Molecular mechanisms of translation initiation in eukaryotes. Proc.Natl.Acad.Sci.U.S.A 98:7029-7036.

164. Pollard, V. W. and M. H. Malim. 1998. The HIV-1 Rev protein. Annu.Rev.Microbiol. 52:491-532.

165. Pooggin, M. M., T. Hohn, and J. Futterer. 2000. Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader. J.Biol.Chem. 275:17288-17296.

166. Prats, A. C., G. De Billy, P. Wang, and J. L. Darlix. 1989. CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus. J.Mol.Biol. 205:363-372.

167. Preiss, T. and M. W. Hentze. 1999. From factors to mechanisms: translation and translational control in eukaryotes. Curr.Opin.Genet.Dev. 9:515-521.

168. Purchase, H. G. and R. L. Witter. 1975. The reticuloendotheliosis viruses. Curr.Top.Microbiol.Immunol. 71:103-124.

169. Rein, A. 1994. Retroviral RNA packaging: a review. Arch.Virol.Suppl 9:513-522.

170. Rein, A. and J. G. Levin. 1992. Readthrough suppression in the mammalian type C retroviruses and what it has taught us. New Biol. 4:283-289.

171. Rhoads, R. E., S. Joshi-Barve, and C. Rinker-Schaeffer. 1993. Mechanism of action and regulation of protein synthesis initiation factor 4E: effects on mRNA discrimination, cellular growth rate, and oncogenesis. Prog.Nucleic Acid Res.Mol.Biol. 46:183-219.

172. Rizvi, T. A., R. D. Schmidt, and K. A. Lew. 1997. Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) functions in a position-dependent manner. Virology 236:118-129. 143 173. Roberts, T. M. and K. Boris-Lawrie. 2000. The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA. J.Virol. 74:8111-8118.

174. Rouault, T. A., M. W. Hentze, S. W. Caughman, J. B. Harford, and R. D. Klausner. 1988. Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA. Science 241:1207-1210.

175. Rozen, F., I. Edery, K. Meerovitch, T. E. Dever, W. C. Merrick, and N. Sonenberg. 1990. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol.Cell Biol. 10:1134-1144.

176. Russell, R. A., Y. Zeng, O. Erlwein, B. R. Cullen, and M. O. McClure. 2001. The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression. J.Virol. 75:6817-6824.

177. Russell, S. J. 1997. Science, medicine, and the future. Gene therapy. BMJ 315:1289-1292.

178. Ryabova, L. A., M. M. Pooggin, D. I. Dominguez, and T. Hohn. 2000. Continuous and discontinuous ribosome scanning on the cauliflower mosaic virus 35 S RNA leader is controlled by short open reading frames. J.Biol.Chem. 275:37278-37284.

179. Saavedra, C., B. Felber, and E. Izaurralde. 1997. The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export. Curr.Biol. 7:619-628.

180. Schmidt-Puchta, W., D. Dominguez, D. Lewetag, and T. Hohn. 1997. Plant ribosome shunting in vitro. Nucleic Acids Res. 25:2854-2860.

181. Schneider, R., M. Campbell, G. Nasioulas, B. K. Felber, and G. N. Pavlakis. 1997. Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation. J.Virol. 71:4892-4903.

182. Schoenberg, D. R. and K. S. Cunningham. 1999. Characterization of mRNA endonucleases. Methods 17:60-73.

183. Schwartz, M. D., D. Fiore, and A. T. Panganiban. 1997. Distinct functions and requirements for the Cys-His boxes of the human immunodeficiency virus type 1 nucleocapsid protein during RNA encapsidation and replication. J.Virol. 71:9295- 9305.

184. Schwartz, S., M. Campbell, G. Nasioulas, J. Harrison, B. K. Felber, and G. N. Pavlakis. 1992. Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. J.Virol. 66:7176-7182. 144 185. Schwartz, S., B. K. Felber, E. M. Fenyo, and G. N. Pavlakis. 1990. Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs. J.Virol. 64:5448-5456.

186. Shen, N., L. Jette, M. A. Wainberg, and M. Laughrea. 2001. Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization. J.Virol. 75:10543-10549.

187. Shimotohno, K. and H. M. Temin. 1981. Formation of infectious progeny virus after insertion of herpes simplex thymidine kinase gene into DNA of an avian retrovirus. Cell 26:67-77.

188. Simpson, S. B., L. Zhang, R. C. Craven, and C. M. Stoltzfus. 1997. Rous sarcoma virus direct repeat cis elements exert effects at several points in the virus life cycle. J.Virol. 71:9150-9156.

189. Skripkin, E., J. C. Paillart, R. Marquet, B. Ehresmann, and C. Ehresmann. 1994. Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc.Natl.Acad.Sci.U.S.A 91:4945-4949.

190. Smith, A. J., M. I. Cho, M. L. Hammarskjold, and D. Rekosh. 1990. Human immunodeficiency virus type 1 Pr55gag and Pr160gag-pol expressed from a simian virus 40 late replacement vector are efficiently processed and assembled into viruslike particles. J.Virol. 64:2743-2750.

