Genome Packaging and Host-‐Pathogen Interactions In

The Pennsylvania State University

The Graduate School

Department of Veterinary and Biomedical Sciences

GENOME PACKAGING AND HOST-PATHOGEN INTERACTIONS

IN PARAMYXOVIRUS ASSEMBLY AND BUDDING

A Dissertation in Pathobiology

by Greeshma Ray

Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

August 2016

The dissertation of Greeshma Ray was reviewed and approved* by the following:

Anthony P. Schmitt Associate Professor of Molecular Virology Dissertation Advisor Chair of Committee Director of Pathobiology Graduate Program

K. Sandeep Prabhu Professor of Immunology and Molecular Toxicology

Robert F. Paulson Professor of Veterinary and Biomedical Sciences

Richard T. Frisque Professor of Molecular Virology Associate Department Head for Equity and Diversity

*Signatures are on file in the Graduate School

iii ABSTRACT

Paramyxoviruses possess non-segmented, negative-sense RNA genomes that are encapsidated by viral nucleocapsid protein (NP), and are packaged into budding virus particles via NP protein interaction with viral matrix (M) proteins, thereby creating infectious viruses. We had previously identified a DLD sequence near the C- terminal end of parainfluenza virus 5 (PIV5) NP protein that was important for interaction with PIV5 M protein, and for enhancing PIV5 virus-like particle (VLP) production. We have shown here that a DLD-containing 15 amino acid sequence derived from PIV5 NP or Nipah virus N proteins is sufficient to direct a foreign protein, Renilla luciferase, into virus-like particles. This short DLD-containing sequence was also able to replace the requirement for NP protein in PIV5 VLP production. Mumps virus NP protein contains a DWD sequence instead of DLD, and consequently, PIV5 NP protein cannot interact efficiently with mumps virus M protein, in spite of the two viruses being very closely related. Altering PIV5 NP DLD sequence to mumps virus NP DWD sequence creates a new PIV5 NP protein that is now compatible with M proteins from both PIV5 and mumps virus. We hypothesize that DLD-like sequences define compatibilities between paramyxovirus M and NP proteins that can be manipulated to drive production of virus-like particles containing therapeutics for delivery to cells.

A hallmark of enveloped virus infection is the hijacking of host cellular pathways during viral egress. To define host factors involved in paramyxovirus budding, a yeast two-hybrid screening approach was used previously, which identified

iv angiomotin-like 1 (AmotL1) as a host factor that binds to the matrix (M) proteins of parainfluenza virus 5 (PIV5) and mumps virus. AmotL1 belongs to a family of proteins that also includes angiomotin (Amot) and angiomotin-like 2 (AmotL2). All three angiomotins harbor PPXY motifs and interact with WW-domain containing proteins such as Nedd4 and YAP. We found that the PIV5 and mumps virus M proteins bind to AmotL1, but not to Amot or AmotL2. Binding was mapped to a 83- amino acid region from the C-terminal portion of AmotL1. Overexpression of M- binding AmotL1-derived polypeptides inhibited production of PIV5 and mumps virus-like particles (VLPs), while overexpression of the corresponding regions of

Amot and AmotL2 had little effect on VLP production. Co-immunoprecipitation studies support the presence of a three-way interaction between Nedd4, AmotL1, and M. Efficient pulldown of M with Nedd4 was observed only when AmotL1 was expressed to bridge the gap. Our findings support a model in which paramyxoviruses indirectly recruit the same WW domain-containing proteins to virus assembly sites through AmotL1 that other enveloped viruses recruit directly via PPXY late domains.

v TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………………………………………vii LIST OF TABLES……………………………………………………………………………………………ix LIST OF ABBREVIATIONS………………………………………………………………………………x ACKNOWLEDGMENTS…………………………………………………………………………………xii

CHAPTER 1……………………………………………………………………………………………………1 LITERATURE REVIEW……………………………………………………………………………………1 1.1 Significance and classification of paramyxoviruses………………………………………...1 1.2 Structure and composition……………………………………………………………………………7 1.3 Life cycle of paramyxoviruses……………………………………………………………………..14 1.4 Paramyxovirus virus-like particles……………………………………………………………...17 1.5 Paramyxovirus matrix-nucleocapsid interactions………………………………………..18 1.6 ESCRT pathway and late domains……………………………………………………………….19 1.7 Angiomotins………………………………………………………………………………………………23 1.8 Preview...…………………………………………………………………………………………………...27

CHAPTER 2………………………………………………………………………………………………….30 MATERIALS AND METHODS……………………………………………………………………...... 30 Plasmids………………………………………………………………………………………………………….30 Antibodies……………………………………………………………………………………………………….32 Membrane co-flotation assays measuring M-NP interactions……………………………..33 Measurements of VLP production……………………………………………………………………..34 Measurement of luciferase activity……………………………………………………………………36 Co-immunoprecipitation assays………………………………………………………………………..36 Immunofluorescence microscopy……………………………………………………………………..37 Amino acid sequence comparisons…………………………………………………………………...38

CHAPTER 3………………………………………………………………………………………………….39 C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles…………………………………………………………………..39 3.1 Introduction……………………………………………………………………………………………….39 3.2 Results……………………………………………………………………………………………………….42 Manipulation of genome packaging interactions to direct a foreign protein into PIV5 VLPs…………………………………………………………………….………………………………………….42 PIV5 NP protein and mumps virus M protein can be engineered for compatibility…………………………………………………………………………………………………...46 Alterations affecting the C-terminal end of mumps virus NP protein impair interaction with M protein and block VLP production………………………………………..52 Manipulation of genome packaging interactions to direct a foreign protein into Nipah VLPs………………………………………………………………………………………………………54

vi Amino acid residues 523 to 528 of N protein are important for RLuc packaging into Nipah VLPs………………………………………………………………………………………………………55 3.3 Discussion………………………………………………………………………………………………….59

CHAPTER 4………………………………………………………………………………………………….63 Role of angiomotin-like 1 in paramyxovirus budding…………………………………63 4.1 Introduction. …………………………………………………………………………………………...... 63 ESCRT and viral late domains…………………………………………………………………………...63 Angiomotins and virus budding……………………………………………………………………...... 67 4.2 Results……………………………………………………………………………………………………….69 PIV5 M binds selectively to AmotL1………………………………………………………………….69 The C-terminal fragment of AmotL1, and not that of AmotL2 or Amotp130, can inhibit PIV5 VLP production..…………………………………………………………………………...70 AmotL1 can bind to different members of the Nedd4 family of E3 ubiquitin ligases…………………………………………………………………….……………………………………….74 PIV5 M, AmotL1 and Nedd4-like proteins interact and form a complex……………...76 PIV5 M, Nedd4L and AmotL1 co-localization within cells………………………………...... 79 Mumps virus matrix protein binds only AmotL1, not AmotL2 or Amotp130…….....85 4.3 Discussion.………………………………………………………………………………………………....86

CHAPTER 5………………………………………………………………………………………………....91 SUMMARY AND FUTURE DIRECTIONS…………………………………………………………91 5.1 Summary…………………………………………………………………………………………………...91 5.2 Future directions…………………………………………………………………………………….....94 Manipulating genome packaging interactions for therapeutic uses……………………94 Examining compatibilities between M-NP proteins from different paramyxoviruses…………………………………………………………………………………………….96 Studying the role of angiomotins and Nedd4 ligases in paramxovirus budding.....97

APPENDICES…………………………………………………………………………………………….100 Appendix A: Packaging of foreign proteins into PIV5 virus-like particles and delivery into cells…………………………………………………………………………………..100 Appendix B: Packaging foreign proteins into mumps virus-like particles………..105 Appendix C: PIV5 M proteins with PPXY late domains can bind directly to Nedd4L………………………………………………………………………………………………………..108

BIBLIOGRAPHY………………………………………………………………………………………..110

vii

LIST OF FIGURES

Figure 1.1. Phylogenetic tree of Paramyxoviridae- subfamily Paramyxovirinae……………………………………………………………………………………………4 Figure 1.2. Gene orders among different paramyxoviruses………………………………8 Figure 1.3. Structure of a paramyxovirus virion………………………………………………8 Figure 1.4. Paramyxovirus life cycle……………………………………………………………...16 Figure 1.5. The ESCRT pathway in viral budding……………………………………………23 Figure 3.1. A total of 15 C-terminal residues of PIV5 NP protein are sufficient to trigger VLP production and to direct a foreign protein into budding particles...44 Figure 3.2. C-terminal ends of paramyxovirus N/NP proteins………………………...45 Figure 3.3. DLD and DWD sequences define compatibilities between PIV5 and mumps virus M/NP protein pairs………………………………………………………………….48 Figure 3.4. Compatibilities of PIV5 and mumps virus M/NP protein pairs measured by membrane coflotation analysis…………………………………………………………………50 Figure 3.5. PIV5 NP L507W protein retains its compatibilities with PIV5 and Nipah virus M proteins…………………………………………………………………………………………...51 Figure 3.6. The DWD-containing C-terminal region of mumps virus NP protein is important for its VLP production function……………………………………………………..53 Figure 3.7. A total of 15 C-terminal residues of Nipah virus N protein are sufficient to direct a foreign protein into budding particles………………………………………………56 Figure 3.8. Amino acid residues 523-NDLDFV-528 within Nipah virus N protein are important for the ability to direct a foreign protein into Nipah VLPs……………...58 Figure 4.1. Late domains in different viruses………………………………………………...66 Figure 4.2. Schematic illustration of different angiomotins and related polypeptides………………………………………………………………………………………………..71

viii Figure 4.3. PIV5 M can bind only to full-length AmotL1 or derivatives, and not to AmotL2 or Amotp130………………………………………………………………………………….72 Figure 4.4. AmotL1 C-terminal fragment can inhibit PIV5 VLP production, whereas AmotL2 or Amotp130 C-terminal fragments do not………………………………………73 Figure 4.5. AmotL1 binds to at least three members of the family of Nedd4 E3 ubiquitin ligases……………………………………………………………………………………….....75 Figure 4.6. AmotL1 functions as an adaptor to link PIV5 M and Nedd4-like proteins together………………………………………………………………………………………………………78 Figure 4.7. AmotL1 functions as an adaptor to link PIV5 M and NedL1 proteins together………………………………………………………………………………………………………79 Fig 4.8. Localization patterns of viral and host proteins, either alone or in combination………………………………………………………………………………………………...82 Figure 4.9. PIV5 M, Amotl1 and Nedd4L co-localization within cells…………...... 83 Figure 4.10. PIV5 M does not associate with either AmotL2 or Amotp130…...... 84 Figure 4.11. Mumps virus matrix protein interacts only with AmotL1, and not the other motins; only AmotL1-derived proteins can inhibit mumps VLP production…………………………………………………………………………………………………...86 Figure A-1. Foreign proteins other than luciferase that contain NP-derived sequences can be directed into PIV5 VLPs…………………………………………………....102 Figure A-2. Using VLPs for luciferase delivery into cells………………………………..104 Figure B-1. Foreign proteins can be packaged into mumps VLPs via manipulation of M-NP protein interactions…………………………………………………………………………...107 Figure C-1. A PPXY-containing PIV5 M protein can bind directly to Nedd4L…..109

ix LIST OF TABLES

Table 1.1. Requirements for VLP production among paramyxoviruses……………18

x LIST OF ABBREVIATIONS

AmotL1 - Angiomotin-like 1 AmotL2 - Angiomotin-like 2 Amot p130/p80 - Angiomotin isoform p130/p80 AP3B1 - Adaptor protein complex AP-3 subunit beta 1 ARG1 - Arginase-1 BAR - Bin/amphiphysin/Rvs Co-IP - Co-immunoprecipitation COX-2 - Cyclooxygenase-2 Ct - C-terminal ESCRT - Endosomal sorting complex required for transport F - Fusion FYVE - Fab1/YOTB1/Vac1/EEA1 G - Glycoprotein Gag - Group-specific antigen GAP - GTPase activating protein H - Hemagglutinin HIV-1 - Human immunodeficiency virus-1 HN - Haemagglutinin-neuraminidase HPIV - Human parainfluenza virus HRSV - Human respiratory syncytial virus IP - Immunoprecipitation L - Large m - minimal domain M - Matrix MeV - Measles virus MMR - Measles,mumps,rubella MuV - Mumps virus MVB - Multi-vesicular body NDV - Newcastle disease virus NEDD4 - Neural precursor cell expressed developmentally downregulated protein 4 NEDL1 - NEDD4-like ubiquitin protein ligase-1 NiV - Nipah virus Nt - N-terminal NP/N - Nucleocapsid P - Phosphoprotein

xi PIV5 - Parainfluenza virus 5 RLuc - Renilla luciferase RNP - Ribonucleoprotein RdRp - RNA-dependent RNA polymerase RSV - Rous sarcoma virus SDS-PAGE - Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM - Scanning electron microscopy SeV - Sendai virus SH - small hydrophobic SOD1 - Superoxide dismutase 1 SV5 - Simian virus 5 TEM - Transmission electron microscopy Tsg101 - Tumor susceptibility gene 101 VLP - Virus-like particle VPS - Vacuolar protein sorting YAP - Yes-associated protein

xii ACKNOWLEDGMENTS

“Time flies like an arrow; fruit flies like a banana!”

-Anthony Oettinger/ Groucho Marx

“All’s well that ends better.”

-J.R.R. Tolkien- The Lord of the Rings

And with that, my Ph.D. journey finally comes to an end…

I wanted to be a nerdy researcher even before I knew what “nerdy” and

“researcher” meant. I was always curious about how and why things worked, and why sometimes they did not. This led to endless questions and multiple remonstrations from my family, because as children, we always ask the most pertinent questions that no one wants to answer. Towards the end of my schooling, all my colleagues were choosing their future areas of study, and almost everyone chose to enter commerce, engineering or medical fields. I was one of the few who wanted to go in for hard-core biological research. Concerned elders would always question my decision, wondering what kind of future existed in this field, especially in India. It was never a difficult decision to make though, in spite of all the doubts cast my way by skeptics. I completed my Bachelor’s and Master’s degrees in Life

Sciences and had a lot of fun learning about different organisms, chemical reactions, and diverse systems. Following this, I applied to enter a Ph.D. program in the USA and was delighted to get an offer from Penn State.

xiii I was in awe as soon as I entered State College. It was summer, and the sun set at 9 pm, which was awesome. The air was fresh and it was clean and green everywhere.

Campus was big and beautiful too and it was always very exciting to walk everywhere and have new experiences. The past 7 years in State College have been delightful; obviously, there were both good and bad times. But looking back, I am very grateful for everything. They have helped me grow as a person, taught me new skills and also made me forget some in the pursuit of research. But overall, I’m in a better place today than I was 7 years back.

The fact that during your Ph.D. journey, you are probably the only person in the world trying to answer a specific question in your field is amazing. Imagine the joy when you find answers to your questions, even though it can feel like an uphill battle most times. Remembering this helped me to finish my degree and I am very thankful for the people who helped me achieve my dreams. Firstly, I would like to thank my advisor Dr. Anthony Schmitt, for his constant encouragement and advice.

He helped me develop my thought process and also gave me enough flexibility to try new things. There are many times a person feels stupid and overwhelmed during their Ph.D. because they didn’t think of simple things. But when you have an advisor who helps you realize errors without being too judgmental that is simply awesome.

Thank you Tony! I would also like to thank my committee members Dr. Sandeep

Prabhu, Dr. Robert Paulson and Dr. Richard Frisque for their tremendous support and patience (and also for not being too scary during qualifying exams). In addition,

I would like to thank Dr. Craig Cameron for his excellent advice on projects during

xiv virology meetings and career advice. I would like to thank all the people I worked with, especially those within the lab. Dr. Phuong Schmitt helped with a lot of advice and extensive troubleshooting with my projects and getting my Ph.D. would have been difficult without her technical expertise. Drs. Megan Harrison, Thomas

McCrory, Weina Sun, Zifei Pei and Ming Li were constantly bothered by me for the duration of their time in the lab, and I am extremely thankful to them for being so nice. A special thanks to my undergrads and younger graduate students in the lab for bearing with me, as I tried to explain to them the finer points of experiments.

Thanks to all the people in the Department of Veterinary and Biomedical Sciences for helping me through my degree, especially the ladies in the office for being so kind and helpful with the crazy paperwork that international students have to go through. Thanks to Webb for being the sweetest puppy ever, and thank you Karen

Hack for letting me play with him as long as I liked.

I would like to thank my family – my father Yohann Ray and mother Shaila Ray for always supporting my decisions. I couldn’t have done it without the hard work and efforts you put into bringing me up. Thanks to my brother Romir Ray who taught me what endurance means. Thanks to my in-laws LR Narayan and Nalini Narayan for their constant encouragement and praise. Thanks to Vishal Narayan for letting me tease you when I felt like. And thanks to the rest of my family and all my friends who were present for me through these years, even though the distance separated us.

The biggest thanks go to my husband Dr. Vivek Narayan. It is only with his love that I made it through the ordeals of a Ph.D. He was ready to troubleshoot

xv experiments with me at any time of day. He would eat anything I cooked and praise it too. My cooking skills improved vastly only because of him. He always encouraged me to try anything that took my fancy, and I am a decent photographer today only because of him. Hell, I overcame my fear of cars and can drive today too. Vivek, I wish you the best of what life has to offer (that includes me :P), and I look forward to spending many more wonderful moments with you by my side!

CHAPTER 1

LITERATURE REVIEW

1.1 Significance and classification of paramyxoviruses

Paramyxoviruses (Greek-para-beyond, myxo-mucus, Latin-virus-poison) are

enveloped, non-segmented, negative-sense, single strand RNA viruses (NNSV),

and include many members that are significant pathogens in humans and

animals. The family Paramyxoviridae (Order-Mononegavirales) is composed of

two sub-families- Paramyxovirinae and Pneumovirinae. The Paramxovirinae is

further composed of seven different genera - Morbilivirus, Rubulavirus,

Avulavirus, Respirovirus, Henipavirus, Aquaparamyxovirus and Ferlavirus [1].

Classification is based on a number of criteria - morphology, genome

organization and similarities between protein sequences and their biological

functions (Fig.1.1).

Morbiliviruses include measles virus, rinderpest virus and canine distemper

virus among others. Measles virus is one of the most contagious viruses known to

man, and is the leading cause of vaccine-preventable childhood mortality.

Infection with measles causes the characteristic rash, fever, cough and runny

nose symptoms [2]. Measles continues to plague the population in spite of the

effective MMR (measles, mumps, rubella) vaccine to prevent infection, with

increased outbreaks all over the world [2–5]. This is mainly due to poor

vaccination coverage and vaccine hesitancy [6]. Japan discontinued MMR usage

due to aseptic meningitis associated with the mumps virus strain used and now

administers separate measles and rubella vaccines. However, the cases of

measles and rubella have only increased [7]. Rinderpest virus has been

eradicated through vaccination [8], but was a significant disease in cattle.

Rubulaviruses include mumps virus, parainfluenza virus 5 (PIV5) and human

parainfluenza viruses 2 and 4. PIV5, previously known as simian virus 5 (SV5),

was first isolated from rhesus and cynomolgus kidney cell cultures in 1956, due

to which it was primarily thought to be a monkey virus [9]. However, it was next

isolated from a human volunteer with acute respiratory symptoms [10], following

which SV5-like viruses were isolated from multiple tissue sources and human

diseases such as respiratory illness, pemphigus skin lesions, infectious hepatitis,

multiple sclerosis and Creutzfeldt-Jacob disease, suggesting that it also infects

humans [9, 11]. In 1967, an SV5-like virus was isolated from dogs afflicted with

respiratory illness. This canine strain of parainfluenza virus (CPI) was closely

related to SV5 and mostly associated with respiratory tissues, however, it was

also present in cerebrospinal fluid of a dog suffering from neurological illness [9].

