UNIVERSITY OF CALIFORNIA RIVERSIDE

A Novel Role for SCAMP3 as an Innate Immune Factor

A Dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

in

Microbiology

by

Vladimir Radic

March 2017

Dissertation Committee: Dr. I-Chueh Huang, Co-Chairperson Dr. Rong Hai, Co-Chairperson Dr. Ilhem Messaoudi

Copyright by Vladimir Radic 2017

The Dissertation of Vladimir Radic is approved:

Committee Co-Chairperson

Committee Co-Chairperson

University of California, Riverside ABSTRACT OF THE DISSERTATION

A Novel Function of SCAMP3 as an Innate Immune Factor

by

Vladimir Radic

Doctor of Philosophy, Graduate Program in Microbiology University of California, Riverside, March 2017 Dr. I-Chueh Huang, Co-Chairperson Dr. Rong Hai, Co-Chairperson

Influenza A Viruses (IAV’s) pose a significant threat to public health. On average, infections account for 25,000 deaths and 250,000 hospitalizations every year in the United States. Despite having been discovered in the 1930’s, and despite decades of study influenza viruses continue to cause significant morbidity and mortality. Furthermore, with the emergence of Highly Pathogenic Avian Influenza

Viruses (HPAIV) in humans, IAV’s pose a greater threat than ever before. The innate immune response is critical to controlling these viral infections, but despite over half a century passing since the discovery of the first innate immune , the full scope of innate immunity has yet to be elucidated. The -Induced Transmembrane

(IFITM) family of proteins has been shown to strongly restrict a broad range of viruses,

iv including IAV5,6,7. However, the exact mechanism of this inhibition as well as the mechanism of IFITM regulation and metabolism is poorly understood. Secretory

Membrane 3(SCAMP3) was first identified in 1997 and has since been implicated in EGFR recycling and Salmonella pathogenesis. Here we demonstrate a new function of

SCAMP3 in the innate immune response. We show that SCAMP3 can function to stabilize by decreasing its lysosomal degradation. We show that SCAMP3 can alter the trafficking of IFITM, resulting in the protein being recycled from early endosomes back to the Golgi apparatus. Upon SCAMP3 depletion, this recycling is ablated and IFITM3 undergoes increased lysosomal degradation. We also show that

SCAMP3 can directly restrict Influenza A Viruses. We show that SCAMP3 and interfere with cleavage of Highly Pathogenic Avian Influenza (HPAIV) (HA) cleavage, resulting in viruses that are deficient in their ability to enter target cells. We also show that SCAMP3 expression, phosphorylation and localization is altered upon viral infection or stimulation with various innate immune proteins, suggesting that

SCAMP3 is an innate immune effector. This research deepens our understanding of the innate immune response and presents a potential therapeutic strategy for treatment or prophylaxis of viral disease.

v Table of Contents

Chapter 1: Introduction ...... 1 1.1 Influenza Viruses ...... 2 1.2 History of Influenza ...... 3 1.3 Influenza Structure and Classification...... 8 1.4 The Influenza Genome ...... 10 1.5 The Influenza Life Cycle ...... 12 1.6 The Hemagglutinin Protein ...... 18 1.7 Innate Immunity to Influenza Viruses ...... 22 1.8 Abrogation of Innate Immunity by Influenza Viruses ...... 24 1.9 Adaptive Immunity to Influenza Viruses ...... 26 1.10 Antiviral Drugs ...... 28 1.11 The Interferon Induced Transmembrane Proteins ...... 31 1.12 The Discovery of IFITM Antiviral Activity ...... 32 1.13 IFITM Protein Structure ...... 34 1.14 Post Translational Modifications of IFITM Proteins ...... 66 1.15 Models of IFITM Mediated Viral Restriction ...... 37 1.16 SCAMP3 ...... 39 1.17 References ...... 45

Chapter 2: SCAMP3 Regulates IFITM3 Expression ...... 58 2.1 Introduction ...... 62 2.2 Results ...... 58 2.3 Discussion ...... 62 2.4 Materials and Methods ...... 68 2.5 Figures ...... 76 2.6 References ...... 90

Chapter 3: SCAMP3 Is and Innate Immune Factor which Inhibits IAV infection by altering Hemagglutinin Trafficking...... 94 3.1 Introduction ...... 95 3.2 Results ...... 100 3.3 Discussion ...... 106 3.4 Materials and Methods ...... 109 3.5Figures ...... 116 References ...... 121

Chapter 4: Discussion ...... 123

vi

List Figures

Chapter 1 Figure 1.1 H5N1 Death Rates...... 2 Figure 1.2 Spanish Flu Death Rates ...... 3 Figure 1.3 Influenza Structure ...... 9 Figure 1.4 The Influenza Life Cycle ...... 12 Figure 1.5 Hemagglutinin Mediated Fusion ...... 19 Figure 1.6 Hemagglutinin Sequence Comparisons ...... 21 Figure 1.7 Innate Immunity to Influenza Viruses ...... 23 Figure 1.8 The IFITM Proteins ...... 31 Figure 1.9 IFITM Topology ...... 35 Figure 1.10 IFITM Restriction Mechanism ...... 38 Figure 1.11 SCAMP3 ...... 40 Figure 1.12 SCAMP3 Relocalizes during Salmonella Infection ...... 32

Chapter 2 Figure 2.1 SCAMP3 Interacts with IFITM3 ...... 76 Figure 2.2 SCAMP3 Contributes to IFITM Restriction of Viral Entry ...... 78 Figure 2.3 SCAMP3 Stabilizes IFITM3 ...... 80 Figure 2.4 SCAMP3 Contributes to IFITM Localization to the Golgi Network ...... 82 Figure 2.5 SCAMP3 Regulates Early-endosome to Golgi Recycling ...... 84 Figure 2.6 IFITM Interacts with SCAMP3 ...... 86 Figure 2.7 SCAMP3 Does not alter Ubiquitination of IFITM3 ...... 88

Chapter 3 Figure 3.1 SCAMP3 Restricts HPAIV Pseudotype Viruses ...... 115 Figure 3.2 SCAMP3 Alters HA Levels ...... 117 Figure 2.3 SCAMP3 and Innate Immunity ...... 119

vii CHAPTER 1

Introduction

1 1.1 Influenza Viruses

Influenza A Viruses have been important human pathogens for hundreds of years1. The first officially recognized influenza pandemic occurred in the summer of

15102. It originated in Asia before spreading to Europe and Africa. An Italian Chronicler described the pandemic:

“On this day [July 13, 1510]…in Modena there appeared an illness that lasts three

days with a great fever, and headache and then they rise…but there remains a

terrible cough that lasts maybe eight days, and then little by little they recover and

do not perish.”3

Over five hundred years later diseases caused by descendants of this virus remain a significant public health concern. Despite almost a century passing since the discovery of

Influenza as the causative agent of these diseases, the flu-associated death toll ranges from 3,000 to 49,000 per year in the United States alone3. Whereas mortality rates for these viruses are generally low in the developed world (around 1.4 deaths/100,000 cases in 2014 in the United States), these viruses pose a much larger threat to people in developing countries, where flu associated mortality rates are higher by orders of magnitude4,5. Recently, Highly Pathogenic Avian Influenza Viruses(HPAIV’s) have acquired the ability to cause accidental infections in humans6. While seasonal Influenza

Viruses typically cause a self-limited infection of the upper respiratory tract, HPAIV’s cause systemic infection, which often leads to multiple organ failure and death in over half of all cases7. This is especially alarming as most victims of HPAIV infections are under 30 years of age with little no underlying health problems8. If these HPAIV strains

2 of influenza acquire the ability to efficiently transmit between humans, the resulting pandemic will be catastrophic. Further research into Influenza viruses and the disease they cause is therefore critical and necessary for avoiding such a public health disaster.