191. Sonenberg, N. 1996. mRNA 5' Cap-binding Protein eIF4E and Control of Cell Growth, p. 245-269. In J. W. B. Hershey, M. B. Mathews, and N. Sonenberg (ed.), Translational Control. Cold Spring Harbor Laboratory Press, Plainview.

192. Sonstegard, T. S. and P. B. Hackett. 1996. Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag. J.Virol. 70:6642-6652.

193. Sorge, J., W. Ricci, and S. H. Hughes. 1983. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J.Virol. 48:667-675.

194. Stoltzfus, C. M., K. Dimock, S. Horikami, and T. A. Ficht. 1983. Stabilities of avian sarcoma virus RNAs: comparison of subgenomic and genomic species with cellular mRNAs. J.Gen.Virol. 64 (Pt 10):2191-2202.

195. Svitkin, Y. V., A. Pause, A. Haghighat, S. Pyronnet, G. Witherell, G. J. Belsham, and N. Sonenberg. 2001. The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure. RNA. 7:382-394.

196. Szilvay, A. M., K. A. Brokstad, R. Kopperud, G. Haukenes, and K. H. Kalland. 1995. Nuclear export of the human immunodeficiency virus type 1 145 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants. J.Virol. 69:3315-3323.

197. Tabernero, C., A. S. Zolotukhin, A. Valentin, G. N. Pavlakis, and B. K. Felber. 1996. The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J.Virol. 70:5998-6011.

198. Tabin, C. J., J. W. Hoffmann, S. P. Goff, and R. A. Weinberg. 1982. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene. Mol.Cell Biol. 2:426-436.

199. Tang, H., G. M. Gaietta, W. H. Fischer, M. H. Ellisman, and F. Wong-Staal. 1997. A cellular cofactor for the constitutive transport element of type D retrovirus. Science 276:1412-1415.

200. Tarun, S. Z., Jr. and A. B. Sachs. 1995. A common function for mRNA 5' and 3' ends in translation initiation in yeast. Genes Dev. 9:2997-3007.

201. Thompson, S. R. and P. Sarnow. 2000. Regulation of host cell translation by viruses and effects on cell function. Curr.Opin.Microbiol. 3:366-370.

202. Tounekti, N., M. Mougel, C. Roy, R. Marquet, J. L. Darlix, J. Paoletti, B. Ehresmann, and C. Ehresmann. 1992. Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA. J.Mol.Biol. 223:205-220.

203. Trono, D. and D. Baltimore. 1990. A human cell factor is essential for HIV-1 Rev action. EMBO J. 9:4155-4160.

204. Truant, R., Y. Kang, and B. R. Cullen. 1999. The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function. J.Biol.Chem. 274:32167-32171.

205. Vagner, S., A. Waysbort, M. Marenda, M. C. Gensac, F. Amalric, and A. C. Prats. 1995. Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor. J.Biol.Chem. 270:20376-20383.

206. Vazquez, D. 1979. Inhibitors of protein biosynthesis. Mol.Biol.Biochem.Biophys. 30:1-312.

207. von Gegerfelt, A. and B. K. Felber. 1997. Replacement of posttranscriptional regulation in SIVmac239 generated a Rev-independent infectious virus able to propagate in rhesus peripheral blood mononuclear cells. Virology 232:291-299.

146 208. Wain-Hobson, S., P. Sonigo, O. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17.

209. Wells, S. E., P. E. Hillner, R. D. Vale, and A. B. Sachs. 1998. Circularization of mRNA by eukaryotic translation initiation factors. Mol.Cell 2:135-140.

210. Wilhelmsen, K. C., K. Eggleton, and H. M. Temin. 1984. Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel. J.Virol. 52:172-182.

211. Yee, J. K., T. Friedmann, and J. C. Burns. 1994. Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol. 43 Pt A:99-112.

212. Yee, J. K., A. Miyanohara, P. LaPorte, K. Bouic, J. C. Burns, and T. Friedmann. 1994. A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc.Natl.Acad.Sci.U.S.A 91:9564-9568.

213. Yu, S. F., T. von Ruden, P. W. Kantoff, C. Garber, M. Seiberg, U. Ruther, W. F. Anderson, E. F. Wagner, and E. Gilboa. 1986. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc.Natl.Acad.Sci.U.S.A 83:3194-3198.

214. Yuan, X., X. Yu, T. H. Lee, and M. Essex. 1993. Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor. J.Virol. 67:6387-6394.

215. Zhou, W., L. J. Parent, J. W. Wills, and M. D. Resh. 1994. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J.Virol. 68:2556-2569.

216. Zolotukhin, A. S., J. B. Harford, and B. K. Felber. 1994. Rev of human immunodeficiency virus and Rex of the human T-cell leukemia virus type I can counteract an mRNA downregulatory element of the transferrin receptor mRNA. Nucleic Acids Res. 22:4725-4732.

217. Zolotukhin, A. S., A. Valentin, G. N. Pavlakis, and B. K. Felber. 1994. Continuous propagation of RRE(-) and Rev(-)RRE(-) human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes. J.Virol. 68:7944-7952.

147