Since SV5/PIV5 seems to be associated with simians as well as domestic animals,

human infections are likely due to a zoonosis [11]. Mumps virus only infects

humans, no animal reservoirs have been found yet. Infection with mumps virus is

characterized by parotid gland swelling and/or respiratory symptoms.

Complications such as oophoritis, orchitis, and encephalitis often occur and are

prominent in adults [12]. Mumps virus caused severe childhood illnesses before

vaccination programs were developed, vaccination with the MMR vaccine helped

reduce incidences of mumps worldwide [13]. However, like measles, there have

been outbreaks in many countries recently, mainly due to non-compliance to

vaccination programs. For instance in Japan, the Urabe strain of mumps virus was

used in the MMR vaccine instead of the widely used Jeryl-Lynn strain. Urabe

strain was associated with increased cases of aseptic meningitis and as a result,

Japan discontinued use of this vaccine. Now only the measles-rubella vaccine is

included in national vaccination programs. Mumps vaccination is no longer

routine, only voluntary and vaccination has dropped, causing an increase in

mumps incidence in the population, with about a million cases reported annually

[7, 13]. Human parainfluenza viruses (HPIVs) cause respiratory illnesses in

children, elderly and immunocompromised individuals [14]. Avulavirus genus

comprises viruses that affect birds, viz. Newcastle disease virus (NDV) and avian

paramyxoviruses. NDV affects many domestic and wild birds and has caused

tremendous losses to the poultry industry [15]. It causes only mild symptoms in

humans. NDV has been shown to be an oncolytic virus and is being explored as an

anti-tumor agent [16, 17]. Respiroviruses mainly comprise Sendai virus and

HPIVs 1and 3. Sendai virus infects mice and other rodent populations, causing

respiratory symptoms in them [18].

Figure 1.1. Phylogenetic tree of Paramyxoviridae- subfamily Paramyxovirinae. The above phylogenetic tree is based on nucleocapsid (NP/N) protein sequences from viruses within the Paramyxovirinae sub-family. Viruses were sub-grouped according to their genus as follows- Rubulavirus: hPIV2 (human parainfluenza virus 2), SV41 (simian parainfluenza virus 41), PIV5 (parainfluenza virus 5), MuV (mumps virus), PorPV (porcine rubulavirus), MprPV (Mapuera virus); Avulavirus: NDV (Newcastle disease virus), APMV6 (Avian paramyxovirus 6); Respirovirus: SeV (Sendai virus), hPIV3 (human parainfluenza virus 3), bPIV3 (bovine parainfluenza virus 3); Henipavirus: NiV-M (Nipah virus-Malaysia strain), NiV-B (Nipah virus- Bangladesh strain), HeV (Hendra virus), CedPV (Cedar virus); Morbilivirus: MeV (measles virus), RPV (rinderpest virus), CDV (canine distemper virus), PPRV (Peste-des-petits ruminants); unassigned paramyxoviruses: TioPV (Tioman virus), MenPV (Menangle virus), hPIV4a (human parainfluenza virus 4a), hPIV4b (human parainfluenza virus 4b), AsaPV (Atlantic salmon paramyxovirus), FdlPV (Fer-de-lance virus), TupPV (Tupaia paramyxovirus), MosPV (Mossman virus), JPV (J virus), BeiPV (Beilong virus), SalPV (Salem virus) [19].

Henipaviruses are among some of the newly emerged paramyxoviruses and

include viruses like Nipah and Hendra virus, as also the recently identified Cedar

virus. Nipah virus, a zoonotic virus, was originally identified in Malaysia in April

1999, when bats from fruit orchards infected pigs bred in piggeries that ate fruit

contaminated with bat saliva and bat droppings. The pigs developed respiratory

symptoms and in turn passed on the infection to their human handlers, who

developed encephalitis. About 1 million pigs had to be culled owing to this

infection; while in humans it was lethal in 105 out of 257 cases. Nipah virus has

no vaccine or drug cure and has about 40% mortality [20, 21]. Recently, a new

strain of Nipah virus-Bangladesh strain has emerged in Bangladesh and

northeast India, where human-to-human transmission is possible [22]. Here,

humans became infected after consuming toddy palm sap contaminated with bat

droppings. Human to human transmission was found to occur in individuals

caring for the sick, or those helping with preparation of bodies for burial. Here

Nipah virus exhibited at least 70% mortality. Similarly Hendra virus emerged in

Australia in 1994, identified when it caused deaths of thirteen horses and their

trainer. Horses and their handlers developed respiratory and/or encephalitic

symptoms and this virus exhibited about 60% mortality. Survey of wildlife in the

area identified pteropid fruit bats as the most likely source for this virus. Hendra

and Nipah viruses have been classified as Category C priority agents and are bio-

safety level 4 (BSL-4) restricted. The Cedar virus however, is not pathogenic to

humans and has been found only in bats till now [19]. There have been

significant advances in vaccine development for these viruses. In November

2012, Commonwealth Scientific and Industrial Research Organization (CSIRO)

introduced Equivac, a vaccine licensed for use in horses [23], based on the

soluble form of Hendra glycoprotein G. In addition to this, neutralizing human

monoclonal antibodies have been developed against the soluble subunit of

Hendra virus G protein, and have shown cross-protection against Nipah virus

infection in African green monkeys and ferrets [24–27]. Soluble subunits of

glycoproteins from both viruses have also been shown to be promising

candidates for vaccines in animal models [28, 29]. Two new genera not shown in

Fig.1 are Ferlavirus genus that comprises only one member, Fer-de-lance

paramyxovirus [30] that affects reptiles and Aquaparamyxovirus genus that also

has only one member, the Atlantic salmon paramyxovirus [31] that infects fish.

Besides all these viruses, there are some recently identified paramyxoviruses

that haven’t been assigned into specific genera yet- J virus, Beilong virus, Tupaia

paramyxovirus, Menangle virus, Mossman virus, Salem virus and Nariva virus

[1].

The other sub-family of paramyxoviruses, Pneumovirinae consists of two

genera- Pneumovirus and Metapneumovirus. Pneumoviruses include human and

bovine respiratory syncytial virus (HRSV/BRSV), while metapneumoviruses

include the human metapneumoviruses [32, 33]. All these viruses cause severe

bronchiolitis and pneumonia in affected individuals, and like the HPIVs are more

dangerous to the immunocompromised, elderly and infants. In fact, HRSV and

HPIVs are the two major causes of hospitalization in children less than 5 years of

age suffering from respiratory tract infections.

1.2 Structure and composition

Virions are typically spherical, but filamentous forms have also been observed.

The viral envelopes are derived from host cell plasma membranes during virus

budding. Embedded in the envelope are two different kinds of transmembrane

glycoproteins - the attachment protein (HN/H/G), and the fusion protein F. The

paramyxovirus genome is ~ 15-19 kb, and is a negative-sense, non-segmented

RNA in the form of a ribonucleoprotein (RNP), encapsidated by the nucleocapsid

(NP/N) protein. It encodes for about 6-10 genes. The gene order is typically 3’-

NP-P/V/W-M-F-HN/H/G-L-5’ (Fig.1.2). Each gene has a start and end signal, and

is separated from other genes by non-coding intergenic regions [1]. The NP-

encapsidated genome, along with phosphoprotein P and RNA dependent RNA

polymerase (RdRp) L, forms the polymerase complex. Between the glycoprotein

layer and the genome lies the peripheral membrane protein matrix (M) (Fig.1.3),

that functions as an adaptor between the viral genome and the glycoproteins at

sites of assembly, helping to form attachment and fusion capable infectious

viruses.

Figure 1.2. Gene orders among different paramyxoviruses. Schematic detailing the order of genes among representative paramyxoviruses from both the Paramyxovirinae and Pneumovirinae sub-families. Not all paramyxoviruses produce the C protein from their P/V genes. Pneumoviruses have additional genes such as M2, NS1 and NS2 not found among viruses of sub-family Paramyxovirinae [34].

Figure 1.3. Structure of a paramyxovirus virion. (A) Schematic showing the overall structure of a paramyxovirus particle. The M protein functions to link the helical RNP and envelope glycoproteins HN/H/G and F together, to form a functional virion. (B) TEM of a mumps virion purified by ultracentrifugation on sucrose gradients. The viral glycoproteins embedded in the envelope are visualized as a spike layer [35].

The nucleocapsid protein is an RNA binding protein that encapsidates the viral

genome to form a helical ribonucleoprotein (RNP) template that is the

biologically active form of the RNA genome [36]. This RNP serves as the template

for genomic transcription and replication [37]. One molecule of NP binds every

six nucleotides and ~13 NP subunits form one turn of the helix [37, 38]. NP

binding to viral RNA protects it from digestion by RNases and also from

recognition by host innate immune responses [37]. NP exists in two forms in the

cell- one associated with the viral genome and the other in a soluble form

termed N0. N0 binds to P protein as seen in a number of paramyxoviruses like

Sendai virus and measles virus [39, 40]. It is also thought to be responsible for

binding to nascent viral RNA during genome and antigenome replication. Except

for measles virus, NP proteins haven’t been shown to interact with any host

proteins. Measles virus N binds to chaperone protein Hsp72 in cells [41], and

this binding could potentially influence morphology of nucleocapsids and viral

RNA synthesis. NP binds to viral M proteins in addition to P protein, and it is this

interaction that helps the NP encapsidated viral genome to be packaged into

newly budding virus particles [42, 43].

The P gene typically encodes for multiple proteins- P/V/W/I/D- that are

produced by RNA editing or C/C’/Y1/Y2 generated by ribosome recognition of

alternative translation initiation sites [1]. RNA editing involves the non-

templated addition of 2-4 guanine nucleotides, causing a frame-shift in the open

reading frame [44]. This generates proteins that have common N-terminal

regions but different C-termini. Serine and threonine residues in the N-terminal

region of P protein are heavily phosphorylated, and this N-terminal region is

responsible for binding to N0 during genome replication. The C-terminal region

binds primarily to N-tail on the N:RNA template and L protein, bringing these

two together to form a functional polymerase complex [45–47]. The V protein is

typically involved with suppression of host anti-viral responses and is also a

negative regulator of viral RNA synthesis [48, 49]. Viruses lacking V protein

show increased RNA synthesis and can also be cleared by the immune system

more efficiently than wt viruses [50]. W/D/I have roles similar to V protein viz.

interfering with RNA synthesis and anti-viral activities in the host [51]. C

proteins- C, C’, Y1,Y2 are produced by leaky scanning mediated by ribosome

recognition of alternative start codons. These proteins can have multiple

functions like antagonizing anti-viral responses in the host, minimizing viral

RNA synthesis, improving viral assembly and egress. For example, Sendai virus C

protein increases VLP budding, via interaction with host proteins involved in the

ESCRT pathway like Alix [52]. Proteins encoded by the P/V/C gene vary among

paramyxoviruses; for example, rubulaviruses do not have C proteins,

henipaviruses and morbiliviruses have one C protein, while respiroviruses like

Sendai virus have all four C proteins. Similar trends have been observed for the

P/V/W/I/D proteins too. Excepting the P protein, other protein products of the

P/V/C gene are not packaged efficiently into virions [1].

Matrix protein is the main driver of virus assembly and is the most abundant

protein in the virion [1]. M proteins are capable of self-oligomerization,

electrostatic and hydrophobic interactions with the plasma membrane and

interaction with viral glycoproteins and NP proteins. M binds to cytoplasmic tails

of both attachment and fusion glycoproteins. Deletion of cytoplasmic tails in

PIV5 F and/or HN leads to loss of their interaction with PIV5 M protein and

failure to produce virus particles efficiently [53]. M binds to NP protein that

encapsidates the viral RNA genome, thus helping the genome to enter particles,

producing infectious virions. If this interaction is disrupted, then empty non-

infectious virus particles are formed. M also hijacks a number of host proteins

during assembly, for example, AP3B1 is needed for efficient henipavirus M-

directed assembly [54] while 14-3-3 [55], AmotL1 [56] and Rabin8 (Zifei Pei,

unpublished data) play a role in PIV5 and mumps virus assembly. Caveolin-1

binds to PIV5 M and is important for assembly of infectious viral particles [57].

Recent reports have shown that actin binding protein cofilin1, caveolae protein

caveolin 2 and zinc finger protein ZNF502 bind to RSV M protein and play a role

in HRSV assembly [58]. In addition to these, Importin β1 [59] and CRM-1 [60],

nuclear import and export factors respectively, also bind to HRSV M and help it

traffic in and out of the nucleus, where it inhibits host protein transcription [58].

NDV M protein binds to B23, a nucleolar phosphoprotein that targets it to the

nucleoli for viral replication [61]. Ubiquitination of M protein is also critical for

normal budding functions and inhibition of the ubiquitin-proteasome machinery

negatively impacts virus budding [62–64]. Viral M proteins have been shown to

often include a protein-protein interaction motif called a “late domain” that

serves to recruit host proteins that are typically involved in the ESCRT

(endosomal sorting complex required for transport) pathway. Host proteins,

thus recruited, help M to traffic properly through the cell and reach its target

microdomains on the plasma membrane. Sendai virus, for example contains a

conventional late domain motif YLDL that binds to Alix, an ESCRT pathway

member [65], and is crucial for proper virus assembly. Paramyxovirus M

proteins however, often possess unconventional late domain motifs whose

binding partners are not known. Some examples of these are FPIV/FPVI motif in

PIV5, mumps and NDV M proteins [66–68] and YMYL [69] and YPLGVG [70]

motifs in Nipah virus M protein.

The attachment proteins are type II integral membrane proteins and are

tetrameric; they can use sialic acid-based or protein-based receptors for

attachment to the cell surface. For example, PIV5 and mumps virus HN proteins

use sialic acid, while measles virus and henipavirus H or G proteins use protein

receptors like SLAM/CD46/ Nectin 4 [71–73] and EphrinB2/B3 [74, 75]

respectively. Attachment proteins from respiroviruses and rubulaviruses have

an additional component- the neuraminidase, which has sialidase activity,

cleaving sialic acid moieties off the hemagglutinin to prevent virus particle

aggregation, or re-attachment of newly released virus particles to the cell

membrane [1]. The fusion protein is a Type I integral membrane protein, is

homo-trimeric and exists as an inactive precursor F0. The F0 form undergoes

cleavage at specific cleavage sites by host enzymes like furin [76] or cathepsin

[77] to form disulfide bond linked F1 and F2. F proteins that are cleaved by furin

undergo cleavage as they are transported through the trans-Golgi, part of the

secretory pathway, on their way to the plasma membrane. Fusion proteins from

Nipah and Hendra viruses are cleaved differently. They first reach the plasma

membrane and get endocytosed, following which they undergo cleavage by

cathepsins in the acidic endosome [77]. Rab-11 containing recycling endosomes

then mediate the trafficking of the cleaved and active F protein to the plasma

membrane (Weina Sun, unpublished data). The cleaved form of F protein, along

with the attachment glycoprotein, mediates fusion of virus with the cell

membrane or fusion between multiple cells, forming multi-nucleated giant cells

called syncytia, a characteristic feature of paramyxoviruses. This fusion activity

has been exploited in creating hybridoma cells to produce monoclonal

antibodies using Sendai virus [78]. Fusion occurs at neutral pH, and releases the

viral genome into the cell cytoplasm. Rubulaviruses like PIV5 and mumps virus

have an integral membrane protein SH (small hydrophobic) that plays a role in

inducing apoptosis. Viruses lacking SH protein display an attenuated phenotype

in cell culture. Pneumoviruses also have SH proteins, with functions similar to

those of rubulaviruses [79–81].

L (large) protein is the viral RNA dependent RNA polymerase (RdRp). It forms

the catalytic unit of the polymerase complex. L protein can form homo-

oligomers, can bind to P protein as well as accessory C proteins. L can also bind

to host proteins; Sendai and measles virus L proteins bind to tubulin [82, 83] and

this enhances L activity. Binding to C proteins can have an inhibitory effect on

RNA synthesis [84]. L is responsible for 5’-end methylation and capping as well

as 3’ poly-adenylation. Poly-(A) tail addition takes place when the viral

polymerase encounters a string of U residues at the end of each viral gene. L

stutters at these regions and adds a long string of adenines forming the poly-(A)

tail. L is found in very low amounts in infected cells and virions.

1.3 Life cycle of paramyxoviruses

The life cycle initiates when virus attaches to a cell. The attachment protein

functions to attach to a specific receptor, either sialic-acid based glycoproteins or

glycolipids or protein receptors. Attachment to the receptor triggers fusion

between the viral envelope and the host cell plasma membrane. Once the virus

enters the cell, either in this manner or by macropinocytosis [1, 85], the M

protein disengages from the NP-encapsidated genome. How the uncoating

occurs is not very clear. Transcription, replication and virus assembly take place

in the cytoplasm of infected cells (Fig.1.4). Since these viruses have a negative-

sense RNA genome, the genome cannot be translated to make proteins directly.

Instead, the viral RdRp has to initially make positive-sense mRNA transcripts,

and then these transcripts can be used for translation into proteins. Non-

segmented negative-strand RNA viruses like paramyxoviruses, filoviruses,

rhabdoviruses employ a method of transcription called stop-start transcription.

The polymerase initiates at the 3’ end of the RNA genome, it then progresses

along the gene till it reaches the gene end. At the end of each gene, there are

poly-U tracts that cause the polymerase to stutter and form a long poly-A tail.

The polymerase then either continues along the intergenic region on to the next

gene or falls off. This leads to a gradient in transcription, where the genes that

are closer to the 3’ end of the genome are transcribed to higher levels than those

at the 5’ end [1]. When sufficient copies of NP are present, replication of the

genome takes place. The negative sense RNA genome is first replicated by the

RdRp to form an antigenome that is positive sense. At this stage, the RdRp is

capable of ignoring gene start and end signals and reads through the entire

genome to form the antigenome. This is simultaneously coupled with NP

encapsidation of nascent viral RNA. The antigenome is then used as a template

by the polymerase to form copies of negative-sense genomes that are NP-

encapsidated to form a helical ribonucleoprotein [1].

When sufficient viral proteins and genomes accumulate in the cell, M protein

co-ordinates virus assembly at the plasma membrane. Here, M binds to

cytoplasmic tails of attachment and fusion glycoproteins and also to

nucleocapsid proteins encapsidating the genome, thus serving to concentrate all

the necessary components needed for an infectious virus. Following this, a bud is

formed at the membrane, which is then pinched off with assistance from ESCRT

family members like Vps4A. Henipaviruses, measles virus and HRSV do not need

ESCRT proteins [70, 86, 87] and how they bud out from the membrane is

unclear. Paramyxoviruses like PIV5, Sendai virus, measles virus and mumps

virus have been shown to bud preferentially from the apical surface of polarized

epithelial cells [88–90]. Apical budding is thought to restrict infections to

epithelial cell layers whereas basolateral budding of viruses can lead to more

systemic infections. However, this is not absolute, as systemic infections have

been observed for viruses that primarily bud from apical surfaces, like measles

virus and henipaviruses [91, 92].

Figure 1.4. Paramyxovirus life cycle. Schematic showing life cycle of paramyxoviruses. Following attachment to and fusion with the cell membrane, viral contents are delivered into cells. Viral proteins are synthesized first, followed by viral antigenomes, which are then used as templates for producing more viral genomes. When sufficient viral proteins and genomes have accumulated, virus assembly is initiated, ultimately leading to release of new infectious virions [93].

1.4 Paramyxovirus virus-like particles

Virus-like particles (VLPs) resemble virions both structurally and

morphologically, however they lack any infectious viral genome. VLPs are useful

to study mechanisms of virus assembly and budding, more so for infectious

viruses that might require handling in BSL-3 or -4 conditions. VLPs are typically

produced by over-expressing a minimal set of viral proteins required for their

formation. This requirement varies between paramyxoviruses. For viruses like

Henipaviruses, only the M protein suffices to produce VLPs [94]. Addition of any

other viral protein alongside M has no effect on VLP production, save to

incorporate those protein into VLPs if they bind to M. However, for some other

paramyxoviruses like PIV5 and mumps virus, the M protein alone is not

sufficient for VLP production (Table 1.1). Co-expression of a viral glycoprotein

and the NP protein are both absolutely essential to produce successful VLPs [68,

95]. These proteins might be inducing M to enter a budding-active state upon

interaction, or else they facilitate interactions with host proteins important for M

budding activities. VLPs have multiple uses, for e.g., they have been used

successfully to produce vaccines against viruses like human papillomavirus and

Hepatitis B virus [96, 97]. VLPs can also be produced in multiple culture systems

like mammalian cell lines, plant cells, insect cells, bacteria and yeast [98–102].