Figure 1.1: An analysis of death rates from H5N1 Influenza with respect to the age of affected people. Full column height denotes the total amount of cases in that age group; dark blue segment represents morbid cases while blue segment represents survivors

1.2 History of Influenza

Influenza-like disease syndromes have been described since antiquity9. Scientific consensus recognizes the earliest recorded influenza pandemic as occurring in the summer of 1510. The virus appeared to have originated in Asia before spreading to

Africa and Europe, where accounts of the pandemic can be found in various countries10.

However it wasn’t until the 1918 Spanish flu pandemic that influenza research became a global health priority. The exact origins of this virus are unknown, but the virus resulted

3 from a genetic shift from a previously circulating strain, which rendered it immune to the herd immunity that had been established among human populations. Several epidemics at

US military training camps were reported in the early spring of 1918. These however were largely ignored due to the fact that World War I was already raging in Europe. The pandemic exploded in the early winter of 1918. It reappeared in Boston, likely brought back by soldiers returning from the front. Soldiers departing for the war brought the disease with them. This resulted in entire battalions of soldiers becoming too sick to fight.

By the end of the war the Spanish flu would have killed more soldiers than the fighting itself did. Vast dissemination of the virus, compounded people throwing large gatherings at Armistice day created a public health disaster. This was further compounded by the fact that hospitals were being taxed to the breaking point by wounded returning from the war. The winter of 1918 bore witness to the worst public health disaster mankind had ever seen. By the time the virus had ran its course, 1/5th of the world population would have been infected and 20-40 million people would have lost their lives. The 1918 virus was incredibly virulent. The estimated death rate was 2.5%, orders of magnitude higher than the average death rate for influenza pandemics (0.1%). Shockingly the death rate for people age 15-34 was over twenty times higher previous strains. This catastrophic pandemic had lasting effects on every country it ravaged and it catapulted influenza viruses into the forefront of public health problems.11,12,13

4

Figure 1.2: Graphical representation of the death rate of the 1918 Spanish flu, compared to the normal influenza strain of the previous year. Both are shown as a function of age of the afflicted.

Influenza Virus was first isolated in 1930 by Richard Shope13. He was able to use chamberlain filters to isolate a strain of Swine influenza and demonstrate that it was the causative agent of “hog flu”. Three years later a human virus was isolated by Wilson

Smith, Sir Christopher Andrewes, and Sir Patrick Laidlaw of the National Institute for

Medical Research in London, England14. The researchers were passaging nasal washes from an influenza patient in ferrets when they discovered that the animals would get

5 infected, display symptoms of influenza-like illness and eventually pass it to their cage mates. They were also able to isolate the virus from a junior researcher who was infected by one of the infected ferrets. The discovery of Influenza as the causative agent of the flu, as well as culture methods utilizing embryonated chicken eggs allowed for an explosion of research on the virus which has continued unabated to this day. The first influenza vaccine was created in 1940 by the US Military15. Since then, better propagation techniques, increased surveillance and reverse genetic systems have been boons to

Influenza research. However, vaccination for these viruses is still hit and miss. Currently, a trivalent influenza vaccine is generated each year against strains that the WHO guesses have potential to be widely disseminated in the next flu season. While this generally results in adequately protective vaccines, oftentimes it is inefficient at controlling epidemics and completely useless against genetic shifts in circulating strains such as one that occurred and caused the 2009 swine flu pandemic16. Further research is necessary to provide better methods of protection against these viruses.

The 1918 Spanish flu pandemic was the first Influenza Pandemic to be reliably recorded. Since then, there have been four verified Influenza pandemics. These outbreaks tend to infect a large portion of the human population in a very short period of time, usually infecting 20 to 40 percent of the population before being stopped or burning out.

They appear to in 10-40 year intervals and are mostly associated with a genetic shift in a circulating strain that nullifies herd immunity to previously circulating viruses. The first pandemic after the 1918 Spanish Flu was the Asian Influenza pandemic of 1957. It was caused by an H2N2 strain of the virus and originated in the Southern Chinese province of

6 Gizhou in February 195717. Genetic and biochemical analysis indicates that it originated from a reassortment between human and avian viruses, as it contained H2 HA, H2 NA and PB1 from avian viral origin. This virus was not extraordinarily pathogenic and poor clinical outcomes from it are associated with lack of existing immunity to these viruses18.

In July of 1968 a virus of the H3N2 subtype was isolated from a patient in Hong

Kong. By the end of the year the virus had spread around the world, infecting an estimated 40% of the global population at the time. Like the 1957 pandemic strain, this virus was not especially deadly and its emergence is credited to it’s HA protein, which was avian in origin. The virus also contained an N2 from a previously circulating human strain which appears to have limited its pathogenicity19,20.

Another pandemic occurred in 1977, originating in the USSR and China. It was caused by and H1N1 strain of the virus, and interestingly morbidity was almost exclusively limited to people younger than 25 years. This is due to the fact that the H1N1 staring responsible for this pandemic was closely related to a strain that had circulated in humans in the 1950’s. The lack of mutations between the 1977 pandemic strain and these previously circulating strains seem to hint at maintenance of these viruses in non-human species leading to a reemergence after herd immunity had dissipated21,22.

Finally, the most recent pandemic to occur happened in 2009 with the Swine Flu.

It began with a flu outbreak in a small Mexican town in mid-February 2009. By June, it had spread from Mexico across north and South America and into Asia and Europe, at which point the World Health Organization declared it a Phase 6 viral pandemic. Within

7 a year the virus completely replaced previously circulating H1 HA containing strains in humans. Genetic analysis revealed that the virus contained PB2 and PA genes of North

American avian origin, a PB1 of a human H3N2 virus, HA, NP and NS genes of classical swine virus original and NA and M genes from Eurasian avian virus origin. This combination of genes allowed the virus to spread incredibly efficiently among humans and this pandemic highlights the ability of Influenza Viruses to utilize genetic material from various sources to escape herd immunity and efficiently spread through human populations. Though decades of research have been dedicated to fighting influenza, the virus still poses a significant threat to public health23,24,2

1.3 Influenza Structure and Classification

Influenza viruses are members of the family , which are defined as viruses with a negative-sense single stranded and segmented RNA genome. There are six genera in this viral family; Influenza A, B and C viruses, Isavirus, and

Quaranjavirus. Influenza A, B and C infect a variety of hosts, including humans. Isavirus is an important pathogen of Atlantic Salmon while Thogotovirus and appear to mainly infect arthropods. However of all the Orthomyxoviridae, subtype A influenza viruses are by far the most clinically relevant in humans26.

8

Figure 1.3: Left- Electron micrograph of influenza viruses generated in MDCK cells. Right- 3D model of a cross section of a single influenza virion.