Table 1.1. Requirements for VLP production among paramyxoviruses. Minimal requirements for efficient VLP production vary between paramyxoviruses. While Nipah virus or NDV need only the M protein for VLP production, PIV5 and mumps virus need the co-expression of a glycoprotein and the NP protein along with the M protein [35].

1.5 Paramyxovirus matrix-nucleocapsid interactions

Interaction between M and NP/N proteins in paramyxoviruses and other

negative-strand RNA viruses is important for the generation of genome-

containing infectious virus particles, however, these interactions are poorly

understood. Biochemical evidence of M-NP interactions has been shown multiple

times in paramyxoviruses [42, 43, 103, 104]. For measles virus, the C-terminal

region of N protein was shown to be important in binding to measles virus M

protein, via a DLLD motif. Measles virus M and N proteins also co-localized at

membranes in transfected or infected cells. Recombinant measles virus that

harbored N protein defective for interacting with M protein exhibited lower

titers than parental virus [43]. Similarly, for PIV5, a DLD motif at the C-terminal

region of NP protein was found to be important for interaction with M protein

[42]. For PIV5, virus-like particle production is optimal only when the M, NP and

glycoprotein/s are co-expressed. In the absence of NP protein, VLP production is

sub-optimal [95]. Disrupting the DLD motif in PIV5 NP protein led to defective

VLP production, due to the loss of M-NP interaction. Recombinant viruses

harboring NP protein mutants adapted by generating second-site mutations in

viral M protein, or proteins involved in the polymerase complex, depending on

the cell-type used to rescue virus. Second-site mutations in M protein often

restored interaction with defective NP protein. Mutations in the polymerase

complex increased transcription leading to higher protein levels of viral

attachment and fusion glycoproteins; this increased cell-cell fusion causing more

efficient virus spread. This indicates that the viral M-NP interaction is very

critical for successful release of infectious virus. In its absence, viruses could

adapt either by mutating their M protein to recover interaction with NP, or by

increasing viral transcription via mutations in the polymerase complex to ensure

efficient cell-cell spread, thus bypassing the need to bud out infectious particles

[42].

1.6 ESCRT pathway and late domains

Viruses do not encode all of the protein machinery required for virus egress.

Instead, they utilize cellular pathways that facilitate functions in the cell similar

to virus budding. One among these is the multi-vesicular body (MVB) pathway.

This pathway is typically used to recycle or degrade transmembrane protein

receptors or transporters. Proteins are first endocytosed at the membrane and

enter early endosomes, from where they are either recycled back to the plasma

membrane via recycling endosomes, or are internalized into intraluminal

vesicles within a multivesicular body [105]. The intraluminal vesicles bud into

the multivesicular body, and this event is topologically similar to the outward

budding of viruses at the plasma membrane. This multivesicular body then fuses

with a lysosome to promote degradation of its cargo. Attachment of ubiquitin to

lysine residues in a protein signals it for lysosomal degradation, either in the

form of multiply monoubiquitinated lysine residues or polyubiquitin chains

linked to K63 in ubiquitin.

Based on studies in yeast, a number of proteins have been discovered to be

involved in MVB biogenesis. These are referred to as “vacuolar-protein sorting”

(Vps) proteins and form a part of the endosomal sorting complex required for

transport (ESCRT) machinery. The ESCRT machinery is made up of 5 complexes-

ESCRT-0, -I, -II, –III and the Vps4 with each complex composed of several

proteins [105, 106]. ESCRT-0 localizes to endosomal membranes via its FYVE

(Fab1/YOTB1/Vac1/EEA1) domain interaction with phosphatidylinositol 3-

phosphate. There, it binds ubiquitin present on cargo proteins and recruits other

complexes to endosomes via interaction with ESCRT-I. ESCRT-I in turn recruits

ESCRT-II; these complexes are also involved in membrane budding and can help

to stabilize the bud neck of a nascent vesicle. ESCRT-III recruits de-

ubiquitinating enzymes, which mediate recycling of ubiquitin attached to cargo

proteins before vesicle formation. Vps4-AAA ATPase is responsible for the final

step involving disassembly of ESCRT complexes from the vesicle and severing

the newly budded vesicle from the endosomal membrane and into the

multivesicular body. In addition to their role in MVB biogenesis, the ESCRTs also

function in cytokinesis. Mutant non-functional forms of Vps proteins lead to

blocks in the ESCRT pathway, leading to defects in MVB biogenesis or

cytokinesis [107, 108]. ESCRT proteins recruit other cellular proteins by

employing protein-protein interaction motifs. For example, Tsg101/ESCRT-I

binds to cellular proteins containing a P(T/S)AP motif, while Nedd4-like E3

ubiquitin ligases contain WW domains that bind to cellular proteins containing

PPXY motifs, forming an indirect link to the ESCRT pathway. Alix binds to YXXL

motifs on proteins and subsequently recruits those proteins to ESCRT-III. The

proteins thus recruited by ESCRT members then carry out their functions within

the desired membrane microdomains.

Virus budding is toplogically equivalent to MVB biogenesis. Viral structural

proteins responsible for the budding process have been shown to contain motifs

similar to those seen in cellular proteins that link to the ESCRT pathway [109].

For example, HIV-1 Gag has a PTAP and YPDL motif and can bind to Tsg101 and

Alix respectively [110, 111]. Ebolavirus VP40 contains a PTAPPEY motif and is

capable of binding to both Tsg101 and Nedd4-like E3 ubiquitin ligase members

[112]. These viruses are dependent on the ESCRT pathway for their budding

(Fig.1.5). Disruption of the PTAP/PPXY/YXXL motifs in these viruses can lead to

a defect at a late step in budding, where the virus particles remain tethered to

the plasma membrane and fail to pinch off. These motifs are therefore referred

to as “late domains”. Late domains have been discovered in a number of negative

strand RNA viruses and retroviruses [113, 114]. However, some NNSVs like

paramyxoviruses do not have conventional late domain motifs. For instance,

members of the rubulavirus genus like PIV5 and mumps contain “FPIV/FPVI”

motifs [66, 68] in their M proteins that have not been shown to interact with any

other protein. However, disruption of these motifs can lead to a severe defect in

M protein function, leading to loss of budding. Moreover, if these motifs are

fused to crippled Gag proteins that lack their late domains, they can rescue Gag

budding activity, leading to the conclusion that they are involved in budding.

Expression of dominant-negative (DN) versions of ESCRT pathway components

can block PIV5 budding. This suggests that FPIV motifs or some as yet

undiscovered protein domain must somehow be involved in the ESCRT pathway.

Similarly, henipavirus M proteins contain late domain motifs YMYL and YPLGVG

[69, 70] that play a role in budding but haven’t been proven to interact with

other cellular proteins. However, henipaviruses do not utilize the ESCRT for

their assembly.

Figure 1.5. The ESCRT pathway in viral budding. Schematic showing the involvement of the ESCRT components in virus budding. Viral late domains PTAP, YXXL and PPXY bind to ESCRT- I/Tsg101, Alix and Nedd4-like E3 ubiquitin ligases respectively, thereby connecting to the ESCRT and facilitating virus budding.

1.7 Angiomotins

Angiomotin was first identified in a yeast two-hybrid screen of a human

placenta cDNA library, using kringle domains 1-4 of angiostatin as bait [115]. It

is expressed at high levels in endothelial cells and mediates endothelial cell

migration. Angiostatin binding to angiomotin disrupts its normal functions and

can inhibit endothelial cell migration. Two isoforms of angiomotin exist within

cells- a p130 and a p80 isoform generated via alternative splicing. Two other

proteins belong to this family based on structural and functional similarities.

These are tight-junction associated Angiomotin-like1 (AmotL1)/ junction-

enriched-and-associated protein (JEAP) and Angiomotin-like 2 (AmotL2)/MAGI-

1-associated coiled-coil tight junction protein (MASCOT) [116]. These proteins

are characterized by LPTY or PPXY motifs in their N–terminal domains (lacking

in Amot p80) that bind to WW domains in a variety of cellular proteins,

primarily members of the Nedd4 family of E3 ubiquitin ligases [117], and yes-

associated protein (YAP) [118–120]. These motifs are highly conserved even

between different species. They also have a conserved coiled-coil (CC) domain

and a PDZ binding domain in their C-terminal regions. The coiled coil regions are

responsible for homo- or hetero-oligomerization between motins, and show

similarities to the amphiphysin BAR (Bin-Amphiphysin-Rvs) domain that is

responsible for either positive or negative membrane curvature via actin

binding. Angiomotin differs from the other two members in that it has an

angiostatin-binding domain in its C-terminal region (between CC and PDZ

domains) that binds to angiostatin and plays roles in endothelial migration. The

conserved consensus motif in the PDZ domain indicates the involvement of

angiomotins in signaling pathways. Motins are expressed in different tissues at

different levels, and within a tissue they show spatiotemporal differences in

expression. For example, AmotL1 is highest in skeletal muscle while AmotL2 is

enriched in mammary tissue. Amot is expressed to high levels in the testis, brain

and thyroid [115].

Angiomotins also share some functional redundancy and are involved in

Hippo/YAP signaling [119], in small G-protein signaling and in endothelial cell

migration [121] and angiogenesis [122]. Angiomotins associate with

Patj/Mupp1 polarity complexes [115] via their PDZ domains and this recruits

Syx, a RhoGEF (RhoA GTPase exchange factor) to the leading edge of migrating

endothelial cells. Knockout of angiomotins in mouse or zebrafish embryos

causes early lethality due to gross defects in formation of vasculature.

Angiomotins associate with tight junctions and can influence epithelial cell

polarity [123, 124]. At tight junctions, Amot binds to Patj and Pals1 which are

cytoplasmic scaffolding proteins; it also binds to a Cdc42/Rac1 GTPase

activating protein called Rich1 and Merlin via their mutual CC/BAR domains.

Binding of Amot to Rich1 inhibits its GAP activity that can impair tight junction

integrity. Merlin inhibits Rac1 activity. Competitive binding between Merlin and

Rich1 for Amot can help to release Rich1 that further inhibits Rac1 through its

GTPase and can modulate downstream signaling pathways like MAPK and p21-

activated kinases, which lead to overall effects on cell polarity. Angiomotins are

downstream regulators of the Hippo pathway that plays roles in cell

proliferation, apoptosis, contact inhibition. Several kinases are involved in this

pathway, viz. the Mst1/2 (Hippo in flies) kinases in complex with WW45 scaffold

protein phosphorylate Lats1/2 kinases and Mob1. These in turn act on Mob1

kinase that phosphorylates YAP, leading to inhibition of its normal function.

Unphosphorylated YAP enters the nucleus and binds to TEAD-domain

transcription elements leading to expression of downstream genes that control

cell proliferation and exhibit anti-apoptotic activity. Overexpression of YAP

consequently displays tumorigenic activity in cells. Phosphorylation of YAP via

the Hippo pathway prevents it from entering the nucleus to exert its function.

Angiomotins have effects on YAP function, both in a Hippo pathway dependent

and independent fashion [125]. A number of Hippo pathway mediators can bind

to angiomotins viz. Merlin, KIBRA, Lats1/2 kinases, leading to its

phosphorylation and increased stability. In addition, angiomotins can also bind

WW domains in YAP via their LPTY/PPXY motifs. Binding of angiomotins to YAP

can sequester it in the cytoplasm and target it for degradation [118, 119].

However, Amotp130 has also been associated with YAP in the nucleus,

suggesting that it can have other effects on YAP function. Angiomotins are

important in virus budding too; AmotL1 was shown to bind to PIV5 M protein.

Knockdown of AmotL1 reduced PIV5 virus budding in 293T cells [56]. In

addition to these, all the three motins were shown to bind HIV-1 Gag. Amotp130

and AmotL2 could function as adaptors between Gag and Nedd4L E3-ubiquitin

ligases and rescue budding of a p6-deleted Gag protein [126]. Nedd4 E3

ubiquitin ligases contain WW domains via which they bind to PPXY motifs in

angiomotins. Nedd4 E3 ubiquitin ligases also have roles in the ESCRT pathway

and they might be able to rescue HIV-1 Gag budding by linking it to other ESCRT

complexes.

1.8 Preview

The M and NP protein interactions are crucial for packaging genomes into

budding paramyxovirus particles, thereby creating infectious viruses. For some

paramyxoviruses like PIV5 and mumps virus, NP protein is also required for

efficient virus-like particle production. Previous work showed that the extreme

C-terminal region of PIV5 NP protein contains a DLD motif that is important for

M binding [42]. Disruption of this motif lead to defects in M binding as well as

PIV5 VLP production, suggesting that the DLD sequence was crucial for binding

with M protein, which in turn stimulated VLP budding. In order to further dissect

the role of NP in stimulating budding, we conducted experiments to determine

how much of the NP protein was sufficient for VLP production. We identified a

minimal region on PIV5 NP protein (~15 amino acids long) that can replace the

requirement for full-length NP protein in VLP enhancement when appended to a

foreign protein, and also served to direct the foreign protein into virus-like

particles. We expanded these studies to Nipah virus N protein (which does not

play a role in stimulating Nipah virus budding, but is directed into particles via

interaction with M), and showed that a 15 amino acid DLD-containing sequence

from the C-terminal region of Nipah virus N protein could direct luciferase into

Nipah VLPs when appended to them. Disruption of this DLD-containing

sequence impaired incorporation of luciferase, indicating that this sequence was

critical for binding to Nipah virus M protein. In addition to this, we showed that a

DWD sequence at the C-terminal end of mumps virus NP protein was important

for M-binding and efficient mumps VLP production. D-x-D type motifs are

present in the C-termini of most paramyxoviral NP proteins and we hypothesize

that NP proteins have evolved along a universal theme to use D-x-D motifs to

guide interactions with M proteins. We also showed that M and NP proteins

from different paramyxoviruses are capable of interacting with each other.

When interaction is poor, mutation to favorable amino acid residues can

strengthen binding. In this way, we managed to create a novel PIV5 NP protein

that is capable of binding to three paramyxovirus M proteins-PIV5 M, mumps

virus M and Nipah virus M proteins.

In addition to viral proteins like NP, certain host proteins are also recruited

during virus budding. AmotL1 for instance, is important for budding of PIV5 as

knockdown resulted in impaired virus release; AmotL1 can form strong

associations with PIV5 M protein [56]. AmotL1 contains PPXY motifs also seen in

viral M protein analogues from other negative strand RNA viruses or

retroviruses. Through their PPXY motifs, these M protein analogues are capable

of binding to WW domain containing members of the Nedd4 E3 ubiquitin ligase

family that might connect them to the ESCRT, eventually facilitating virus

budding. PIV5 budding utilizes ESCRT pathways, as expression of dominant

negative mutants of ESCRT proteins impair virion release [66]. However, how

PIV5 proteins connect to the ESCRT is not understood. We hypothesize that PIV5

M might indirectly bind to Nedd4 proteins via the AmotL1 protein, thus using

the Nedd4 ligases as links to the ESCRT for budding. We have successfully

shown here that AmotL1 can function as a linker between PIV5 M and Nedd4

proteins. Surprisingly, binding of PIV5 M is restricted only to AmotL1 and not to

any of the other two motins. This effect is observed for mumps virus M protein

too. Further studies need to be carried out to understand exactly how AmotL1

and the Nedd4 proteins can facilitate PIV5 and mumps virus budding as also

whether they are involved in directing virus egress in other paramyxoviruses.

CHAPTER 2

MATERIALS AND METHODS

Plasmids.

Packaging of foreign proteins into VLPs and M-NP interaction. cDNA for coding sequences of PIV5 NP, PIV5 M, PIV5 HN and PIV5 F were derived from the

SV5 infectious clone pBH276 (GenBank accession no. AF052755) and were subcloned into eukaryotic expression vector pCAGGS [95]. Plasmids pCAGGS-MuV

M, pCAGGS-MuV NP and pCAGGS-MuV F (Iowa strain) are kind gifts of Dr. Biao He

(University of Georgia, Athens, GA). pCAGGS-NiV M and pCAGGS-NiV N are kind gifts of Dr. Paul Rota ( Centers for Disease Control and Prevention, Atlanta, GA). Site- specific mutants of PIV5, mumps virus and Nipah virus NP/N genes were created by

PCR mutagenesis of the wt sequences and the resulting mutant cDNAs were subcloned into pCAGGS [127]. Renilla luciferase cDNA was obtained by PCR amplification from plasmid pSMG-RLuc, which was a kind gift of Dr. Biao He

(University of Georgia, Athens, GA). This sequence was modified via PCR to include

C-terminal sequences derived from PIV5 NP, Nipah virus N and mumps virus NP, with a double glycine (-GG-) linker inserted between the luciferase gene and virus- derived sequences. The resulting modified luciferase cDNAs were subcloned into pCAGGS. pCAGGS-RLuc-NP15 (derived from PIV5 NP) was then modified by PCR mutagenesis to remove the sequence encoding for the C-terminal four amino acid residues (-DLDI) to generate pCAGGS-RLuc-NP15∆4, while pCAGGS-RLuc-N15

(derived from NiV N) was modified by PCR mutagenesis to generate a set of alanine

31 substitution mutants. cDNAs for SOD1, SerpinB3 and ARG1 were purchased from

Transomic technologies (Huntsville, AL), these cDNAs were amplified using PCR and then cloned into pCAGGS, with a myc tag (EQKLISEEDL) at their N-terminal ends.

Myc-SOD1, ARG1 and SerpinB3 were further modified by subcloning to incorporate

PIV5 NP derived sequences, using the same cloning strategy used earlier for the

RLuc-NP fusions. The resulting modified cDNAs were subjected to PCR mutagenesis to create DWD versions of the wt DLD sequences derived from PIV5 NP. DWD versions of RLuc-PIV5 NP15 and RLuc-PIV5 NP30 were also generated.

Matrix-Angiomotin-Nedd4 interactions. cDNA for Nedd4-1 was purchased from

Transomic technologies (Huntsville, AL), cDNAs for Nedd4L, Nedd4L C942A,

Nedd4LΔWW were obtained from the plasmid repository at DNASU (Arizona State

University, Tempe, AZ); these were PCR amplified and inserted into pCAGGS with a myc tag at their N-termini. cDNA for NedL1 was a kind gift of Dr.Wes Sundquist

(University of Utah, Salt Lake City, UT) and was also PCR-amplified and cloned into pCAGGS with a myc tag at its N-terminal end. cDNAs for AmotL2, Amotp130,

Amotp80 were obtained from DNASU (Arizona State University, Tempe, AZ), and were PCR amplified and cloned into pCAGGS with a Flag (DYKDDDDK) tag at their

N-termini. Various truncated forms of these genes were created with either N- or C- terminal Flag tags and inserted into pCAGGS. Constructs for AmotL1 have been described before [56]; briefly cDNA for AmotL1 was cloned into pCAGGS with N- or

C-terminal FLAG tags. DNA sequencing of the entire genes was carried out to verify their identities (Genomics Core Facility, The Pennsylvania State University).

Antibodies.

PIV5 antibodies M-f, M-h, NP-a, NP125, HN1b [128] were kind gifts of Richard

Randall (St. Andrews University, St. Andrews, Scotland, UK). M-f was used for IP and western blotting experiments to pulldown or detect PIV5 M, while M-h was used to detect M in immunofluorescence microscopy. NP125 was used for western blotting to detect PIV5 NP while NP-a was used for NP pulldown in IP experiments. HN1b was used for IP of PIV5 HN. PIV5 HN protein was detected on western blots using the polyclonal antibody SDS-HN, a kind gift of Robert Lamb (Northwestern

University, Evanston, IL). Mumps virus M, NP, F and HN proteins were detected and/or pulled down using polyclonal anti-peptide antibodies that have been described before [68]. Nipah virus M and N proteins were detected using polyclonal antibodies raised against the full-length recombinant proteins [68]. Renilla luciferase was detected using a polyclonal antibody purchased from MBL

International (Woburn, MA). AmotL1 was detected using a polyclonal antibody described before [56]. All myc-tagged proteins were detected or pulled down using a monoclonal antibody against the myc tag 9E10 that was produced in-house. All flag-tagged proteins were detected by an anti-Flag (clone M2) mouse monoclonal antibody purchased from Stratagene (La Jolla, CA). Flag-tagged proteins were pulled down using anti- Flag M2 magnetic beads purchased from Sigma Aldrich

(St.Louis, MO). Flag-tagged angiomotin proteins in immunofluorescence studies were visualized using anti-DDK monoclonal antibody specific for flag epitopes

(Origene, Rockville, MD).

Membrane co-flotation assays measuring M-NP interactions.

293T cells in 6-cm diameter dishes were grown to ~70-80% confluency in

Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and were then transfected with M and/or NP proteins from PIV5, NiV or MuV

(1.2 µg of M and 0.8 ug of NP). Lipofectamine-Plus reagents in Opti-MEM were used for transfection (Invitrogen, Carlsbad, CA). 24h post transfection, cells were harvested and first rinsed with 1X PBS. They were then scraped off the dishes and re-suspended in 0.6 ml of hypotonic buffer (25 mM NaCl, 50 mM Na2HPO4, pH 7.4; 1 mM phenylmethyl-sulfonyl fluoride) with rocking for 20 mins/4C. Cells were then passaged about 20x through a 23-gauge needle to lyse them. Nuclei and other debris were cleared through a 5 min centrifugation step at 1500 x g/4C. Supernatants containing cellular membranes were then mixed with 1.5 ml of 80% sucrose in NTE

(0.1 M NaCl; 0.01 M Tris-HCl, pH 7.4; 0.001 M EDTA). This was then overlaid with

2.4 ml of 50% sucrose and 0.6 ml of 10% sucrose to form a gradient. Samples were spun in an ultracentrifuge at 160,000 x g/4h/4C using a Sorvall AH-650 swinging bucket rotor (Thermo Fisher Scientific, Waltham, MA). 4 x 1.25 ml fractions were then collected beginning from the tops of the gradients using an Auto Densi-Flow fraction collector (LabConco, Kansas City, MO). 2% of each fraction was mixed with an equivalent volume of 1X PLB and loaded onto a 10% SDS-PAGE gel. Proteins were next subjected to western blotting and detection was carried out using antibodies specific for M/NP proteins. Bands were visualized and quantified using a

Fuji FLA-7000 laser scanner (FujiFilm Medical Systems, Stamford, CT). NP protein in

34 membrane fractions was quantified by measuring the amount of NP protein in the top two fractions divided by the sum of NP protein in all four fractions.

Measurements of VLP production.

293T cells in 6-cm-diameter dishes were cultured at about 70-80% confluency in

DMEM supplemented with 10% fetal bovine serum and were transfected with pCAGGS plasmids encoding proteins for PIV5, mumps virus, or Nipah virus for generating PIV5, mumps, or Nipah virus-like particles (VLPs). In some cases, host proteins were also expressed along with the viral proteins. Lipofectamine-Plus reagents in Opti-MEM were used for plasmid transfection. Plasmid quantities per dish were as follows- pCAGGS-PIV5 M – 0.4 ug, pCAGGS-MuV M – 0.4 ug, pCAGGS-

NiV M – 0.4 ug, pCAGGS-PIV5 NP and derivatives – 0.1 ug, pCAGGS-MuV NP and derivatives – 0.1 ug, pCAGGS-NiV N and derivatives – 0.1ug, pCAGGS-RLuc – 0.1 ug, pCAGGS-RLuc-PIV5 NP/NiV N/MuV NP fusions – 0.1 ug, pCAGGS-PIV5 HN - 1.5ug, pCAGGS-MuV F – 0.1 ug, pCAGGS-FL. AmotL1/AmotL2/Amotp130 and variants-0.1 ug. An empty pCAGGS plasmid that does not encode any viral protein was included wherever required in order to equalize plasmid amounts across samples.

At 24 h p.t., the transfection medium was substituted for DMEM with 2% fetal bovine serum, or for metabolic labeling experiments, DMEM containing one-tenth the normal amount of cysteine and methionine, along with 37 µCi of (35S)

Promix/ml (Perkin Elmer, Waltham, MA) was used. 16-18 h later, cells and media were harvested. Culture media were initially centrifuged at 8,000 x g for 2 min to remove cell debris. Supernatants were then layered onto 20% sucrose cushions (4

35 ml in NTE). Samples were now subjected to ultracentrifugation at 140,000 x g for

1.5 h, after which VLP pellets were re-suspended in 0.9 ml of 1X phosphate-buffered saline (PBS) (0.13 M NaCl; 2.6 mM KCl; 1.4 mM KH2PO4; 8.0 mM Na2HPO4.7H20; pH

7.4), and mixed with 2.4 ml of 80% sucrose in NTE. Layers of 50% sucrose in NTE

(3.6 ml) and 10% sucrose in NTE (0.6 ml) were applied to the tops of the gradients, and these were then centrifuged at 140,000 x g for 3 h. 4 ml was collected from the top of each gradient using a fraction collector, and the VLPs contained in this fraction were pelleted by centrifugation at 190,000 x g for 1.5 h. VLP pellets were then re-suspended in SDS-PAGE loading buffer containing 2.5% (wt/vol) dithiothreitol.

For cell lysates preparation, cells from each sample were lysed with 0.15 ml of

SDS-PAGE loading buffer. The lysates were centrifuged through QIAshredder homogenizers (Qiagen, Germantown, MD) to break up cell debris. Lysates and purified VLPs were fractionated by SDS-PAGE using 10% gels, and proteins were detected by immunoblotting using antibodies specific to the viral proteins and/or host proteins. Imaging and quantification was performed using a FUJI FLA-7000 laser scanner. PIV5 and mumps VLP production was measured by calculating the amount of M protein in VLPs, normalized to the amount of M protein present in cell lysates. Luciferase protein or NiV N incorporation into Nipah VLPs was measured by calculating the amount of either protein in particles, divided by the amount of M protein in the same particles.

Measurement of luciferase activity.

PIV5 VLPs, generated as described earlier, were re-suspended in 100 ul of passive lysis buffer (Promega, Madison, WI). 1/3rd of transfected cells were also lysed in 100 ul passive lysis buffer. 5 ul of cell lysate and 20 ul of lysed VLPs were subjected to dual luciferase assay, as per the manufacturer’s protocol (Promega). For experiments to test delivery of luciferase-NP fusions into cells, PIV5 VLPs were generated in 10-cm dishes under sterile conditions and purified using a single ultracentrifugation step at 140,000 x g/1.5h/4C. They were then re-suspended in

PBS and applied to 293T cells in 12-well plates overnight. ~16-20 hrs later, cells were harvested and lysed in 100ul passive lysis buffer, 20ul of which was used for the assay. Activity of Renilla luciferase was measured using a Veritas microplate luminometer (Turner BioSystems, Sunnyvale, CA). Enzymatically active Renilla luciferase incorporated into VLPs was calculated by measuring the luciferase activity in VLPs, normalized to the luciferase activity in cell lysates.

Co-immunoprecipitation assays.

293T cells in 10-cm diameter dishes were transfected with 0.8ug PIV5/MuV M,

1.0ug AmotL1/AmotL2/Amotp130 or variants, and/or 1.5ug Nedd4-1/Nedd4L

C942A/NedL1. 24h p.t., cells were harvested, rinsed with 1X PBS, then lysed in 1ml of Co-IP buffer (10mM Tris, pH 7.4, 150mM NaCl, 0.1mM EDTA, 0.1% Triton X-100,

0.1mM PMSF) for 15-20 mins on ice. Cell debris was then cleared by centrifuging lysates for 30 mins at 14,000 x g/4C. 2% of lysates were taken as inputs, while the rest were subjected to immunoprecipitation with specific antibodies for 3-4 hours.

Protein A Sepharose beads were added next for half an hour to pull down complexes. Alternatively, for immunoprecipitation of Flag-tagged proteins, Flag-M2 magnetic beads were used and addition of protein A sepharose was unnecessary.

Beads were then washed thrice with lysis buffer, and then re-suspended in 1X PLB and boiled. Samples were loaded on a 10% SDS-PAGE gel and blots were then probed with antibodies specific to viral and host proteins.

In cases where proteins were metabolically labeled, 24h after transfection, cells were starved for 20 mins in DMEM containing one-tenth the normal amount of cysteine and methionine. Following this, labeling media containing DMEM deficient in Cys and Met along with 37 µCi of (35S) Promix/ml (Perkin Elmer, Waltham, MA) was added to cells for 3 hours. The same procedures were then followed for co- immunoprecipitation as detailed above. Membranes post blotting or dried gels containing labeled proteins were then imaged using a Fuji FLA-7000 laser scanner.

Immunofluorescence microscopy.

A549 cells were seeded on poly-D-lysine coated cover slips at ~60-70% confluency. They were then transfected with 0.2ug each of PIV5 M,

AmotL1/L2/p130 and Nedd4L in different combinations using Lipofectamine-Plus reagents. ~16-20 hrs p.t., cells were washed thrice with warm 1X PBS. They were then fixed with 4% p-formaldehyde in 1X PBS for 15 mins. Following fixation, 3 x 10 min washes were carried out with 1X PBS. Cells were then permeabilized with 0.5.%

Triton X-100 in 1X PBS for 5 min and washed thrice again. Blocking was then done using a solution containing 0.1% fish gelatin and 1% BSA for 1 hr. Primary and

38 secondary antibodies were then added for 2 hrs and 1 hr respectively, with washes after each incubation. Coverslips were lastly mounted in Prolong Gold antifade reagent containing nuclear stain DAPI (Invitrogen), and sealed with nail polish.

Fluorescently labeled proteins were then visualized using a Zeiss Axioimager M1 fluorescence microscope (Carl Zeiss Inc. Thornwood, NY). Images were captured using an Orca R2 digital camera (Hamamatsu Photonics, Bridgewater, NJ). Images were processed and deconvolved using iVision software (BioVision Technologies,

Exton, PA).

Amino acid sequence comparisons.

Comparison of C-terminal sequences of paramyxovirus nucleoproteins was carried out by deriving data from GenBank files with the following accession numbers: PIV5, AF052755; mumps virus, JN012242; Nipah virus, AF212302; human parainfluenza virus type 2 (HPIV2), M55320; measles virus, AB016162; Sendai virus, M30202; Newcastle disease virus (NDV), AF064091; Hendra virus,

AAC83187; human respiratory syncytial virus (HRSV), AEO45904.

CHAPTER 3

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

[This is a reprint of the published paper (Ray G, Schmitt PT, Schmitt AP. 2016. C-Terminal DxD- Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles. Journal of Virology, 90 (7), 3650-60)].

3.1 Introduction

The paramyxoviruses comprise a group of enveloped viruses that harbor nonsegmented, negative-sense RNA genomes [1]. Included among the paramyxoviruses are a number of human and animal pathogens, including measles virus, mumps virus, Nipah virus, respiratory syncytial virus (RSV), and Newcastle disease virus (NDV). Paramyxovirus infections are spread via particles which bud from plasma membranes of infected cells. Formation of these particles is driven by the viral matrix (M) proteins which can self-assemble to form ordered yet flexible arrays [129, 130] that likely play key roles in generating the membrane curvature required for budding. M proteins also organize the particle assembly process by interacting with the viral glycoproteins via their cytoplasmic tails and also with the viral ribonucleoprotein (vRNP) complexes via the nucleocapsid (N or NP) proteins

{reviewed in references [35, 131]}. These interactions bring together and concentrate all of the viral structural components onto specific sites underlying infected cell plasma membranes, enabling infectious virions to subsequently bud from these locations.

For many paramyxoviruses, expression of M protein in the absence of any other viral components is sufficient to induce the assembly and release of virus-like particles (VLPs) from transfected cells. M proteins of Sendai virus [132, 133], measles virus [134, 135], Nipah virus [69, 94], Hendra virus [54], Newcastle disease virus [136], and human parainfluenza virus 1 (HPIV1) [137] are all capable of directing VLP production and release from transfected cells when expressed alone.

In these cases, additional viral components, including the viral glycoproteins and the nucleocapsid-like structures that form upon expression of paramyxovirus N/NP proteins, can be efficiently packaged into the VLPs if they are co-expressed along with the M proteins [35]. For other paramyxoviruses, including mumps virus [68] and parainfluenza virus 5 (PIV5) [95], the viral M proteins do not induce significant

VLP production when expressed alone in transfected cells. In these cases, co- expression of M proteins together with viral glycoproteins and NP proteins is necessary for VLP production to occur. Such an arrangement could in theory provide a benefit to viruses by preventing the release of empty virions that lack vRNPs. Other negative-strand RNA (nsRNA) viruses, including Ebolavirus [138] and

Tacaribe virus [139], for which enhancements to VLP production were observed upon co-expression of the viral nucleocapsid proteins may employ similar strategies.

Paramyxovirus N/NP proteins function to bind and encapsidate viral genomic and antigenomic RNAs, forming helical nucleocapsid structures that serve as the templates for the viral polymerase [36]. Encapsidation, which is directed by the

RNA- binding, N-terminal core regions of the N/NP proteins, also protects viral

RNAs from RNase digestion and impairs recognition of viral RNAs by host innate immune responses [1]. Flexibility between domains of the N/NP structures is thought to allow for presentation of the RNA bases and access by the viral polymerase only when needed [37, 140, 141]. The C-terminal tail regions of paramyxovirus N/NP proteins are dispensable for RNA binding and instead function to direct interactions with a variety of viral and host proteins, including viral M proteins [42, 43, 103] and viral P proteins [46, 142, 143], although P protein binding and polymerase docking can in some cases be mediated instead by the N-terminal core region of N [45, 47, 144].

Interactions between matrix and nucleocapsid proteins of negative-strand RNA viruses are universally important for generation of infectious, genome-containing virus particles [145], but the details of these interactions are poorly understood.

Studies with measles virus have defined a region very close to the C-terminal end of

N protein that is necessary for M protein binding [43]. For PIV5, the sequence DLD near the C-terminal end of NP protein is critical for its virus assembly functions [42].

Mutations to DLD abolished particle formation function and disrupted M-NP interaction. Building on these observations, we showed in this study that the DLD- containing C-terminal residues of PIV5 and Nipah virus nucleocapsid proteins are sufficient to direct a foreign protein into budding PIV5 or Nipah VLPs. Moreover, we demonstrated that DLD-like sequences can act as the key determinants that define compatibilities between M and NP proteins of different paramyxoviruses. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions, but with important variations, controlled by DLD-like

42 sequences, that play key roles in determining M/NP compatibilities.

3.2 Results

Manipulation of genome packaging interactions to direct a foreign protein into PIV5 VLPs.

We recently identified a DLD sequence near the C-terminal end of PIV5 NP protein that is critical for M binding and for efficient VLP production [42] (Fig. 3.1A). To further investigate the role that these residues play in virus assembly, we transplanted segments from the C-terminal end of PIV5 NP onto the C-terminal end of Renilla luciferase (RLuc), as illustrated in Fig. 3.1A. The luciferase proteins were expressed together with PIV5 M and HN proteins in 293T cells for VLP production.

The unmodified RLuc reporter protein completely lacked VLP assembly functions, as expression of RLuc together with PIV5 M and HN proteins led to poor VLP production, similar to that observed when M and HN proteins were expressed alone

(Fig. 3.1B, lanes 1 and 3). In contrast, expression of M and HN proteins together with

PIV5 NP protein led to highly efficient VLP production (Fig. 3.1B, lane 2), consistent with earlier findings [42]. VLP production was quantified based on the amount of viral M protein detected in sucrose gradient-purified VLPs, normalized to the amount of M detected in cell lysate fractions (Fig. 3.1C). Fusion of either 5 residues or 10 residues from NP to the C-terminal end of RLuc had little impact on VLP production. However, when 15 or more residues were appended to RLuc, the modified RLuc gained the ability to stimulate VLP production (Fig. 3.1B and C).

RLuc- NP15 expression led to VLP production that was about 60% of that observed with the authentic viral NP protein, and expression of each of RLuc-NP30 and RLuc-

NP50 led to VLP production that was roughly equivalent to that observed with NP.

Moreover, substantial quantities of modified RLuc were found within the purified

VLP preparations (Fig. 3.1B). To more directly assess the incorporation efficiency,

VLPs were produced in cells that were metabolically labeled with 35S amino acids.

VLPs were purified and loaded directly on SDS gels, and proteins were detected using a phosphorimager for visualization of VLP polypeptide composition (Fig.

3.1D). The result indicated that RLuc-NP30 was abundantly packaged into VLPs

(1.1:1 ratio of RLuc-NP30/M, taking into account the numbers of methionine and cysteine residues present in the respective proteins).

Additional experiments were carried out to test if the modified RLuc retains its enzymatic activity even after it has been packaged into VLPs. Cells were transfected to produce VLPs as before, and the amounts of enzymatically active luciferase in the

VLP and cell lysate fractions were measured using a luminometer (Fig. 3.1E).

Consistent with the results obtained by Western blot detection shown in Fig. 1B and

C, we found that expression of RLuc-NP15 or RLuc-NP30 (but not unmodified RLuc) together with the viral M and HN proteins led to substantial release of VLPs containing enzymatically active luciferase (Fig. 3.1E). Luminometer-based VLP quantification revealed a difference of more than 15-fold between VLP production in the presence of RLuc-NP30 and VLP production in the presence of unmodified

RLuc. In addition to providing an alternative method of VLP quantification, these

Figure 3.1. A total of 15 C-terminal residues of PIV5 NP protein are sufficient to trigger VLP production and to direct a foreign protein into budding particles. (A) (Top panel) C-terminal amino acid sequences of three paramyxovirus NP/N proteins. The DLD sequence critical for virus assembly functions of PIV5 NP protein is highlighted in bold. (Middle panel) Schematic illustration of Renilla luciferase, appended with residues derived from a paramyxovirus NP protein, being packaged into budding VLPs using the same interactions that would normally direct the packaging of vRNPs into virions. (Bottom panel) Illustration of Renilla luciferase proteins appended with residues derived from PIV5 NP protein. In the cases of RLuc-NP15 and RLuc-NP15Δ4, the NP-derived amino acid sequences are shown in full. (B) 293T cells were transfected to produce PIV5 M and HN proteins together with the indicated luciferase-NP fusions. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (C) Relative levels of efficiency of VLP production were calculated as the amount of M protein detected in VLPs divided by the amount of M protein detected in cell lysates, normalized to the value obtained with NP protein. Error bars indicate standard deviations (n = 3). **, P < 0.005. (D) 293T cells were transfected to produce PIV5 M and HN proteins together with either unmodified luciferase or RLuc-NP30. Cells were metabolically labeled with 35S, and sucrose-gradient purified VLPs were loaded directly onto SDS gels. VLP-derived proteins were detected using a phosphorimager. (E) VLPs were generated and purified as described for Fig. 3.1B. Enzymatically active luciferase contained within VLP and cell lysate fractions was measured using a luminometer. Values were calculated as luciferase activity of VLPs divided by luciferase activity of cell lysate, normalized to the value obtained with RLuc-NP30 (n = 2). (F) 293T cells were transfected to produce PIV5 M and HN proteins together with the indicated RLuc-NP fusions. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting.