Influenza A viruses have a complex structure. The virus possesses a lipid membrane derived from host cells. The envelope harbors three viral proteins: hemagglutinin (HA), (NA) as well as the Matrix protein M2. Under the lipid membrane lies the M1 Matrix protein. The core of the viral particle contains the ribonucleoprotein (RNP) complex. This complex contains the viral polymerase proteins

PB1, PB2 and PA, the viral nucleoprotein (NP) that surrounds eight negative sense RNA genome segments. The NEP/NS2 (Nuclear export protein/nonstructural protein 2) can also be found inside the influenza virion. Viral morphology is characterized by distinctive spikes of 10-14nm which are easily observable in electron micrographs27. The virus is pleomorphic, existing as spherical particles with a diameter of about 100nm, or as filamentous structures with a length of up to 300mm28. The filamentous viruses are frequently observed in fresh clinical isolates, but also seem to correspond with viruses containing specific M1 or M2 proteins29. The virus is categorized by the two major antigenic , the hemagglutinin and neuraminidase30. Currently, there are

9 currently 21 different HA and 11 different NA. Viral nomenclature consists of: type of host, in case the virus has not been isolated from humans; geographical region of origin; number of lineage; year of isolation and; protein antigen type (ex.

A/California/04/2009(H1N1)) 31.

1.4 The Influenza Genome

Influenza A viruses contain an eight-segmented negative sense RNA Genome.

These segments range from about 900 to over 2300 base pairs in length and utilize alternative open reading frames code for a total of 11 proteins31. The segments are, in order from longest to shortest:

PB2, ~2300bp which codes for PB2, a component of the viral polymerase

responsible for cap recognition. 32

PB1, ~2300bp which codes for PB1, a component of the viral polymerase

responsible for elongation. This gene also contains an alternate open reading

frame which codes for PB1-F2, a protein with pro apoptotic activity which also

promotes viral infection by serving as an Interferon agonist.33

PA, ~2300bp, which codes for PA, the final protein of the viral polymerase,

which has endonuclease activity and functions in cap snatching from cellular pre

mRNA. 34

10 HA, ~1800bp. Codes for hemagglutinin one of the major antigenic proteins of

influenza. Hemagglutinin is responsible for receptor binding and is responsible

for fusion between the virion and host cell membranes. 35

NP, ~1600bp. Codes for the viral nucleoprotein which encapsidates the viral

genome and aids in transcription, replication and assembly. 36

NA, ~1400bp. Codes for the viral neuraminidase. This is the other major

antigenic and is necessary for efficient budding of daughter virions

from an infected cell. 37

M, ~1000bp. Codes for M1 and M2 matrix proteins via alternative splicing of

viral mRNA. M1 interacts with the RNP complex and glycoproteins and is

necessary for RNP nuclear export, assembly and budding. M2 is a membrane-

associated protein which functions as an ion channel. It also plays a role in

assembly and budding of nascent virions. 38

NS, ~900bp. Codes for the NS1 and NEP/NS2 proteins. NS1 functions in

suppressing the innate immune response. Whereas NS2 functions in RNP nuclear

export as well as regulation of RNA synthesis. 39

The alternate spliced variants of the proteins expressed by PB1, and NS are not expressed by all strains meaning that these are true accessory proteins. Each viral segment contains has non-coding regions at the 3’ and 5’ ends. The extreme ends of each of these segments are conserved and a segment-specific noncoding region follows this.

These non-coding regions vary between strains and the biological significance of this variation is not well understood. However these non-coding regions have been

11 previously demonstrated to be critical for viral polymerase binding, cap-snatching, transcription initiation, packaging, and replication. 40

1.5 The Influenza Life Cycle

Figure 1.4: A diagram of the life cycle of an Influenza virus. Left: A graphic of an influenza virion as well as the assembled RNP complex. Right: A schematic of influenza infection from receptor binding though budding of daughter virions from the infected cell. Influenza infection begins with receptor binding to sialic acids on the surface of target cells. residues are ubiquitous among all species as well as cell types, but this binding action is constrained by the fact that HA’s of viruses bind specifically to different sialic acid linkages. Human viruses preferentially bind to N-acetylneuraminic acid attached to a galactose molecule via an α2,6 linkage (SAα2,6Gal), where as avian viruses bind to sialic acids with an α2,3 linkage41. Congruent with this observation,

12 human tracheal epithelium cells contain mostly α2,6 linked sialic acids while the gut epithelium of most migratory waterfowl contains mostly α2,3 linked sialic acids. This is not an absolute barrier between species-specific viruses however, as each of these tissues contain a smaller number of the other sialic acid linkages. Also, studies have shown that factors such as the type of carbohydrate backbone, chain length and branching patter, as well as other variation can influence a sialic acid residue’s ability to interact with a given viral HA42.

Once an influenza virion binds a sialic acid residue, it is internalized into the cell.

Clatherine mediated endocytosis is has traditionally been believed to be the mechanism of influenza viral entry, but recently non-clatherine, non calveolae-mediated internalization and macropinocytosis of influenza virions have been described as well43,44. Once internalized into the cell, the virus requires low pH to initiate fusion with the host cell membrane. The low pH of the late endosome causes a structural change in

HA to occur, which allows the fusion peptide at the n terminus of the protein to to insert into the endosomal membrane. As pH drops further, the concerted conformational changes of several HA molecules (viral associated HA exists as a trimer of three single

HA molecules) brings the viral and host cell membranes together, ultimately opening up a pore through which the RNP can cross into the cytosol45.

Unlike other RNA viruses, influenza depends on nuclear functions for replication.

All viral RNA Synthesis occurs in the nucleus. The viral RNP (the influenza genome never exists as naked RNA) is imported into the nucleus via an energy-dependent process. This process begins when the nuclear localization sequence on the viral NP is

13 recognized by kareopherin α. Kareopherin α then recruits kareopherinβ, forming a trimeric complex which then docks to the nuclear pore and is imported into the nucleus46.

Once inside the nucleus, the viral polymerase can begin synthesizing the viral mRNA and replicating the viral genome. The influenza genome is first transcribed into positive sense copies (cRNA), which are then used as templates to generate new vRNA which can serve as a template for mRNA47. Replication is wholly dependent on the viral polymerase complex48. This complex is composed of three subunits, PB1, PB2 and PA.

PB1 catalyzes nucleotide addition during RNA chain elongation. It contains conserved motifs, which are characteristic of RNA dependent RNA polymerases. The PB2 protein is critical for initiation of transcription as it is able to bind host pre-mRNA molecules. The polymerase complex requires a 5’ capped primer, which it steals from host pre-mRNA molecules, for both elongation of viral RNA as well as export of viral RNA from the nucleus for eventual transcription or packaging into daughter virions. The PA protein has exonuclease activity and is necessary for cleaving the 5’ cap from host pre-mRNA, thus completing the cap-snatching event. All influenza RNA segments contain 5’ and 3’ noncoding sequences. 48 These sequences vary between strains and segments, however the terminal 13 nucleotides at the 5’ end and the terminal 12 nucleotides at the 3’ end are conserved among all segments and all strains49. These conserved ends are complementary which allows for base paring between the 3’ and 5’ ends of each influenza genomic segment resulting in a panhandle structure. The viral polymerase complex binds this panhandle structure, allowing for initiation of transcription to occur after cap snatching of host pre-mRNA. Once RNA transcription is complete, the viral polymerase can catalyze

14 polyadenylation, which is dependent on a stretch of five to seven uracil residues on the 5’ end of the vRNA template adjacent to the double stranded region of the vRNA promoter50. As the polymerase approaches the vRNA panhandle, steric hindrance prevents it from elongating the entire sequence and it stutters on the uracil sequence, resulting in a polyadenylated transcript49. This along with the cap taken from host pre- mRNA results in a transcript, which can use host machinery to be translocate to the ER and utilize host translational machinery to generate viral proteins. Due to cap snatching, host cell mRNA can’t exit the nucleus, resulting in a down regulation of host in favor of viral replication. Influenza viruses can increase the coding capacity of their genomes using alternate splicing. Primary transcripts of the M, NS and PB1 genomic segments have 5’ and 3’ splice sites which fit the consensus sequence for the exon/intron boundaries of cellular transcript50. The virus can therefore utilize host cellular machinery to undergo alternative splicing, which has been shown to occur in the absence of any viral proteins51.

Once the genome has been replicated and production has begun, the viral genome must switch from primarily generating viral mRNA to generating viral genomic RNA. Current understanding is that the availability of free NP controls the switch between mRNA and cRNA synthesis. Unlike viral mRNA, vRNA and cRNA are encapsidated after transcription therefore as NP accumulates it stabilizes cRNA, promotes its transcription into vRNA, which is then encapsidated and transported out of the nucleus for virion assembly and budding. However, other studies have shown that

NEP/NS2 is required for the switch between transcription and replication. These proteins

15 are required for generation of small viral RNA’s (svRNA’s), which appear to promote synthesis of vRNA from cRNA52,53.

Following viral genome replication, the nascent RNP complexes are assembled in the nucleus and exported to the cytoplasm in a process which requires interaction with

M1 and NEP/NS2. M1 facilitates the interaction between the RNP and NEP/NS2, while

NEP/NS2 interacts with Crm1, a nuclear export receptor and several nucleoporins. Once outside of the nucleus the NLS on M1 is masked by its interaction with NEP/NS2, preventing re-entry of the RNP into the nucleus. Furthermore, NP has been shown to bind to filamentous actin further promoting the retention of the RNP in the cytoplasm in the later stages of infection54.

Following synthesis on membrane-associated ribosomes, the three influenza membrane proteins, HA, NA and M2 enter the ER where they are folded and glycosylated. From here, these three proteins are directed to the apical plasma membrane via apical sporting signals present in the transmembrane domains of each of these proteins. These transmembrane domains also contain signals for association with lipid rafts, cholesterol and sphingolipid rich microdomains on the cell surface that have been shown to be the primary sites of viral budding. The exact mechanism of how the RNP reaches the assembly site is currently poorly understood. The current hypothesis is that

M1 acts to recruit the non-membrane-bound viral components to the assembly site, which is supported by the fact that it bridges the internal viral components and the membrane bound proteins55. Several groups have proposed models for how this interaction occurs. It has been proposed that M1 becomes associated with the surface glycoproteins as they are

16 being trafficked through the exocytic pathway. M1 can then interact with them on their way to the cell surface and bring the RNP complex along with it. An alternative hypothesis proposes that the M1/RNP complex can interact with cytoskeletal components and directly traffic to the plasma membrane to initiate budding, however more research is necessary to elucidate the exact method of RNP trafficking56.

A daughter virion needs to have all eight genome segments present in order for it to be infectious. The exact mechanism of how this happens is currently unknown but two models exist to explain how influenza virions can incorporate all necessary genomic segments. The first model to be proposed assumes that RNP’s containing each of the eight genomic segments are randomly incorporated into virions. Assuming that each virion only contains 8 segments then only 0.24% of virions would be infectious. This doesn’t line up with what can be observed experimentally. However if it is assumed that

12 segments are packaged per virion then approximately 10% of virions would be infectious, which is compatible with experimental data and observations that virions can contain more than just 8 genomic segments. The second model assumes selective incorporation of each genomic segment. This has been described in other viruses and assumes that each vRNA segment has a unique packaging signal allowing for selective incorporation of exactly eight genomic segments into each daughter virion. Serial sectioning and imaging support this model and have shown that each nascent virion appears to have vRNP’s arranged in a distinct pattern, one in the center of the virion and the other seven parallel to it with three on either side of the middle segment. Also, the presence of packing signals at the 3’ and 5’ ends of each genomic segment have been

17 experimentally confirmed, and mutations in these packing signals have been shown to decrease viral integration of the affected segments57.

Viral budding requires outward curvature of the cell membrane. The virus then buds out until the inner core is enveloped before the membrane at the base of the nascent virion undergoes fission from the cell membrane. Several influenza structural proteins have been implicated in this process. HA, NA and M2 when expressed alone in transfected cells are all sufficient to allow for budding of virus-like particles. All of these except can interact with components of the host ESCRT complex providing an explanation on how they’re able to facilitate budding. Budding virions are either spherical or filamentous and this morphology appears to depend on several residues on the M proteins58.

Once the virus has budded out, it needs to be actively released from the cell surface. This is because HA on daughter virions will bind to sialic acids on the cell surface preventing the virus from being released into the extracellular space. This requires Neuraminidase activity to cleave these residual sialic acids from the cell surface.

Neuraminidase inhibitors target this step of viral replication and prevent virions from being released from the cell. Once the virus has been relased from the host cell it can infect other target cells and restart the infection cycle anew59.

1.6 The Hemagglutinin Protein

Hemagglutinin is in many ways the main determinant of influenza infection. It is necessary for both receptor binding and fusion and subtle differences in the influenza

18 sequence can mean the difference between a strain of virus that causes mild disease and a strain that causes death. Hemagglutinin is a class I fusion peptide, meaning that it requires proteolytic processing to become fusion competent, it forms a trimer and its major secondary structure consists of αhelices. The Hemagglutinin gene is translated into a single proprotein (HA0) which is later cleaved by cellular proteases into two subunits

(HA1 and HA2) linked by a disulfide bridge. The fusion peptide is located at the N- terminus of HA0 and stays buried in the subunit interface once post-translational processing has been completed. Once pH drops in the late endosome, the structure changes to a trimer of hairpins with a central αhelical coiled coil60,61.