45 results also suggest that biologically active foreign proteins can be made to efficiently incorporate into PIV5 VLPs through the addition of short sequences to their C-terminal ends. To confirm that incorporation of modified RLuc into PIV5

VLPs is dependent on the DLD residues near the C-terminal end of the NP-derived transplanted sequence, RLuc-NP15 was further modified to create RLuc-NP15Δ4, in which the C-terminal 4 residues (including DLD) have been removed (Fig. 3.1A). In contrast to RLuc-NP15, RLuc-NP15Δ4 failed to stimulate VLP production and was poorly incorporated into the few VLPs that were made, similarly to unmodified RLuc

(Fig. 3.1F). Hence, the DLD sequence near the C-terminal end of PIV5 NP is critical for VLP assembly functions, both in its natural context [42] and in the context of a foreign RLuc reporter protein.

Figure 3.2. C-terminal ends of paramyxovirus N/NP proteins. The upper portion includes sequences derived from paramyxoviruses within the Rubulavirus genus, while the lower portion includes sequences derived from paramyxoviruses outside the Rubulavirus genus. DLD-like sequences are highlighted in bold. MuV, mumps virus; NiV, Nipah virus; SeV, Sendai virus; MeV, measles virus.

PIV5 NP protein and mumps virus M protein can be engineered for compatibility.

In contrast to the PIV5 nucleocapsid protein, which harbors a DLD sequence near its C-terminal end, the NP protein of mumps virus lacks DLD near its C-terminal end

(Fig. 3.2). This raised the possibility that PIV5 and mumps virus might be incompatible with respect to M-NP interactions, despite being very closely related viruses overall (both are within the Rubulavirus genus of the paramyxoviruses). To test this, mumps virus M, F, and NP proteins were expressed together in 293T cells for VLP production. Consistent with earlier results [68], VLP production was efficient when all three of these proteins were co-expressed but was poor (more than 10-fold reduced) when NP expression was omitted (Fig. 3.3A and B). When

PIV5 NP protein was expressed together with the mumps virus M and F proteins,

VLP production was markedly impaired (Fig. 3.3A and B). This result suggests that the PIV5 NP protein is incompatible with the mumps virus M protein for particle assembly. To further explore the parameters that govern compatibilities between these proteins, a series of chimeric NP proteins was generated as illustrated in Fig.

3.3C. These proteins are based on PIV5 NP protein, but the C-terminal ends were progressively replaced with sequence derived from the mumps virus NP protein.

None of these alterations had significant effects on NP protein expression levels (Fig.

3.3D). The chimeric NP proteins were expressed together with mumps virus M and

F proteins, and VLP production was measured (Fig. 3.3D and E). Chimera M1, which affects just a single amino acid residue at the very C-terminal end of NP protein, did not restore mumps VLP production. However, chimeras M3, M6, M7, M8, and M9 all

47 restored mumps VLP production to levels similar to that obtained with authentic mumps virus NP protein (Fig. 3.3D and E). The M1 and M3 chimeras are markedly different in terms of mumps VLP production function (compare lanes 4 and 6) and yet differ from one another by only a single amino acid residue substitution. This change affects the DLD sequence of PIV5 NP, converting it to the DWD sequence that is found at the corresponding position in mumps virus NP. To determine if this single amino acid residue change is sufficient to induce M/NP compatibility, PIV5 NP

L507W was generated by site-directed mutagenesis. This protein was fully functional for mumps VLP production (Fig. 3.3D, lane 5, and E). Hence, a single amino acid change converting DLD to DWD induced PIV5 NP protein to gain compatibility with mumps virus proteins for VLP production.

Compatibilities between PIV5 and mumps virus M/NP proteins were further analyzed using a membrane coflotation assay. This assay is based on the observation that paramyxovirus M proteins intrinsically bind to cellular membranes, whereas NP proteins do not. M-NP protein interaction indirectly recruits NP protein to membranes, allowing coflotation of NP with membranes on sucrose gradients. This assay has been used previously to monitor M-NP protein interactions of measles virus [135] and PIV5 [42]. As anticipated based on these earlier studies, we found that mumps virus NP protein expressed alone did not float with membranes, whereas a significant fraction of M protein expressed alone floated with membranes (Fig. 3.4). Co-expression of M with NP induced significant

Figure 3.3. DLD and DWD sequences define compatibilities between PIV5 and mumps virus M/NP protein pairs. (A) 293T cells were transfected to produce mumps virus M and F proteins together with the indicated NP proteins for mumps VLP production. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (B) Relative levels of efficiency of VLP production calculated as described for Fig. 3.1C. Error bars indicate standard deviations (n = 3). (C) Schematic illustrating chimeric NP proteins. Substitutions shown in bold progressively convert the C-terminal end of PIV5 NP to match the sequence of mumps virus NP. (D) 293T cells were transfected to produce mumps virus M and F proteins together with the indicated NP protein chimeras for mumps VLP production. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (E) Relative levels of efficiency of VLP production calculated as described for Fig. 3.1C. Error bars indicate standard deviations (n = 3).

49 coflotation of NP (about 47% recovered in membrane-bound fractions). However, co-expression of mumps virus M with PIV5 NP resulted in a substantially lower level of NP coflotation (about 27% recovered in membrane-bound fractions), consistent with the incompatibility that was observed between these proteins in VLP production experiments. The L507W single amino acid change significantly improved compatibility with mumps virus M protein (about 42% recovered in membrane-bound fractions). Together, these results indicate that PIV5 NP protein is incompatible with mumps virus M protein both for binding and for VLP production and that compatibility can be induced through a single amino acid change that converts the C-terminal DLD sequence to DWD.

We next considered the possibility that the L507W change to PIV5 NP, while creating new compatibility with mumps virus M, might at the same time disrupt the existing compatibility with its natural binding partner, PIV5 M. To test this, PIV5 NP

L507W was analyzed for PIV5 M interaction using coflotation (Fig. 3.5A and B) and for the ability to induce production of PIV5 VLPs (Fig. 3.5C and D). The results indicate that PIV5 NP L507W functions just as well as wt PIV5 NP, both for PIV5 M interaction and for PIV5 VLP production. Hence, this altered NP protein gained compatibility with mumps virus M without losing its original compatibility with

PIV5 M. We also tested if the L507W change altered compatibility with the Nipah virus M protein. Nipah virus M protein is compatible with the wt PIV5 NP protein both for binding (Fig. 3.5A and B) and for incorporation into budding VLPs (Fig.

Figure 3.4 Compatibilities of PIV5 and mumps virus M/NP protein pairs measured by membrane coflotation analysis. (A) The indicated pairs of M and NP proteins were expressed in 293T cells, and detergent-free lysates were fractionated on sucrose flotation gradients. Viral proteins were visualized by immunoblotting. (B) The fraction of membrane-associated NP was calculated as the amount of NP in the top two fractions divided by the total NP protein signal in all four fractions. Error bars indicate standard deviations (n = 3). **, P < 0.005.

3.5E). The L507W change to PIV5 NP protein did not impair this compatibility (Fig.

3.5A, B, and E). These results indicate that the DWD-containing PIV5 L507W protein is compatible with the M proteins of at least three different paramyxoviruses—PIV5, mumps virus, and Nipah virus.

Figure 3.5. PIV5 NP L507W protein retains its compatibilities with PIV5 and Nipah virus M proteins. (A) The indicated pairs of M and NP proteins were expressed in 293T cells, and detergent- free lysates were fractionated on sucrose flotation gradients. Viral proteins were visualized by immunoblotting. (B) The fraction of membrane-associated NP was calculated as the amount of NP in the top two fractions divided by the total NP protein signal in all four fractions. Error bars indicate standard deviations (n = 3). (C) 293T cells were transfected to produce PIV5 M and HN proteins together with either PIV5 NP or PIV5 NP L507W. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (D) Relative levels of efficiency of VLP production calculated as described for Fig. 3.1C. Error bars indicate standard deviations (n = 3). (E) 293T cells were transfected to produce Nipah virus M protein together with either PIV5 NP or PIV5 NP L507W. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting.

Alterations affecting the C-terminal end of mumps virus NP protein impair interaction with M protein and block VLP production.

The findings on M/NP compatibility described above suggested a critical role for the DWD residues near the C-terminal end of mumps virus NP protein. To directly test the importance of these residues within the context of full-length mumps virus

NP protein, site-directed mutagenesis of mumps virus NP protein was carried out as illustrated in Fig. 3.6A. We found that mumps VLP production was severely impaired when residue D546 or residue W547 was changed to alanine (Fig. 3.6B and C). A similar negative effect on VLP production was also observed for NPΔ4, in which the four C-terminal residues (including D546 and W547) have been removed

(Fig. 3.6B and C). Residue W547 was also changed to leucine, creating a DLD sequence in place of DWD. This severely impaired VLP production, reinforcing the observation that DLD sequences are incompatible with mumps virus M protein (Fig.

3.6B, lane 5, and C). In each of these cases, the low level of VLP production was similar to that of the negative control (absence of NP expression), indicating a nearly complete loss of VLP production function. On the other hand, alanine substitutions that targeted the adjacent D548 and E549 residues resulted in no significant impairments to VLP production (Fig. 3.6B and C). The altered mumps virus NP proteins were also tested for the ability to assemble together with mumps virus M protein in membrane coflotation experiments (Fig. 3.6D and E). We found that the same mutations that disrupted VLP production also impaired membrane coflotation with M protein. Together, these results indicate that virus assembly functions of mumps virus NP protein are critically dependent on residues D546 and W547

53 within its C-terminal DWD motif.

Figure 3.6. The DWD-containing C-terminal region of mumps virus NP protein is important for its VLP production function. (A) Schematic illustrating mutations made to mumps virus NP protein. (B) 293T cells were transfected to produce mumps virus M and F proteins together with the indicated NP protein mutants for VLP production. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (C) Relative levels of efficiency of VLP production calculated as described for Fig. 3.1C. Error bars indicate standard deviations (n = 3). (D) The indicated pairs of M and NP proteins were expressed in 293T cells, and detergent-free lysates were fractionated on sucrose flotation gradients. Viral proteins were visualized by immunoblotting. (E) The fraction of membrane-associated NP was calculated as the amount of NP in the top two fractions divided by the total NP protein signal in all four fractions. Error bars indicate standard deviations (n = 3).

Manipulation of genome packaging interactions to direct a foreign protein into Nipah VLPs.

Sequences within the Nipah virus N protein that function to coordinate with M protein during virus assembly have not been defined. Here, we hypothesized on the basis of analogy with PIV5 that such sequences might be located near the C-terminal end of N protein. To test this hypothesis, we transplanted segments from the C- terminal end of Nipah virus N onto the C-terminal end of RLuc. The luciferase proteins were expressed together with Nipah virus M protein, and incorporation of

RLuc into the budding VLPs was measured (Fig. 3.7). Note that in the case of Nipah virus, VLP production does not depend on expression of N protein or glycoproteins—VLPs are produced efficiently upon expression of Nipah virus M protein alone [69, 94]. However, N protein was incorporated into the M-containing

VLPs when it was coexpressed [94] (Fig. 3.7A, lane 2). We found that unmodified

RLuc was incorporated poorly into M-VLPs (Fig. 3.7A, lane 3). Fusion of 5 residues derived from N to the C-terminal end of RLuc did not improve its incorporation into

VLPs. However, when 10 residues were appended to RLuc, incorporation into VLPs improved, and when 15 or 30 residues were appended to RLuc, VLP incorporation was improved still further, to a level that was approximately 2.5 times greater than that observed with the unmodified RLuc control (Fig. 3.7A and B). RLuc-N50 was incorporated somewhat less efficiently into VLPs than RLuc- N15 or RLuc-N30, but its incorporation was still approximately 1.5-fold higher than that observed with the unmodified RLuc control. These findings with respect to Nipah virus parallel those shown in Fig. 1 with PIV5, even though these two viruses come from separate

55 genera within the Paramyxoviridae and have M proteins with relatively poor homology (less than 25% amino acid identity). Overall, our results support the general idea that foreign proteins can be engineered for packaging into budding paramyxovirus VLPs through addition of small (10-to-15- residue) appendages to their C-terminal ends.

Amino acid residues 523 to 528 of N protein are important for RLuc packaging into Nipah VLPs.

The C-terminal 15 residues of Nipah virus N protein were targeted by alanine scanning mutagenesis to determine which of these residues are necessary for efficient direction of the RLuc reporter into Nipah VLPs (Fig. 3.8A). The altered RLuc proteins were co-expressed together with Nipah virus M protein, and RLuc incorporation into VLPs was measured. Substitutions at any of the positions from residue 523 to residue 528 caused a significant reduction in luciferase incorporation

(Fig. 3.8B and C). The most severe defects were associated with changes at positions

N523 and D524. In those cases, luciferase incorporation into VLPs was similar to that observed with the unmodified luciferase control (Fig. 3.8B and C). Alanine substitutions targeting the surrounding residues, outside the sequence 523-

NDLDFV-528, had little impact on luciferase incorporation into VLPs.

Figure 3.7 A total of 15 C-terminal residues of Nipah virus N protein are sufficient to direct a foreign protein into budding particles. (A) 293T cells were transfected to produce Nipah virus M protein together with the indicated Renilla luciferase-Nipah N fusions. Viral proteins from cell lysates and from sucrose gradient-purified VLPs were detected by immunoblotting. (B) Relative efficiencies of luciferase incorporation into VLPs were calculated as the amount of luciferase detected in VLPs divided by the amount of M protein detected in VLPs, normalized to the value obtained with RLuc- N15. Error bars indicate standard deviations (n = 3). **, P < 0.001.

During the course of these experiments, we noticed that RLuc- N15, RLuc-N30, and RLuc-N50 (but not RLuc-N5 or RLuc-N10) could in some cases be detected on immunoblots using an N-specific polyclonal antibody [68] that had been raised against purified N protein expressed in Escherichia coli (Fig. 3.8B, marked with asterisks, and data not shown). Detection was impaired or lost when any of residues

N523, D524, L525, or D526 was changed to alanine (Fig. 3.8B, top panels). These are the same residues that were the most critical for RLuc incorporation into VLPs. Our findings suggest that these residues, in addition to directing VLP incorporation, also define an epitope that is recognized by a polyclonal antibody raised to full-length N protein.

Figure 3.8. Amino acid residues 523-NDLDFV-528 within Nipah virus N protein are important for the ability to direct a foreign protein into Nipah VLPs. (A) Schematic illustrating variations on RLuc-N15, generated by site-directed mutagenesis. Only the 15 residues derived from Nipah virus N protein are shown. (B) 293T cells were transfected to produce Nipah virus M protein together with the indicated variants of RLuc-N15. Viral proteins from cell lysates and from sucrose gradient- purified VLPs were detected by immunoblotting. The asterisk denotes the position of RLuc-N15 variants that could be detected using a polyclonal antibody raised against Nipah virus N protein. WB, Western blot. (C) Relative efficiencies of luciferase incorporation into VLPs were calculated as the amount of luciferase detected in VLPs divided by the amount of M protein detected in VLPs, normalized to the value obtained with RLuc-N15. Error bars indicate standard deviations (n= 3). *, P < 0.05; **, P < 0.005.

3.3 Discussion

During assembly of negative-strand RNA viruses, viral genomes, in the form of vRNPs, are packaged into budding particles via M-NP interactions [145, 146]. Hence, these interactions are fundamentally important for the production of genome- containing, infectious viral particles. Here, we have defined regions near the C- terminal ends of paramyxovirus NP proteins that direct their virus assembly functions. A 15-residue DLD-containing sequence derived from the C-terminal end of PIV5 NP protein was capable of directing a foreign protein (Renilla luciferase) into PIV5 VLPs. Likewise, a 15-residue DLD-containing sequence derived from the C- terminal end of Nipah virus N protein was sufficient to direct Renilla luciferase into

Nipah VLPs. Other paramyxoviruses harbor similar DLD-like sequences near their C- terminal ends as well (illustrated in Fig. 3.2). For example, the HPIV2 NP protein contains DFD specifically in place of the DLD found in PIV5. NDV and HRSV N proteins have DND sequences near the C-terminal ends. Measles virus N protein contains the sequence DRDLLD at the C-terminal end, and alterations that affect this sequence have been found to disrupt virus assembly functions [43]. The C-terminal portion of Sendai virus NP protein has also been implicated in virus assembly [103].

The mumps virus NP protein harbors DWD in place of the DLD sequence found in

PIV5 NP. We found this DWD sequence to be critical for efficient mumps VLP production. Interestingly, DWD and DLD sequences did not function equivalently for

VLP production but were instead the key determinants that defined compatibilities between PIV5 and mumps virus M/NP protein pairs. Mumps VLP production was

60 efficient only in the presence of DWD-containing NP proteins, such as wt mumps NP protein or PIV5 NP L507W that was engineered to contain DWD in place of DLD.

Mumps VLP production was poor in the presence of DLD-containing NP proteins, such as wt PIV5 NP protein and mumps NP W547L that was engineered to contain

DLD in place of DWD. Based on these collective findings, we propose that paramyxoviruses share an overall common strategy for directing M-NP interactions, but with important variations, controlled by DLD-like sequences, that play key roles in defining M/NP compatibilities.

For a subset of the nsRNA viruses, including PIV5 [95], mumps virus [68],

Ebolavirus [138], and Tacaribe virus [139], matrix-nucleocapsid protein interactions appear to function not only as a means of recruiting vRNPs into particles but also as a signal to trigger particle release itself. The mechanism(s) by which nucleocapsid proteins enhance particle production in these cases is not clear.

It is possible that a nucleocapsid requirement for the completion of particle assembly could benefit these viruses by minimizing the release of noninfectious, empty virions that lack viral genomes. Here, we found that luciferase proteins appended with short NP-derived, DLD-containing sequences completely replaced the requirement for NP protein in PIV5 VLP production. Hence, we believe that the mechanism for enhanced PIV5 particle release in the presence of NP protein has nothing to do with assembly of M protein onto encapsidated RNAs, as the appended sequences are all derived from the C-terminal tail region of NP and not from the N- terminal core which is responsible for RNA binding and encapsidation [37]. It is possible that NP stimulates PIV5 particle production through direct occupation of a

61 binding pocket on M protein by the DLD-containing sequence, which could then induce M protein to enter a budding-active state. An alternative possibility is that one or more host factors, essential for particle budding, could be recruited to PIV5 assembly sites via NP protein.

We found that a luciferase reporter protein could be induced to package into

Nipah VLPs if it was appended with a 10-to-15- residue sequence derived from the

C-terminal end of Nipah virus N protein. Here, the sequence 523-NDLDFV-528 within this region was critical for directing luciferase into the budding Nipah VLPs.

However, mutagenesis of these residues in the context of full-length N protein did not substantially impair N protein incorporation into VLPs (data not shown). This is in contrast to the DLD-containing sequence near the C-terminal end of PIV5 NP protein, which not only was sufficient to direct a luciferase reporter into VLPs (Fig.

3.1C) but also was necessary for VLP assembly in the context of full-length NP protein [42]. Likewise, the mumps DWD sequence was critical for mumps VLP release in the context of the full-length mumps virus NP protein (Fig. 3.6). It is possible that multiple, redundant interactions drive incorporation of Nipah virus N protein into budding particles, with one interaction based on 523-NDLDFV-528 near the C-terminal end and one or more additional interactions directed by sequences located elsewhere within N protein.

The ability to manipulate viral M-NP protein interactions could prove useful for future development of VLP-based protein delivery tools. In this scenario, foreign proteins of interest would be tagged to induce their interaction with M protein and

62 subsequent incorporation into fusion-competent VLPs, which would then deliver the contents to target cells. Although incorporation of target proteins into VLPs has been demonstrated in the past, the approaches used typically require direct fusion of the target protein amino acid sequence to the viral Gag or M protein that directs particle budding [147–149]. Here, we have instead achieved efficient incorporation of an enzymatically active foreign protein into paramyxovirus VLPs by harnessing the M-NP protein interactions that normally direct viral RNPs into budding virions.

This approach is highly flexible, as it requires no modification at all to the viral matrix protein component, and the target protein in this case was modified only through the addition of a 15-amino-acid NP-derived binding sequence to its C- terminal end. Further modification of this approach to include the paramyxovirus fusion and attachment glycoproteins would in theory result in particles capable of transmitting the foreign proteins to target cells that would be similar to the

“infectious” paramyxovirus VLPs that have been studied in the past and that are capable of delivering their NP- encapsidated minigenome cargos [150–154]. Such

VLP-based delivery vehicles could provide a highly flexible and safe platform for therapeutic delivery of functional proteins or toxins to cells.

Chapter 4

Role of angiomotin-like 1 in paramyxovirus budding

4.1 Introduction

ESCRT and viral late domains. Enveloped viruses acquire their lipid envelopes as they bud through membranes, and in doing so hijack a number of cellular pathways. Notable among these is the ESCRT (endosomal sorting complex required for transport) pathway. The ESCRT pathway is involved in multivesicular body

(MVB) formation that functions to degrade or recycle transmembrane proteins, and in cytokinesis and virus budding. Virus budding is topologically similar to MVB formation. Vesicles or virus particles bud outwards from the cytoplasm, and host factors that function to constrict and sever the vesicle must work from within the bud neck. In contrast to this, cellular processes like endocytosis are the topological inverse. Here, vesicle membranes bud towards the cytoplasm, and host factors like dynamin must work to sever the vesicle from outside the bud neck [109].

The ESCRT machinery is made up of 5 complexes- ESCRT-0, -I, -II and –III, and

Vps4. ESCRT-0 binds to endosomal membranes where it recruits ESCRT-I and

ESCRT-II, which are involved in vesicle budding and can help in stabilization of the bud necks of nascent vesicles [107]. ESCRT-III enlists de-ubiquitinating enzymes that assist in recycling of ubiquitin bound to cargo proteins, while Vps4-AAA ATPase is responsible for the final step involving ESCRT disassembly and fission of the vesicle from the endosomal membrane, into the multivesicular body. One protein especially important in ESCRT-mediated cytokinesis is Bro1/ALIX that directs

ESCRT machinery to the midbody for membrane abscission. ESCRT proteins recruit a variety of cellular proteins by employing protein-protein interaction motifs. For example, Tsg101, a subunit of ESCRT-I binds to cellular proteins containing a

P(T/S)AP motif via its UEV domain [107, 109, 155], while Alix binds to YXXL motif containing proteins via its central V-domain, and connects them to ESCRT-III.

Another set of proteins - Nedd4 (neural precursor cell expressed developmentally downregulated protein 4) family of HECT (homologous to the E6AP carboxyl terminus) ubiquitin E3 ligases, contain WW domains that bind to cellular proteins containing PPXY motifs. PPXY motifs were intitially identified in proteins that are ubiquitylation substrates of Nedd4 in addition to binding to them, and are also sorted via the MVB pathway [156]. The ubiquitin ligase function of Nedd4 proteins is crucial for its role in sorting PPXY containing proteins [126, 157–160]. However, it is not clear how Nedd4s link to the ESCRT; recent evidence points to the involvement of arrestin-related trafficking adaptors [161].

Viral structural proteins critical for budding have been shown to contain motifs similar to those seen in cellular proteins that link to the ESCRT pathway [109]. For example, HIV-1 Gag (group-specific antigen) has PTAP and YPDL motifs in its p6 domain that bind to Tsg101 and Alix, respectively. Many other retroviruses like human T-cell leukemia virus type 1 (HTLV-1) [162] and Rous sarcoma virus (RSV)

[163] have PPXY motifs that can interact with Nedd4 ubiquitin ligases, which ultimately feedback onto the ESCRT pathway . Ebolavirus VP40 contains a PTAPPEY motif and is capable of binding to both Tsg101 and Nedd4 E3 ligase members [113,

114], while rhabdovirus M proteins contain a PPXY motif and can bind to Nedd4-like

E3 ubiquitin ligases [164]. Disruption of the PTAP/PPXY/YXXL motifs in these viruses can lead to a defect at a late stage in budding, where the virus particles remain tethered to the plasma membrane and fail to pinch off. These motifs are therefore referred to as “late domains”. Typically, over-expression of proteins that bind to late domains improve virus budding. Late domains have been discovered in a number of negative strand RNA viruses and retroviruses (Fig.1) [110, 112–114,

163–166]. In addition to these, there are also instances where the ESCRT binding partners of late domains have not been discovered. Paramyxoviruses, for example, do not have conventional late domain motifs. Members of the Rubulavirus genus like

PIV5, mumps and NDV contain “FPIV/FPVI” motifs [66–68] in their matrix proteins that have not been shown to interact with any other protein yet. However, disruption of these motifs can lead to a severe defect in M protein function, leading to loss of budding. Moreover, if these motifs are fused to crippled Gag proteins that lack their late domains, they can restore Gag budding functions, leading to the conclusion that they must behave as late domains in paramyxovirus budding [66].

Expression of DN versions of ESCRT pathway components can block PIV5 and mumps virus budding [66, 68]. This suggests that these viruses need the ESCRT machinery for budding, and they could be facilitating this via the FPIV/FPVI motifs that reside in their M proteins.

Figure 4.1. Late domains in different viruses. (A) Conventional late domains – PTAP, YXXL and PPXY seen in retroviral Gag, rhabdoviral M and filoviral VP40 proteins, that bind to Tsg101, Alix and Nedd4-like E3 ubiquitin ligases respectively [114]. (B) Unconventional late domain motifs observed in paramyxoviruses, within the red box [67].

Angiomotins and virus budding. Angiomotins are tight junction-associated proteins [167] that mediate endothelial migration, Hippo/YAP signaling, small G- protein signaling and angiogenesis [115]. There are three members in this family-

Amot with two isoforms p130 and p80, AmotL1 and AmotL2. Angiomotins are characterized by an N-terminal domain consisting of LPTY and two PPXY motifs that bind to WW-domain containing proteins, a coiled-coil domain responsible for oligomerization between motins and a C-terminal PDZ binding domain involved in signaling pathways [115]. Previous research by our group has shown that in a yeast two-hybrid assay, PIV5 M could interact with AmotL1 when used as bait against prey derived from a human cDNA library. This assay was conducted in order to find cellular binding partners for PIV5 M, with the hope of understanding M function in virus assembly [56]. The interaction between PIV5 M and AmotL1 was also observed in mammalian cells. The AmotL1 protein was next split in two portions- an

N-terminal portion containing LPTY and PPXY motifs and a C-terminal region containing the PDZ-binding domain, in order to localize the M-binding region on

AmotL1. Co-IP analysis showed that PIV5 M bound to the C-terminal fragment, and this was mapped to an 83 aa region (aa 667-749) within it. This 83 aa region, now called minimal domain, could bind to PIV5 M just as well as the C-terminal fragment.

When over-expressed along with PIV5 M, HN and NP proteins for VLP production, the minimal domain was found to potently inhibit VLP release in transfected cells and virus production in infected cells. This suggests that AmotL1 is likely important in PIV5 budding pathways. Another important experiment that reinforced this

68 observation is the siRNA-mediated knockdown of AmotL1, which negatively affected PIV5 virion production in infected cells.

Another viral protein that interacts with angiomotins is HIV-1 Gag. Earlier observations had shown that over-expression of Nedd4L could rescue budding function of a p6-deleted Gag [157, 159]. This is puzzling as Gag doesn’t have PPXY motifs and should not be able to bind Nedd4L. This strongly points to the potential involvement of a third protein, via which Nedd4L could bind to Gag. A recent report by Wes Sundquist et. al. [126] has shown that this intermediate protein is angiomotin, specifically its p130 isoform. Amot p130 can bind to Nedd4L via its

PPXY motifs, and can also bind Gag. The other two angiomotin members Amot-L1 and AmotL2 perform the same binding functions as Amot. Over-expressing both

Amotp130 and Nedd4L along with a crippled Gag provirus led to its rescue in mammalian cells, indicating that this interaction serves as an auxiliary budding function in addition to the late domains of Gag. Amotp130 was also required for efficient release of wt virus as knockdown led to a decrease in budding, similar to that seen with Tsg101 depletion. Transmission electron microscopy showed that in cells depleted of Tsg101, HIV-1 particles arrested at a late step in virus budding, displaying a lollipop morphology. However, when Amot was knocked down in HIV-1 infected cells, particle formation was arrested at a step earlier than Tsg101, as evidenced by measuring curvature of the Gag shell.

We show here that unlike Gag [126], PIV5 M binds exclusively to AmotL1, and not to AmotL2 or Amotp130. We also show that C-terminal fragments derived from motins other than AmotL1 are incapable of blocking PIV5 VLP production. Only

AmotL1 C-terminal region that binds PIV5 M had a negative impact on VLP production. We demonstrate that AmotL1 can function as a linker between PIV5 M and at least three Nedd4 proteins, bringing them together in a complex, the exact function of which remains to be elucidated. This identifies a potential new connection for paramyxoviruses with the ESCRT machinery. Further elucidation of the mechanism by which AmotL1 and Nedd4L impact PIV5 budding could help us define successful virus assembly in PIV5.

4.2 Results

PIV5 M binds selectively to AmotL1.

HIV-1 Gag was shown to bind to all three angiomotins [126]. We were curious to see if PIV5 M could also bind to all the motin family members, and set out to test this possibility. cDNAs for Amot p130, its shorter isoform Amotp80 and AmotL2 were acquired and inserted with an N-terminal flag tag into pCAGGS (Fig.4.2). Co-IP assays were then conducted in 293T cells by over-expressing these motins with

PIV5 M protein. Surprisingly, we found that PIV5 M bound only to AmotL1 and not to the other two motins (Fig.4.3A). The C-terminal and minimal fragments of

AmotL1 bind very strongly to M protein [56]. We decided to construct similar fragments for Amotp130 and AmotL2 and test them in a Co-IP assay with PIV5 M, to test if these shorter fragments might bind M better than the full-length protein.

However, even here, we found that only AmotL1 derived proteins were capable of binding PIV5 M. No peptides derived from Amotp130 or AmotL2 could bind M

(Fig.4.3B and C). This is intriguing and it now becomes important to understand why PIV5 M evolved to bind such a specific target, given the similarity between the different motins both structurally and functionally.

The C-terminal fragment of AmotL1, and not that of AmotL2 or Amotp130, can inhibit PIV5 VLP production.

Over-expression of either the C-terminal portion or minimal fragment of AmotL1 led to an acute depletion in PIV5 VLP production [56]. The N-terminal portion that does not bind to PIV5 M protein had no effect on PIV5 VLPs when over-expressed.

Here, attempts were made to over-express C-terminal (Fig.4.4A) fragments of

Amotp130 and AmotL2 to test if they had any effect on PIV5 VLPs. Over-expression of only the AmotL1 C-terminal fragment could inhibit PIV5 VLPs at the lowest amounts tested (Fig.4.4A, lane 3). Amotp130Ct or AmotL2 Ct had no effects on VLP production (Fig.4.4A, lanes 4 and 5). However, this effect was dose dependent as using higher amounts of these C-terminal fragments led to a drop in PIV5 VLPs irrespective of the motin used, possibly owing to disruptions caused by these proteins in downstream cellular pathways. Over-expression of minimal fragments

Figure 4.2. Schematic illustration of different angiomotins and related polypeptides. Schematic detailing different domains and motifs in the angiomotins and their derivatives. Numbers correspond to amino acid positions in the full-length proteins. BAR denotes the Bin/Amphiphysin/Rvs domain.

Figure 4.3. PIV5 M can bind only to full-length AmotL1 or derivatives, and not to AmotL2 or Amotp130. (A-C) 293T cells were transfected to produce different full-length (A), C-terminal fragment (B) and minimal fragment (C) motin proteins, along with PIV5 M. Cells were metabolically labeled with 35S. Co-IP analysis was carried out by pulldown with M2 flag beads, directed against an epitope on flag-tagged motin proteins. Proteins in inputs were pulled down using flag beads and a PIV5 M specific antibody. Viral and host proteins were detected using a phosphorimager.

73 of all motins, especially AmotL1 and AmotL2, led to significant drops in VLP production (Fig.4.4B). In the case of AmotL1-m, this effect might be due to tight binding to PIV5 M (Fig.4.4B, lane 3), sequestering it away from other important viral or host factors. However, AmotL2-m and Amotp130-m do not bind to PIV5 M and their inhibitory effect on VLP production may be indirect (Fig.4.4B, lanes 4 and 5).

Figure 4.4. AmotL1 C-terminal fragment can inhibit PIV5 VLP production, whereas AmotL2 or

Amotp130 C-terminal fragments do not. (A) 293T cells were transfected to produce PIV5 M, HN and NP along with indicated motin-derived C-terminal fragments. Cells were metabolically labeled with 35S; VLPs were purified on sucrose gradients and directly loaded onto SDS-PAGE gels, while proteins in cell lysates were pulled down with specific antibodies. Proteins were visualized using a phosphorimager. Relative efficiency of VLP production was calculated by measuring M protein in

VLPs divided by M protein in cell lysates, normalized to the value obtained with VLPs produced in the absence of motin-derived fragments. (B) 293T cells were transfected and VLPs were purified and quantified as described in 4.4(A), however, motin-derived C-terminal fragments were replaced with motin-derived minimal fragments.

AmotL1 can bind to different members of the Nedd4 family of E3 ubiquitin ligases.

Amotp130 and other angiomotins that contain LPTY/PPXY motifs can bind to

WW-domain containing Nedd4-like E3 ubiquitin ligases [115, 117, 126]. Amotp130 is capable of binding to different members of the Nedd4 family- Nedd4-1, Nedd4-

2/L and Itch [117, 126]. Here, we have shown that AmotL1, like Amotp130 can bind

Nedd4-1 and Nedd4-L, and in addition to these two, it can also bind NedL1 (Fig.4.5, lanes 2, 4 and 6). AmotL1 levels in the inputs are very low when co-expressed with

Nedd4L; this is likely because Nedd4L ubiquitinates AmotL1 more efficiently than other Nedd4-like ubiquitin ligases, leading to its degradation (Fig.4.5B, inputs-lane

4). When the LPTY and two PPXY motifs are mutated in AmotL1, then AmotL1 is no longer capable of binding to any of the Nedd4-like E3 ubiquitin ligases such as

Nedd4-1, Nedd4L and NedL1 (Fig.4.5A and B, lanes 3, 5 and 7). Other Nedd4-like proteins were not tested here, and it is possible that AmotL1 has more binding partners within this family.

Figure 4.5. AmotL1 binds to at least three members of the family of Nedd4 E3 ubiquitin ligases. (A) Schematic illustrating mutations made to the PPXY motifs in AmotL1. (B) Indicated AmotL1 and Nedd4-like proteins were expressed in 293T cells and co-IP assay was conducted by pulldown with an antibody towards the myc epitope on myc-tagged Nedd4-like proteins. Upper panels were probed with a rabbit polyclonal AmotL1 antibody, while the lower panels were probed with the same myc antibody used to pull down Nedd4 complexes.

PIV5 M, AmotL1 and Nedd4-like proteins interact and form a complex

PIV5 M binds to AmotL1, and AmotL1 binds to different WW domain containing proteins. Two among these are members of the Nedd4 family of E3 ubiquitin ligases and yes-associated protein (YAP). From this, it follows that PIV5 M should be able to bind indirectly to either Nedd4 proteins or YAP, especially if either of these interactions can assist in PIV5 budding. A likelier candidate here is Nedd4, as viral M protein counterparts containing PPXY motifs have been shown to bind Nedd4 proteins, and over-expression of Nedd4-like proteins leads to increased budding for these viruses [112, 162, 163]. Ablation of PPXY motifs in these other viral proteins leads to loss of Nedd4 binding and also impairs virus budding, leading to a late domain phenotype. To begin with, Nedd4-1, PIV5 M and AmotL1 were expressed together in 293T cells (Fig.4.6A). As negative controls, full-length AmotL1 was replaced with L/PPXY mutated AmotL1, AmotL1-Nt or AmotL1 Ct (Fig.4.6A, lanes 4-

6). Both AmotL1 and Nedd4-1 were also individually expressed with PIV5 M

(Fig.4.6A, lane 3). Pulldowns were carried out with myc antibody against the myc tag on Nedd4-1. Proteins were blotted on membranes and probed with antibodies to all three over-expressed proteins. It was seen that only one combination, that of the three wild type proteins expressed together, could pull down PIV5 M protein

(Fig.4.6A, lane 3). Expressing PIV5 M and AmotL1 together and pulling down with myc antibody could not pull down M (Fig.4.6A, lane 1). Similarly, when Nedd4-1 and

PIV5 M were expressed together, no M was pulled down even though Nedd4-1 was, as the two proteins do not interact (Fig.4.6A, lane 2). Using L/PPXY mutated AmotL1 led to loss of AmotL1-Nedd4-1 interaction, so no M was pulled down. AmotL1-Nt

77 contains its L/PPXY motifs and is pulled down with Nedd4-1 but doesn’t bind M, whereas AmotL1-Ct does not contain L/PPXY motifs and cannot bind to Nedd4-1, so no M is present there. Only when full-length AmotL1 is present, it can bind to both

Nedd4-1 and PIV5 M, and here pulldown with Nedd4-1 leads to IP of PIV5 M in the same complex. This leads to the conclusion that M, via interaction with AmotL1, can bind to Nedd4-1 and by doing so can probably connect to the ESCRT pathway essential for virus assembly.

This experiment was next tested with Nedd4L wt and NedL1. Nedd4L is a strong ubiquitinator of AmotL1, leading to its degradation. This leads to insufficient levels of AmotL1 within cells, and consequently very poor PIV5 M pulldown. To bypass this effect, a mutant of Nedd4L - NEDD4L.C942A, that has its HECT ubiquitin ligase activity ablated, was used. This mutant can bind AmotL1 without causing significant degradation. The results observed here for M pulldown were similar to those seen with Nedd4-1 (Fig.4.6B). Another Nedd4L mutant, ΔWW, that has its WW domains mutated, when tested in a pulldown, could not bind AmotL1 and consequently could not pulldown M (data not shown).

NedL1, a different member of the Nedd4-like E3 ubiquitin ligases, was also tested in a co-IP assay with AmotL1 and PIV5 M (Fig.4.7). NedL1 binds to AmotL1 (Fig.4.5), and it follows that it should be present in a complex with AmotL1 and PIV5 M, just like the other Nedd4-like proteins tested. 293T cells were transfected as described earlier, but here NedL1 was used instead of Nedd4-1/Nedd4L. NedL1 pulled down

AmotL1, and with it PIV5 M protein. AmotL1 derivatives that were defective in binding NedL1 or PIV5 M could not pull down any M protein. Here, a different

AmotL1 mutant was used- Δ310,367 PPXY, instead of the ΔL/PPXY. This mutant protein abolished AmotL1 interaction with NedL1 (Fig.4.7, lanes 4 and 8). However, for the Nedd4-1 or Nedd4L proteins, AmotL1 binding was depleted only after all

PPXY motifs were mutated. The above experiments point to the conclusion that it is only through full length AmotL1 that PIV5 M can bind to Nedd4 family members. It remains to be seen whether this complex can be reconstituted with other proteins that bind to AmotL1, like YAP, or whether this activity is only specific to Nedd4 proteins as these are likelier to form relevant and useful interactions.

Figure 4.6. AmotL1 functions as an adaptor to link PIV5 M and Nedd4-like proteins together. (A) 293T cells were transfected to produce PIV5 M, AmotL1 and derivatives and/or Nedd4-1. Protein complexes were pulled down with an antibody targeted against the myc epitope on myc-tagged Nedd4-1. Viral and host proteins were detected by immunoblotting. (B) 293T cells were transfected as described in Fig.4.6A, except this time a ubiquitin ligase defective Nedd4L mutant, Nedd4L C942A was used in place of Nedd4-1. Pulldown of protein complexes and detection of proteins was conducted as described in Fig.4.5A.