Figure 1.5: Schematic of Hemagglutinin mediated fusion. A depicts the prefusion confirmation. At this point the hemagglutinin trimer would still be associated with a sialic acid residue on the cell membrane. B as pH drops the receptor binding domain dissociates from the sialic acid. C the loop between the shorter and longer helices of HA2 becomes a helix, extending the HA2 towards the cell membrane and allowing for insertion of the fusion peptide (which until this point has been sequestered on the inside of the trimer) to insert into the host cell membrane. D the prefusion structure collapses on itself to form a trimer of coiled coils, resulting in the creation of the fusion pore. Cleavage of HA0 is the most important determinant of whether a virus will be infectious or not and also determines whether a virus will have a low pathogenicity or high pathogenicity phenotype. If HA0 isn’t cleaved into HA1 and HA2, the fusion

19 peptide remains setrically locked and is unable to insert into the host cell membrane and mediate fusion, resulting in the virion undergoing lysosomal degradation. The cleavage site between HA1 and HA2 on the proprotein in low pathogenicity IAV strains is a single

Arginine residue preceding the fusion peptide. This needs to be cleaved by Trypsin like proteases, which in mammals are expressed mainly in the lumen of the upper respiratory tract. Therefore these viruses must first bud from the tracheal epithelium and be cleaved by extracellular proteases before they can infect another host cell. In contrast, high pathogenicity strains contain a polybasic cleavage site (Consesnsus R-X-K/R-R). This allows the HA of HPAIV’s to be cleaved in the trans face of the Golgi apparatus by

Furin. Furin is ubiquitously expressed in vertebrates and this fact explains the difference in disease caused by these two kinds of viruses in humans. Since HA of low pathogenicity influenza viruses can only be cleaved in the upper respiratory tract, the virus can only replicate efficiently in this area, resulting in the symptomology classically associated with influenza infection as well as the relatively low death rate. In contrast, highly pathogenic influenza virus HA can be cleaved in all tissue, allowing it to have a much broader tropism and giving it the ability to cause systemic infection and death via multi organ failure. 62,63,64

20

Figure 1.6: Comparison of the protein sequences of High and Low Pathogenicity Avian influenza. Hemagglutinin is also a major determinant of host range restriction of influenza viruses. Avian viruses preferentially bind α2,3 linked sialic acids while human viruses bind α2,6 linked sialic acids. A switch in receptor specificity is necessary for avian viruses to efficiently infect humans. In H3 hemagglutinin containing viruses a glutamate at position 226 provides binding specificity to 2,3 linked sialic acids whereas a leucine at this location allows for preferential binding to 2,6 linked sialic acids. Recently, a group of researchers was able to generate an airborne transmissible form of highly pathogenic avian influenza H5N1 by serial passaging in ferrets65. These airborne viruses needed only

5 substitutions, three of which were in the HA to both switch receptor specificity and to acquire the ability to transmit in aerosols, underscoring the importance of the HA molecule in influenza pathogenesis. HA cleavage is an attractive therapeutic

21 target given the fact that the protein is exposed to the extracellular environment rather than sequestered and also given the importance of HA cleavage to viral pathogenesis66.

1.7 Innate Immunity to Influenza Viruses

The innate immune response to influenza virus infection is critical in controlling the virus. Deficiencies in innate immune sensors and signaling molecules has been shown to be associated with increased susceptibility the virus in vivo as well as poor clinical outcomes in humans. The innate immune response to influenza can broadly be divided into three steps. First a pathogen associated molecular pattern (PAMP) is recognized by cellular pathogen recognition receptors (PRR’s) resulting in the expression of type I interferon, as well as chemokines and cytokines. Next, interferon signaling pathways are activated, resulting in the production of thousands of interferon stimulated genes, many of which have antimicrobial functions. Finally the proteins produced by these genes enact their antimicrobial functions, limiting influenza replication. The interferon system acts in a autocrine and paracrine manner, limiting viral replication in infected cells and making downstream non-infected cells resistant to viral infections67.

22

Figure 1.7: Graphical representation of the various ways Influenza viruses activate innate immunity as well as the signaling pathways involved in interferon induction.

Influenza is sensed in infected cells by TLR3, TLR7 and RIG-I. TLR3 senses double-stranded RNA and acts through the TRIF adaptor molecule. This leads to the activation of IRF3, NFκB and the activator protein AP1 transcription factors. All of these are critical in stimulating the expression of type I interferon and pro-inflammatory cytokines. TLR7 recognizes single stranded RNA in endosomes, which is a common feature of viral genomes, which are internalized in macrophages and dendritic cells. RIG-

I senses 5’-triphopshpate groups on viral RNA’s. Once activated, RIG-I interacts with the interferon β promoter stimulator-1 protein at the mitochondrial membrane. This leads to the stimulator of IRF3, IRF7 and NFκB activity. RIG-I is upregulated as a response to influenza infection. In mouse embryonic fibroblasts, the expression of RIG-I and IPS-1

23 has been shown to be essential for the induction of type I interferon and the up regulation of interferon-stimulated genes. Also, RIG-I is critical for the recognition of influenza virus recognition and type-1 interferon in fibroblasts, dendritic cells and epithelial cells.

Influenza virus also activates the inflammasome, a complex of a NOD-like receptor and the ASC adaptor protein. This activation appears to involve only the M2 ion channel.

Inflammasome activation leads to the cleavage and activation of procaspase-1, which goes n to activate certain cytokines. Of these, IL-18 and IL-1β. In vivo studies have shown that mice deficient in various inflammasome components show increased mortality and delayed viral clearance after influenza infection. Together these molecules provide the first line of defense against influenza viruses68,69.70.

1.8 Abrogation of Innate Immunity by Influenza

Viruses

While these innate immune sensors provide a critical function in recognizing influenza infection and stimulating productions of proteins to stop it, the virus has evolved mechanisms to combat this. They accomplish this primarily through the actions of NS1, and viruses with truncated, deleted or mutated NS1 stimulate higher interferon production than their wild-type counterparts. NS1 can regulate innate immunity via two mechanisms: interference with RIG-I activation and signaling and by interfering with the processing of cellular pre-mRNA71.

24 Early studies suggested that NS1 may inhibit RIG-I activation by sequestering dsRNA, preventing its recognition by host PRR molecules. However, dsRNA is produced in relatively small amounts in viral infected cells, and mutation of dsRNA binding sites on NS1 has been shown to have a marginal effect on interferon production. Instead, NS1 blocks RIG-I activation by interfering with its ubiquitination by TRIM25. The RIG-I molecule consists of a central DEAD box helicase, two caspace activation and recruitment (CARD) domains at the N-terminus and a CARD repressor domain at the C- terminus. Binding of dsRNA to the helicase releases the repressor domain from the

CARD-CARD domain, allowing for ubiquitination of the CARD domains, by TRIM25 which can then interact with IPS-1 to induce interferon expression. NS1 interacts with the coiled-coil domain of TRIM-25, blocking its ability to ubiquitinate RIG-I and preventing downstream RIG-I signaling. In addition to suppressing TRIM25 mediated RIG-I activation, NS1 can also interfere with interferon levels by blocking splicing, polyadenylation and nuclear export of cellular pre-mRNA’s. This activity requires the interaction of NS1 with the cleavage and polyadenylation specificity factor (CPSF). This interaction prevents nuclear export and polyadenylation of non-viral mRNA. This allows the virus to effectively replicate even if it is sensed by cellular PRR’s72.

NS1 also has an apparent PDZ ligand domain motif at its c-terminus. This motif is recognized by PDZ domain proteins, a large that plays multiple roles in cellular processes. Though the exact function of this motif is still under debate, several studies have shown that this PDZ ligand domain motif can interact with the PDZ domain protein Scribble. Scribble has a proapoptotic factor, and interaction of NS1 and scribble

25 has been shown to inhibit apoptosis. Furthermore mutations in the PDZ domain have been shown to decrease virulence in several strain backgrounds. This domain is believed to regulate apoptosis in virus-infected cells, and has also been shown to limit the levels of

Mx (an antiviral protein) in infected cells73.