Figure 4.7. AmotL1 functions as an adaptor to link PIV5 M and NedL1 proteins together. 293T cells were transfected to produce NedL1, derivatives of AmotL1 and PIV5 M in different combinations. Complexes were pulled down using antibody against the myc tag on NedL1. Proteins were then detected by immunoblotting.

PIV5 M, Nedd4L and AmotL1 co-localization within cells.

PIV5 M, AmotL1 and Nedd4L were over-expressed in A549 cells grown on coverslips, either by themselves, in combinations of two or all three proteins together

(Fig.4.8 and 4.9). A549 cells are a human respiratory epithelial cell line and were used here, as PIV5 infections typically affect the respiratory system. Besides, these cells have a low nucleus:cytoplasm ratio, making them optimal for visualizing cytoplasmic compartments. PIV5 M by itself is diffusely distributed in the cytoplasm,

80 and is not present in the nucleus. Nedd4L is present all over the cell in a diffuse pattern. AmotL1 distributes differently and forms large circular puncta inside the cell (Fig.4.8A). It remains to be seen whether these are associated with lysosomes, representing large amounts of over-expressed AmotL1 that cells need to dispose of, or whether this is simply characteristic of endogenous AmotL1. When expressed in combinations of two (Fig.4.8B), PIV5 M and AmotL1 co-localize in large punctae in the cytoplasm and also at the plasma membrane (Fig.4.8B, upper panel). This distribution is different from that observed when the two proteins are expressed alone (Fig.4.8A). Nedd4L and AmotL1 also co-localize in tiny punctae, however this is difficult to observe as ubiquitination by Nedd4L potently leads to degradation of the over-expressed AmotL1 (Fig.4.8B, middle panel). Very few transfected cells showed sufficient levels of AmotL1 in the presence of Nedd4L. No co-localization was expected between PIV5 M and Nedd4L as these proteins do not interact via co-

IP in 293T cells. Surprisingly, PIV5 M and Nedd4L co-localized in A549 cells

(Fig.4.8B, lower panel). This interaction might be brought about by trace levels of endogenous AmotL1, or an as yet unknown protein. Clearly, this interaction is too weak to be retained under co-IP conditions in 293T cells, providing it exists there too.

When all three proteins were expressed together and stained in combinations of two, it was seen that PIV5 M and AmotL1 display roughly the same punctate pattern, as evidenced in the absence of Nedd4L (Fig.4.9, upper panel). However, Nedd4L was also present in these large punctae now that co-localized with both M and AmotL1

(Fig.4.9, middle and lower panel). Abundant co-localization was also seen at

81 membranes. The pattern of the M-Nedd4L distribution was different from that seen when M and Nedd4L were expressed in the absence of over-expressed AmotL1

(Figure 4.8B, bottom panel). The distribution of AmotL1 with Nedd4L was also radically different – AmotL1 was more abundant in cells transfected with all three proteins, and was found to be present with Nedd4L in the same cellular compartments containing PIV5 M. The tiny punctae observed in the absence of M expression were no longer visible. The same Nedd4L mutant used for co- immunoprecipitation experiments – Nedd4L.C942A, that is defective in ubiquitin ligase activity and can no longer ubiquitinate AmotL1, was also tested (data not shown). Cells transfected with mutant Nedd4L showed higher levels of AmotL1 compared to cells transfected with wt Nedd4L. However, the results with mutant

Nedd4L were similar to those with wt Nedd4L, AmotL1 and PIV5 M, with the major difference being an increase in AmotL1 levels within cells. All of these observations suggest that Nedd4L, PIV5 M and AmotL1 are present within the same cellular compartments in complexes that can be visualized by immunofluorescence, corroborating results from co-IP analysis.

Fig 4.8. Localization patterns of viral and host proteins, either alone or in combination. 293T cells were transfected to produce PIV5 M, Nedd4L and AmotL1 either by themselves (A) or in combinations of two (B). Proteins were detected by immunofluorescence with specific antibodies. Labels in each panel indicate the protein stained. Above images are representative of at least two experiments.

Figure 4.9. PIV5 M, AmotL1 and Nedd4L co-localization within cells. 293T cells were transfected to produce PIV5 M, AmotL1 and Nedd4L and proteins were detected by immunofluorescence. Staining was done for two proteins at a time. Labels in each panel indicate the protein stained. Above images are representative of at least two experiments.

PIV5 M and AmotL2 or Amotp130 were also expressed in A549 cells (Fig.4.10).

Immunofluorescence analysis confirmed co-IP results here. It was observed that

PIV5 M could not co-localize with either Amotp130 or AmotL2. AmotL2 shows a

84 distribution pattern similar to AmotL1; however, no M was routed to AmotL2 containing structures. Amotp130 interestingly displays a very different distribution pattern from AmotL1 and AmotL2. It can be visualized at the membrane and also intracellularly in a diffuse pattern. However, Amotp130 still does not co-localize significantly with M protein like AmotL1 does. These observations strengthen the conclusion that PIV5 M can bind only to one out of three cellular motins, in spite of structural and functional conservation between motin proteins. This makes it both important and interesting to study the role played by AmotL1 in PIV5 and mumps budding.

Figure 4.10. PIV5 M does not associate with either AmotL2 or Amotp130. 293T cells were transfected to produce PIV5 M and AmotL2 or Amotp130. Proteins were detected by immunofluorescence with specific antibodies. Labels in each panel indicate the protein stained.

Mumps virus matrix protein binds only AmotL1, not AmotL2 or Amotp130.

We had previously shown that mumps virus M protein binds to AmotL1, albeit weakly [56]. The C-terminal region of AmotL1 bound more efficiently to mumps virus M protein. Here, we repeated the original results and showed that mumps virus M protein binds both AmotL1 and its C-terminal region (Fig.4.11A, lanes 2,3 and 6). However, when tested for binding with AmotL2 and Amotp130, mumps virus M protein behaved like PIV5 M; it could bind neither the full-length AmotL2 and Amotp130, nor C-terminal fragments derived from these motins (Fig.4.11A, lanes 4,5,7,8).

We also tested the effects of over-expression of full-length and C-terminal motin fragments on mumps VLP production (Fig.4.11B). We observed that only AmotL1- derived proteins had a negative effect on mumps VLP production (Fig.4.11B, lanes 3 and 6). This was not observed earlier [56], since different conditions were used then for mumps VLP production. There, full length AmotL1 did not seem to affect mumps

VLP production, although the C-terminal fragment lowered it about two-fold. Here, we optimized VLP conditions for mumps and this assay was more sensitive to minor perturbations. Over-expression of both the full-length and C-terminal fragment of

AmotL1 lowered the VLP yield. Over-expressing AmotL2 or Amotp130 derived motins had no effect on mumps VLP production. The co-IP and VLP experiments indicate that like PIV5 M, mumps virus M protein evolved to bind only to AmotL1 and not to the other motins. Similar to PIV5, the exact role that AmotL1 plays in mumps virus assembly needs to be investigated further.

Figure 4.11. Mumps virus matrix protein interacts only with AmotL1, and not the other motins; only AmotL1-derived proteins can inhibit mumps VLP production. (A) 293T cells were transfected to produce MuV M along with full-length motins or their C-terminal regions. Co-IP was carried out using flag beads; proteins of interest were detected by immunoblotting. (B) 293T cells were transfected for mumps VLP production; MuV M, F and NP were expressed along with full-length motins or their C-terminal regions. Sucrose gradient purified VLPs and cell lysates were loaded on SDS-PAGE gels and proteins were detected by immunoblotting. Relative efficiency of VLP production in the presence of motin over-expression was quantified as described in Fig. 4.4.

4.3 Discussion

Viruses co-opt a variety of host proteins for their own benefit as they carry out their life cycle within cells. We previously demonstrated host protein involvement in budding of paramyxoviruses - PIV5, mumps virus and Henipaviruses, via interactions with viral M proteins [54–56]. One of these host proteins, angiomotin- like 1 (AmotL1), could interact with PIV5 and mumps virus M proteins, depletion of which negatively affected PIV5 virion release, indicating a potential role for AmotL1

87 in PIV5 budding. Over-expression of a small M-binding fragment of AmotL1 also strongly impaired PIV5 VLP production. Angiomotins are tight junction-associated proteins that play multiple roles in different cellular signaling pathways, and consist of three members – Amot, AmotL1 and AmotL2 [115]. We demonstrated here that

PIV5 M and mumps virus M protein are capable of binding exclusively to AmotL1, and not to Amot or AmotL2. This is intriguing as all the three motins are highly conserved both structurally and functionally (Fig. 4.2) [115].

Like many other viruses, PIV5 and mumps virus require the ESCRT machinery for efficient egress [66, 68]. Most viruses contain late domain motifs in their structural proteins via which they connect to the ESCRT [110–112, 162–164, 168, 169].

However, paramyxoviruses do not possess conventional late domain motifs and it is not known how they utilize the ESCRT machinery for budding. The sole late domain in PIV5 M - “FPIV” does not bind to known ESCRT members. Here, we have shown that AmotL1 functioned to link PIV5 M protein to at least three different Nedd4 ligases- Nedd4.1, Nedd4L and NedL1. Only full-length AmotL1 capable of binding to both PIV5 M and Nedd4 ligases could bridge the gap between M and Nedd4 proteins.

AmotL1 and other motins contain one LPTY and two PPXY motifs in their N- terminal regions, via which they can bind to WW domain containing proteins like

YAP and Nedd4-like ubiquitin E3 ligases, of which Nedd4 ligases are actively involved in the ESCRT pathway. We speculate that PIV5 M protein recruits, through

AmotL1, the same WW domain containing Nedd4 ligases that other viruses recruit through their PPXY late domains, thus allowing the host ESCRT machinery to help facilitate the late stages of virus budding.

This raises the question of why PIV5 has evolved to recruit the ESCRT machinery indirectly through AmotL1, instead of simply including PPXY motifs directly into its proteins as other viruses do. There are several conceivable advantages for indirect recruitment of ESCRT via AmotL1. First, AmotL1-containing tight junctions are concentrated around apical regions of polarized epithelial cells, separating the apical and basolateral surfaces. PIV5 and mumps viruses bud from these same apical surfaces [89, 90], and AmotL1 might function to guide M proteins to virus assembly sites, one among which might be these tight junctions. In its absence, M could possibly be mistargeted, leading to reduction in virion production. A number of viruses bud from apical surfaces and it remains to be seen whether they interact with angiomotins at these budding interfaces [88–90]. Secondly, angiomotins associate with F-actin [170]; they also contain BAR-like domains that are involved in membrane remodeling [171]. It is possible that PIV5 and mumps virus M proteins recruit AmotL1 at virus assembly sites, where the BAR domain activity of AmotL1 further boosts bud formation initiated by PIV5 M. Actin is abundant in PIV5 virions

[56] and AmotL1 might play a role in F-actin recruitment at virus assembly sites, which might help in release of virus particles. Knockdown of AmotL1 however did not reduce actin in virions [56] and it would be interesting to explore the interplay between AmotL1 and actin in PIV5 infected cells. Angiomotins were recently shown to bind to HIV-1 Gag, and were needed in concert with Nedd4L to rescue budding of a HIV-1 provirus containing a late-domain depleted Gag protein [126]. The PPXY-

WW domain interaction between motins and Nedd4L, as well as the HECT ubiquitin ligase activity of Nedd4L were essential for the recovery of virus budding functions.

Angiomotins thus functioned to link HIV-1 Gag that is devoid of PPXY motifs to

Nedd4L and the ESCRT, thereby serving an auxiliary budding function, in addition to the PTAP and YPDL motifs in Gag. Depletion of Amot in HIV infected cells caused budding to arrest at a “half-moon” stage [126]; depletion of PPXY motifs in Gag protein of Mason-Pfizer monkey virus (MPMV) causes virions to stack up against the plasma membrane, accompanied by a failure to initiate membrane envelopment

[172]. These similar phenotypes suggest that viruses might need Nedd4-motin complexes at virus assembly sites for proper bud formation and recruit either member of this complex to boost the budding process.

AmotL1 thus links PIV5 M to Nedd4 ligases, and potentially to the ESCRT. Unlike

HIV-1 Gag though, overexpressing these proteins does not improve PIV5 or mumps

VLP production. Overexpression of AmotL1 and Nedd4 either singly or in combination led to decreases in PIV5 VLP production [56 and data not shown].

Overexpression of full-length AmotL1 also impaired mumps VLP production. This is quite intriguing and it is possible that increasing the AmotL1 or Nedd4 levels in cells might have adverse effects on other cellular pathways that are critical for PIV5 and mumps virus budding. When co-expressed within cells and observed via immunofluorescence, PIV5 M, AmotL1 and Nedd4L were found in huge punctae within cells. It is unknown what cellular compartments these punctae correspond to and further work needs to be conducted in this regard towards obtaining a clearer picture of AmotL1 involvement in PIV5 budding.

It would be interesting to find out if other WW domain proteins that bind to

AmotL1 can also indirectly bind to PIV5 M protein, or whether this interaction is

90 specific to Nedd4 ligases. One among these is YAP that binds to angiomotins [118–

120, 125]. YAP is a part of the Hippo pathway and mediates cell proliferation, apoptosis and contact inhibition. YAP has been known to recruit c-Abl that phosphorylates Nedd4L [173], preventing it from ubiquitinating AmotL1, thereby increasing AmotL1 cellular levels. Since YAP and Nedd4 ligases both utilize the PPXY motifs in AmotL1, there might be competition between these two proteins for binding to AmotL1. It will be of interest to study how this dynamic influences PIV5 budding.

The PIV5 M – AmotL1 – Nedd4 binding interfaces offer prime druggable targets for the creation of antivirals. The M binding site on AmotL1 is known, and a minimal

M-binding fragment derived from AmotL1 potently inhibits PIV5 budding. However, the AmotL1 binding site on M protein has still eluded identification. The FPIV motif was initially tested for binding to AmotL1, but it is not involved and might be essential for interaction with other host proteins [56]. Recent advances on Nedd4-

PPXY interfaces have led to the development of small molecule inhibitors that impair budding in filoviruses, rhabdoviruses and arenaviruses [174]. Exploring these varied binding interfaces between viral and host proteins further holds promise towards developing antivirals to combat pathogenic paramyxoviruses.

CHAPTER 5

SUMMARY AND FUTURE DIRECTIONS

5.1 Summary

Genome packaging into virus particles is crucial for maintaining infectivity and successive cycles of infection and replication. In paramyxoviruses, the matrix (M) and nucleocapsid (NP) protein are primarily involved in packaging the viral RNA genome into nascent virions. Studying M-NP binding interfaces is thus essential as they offer an attractive drug target for antiviral design. Previous work has revealed that for a number of paramyxoviruses, the M-binding site on NP is located in its C- terminal region, often at the very C-terminal end [42, 43]. Binding was localized to a

DLD containing motif in PIV5 NP and to a DLLD motif in measles virus N protein.

Any disruptions to these DLD-like motifs abolished interaction with M protein. We have shown here that these genome packaging interactions can be manipulated to package foreign proteins into VLPs, with the potential of using them as delivery vehicles. This was carried out by appending different sized fragments of the C- terminal region of PIV5 NP protein to Renilla luciferase, followed by testing the modified luciferases for PIV5 VLP production. We found that as little as 15 amino acids from the DLD-containing C-terminal region of NP protein were sufficient to direct luciferase into PIV5 VLPs. Successful PIV5 VLP production depends on the presence of NP protein in addition to M and HN/F; these 15 amino acids appended to luciferase also replaced the requirement for full length NP protein in PIV5 VLP

92 production. PIV5 and mumps virus are both rubulaviruses, and hence are closely related. PIV5 NP, however, cannot substitute for mumps virus NP protein to produce mumps VLPs. PIV5 NP contains a DLD sequence critical for M binding, while mumps virus NP protein has a DWD sequence. We demonstrated that a single amino acid substitution on PIV5 NP, L507W, could induce it to be compatible with mumps virus

M protein and replace mumps virus NP protein for efficient generation of mumps

VLPs. Moreover, this PIV5 NP protein did not lose compatibility with PIV5 M protein. It could also bind to Nipah virus M protein, similar to wt PIV5 NP protein, and be incorporated into Nipah VLPs. DWD residues were also critical in the context of mumps virus NP protein, as mutation or deletion of this motif led to defects in NP interaction with M and consequently, poor mumps VLP production. The information about DWD motifs was used to generate foreign proteins appended to PIV5 NP sequence where the DLD was changed to DWD, with the result that these proteins were now compatible with mumps virus M protein and could be packaged into mumps VLPs (Appendix B). 15 amino acids from Nipah virus N protein were also sufficient to direct luciferase into Nipah VLPs. Mutant modified luciferases with 15 amino acids from N sequence were created, where N residues were changed to alanines. This revealed that a DLD-containing motif NDLDFV was critical for directing luciferase into Nipah VLPs and ablating it led to poor luciferase incorporation, indicating that this motif is a possible M-binding site on Nipah virus

N protein. We hypothesize that paramyxovirus NP/N proteins have evolved along a universal theme and employ DLD-like motifs for interaction with M proteins and subsequent genome packaging for generation of infectious viruses.

Paramyxoviruses hijack a number of cellular proteins as they bud out of cells. One among these is AmotL1, which was found to be important in PIV5 budding pathways. Yeast two-hybrid analyses previously revealed that PIV5 M binds to

AmotL1 [56], and depletion of AmotL1 in mammalian cells impaired PIV5 virus production. An M-binding, minimal fragment derived from AmotL1 could block PIV5

VLP production potently. We have shown here that both PIV5 and mumps virus M proteins exclusively bind to AmotL1, and not to motins Amot or AmotL2. Only M- binding C-terminal fragments derived from AmotL1 were capable of blocking PIV5 and mumps VLP production; Amot or AmotL2 derivatives had no effect. PIV5 and mumps viruses also utilize the ESCRT machinery for budding [66, 68], however no

ESCRT binding partners have been isolated yet for these viruses. We showed here that AmotL1 might help PIV5 M protein make connections to the ESCRT by facilitating interactions with Nedd4 E3 ubiquitin ligases. M and Nedd4 proteins were in complexes together only when AmotL1 was present to bridge the gap. These proteins were also observed in the same cellular compartments by immunofluorescence, while PIV5 M and Amot or AmotL2 distributed differently within cells. These findings support a model whereby M recruits the same WW domain containing proteins to virus assembly sites indirectly via AmotL1, that other viruses recruit directly through PPXY motifs on their structural proteins.

5.2 Future directions

Manipulating genome packaging interactions for therapeutic uses.

We have shown here that a foreign protein like luciferase can be packaged into

PIV5 and Nipah VLPs. We have also shown that a variety of other proteins like superoxide dismutase-1 (SOD1) and/or SerpinB3 can be directed into mumps and

PIV5 VLPs (Appendix A-1A and B-1B). These proteins were packaged into VLPs by manipulating the same M-NP interactions that are involved in packaging viral RNA genomes into particles. Here, 15-30 amino acids from the C-terminal region of PIV5

NP or Nipah virus N protein when appended to luciferase, could direct it into VLPs.

In the case of PIV5, modified foreign proteins could even replace the requirement for full-length NP protein in VLP production. The foreign proteins with PIV5 NP sequence can be induced to package into mumps VLPs by mutating the DLD motif in the PIV5 NP sequence to DWD, making them compatible with mumps virus M protein. This leads to the development of three different VLP systems that can potentially be utilized for delivering foreign proteins. The next step towards making this possible is to generate VLPs that are capable of both attachment and fusion to cells. PIV5 and mumps VLPs are typically generated with only one glycoprotein and

NP protein, making them capable of either attachment or fusion. Nipah VLPs on the other hand, are generated by expressing only the M protein; expressing other viral proteins has no effects on VLP production. Nipah VLPs have been produced in the past with M, N, G and F proteins [94]; optimal conditions for packaging foreign proteins into such VLPs need to be deduced. We have taken steps towards creating attachment and fusion capable PIV5 and mumps VLPs that contain foreign proteins

95 appended to either wt or mutant PIV5 NP sequence (Appendix A-1A and B-1B). In order to demonstrate efficient delivery, initial experiments were conducted where

PIV5 VLPs containing M, HN and F along with the luciferase-NP were generated.