1.9Adaptive Immunity to Influenza Viruses

While the innate immune system provides an important barrier to Influenza infection, once active infection is established the adaptive immune system is necessary for viral clearance. In vivo studies have shown that mice that lack both B and T cells will succumb to influenza infection. However mice lacking a single of these cell types can control, although not necessarily clear influenza infections. Once the virus has been cleared, strain specific immunity can be long lasting. This is evidenced by are specific attack rates of several of the past pandemic strains. For example, the Russian flu in 1977 caused much more severe disease in younger people (<25 years old) than older adults74, which very unusual for influenza viruses which tend to cause significant morbidity and mortality in older people. This was due to the fact that it was cased by and H1N1 strain of the virus and most older people had immunity from it due to the circulation of the

Spanish Flu several decades before. Immune selection is an important selective force for human circulating influenza viruses. Particularly, B-cell epitopes in influenza viruses appear to drift rapidly. Innate immunity against these viruses is strongly strain-specific, although cross-reactive immunity has been reported. Generation of a vaccine, which can

26 provide cross-strain immunity, is a major goal of influenza research however it has proved elusive75,76.

The humoral response plays a critical role in limiting and clearing influenza infections. Upon viral infection, antibodies to the HA, NA, NP and M proteins are produced. The anti HA antibodies have been shown to be the most important for viral neutralization. HA and NA are the primary antigenic molecules of influenza viruses and the antibodies to these are the main mediators of resistance to viral challenge. HA antibodies can prevent viral infection by neutralizing the viruses ability to bind to host cells or interfere with the proteins ability to mediate fusion77. Without the ability to bind cells or fuse in the late endosome, these virions either stay in the extracellular space or get degraded in the lysosome, in either case not causing productive infection. NA specific antibodies also reduce infectivity, but in contrast with HA specific antibodies these minimize viral release from host cells by interfering with NA’s ability to cleave sialic acids. Since daughter virions can’t be released from host cells, they are effectively rendered noninfectious78. Antibodies to M and NP also develop over the course of infection and are able to provide passive immunity when injected into immunologically naïve individuals. Natural titers of these antibodies are fairly low, however, and they don’t appear to be as important to viral clearance as HA and NA antibodies79.

Cellular immunity to Influenza also serves an important role in clearing influenza infections, however its exact role in humans has yet to be fully elucidated. Cellular immunity to influenza is mediated by two cells types; CD4+ and CD8+ T cells. CD8 T cells eliminate virus via two mechanisms: direct lysis of infected cells and secretion of

27 proinflammatroy cytokines. CD8+ T cells can be detected in the blood of influenza infected individuals on days 6 to 14 post-infection and are largely gone by day 2180.

Memory CD8+ T cells can remain in the blood for decades after infection and aid in clearing subsequent infections with little or no obvious signs of disease appearing. CD4+

T cells function in aiding proliferation of B cells and CD8+ T-cells. In CD8+ T cell deficient mice, influenza can be cleared by a solely CD4+ T-cell dependent mechanism.

However, passively transferred, virus-specific CD4+ T-cells can only clear influenza infection in mice which have a functional humoral immune response81. This indicates that the major function of this cell type is to help B cells produce antiviral antibodies.

Likewise, mice deficient in CD4+ T-cells can still clear infection, however mice deficient in both have been shown to succumb to infection. However, in addition to their importance in control and clearance of influenza infections, CD8+ T-cells also have the potential to cause significant immunopathology, and they have been implicated to play a significant role in HPAIV pathogenesis82

1.10 Antiviral Drugs

The search for anti-influenza therapy has been ongoing since the virus was found the be the causative agent of the flu in the 1930’s. Despite this, there have only been two classes of antiviral drugs approved for use in humans. The first to be discovered was

Amantidine, an ion channel inhibitor. This drugs acts by blocking the acid activated ion channel formed by the M2 protein on the viral membrane83. By blocking the flow of

Hydrogen ions from the acidified endosome into the interior of the virion. This process is

28 necessary for release of the RNP into the cytosol so blocking it leads to lysosomal degradation of viral components. and an analogous molecule called

Rimantidine were licensed for use in humans in the 1960’s. They are effective against non-resistant strains and they manage to accelerate clearance of the virus and reduce the duration of symptomology by about 1 day. They were widely used until 2004 when resistance strains became alarmingly common. During the 2003-2004 flu season, only about 12% of circulating H3N2 viruses were resistant to the drug. One year later over

90% of H3N2 strains recovered from patients were amantadine resistant. This resistance is typically conferred by an S to A mutation at position 31 of the M2 protein.

Additionally, the 2009 pandemic H1N1 strains, which now dominate the circulating population of H1 containing viruses are also resistance to Amantidine and Rimantidine.

As ion channel inhibitor resistance has become practically ubiquitous among circulating influenza viruses, this class of drugs is no longer recommended for use in treating influenza infection. The fact that human-infecting influenza viruses acquired basically universal resistance to these drugs over a span of 5 years highlights the propensity of this virus to develop drug resistance84. It also highlights the need for new and better drugs to be developed to treat future pandemics.

The other class of drugs that are effective in treating influenza are the neuraminidase inhibitors. The drugs and were approved for use in the United States in 1999, and since then several other neuraminidase inhibitors were granted special approval for use during the 2009 swine flu pandemic. These drugs function by binding to the of the viral neuraminidase, ablating its ability to

29 cleave sialic acid residues. While this doesn’t directly inhibit a virus’s ability to replicate once inside a cell, it does prevent daughter virions form being released into the extracellular space, as hemagglutinin on the new virions will stay bound to sialic acids, preventing detachment. These drugs are effective against influenza, limiting viral shedding as well as reducing the course of disease by about one day. They are also effective prophylactics against influenza virus infection. However, it is critical that treatment is started within 48 hours of onset of symptoms, otherwise the effectiveness of these drugs is limited. Resistance to Neuraminidase Inhibitors has been observed, but it is not as widespread as ion channel inhibitor resistance. Mutations that confer resistance to neuraminidase inhibitors mainly map to the catalytic site of NA. or framework sites that function in stabilizing the active site85. However most strains which are resistant to one will remain sensitive to others, and multi-drug resistance is mainly seen in immunocompromised individuals who undergo drug treatment for a long period of time86. Although widespread resistance to neuraminidase inhibitors isn’t currently a threat to global health, the rapid emergence of ion channel inhibitor resistance should serve as a warning against relying only on these drugs to treat the disease.

30 1.11 The Interferon Induced Transmembrane Proteins

Figure 1.8: IFITM mediated restriction of various viruses. The host innate immune response consists of a wide variety of factors, which can limit a virus’s ability to infect and replicate in target cells. Among these proteins, the

Interferon Induced Transmembrane (IFITM) protein family is unique in that they block viral entry into host cells. In humans, the IFITM protein family consists of four members,

IFITM1, IFITM2 IFITM3 and IFITM5. This protein family was identified as viral restriction factors in 200987. These proteins are interferon induced cell-intrinsic

31 restriction factors, which exhibit high levels of constitutive expression, many cells, including barrier epithelial cells. The expression of IFITM1, IFITM2 and IFITM3 are strongly upregulated by type I and II . They are able to restrict a wide assortment of viral pathogens, including Dengue Virus, Influenza A Virus, HIV-I and

Flaviviruses, among others. However, they are unable to restrict other viral pathogens such as Lassa fever virus and . Though much progress has been made in understanding how these proteins function since their discovery as viral restriction factors in 2009, the exact molecular mechanisms mediating their antiviral activity are still largely unknown88.