These VLPs were then applied to cells which were later harvested. Lysates from these cells were analyzed in a luminometer (Appendix A-2) and the presence of luciferase was detected, indicating the PIV5 VLPs could both attach and fuse to cells to deliver their cargo successfully. PIV5 VLPs generated in a similar fashion but without fusion protein could not fuse to cells, and lysates from these cells did not contain modified luciferase. Efforts are now being made to develop assays to test efficient delivery of SOD1 or SerpinB3 to cells. It is also important to determine whether cargo contained in VLPs is delivered to the intended cellular compartments, and not just shunted towards lysosomes for degradation. Efforts should also be made towards identifying new candidate proteins that can be packaged into particles and have therapeutic benefits for cells upon delivery.

We are also interested in testing whether NP/N sequences appended to RLuc or other foreign proteins can competitively inhibit full-length NP proteins for binding to their respective M proteins. This would offer an important strategy for antiviral design, as this would possibly lead to the release of empty, non-infectious particles that lack viral RNA genomes. This could be tested by creating stable cell lines that express the M-binding modified foreign proteins, which could then be infected with

PIV5 or other paramyxoviruses of interest. Following this, growth curve experiments could be conducted to test for antiviral activity.

Examining compatibilities between M-NP proteins from different paramyxoviruses.

We have shown here that M-NP proteins can be compatible both between closely and distantly related paramyxoviruses. For instance, PIV5 M protein is compatible with mumps virus NP protein (data not shown), and PIV5 NP is compatible with

Nipah virus M protein. In cases where compatibility is poor or non-existent, it can be induced. PIV5 NP protein is not compatible with M protein from mumps virus.

However, changing only a single amino acid Leu at position 507 to Trp makes it compatible with mumps virus M protein. The above change creates a PIV5 NP protein that has DWD residues instead of DLD; DWD residues in mumps virus NP protein are important for its interaction with M protein and consequently for NP function in VLP production. In this manner, PIV5 NP that doesn’t bind to M protein from mumps virus can be induced to be compatible with this M protein. At the same time, it does not lose interaction with PIV5 M and Nipah virus M proteins. We aim to study compatibilities between M and NP proteins from other paramyxoviruses, either by membrane coflotation or VLP assays. Some candidate viruses are hPIV2,

HRSV, HMPV and Sendai virus. We are in the process of gathering reagents for studying M-NP proteins from these viruses. We hypothesize that the DLD-like motifs define compatibility between M-NP proteins and we will test if this holds true for the above viruses, by alanine substitutions and truncations. In cases where the

DLD-like motif is not important for M-NP binding, it will be exciting to test how that paramyxovirus differs in behavior from others of its kind. M-NP proteins can also be interchanged between these viruses and tested for compatibility by membrane

97 flotation. If there is no compatibility, attempts can be made to induce it. This could, in the future, lead to the development of novel recombinant paramyxoviruses with foreign envelope proteins that might have potential use as vaccines, in addition to helping obtain a clearer picture of rules that govern M-NP interactions in paramyxoviruses.

Studying the role of angiomotins and Nedd4 ligases in paramxovirus budding.

We have shown here that M proteins from PIV5 and mumps virus bind exclusively to AmotL1, and not to Amot or AmotL2. AmotL1 knockdown impaired PIV5 budding

[56], while M-binding fragments derived from AmotL1 could block PIV5 and mumps

VLP production. This suggests that AmotL1 has an important role in the budding processes of these viruses. We are in the process of conducting experiments to understand this. CRISPR technology is being used for constructing 293T cells depleted of either AmotL1/AmotL2/Amot. Once these cells become available, they will be transfected to produce PIV5 or mumps VLPs, or infected with PIV5 and the phenotype of these VLPs or viruses will be determined. Viruses can also be tested for infectivity by plaque or tissue culture infectious dose 50 (TCID50) assays.

AmotL1 depletion should have negative effects on virus or VLP production; however, knockout of Amot or AmotL2 should not have negative effects as these do not bind to viral M proteins from PIV5 or mumps virus. If VLP or virus production is severely impaired in Amotl1 knockout cells, further experiments can be done to overexpress AmotL1 in these cells by transfection to check whether virus/VLP production can be rescued. If virus production is reduced, we could also passage this

98 virus several times and check to see if virus production improves. If it does, the complete virus genome can be sequenced to look for adaptive mutations. Infected cells depleted for AmotL1 can also be subjected to SEM and TEM analysis, to figure out if the phenotype is similar to that seen for HIV-1 Gag in Amot depleted cells.

Amot depletion caused HIV-1 Gag to arrest at a half-moon stage and led to the conclusion that motins are required for HIV-1 budding at a stage earlier than

Tsg101 [126].

Nedd4 HECT E3 ubiquitin ligases are WW domain proteins that bind to cellular proteins with PPXY motifs and ubiquitinate them, among which are members of the motin family. Previous studies have shown that viral proteins with PPXY motifs also require Nedd4 ligases, where they assist in virus budding [112, 162–164, 168].

Nedd4 ligases can connect to the ESCRT machinery possibly via Tsg101 [169] or arrestin-related complexes [161], and thus link virus assembly to ESCRT complexes.

We have shown here that AmotL1 can function as an adaptor, linking at least three

Nedd4 ligase members to PIV5 M. These complexes can also be visualized in cells via immunofluorescence. The AmotL1-Nedd4 connection possibly forms an indirect link to the ESCRT for PIV5, and needs to be studied further. To do this, we have developed a PIV5 M protein that contains the p2b region from Rous sarcoma virus

Gag that includes a PPXY late domain motif (Schmitt AP, unpublished data). We have shown that PIV5 M.RSV can now bind to Nedd4L because of its PPXY motif, whereas wild-type PIV5 M cannot. PIV5 M.RSV however, does not lose compatibility with

AmotL1 (Appendix C-1A and B). We are in the process of creating a recombinant

PIV5 virus that contains the M.RSV. Infecting AmotL1 knockout cells with this virus

99 should theoretically not disrupt budding pathways, as it should be able to directly use Nedd4 ligases to facilitate budding, instead of indirectly via AmotL1. However, since it can still bind AmotL1, it will be interesting to check its phenotype.

Immunofluorescence studies can be carried out to identify the cellular compartments involved in localization of AmotL1, PIV5 M and Nedd4L. This information would offer further insights about the mechanisms involved in AmotL1 and Nedd4L roles in budding pathways of PIV5. In addition to this, the 3-way co- immunoprecipitation experiments involving PIV5 M, Nedd4 and AmotL1 should also be extended to mumps virus M protein. It is important to look into other WW domain proteins that bind AmotL1, like YAP. YAP plays important roles in the Hippo pathway [118, 125, 175] and it is reasonable to assume that competition between

YAP and Nedd4 proteins to bind AmotL1 might have effects on virus budding efficiencies.

100

APPENDICES

Appendix A: Packaging of foreign proteins into PIV5 virus-like particles and delivery into cells.

Attempts were made to package a number of other foreign proteins besides luciferase into PIV5 VLPs and these are discussed below. Gaussia luciferase, an enzyme similar in function to Renilla luciferase was appended to NP sequence and tested for PIV5 VLP production. EGFP and mCherry with 15 or 30 amino acids from

PIV5 NP sequence were also prepared and tested for VLP production. However, these fusion proteins did not work like the Renilla luciferase and failed to trigger

VLP production (data not shown). This failure to trigger efficient VLP production could be due to a number of reasons – the localization of Gaussia luciferase/

EGFP/mCherry within cells might be different from that of RLuc or the beta-barrel structure of EGFP/mCherry might not expose the disordered tail of PIV5 NP protein appropriately, making interactions with M protein difficult.

Four more proteins were tested – cyclooxygenase-2 (COX-2), superoxide dismutase-1(SOD1), serpinB3 and arginase-1(ARG1). COX-2, the inducible isoform of COX, is involved in production of prostaglandins and thromboxanes, and like COX-

1, is a major target of NSAIDs (non-steroidal anti-inflammatory drugs) [176]. SOD1 is one of the three superoxide dismutases, binds Cu and Zn and can destroy free superoxide radicals in the body. It plays important roles in apoptosis, and mutations in this enzyme have been implicated in amyotrophic lateral sclerosis (ALS) [177].

SerpinB3 is a serine proteinase inhibitor involved in squamous cell carcinoma and

101 other cancers [178]. ARG1 is involved in the urea cycle and catalyzes the conversion of arginine to ornithine and urea. Arginase deficiency can cause arginemia, characterized by growth and neurological defects [179]. All of these proteins were tagged with a myc-tag at their N-terminal ends, and were tested for expression. 15 or 30 amino acid sequences from PIV5 NP protein were also appended to the C- terminal ends of these different host proteins. All the COX-2 related proteins, with or without fused NP sequence, were defective in expression and could not be detected using an antibody to the myc epitope. Addition of the myc tag might have destabilized the enzyme, leading to degradation. The ARG1 based constructs were also defective in expression; only the ARG1-NP30 protein expressed at reasonable levels. The SOD1 and SerpinB3 based NP fusions were all expressed well and were further tested for their role in PIV5 VLP production. Mutant versions of the SOD1 and SerpinB3-based constructs were generated, where the DLD in the appended NP sequence was changed to DWD. Of these, only the SOD1-NP fusions could function with PIV5 M, F and HN to produce PIV5 VLPs (Fig.A-1A and B). ARG1-NP and

SerpinB3-NP fusions did not function efficiently for PIV5 VLP production (data not shown). SOD1 could be packaged into PIV5 VLPs; SOD1-NP30 mutant protein with

DWD sequence worked better than any other SOD1-NP fusion protein (Fig.A-1A and

B). Efforts are now underway to demonstrate whether the SOD1-NP fusion proteins still retain their biological activity.

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Figure A-1. Foreign proteins other than luciferase that contain NP-derived sequences can be directed into PIV5 VLPs. (A) 293T cells were transfected to produce PIV5 M, HN and F along with indicated SOD1-NP fusions or full-length NP proteins. Viral proteins in cell lysates and VLPs purified on sucrose gradients were detected by immunoblotting. (B) Relative efficiency of PIV5 VLP production was quantified by measuring amount of M protein in VLPs divided by amount of M in lysates, normalized to the value obtained with full-length NP protein. Error bars indicate standard deviations (n=3).

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PIV5 VLPs containing M, HN and F proteins along with RLuc-NP30.DWD were generated in 293T cells under aseptic conditions. These VLPs were then purified and applied to 293T cells, with the hope of delivering the luciferase into these new cells. Cells exposed to VLPs were lysed and the enzymatically active luciferase in lysates was measured by using a luminometer (Fig.A-2). Only cells exposed to VLPs containing both glycoproteins along with M and modified luciferase contained significant amount of luciferase, as evidenced by the activity readout on the luminometer. As controls, VLPs containing unmodified RLuc or VLPs without F protein were generated. VLP production should be poor with unmodified Rluc, however these VLPs should still be delivered into cells as they contain both glycoproteins and consistent with this, there were low levels of luciferase in 293T cell lysate exposed to such VLPs. However, VLPs generated without any F protein should not be able to enter cells at all. In this case, VLPs with M, HN and modified luciferase should be abundantly produced but should be incapable of fusing with cells to deliver their cargo. We noticed that cells exposed to such VLPs displayed only background levels of luciferase when lysed, which is consistent with failure to deliver luciferase into cells. This experimental technique should be explored further with VLPs generated using other foreign proteins as a potential therapy that can deliver proteins or protein-based toxins to specific cells.

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Figure A-2. Using VLPs for luciferase delivery into cells. 293T cells were transfected to produce PIV5 M, HN and/or F, along with indicated RLuc-NP fusions. Resulting VLPs were applied to 293T cells overnight and cells were then lysed to assess the ability of VLPs to deliver luciferase into cells. Luciferase amounts present in cell lysates were represented as fold changes over the value obtained for cells exposed to VLPs containing unmodified RLuc, along with M, HN and F proteins.

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Appendix B: Packaging foreign proteins into mumps virus-like particles.

Packaging foreign proteins into PIV5 virus-like particles was accomplished successfully. Attempts were made to use the same strategies to direct foreign proteins into mumps VLPs. Accordingly, mumps virus NP protein C-terminal sequences were appended to the C-terminal ends of Renilla luciferase. We hypothesized that the DWD containing NP sequence should function to direct foreign proteins into mumps VLPs via binding to mumps virus M protein, similar to the function of PIV5 NP sequences linked to foreign proteins. However, the luciferase-mumps virus NP fusions were impaired in their ability to trigger mumps

VLP production (Fig.B-1A, lanes 5 and 6). Expressing different amounts of these proteins than what was typically used for PIV5 VLP experiments also did nothing to improve mumps VLP release (data not shown). This failure to stimulate efficient mumps VLPs could be a result of the amino acid sequences selected from mumps virus NP protein that might have different folding properties when attached to a foreign protein, even though the C-terminal tail of mumps virus NP is highly disordered, just like that of PIV5 NP protein. As a control in these experiments, an

RLuc-PIV5 NP15 fusion protein was generated, where the DLD was replaced with the DWD motif found in mumps virus NP protein. The hypothesis was that both the

RLuc-PIV5 NP15 L507W and the RLuc-mumps virus NP fusions should work for mumps VLP production owing to the presence of the DWD motif. Interestingly, the

RLuc-NP fusion containing the sequence from PIV5 NP mutated to DWD worked successfully to rescue mumps VLP production (Fig.B-1A, lane 4), even though the

RLuc-mumps virus NP fusions could not. This difference in function could be a result

106 of the amino acid sequences surrounding the DLD/DWD motif in PIV5/mumps virus

NP proteins, leading to differences in folding and/or binding.

Since the RLuc-PIV5 NP L507W protein worked for mumps VLP production, we decided to use the other host protein-PIV5 NP fusions we had generated, with a

DWD motif instead of the wt DLD motif for VLP production. The SOD1 and

SerpinB3-PIV5 NP15/30 fusions with DWD mutations were expressed with mumps virus M, F and HN proteins in 293T cells (Fig.B-1B). VLPS were purified on sucrose gradients. Results from these experiments showed that the SOD1-PIV5 NP15/30

DWD proteins were the best at triggering mumps VLPs (Fig.B-1B, lanes 9 and 11) and could replace the requirement for MuV NP protein. SOD1-NP fusions without

DWD sequence were ineffective at triggering mumps VLP production (Fig.B-1B, lanes 8 and 10). SerpinB3-PIV5 NP fusions did not function as well as the SOD1 fusions, but efforts could be made to optimize this further. All proteins with DWD containing motifs could be packaged into mumps VLPs to different levels, in spite of the fact that they varied in their abilities to trigger efficient mumps VLP production, indicating ability to bind to mumps virus M protein. However, proteins with DLD motifs could not bind to mumps virus M protein and could not direct foreign proteins into mumps VLPs.

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Figure B-1. Foreign proteins can be packaged into mumps VLPs via manipulation of M-NP protein interactions. (A) 293T cells were transfected to produce mumps virus M and F proteins along with indicated luciferase-PIV5/MuV NP fusion proteins. Viral proteins from cell lysates and purified VLPs were detected by immunoblotting. VLP production efficiency was calculated as described in Fig.A-1B. (B) 293T cells were transfected to produce mumps VLPs with the indicated SOD1/Serpin B3-PIV5 NP fusion proteins, MuV M, F and HN. Viral proteins were detected by immunoblotting and relative VLP production efficiencies were quantified as described in A-1B.

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Appendix C: PIV5 M proteins with PPXY late domains can bind directly to

Nedd4L.

PIV5 M proteins with late domain regions from Ebola VP40 and Rous sarcoma virus Gag were generated (Schmitt AP, unpublished data). The Ebola VP40 protein contains overlapping late domain motifs PTAP and PPEY, whereas the RSV Gag contains a PPXY motif. These modified M proteins were tested for expression and

PIV5 VLP production (data not shown), and they functioned similarly to wt PIV5 M.

Co-IP experiments were carried out to test if these modified M proteins could interact with the same host proteins as wt PIV5 M. AmotL1 and 14-3-3 were co- expressed with these M proteins and complexes were pulled down (Fig.C-1B). Both

PIV5 M.Ebola and PIV5 M.RSV functioned similarly to the wt PIV5 M in binding

AmotL1 and 14-3-3. Since both modified M proteins have PPXY motifs they should be able to bind directly to WW domains in Nedd4 ligases, and they were tested for binding to Nedd4L (Fig.C-1A). Surprisingly, only the M.RSV was capable of binding to Nedd4L (Fig.C-1A, lane 4). PIV5 M wt doesn’t bind Nedd4L and behaved as expected, but M.Ebola with a PPXY motif should bind Nedd4L and it does not.

M.Ebola might be better adapted to bind other Nedd4 ligases and should be tested for binding to them. Not all PPXY containing proteins can bind all WW domain proteins; for instance, Amotp130 can bind only Nedd4.1, Nedd4L and Itch, but not most other members of the Nedd4 ubiquitin ligase family [117].

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Figure C-1. A PPXY-containing PIV5 M protein can bind directly to Nedd4L. (A) 293T cells were transfected to produce wt and modified PIV5 M proteins along with Nedd4L. Lysates were subjected to co-IP analysis via pulldown with a myc antibody. Proteins were then detected by immunoblotting. (B) 293T cells were transfected to produce wt and modified PIV5 M proteins along with flag-tagged AmotL1 or 14-3-3. Complexes were pulled down with flag beads and proteins were detected by immunoblotting.

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VITA Greeshma Ray

Education 2009-2016 Ph.D. in Pathobiology, The Pennsylvania State University, University Park, PA.

2005-2007 M.Sc in Life Sciences, University of Mumbai, Mumbai, India.

2002-2005 B.Sc in Life Sciences, Ramnarian Ruia College of Arts and Sciences, Mumbai, India.

Conferences and Presentations June 2016 Workshop talk “Recruitment of angiomotin-like 1 for paramyxovirus budding” at the annual American Society for Virology Meeting, Virginia Tech, Blacksburg, VA. March 2016 Poster presentation “Manipulation of paramyxovirus genome packaging interactions to direct foreign proteins into virus-like particles” at Graduate Exhibition of The Pennsylvania State University, University Park, PA. November 2015 Co-organizer at the second Penn State Intercampus Virology Meeting, The Pennsylvania State University, University Park, PA. June 2015 Research talk at the first Penn State Intercampus Virology (PSiV) Meeting. Manipulation of protein- protein interactions that lead to paramyxovirus genome packaging. The Pennsylvania State University, Hershey, PA. July 2013 Volunteer at the American Society for Virology Meeting, The Pennsylvania State University, University Park, PA. July 2012 Poster presentation “Interactions that drive the packaging of RNPs into budding paramyxovirus particles “ at the annual American Society for Virology meeting, University of Wisconsin-Madison, Madison, WI.

Publications and patents: Process begun for patent application titled “ Paramyxovirus virus-like particles as protein delivery vehicles”- Anthony P Schmitt, Phuong T Schmitt, Greeshma Ray, Jan 2016.

Ray G, Schmitt PT, Schmitt AP. C-terminal DxD containing sequences within paramyxovirus nucleocapsid proteins determine matrix protein compatibility, and can direct foreign proteins into budding particles. J.Virol. doi. 10.1128/ JVI.02673-15. Selected as a JVI spotlight feature.

Schmitt PT, Ray G, Schmitt AP. The C-terminal end of PIV5 NP protein is important for virus-like particle production and M-NP protein interaction. J.Virol.84, 12810-12823, 2010.