1.12 The Discovery of IFITM Antiviral Activity

The first indication that these proteins may have antiviral activities was published in 1996. Alber & Staeheli observed that when IFITM1 was exogenously expressed at high levels, it was able to restrict the replication of Vesicular Stomatitis Virus, although less potently than other known restriction factors. These results were marginal and it took another 13 years before the full extent of IFITM mediated viral restriction would begin to be elucidated89.

In 2009, two separate studies determined that IFITM3 was a potential Influenza restriction factor. These studies used RNA interference screens to show that depletion of

IFITM3 strongly promoted H1N1(A/PR/8/43) replication in U2OS cells, and that specific siRNA could overcome suppression of viral replication by the interferon response.

Overexpression of IFITM1, IFITM2 or IFITM3 was shown to suppress replication of

32 H1N1(A/PR/8/34) and H3N2(A/Udorn/72). However overexpression of these proteins had no effect on the replication of Murine Leukemia Virus in a variety of cell lines.

Embryonic fibroblasts (MEFs) from IFITM knockout mice were shown to be much more susceptible to influenza infection than MEFs from wild type mice. Finally, when envelope glycoprotein-deficient were pseudotyped with H1, H3, H5 and H7 hemagglutinin were efficiently restricted by IFITM1, 2 and 3. Indicating that these proteins were able to restrict a hemagglutinin specific process of influenza infection, presumably viral entry90,01.

Since the discovery of the IFITM family of proteins as influenza restriction factors, several in vivo studies have confirmed these findings. Several strains of IFITM knockout mice have been established, including IfitmDel-/- mice, which contain a 120kb deletion of the entire IFITM locus. These mice are viable and display no obvious developmental defects, however study of Ifitm-5 knockout mice revealed subtle developmental changes in skeletal morphology so an ifitm3-/- strain was developed which showed no such defects. Two studies using these mice characterized the importance of

IFITM3 in preventing viral infection. The first showed that when challenged with a fairly non-pathogenic, murine adapted strain of H3N2 influenza (A/X-31) as well as with a

2009 pandemic strain(A/09 Eng/195) IFITM3 knockout mice consistently lost over a quarter of their bodyweight by day 6 post infection had had to be euthanized. Wild type mice generally recovered from infection and an intermediate disease phenotype was noted in heterozygous mice, demonstrating a dosage dependent effect for IFITM392,93.

Interestingly, no difference was noted between the IfitmDel-/- mice and ifitm3-/- mice,

33 highlighting the importance of IFITM3 in controlling influenza infections. These studies also demonstrated constitutive expression of IFITM3 in the lungs of non-infected, wild- type mice, particularly in the respiratory epithelium. These tissues constitute a barrier between the host and the environment, so this work suggests that IFITM3 prevents viral dissemination in the host by making these cells non-permissive to viral infection. They also found that IFITM3 is expressed in high levels in type II alveolar pneumocytes

(another important influenza target cell) after viral infection. There has been an IFITM3 polymorphism detected in humans which results in a 20 amino acid deletion in the N- terminus. This single nucleotide polymorphism(termed rs12252-C) appears to be enriched in patients who were hospitalized by the 2009 swine flu pandemic. However, the sample size of this study is fairly small and to this point these findings have yet to be experimentally verified. Together, this work suggests that that the IFITM proteins, and

IFITM3 in particular play a critical role in the control and clearance of influenza infections94.

1.13 IFITM Protein Structure

The IFITM proteins can be divided into five domains which are characterized by their hydrophobicity and conservation. These include a variable N terminal domain from residues 1-57, a conserved hydrophobic intramembrane domain from residues 58-80, a conserved intracellular loop from residues 81-104, a variable hydrophobic transmemberane domain from residues 105-126 and a short, highly variable c terminal domain from residues 127-13395.

34

Figure 1.9: Schematics of the three different models of IFITM topology.

The exact topology of these proteins has been under considerable debate.

Specifically, evidence for the exact orientations of the N and C termini of the protein have been conflicting. Early literature suggested that IFITM’s were type III membrane proteins with a two pass transmembrane topology which oriented both the N and C termini to ER lumen or the extracellular space. This was supported by the fact that antibodies that bound to the N terminus of IFITM1 caused aggregation of cells, IFITM could be recovered by immunopercipitation after radiolabelling of the cell surface and that IFITM proteins with tags on their N or C termini could be recognized at the plasma membrane by flow cytometry96. However, conflicting studies have shown that Lys24 of the N terminal domain can be ubiquitinated by cellular ubiquitin . Furthermore other groups have demonstrated phosphorylation of the N terminus of IFITM3, consistent with this region being accessible to cellular kinases97. Despite these early conflicting results, recent studies have elucidated the topology of IFITM3. Several novel studies have shown that the C terminal domain localizes to the ER lumen and contains a KDEL

ER retention motif. Also greater antibody recognition od c terminal epitope tags

35 compared to N terminal tags has been observed in non-permiabilized cells, with this difference disappearing after permeabilization. Furthermore, these studies revealed the presence of an alternative topology IFITM3 where the N terminal domain was facing outside of the ER lumen, its unclear what function this alternatively oriented IFITM3 serves, but the presence of two alternative topology IFITM proteins reconciles earlier findings with clear evidence for a predominantly cytosolic orientation of the N-terminus of the protein.98,99

1.14 Post Translational Modifications of IFITM

Proteins

IFITM proteins contain many posttranslational modification sites that contribute to their antiviral activity as well as their stability. IFITM proteins have two highly conserved palmitoylation sites at cysteines 71 and 72, with a more poorly conserved one at cysteine

105. These sites are necessary for the antiviral activity of IFITMs. With Cys72 playing an especially important role. It seems likely that these palmitoylation sites contribute to the stability of the IFITM proteins as well as their subcellular localization and association with lipid rafts. Furthermore, the IFITM proteins have 4 ubiquitination sites: Lys34,

Lys83, Lys88 and Lys104. Research has shown that these can all be mutated to alanines and have no effect of IFITM-mediated viral restriction in an overexpressing system, indicating that their role is more likely to be in regulation of intracellular IFITM levels.

Tyroseine 20 is also highly conserved among in IFITM 2 and 3, but not IFITM 1.

36 Mutation of this site alters IFITM subcellular localization and appears to ablate IFITM’s ability to restrict viral entry100.

1.15 Models of IFITM Mediated Viral Restriction

Since the discovery of the IFITM proteins significant effort has been made to discern the exact mechanism of IFITM-mediated viral restriction. Despite this the exact mechanism of IFITM proteins is not fully understood. Perhaps the most important observation in IFITM protein research has been that these proteins not only inhibit the replication of infectious viruses, but also that they prevent the entry of retroviral particles pseudotyped with entry glycoproteins of these same viruses. Conversely, membrane proteins from viruses that aren’t sensitive to IFITM-mediated restriction can be used to generate pseudotyped viral particles that also don’t show any IFITM-mediated entry inhibition101. A compelling explanation of this phenomenon is that the IFITM proteins are able to block viral entry at specific sites of fusion. In agreement with this explanation, several studies have found that IFITM-restricted viruses mostly fuse in a pH-dependent or cathepsin-dependent manner, whereas viruses which are resistant to IFITM mediated restriction tend to fuse on the plasma membrane. Evidence for this model includes the fact that inhibiting IFITM’s internalization into endolysosomal compartments (By mutating a signal sequence) impairs its ability to restrict flu viruses. Furthermore, other studies on SARS-CoV and reovirus support the idea that IFITM has a site-specific restriction mechanism. Fusion of these viruses depends on acid-dependent proteases which are only active in the late endosome. Under normal conditions, these viruses are

37 sensitive to IFITM-mediated restriction, however if they are treated with exogenous proteases and then allowed to infect cells(in which case fusion occurs at the plasma membrane) IFITM-mediated restriction is ablated102.

Figure 1.10: A potential mechanism for IFITM mediated viral restriction. Researchers found that adding amphotericin to cells before infecting with psedudotype virus would ablate IFITM’s ability to prevent viral infection. Likewise treatment with amphotericin caused mice to succumb to influenza infection rather than surviving like their non-treated counterparts. Amphotericin increases membrane fluidity by its interaction with cholesterol, suggesting a potential mechanism for how IFITM blocks fusion. Considering these findings it appears apparent that the IFITM proteins restrict viruses at the site of viral fusion and are far less effective at the cell membrane than they are in the maturing endosome. The exact mechanism for how IFITM is able to restrict

38 viruses has yet to be elucidated. Several models have been proposed, however, which postulate that IFITM proteins can alter endosomal membranes in such a manner that they prevent the formation of fusion pores. Several studies have shown that accumulation of

IFITM proteins causes the loss of membrane fluidity. Other studies have demonstrated that IFITM3 overexpression results in the accumulation of cholesterol in late endosomes104. Furthermore, treatment of cells with amphotericin B or nystatin has been shown to counteract IFITM mediated restriction of Influenza viruses. Both of these drugs bind membrane sterols and are able to restore membrane fluidity in high-cholesterol membranes. These findings imply a possible mechanism where IFITM proteins cause an enrichment of cholesterol at the endosomal membrane, decreasing membrane fluidity and thus preventing fusion pores from forming105. However more work must be done to validate this hypothesis.

1.16 SCAMP3

Secratory Carrier Membrane Protein 3 is a tetraspanning membrane protein. It is part of a 5 member family of SCAMP proteins (SCAMP1-5). These proteins are all ubiquitously expressed except for SCAMP 4 which is primarily neuronal106. SCAMP3 is unique among the other proteins in that it contains a proline-rich domain near its N- terminus. This domain contains a PSAP as well as a PY motif. These two motifs can interact with TSG101 and NEDD4 respectively, and several studies have provided evidence that SCAMP3 interacts with these two proteins in the cell107. As NEDD4 and

TSG101 are fairly important to the innate immune response and protein degradation

39 respectively, it is possible that SCAMP3 may play some role in these processes although that has not yet been investigated.

SCAMP3 was discovered in 1997 when researchers compared predicted sequences from an expressed sequence tag library, and were later able to clone out

SCAMP1, SCAMP2 and SCAMP3 from a HeLa library.108 These researchers also discovered that all three of these SCAMP proteins were expressed ubiquitously in various tissues. Furthermore they were able to use confocal microscopy to demonstrate that each of these three proteins had a similar subcellular distribution109. Since then several studies have aimed at elucidating the cellular function of SCAMP3.

Figure 1.11: Represenatation of SCAMP3 structure and topology. Insert shows the sequence of the N-terminal proline rich motif. Sequences of SCAMP3 orthologs from different species are added to highlight te conserved PY and PSAP motifs found in this protein.

40 A study performed in 2009 attempted to determine if SCAMP3 played a role in

Epidermal Growth Factor Receptor (EGFR) recycling. These researchers were the first to note that the proline rich N terminal domain of SCAMP3 contained a PY and PSAP motif. They were also able to show that this motif interacted with NEDD4 and TSG101 using immunopercipitation and a yeast two-hybrid screen. They were also able to show that SCAMP3 co-percipitated with hepatocyte growth factor-regulated tyrosine kinase substrate (hrs). They postulated since hrs is important in sorting proteins in to transport vesicles from the cell surface, maybe SCAMP3 could play a role in receptor recycling.

To test this they looked at the Epidermal Growth Factor (EGF) and its receptor (EGFR).

They found that upon EGF stimulation, the EGFR could be found localizing to endosomes with SCAMP3. Furthermore, once SCAMP3 was depleted from cells, it appeared to increase EGFR degradation upon EGF stimulation and conversely overexpression of SCAMP3 resulted in more EGFR being recycled to the plasma membrane than cells expressing physiological levels of SCAMP3. They attempted to narrow down this function to an interaction of SCAMP3 with the ESCRT proteins, however their results were inconclusive and they concluded that SCAMP3 has some role that it plays in parallel with the ESCRT proteins to facilitate receptor recycling.110

While this first look into SCAMP3’s cellular function, another study published three years later challenged these findings111. Using electron microscopy, this group showed that SCAMP3 promoted multivesicular body formation and the sorting of EGFR into endosomes destined for lysosomal degradation. They showed that in SCAMP3 knock down cells this sorting was ablated and EGFR degradation was reduced. This was in

41 direct conflict with previous work that showed that depletion of SCAMP3 resulted in increased EGFR turnover. The group explained this by stating that differences in

SCAMP3 ubiquitination could result in the protein functioning differently. They stated that previous experiments were performed in conditions that promoted SCAMP3 ubiquitination, which could have altered the function of the protein from facilitating

EGFR degradation to increasing its intracellular turnover. They stated that since their experimental conditions didn’t result in ubiquitination of SCAMP3, this was a sufficient explanation for the difference in their observations. There was however no experimental data provided to substantiate these claims so it remains conjecture. To this day no other groups have been able to build on the understanding of SCAMP3’s function in the cell, thus its exact intracellular function remains a mystery.

Figure 12: Confocal micrographs of naïve cells and sells infected with Salmonella Enterica. Cells were either unstained or stained for SCAMP2, SCAMP3 TGN42, a Trans-Golgi Network marker, LAMP1, a lysosome marker and salmonella.

42 Interestingly SCAMP3 was also reported to play a role in Salmonella pathogenesis. Salmonella replicates intracellularly in a membrane bound compartment known as a Salmonella Containing Vacuole(SCV). A hallmark of salmonella infection of epithelial cells in the appearance of long tubules which contain a wide variety of endosomal makers. These are known as Salmonella Induced Filaments(SIF’s). The exact function of these filaments is unknown but it has been shown that inhibition of filament formation by mutating genes responsible for their formation results in ablated bacterial replication and the dissolution of the SCV. Using confocal microscopy and live cell imaging, this group was able to demonstrate that SCAMP3 co localized to a certain subset of these filaments. While previous studies postulated that these filaments arose from Salmonella’s interaction with the endosomal pathway, this group was able to show that there exists a subset of these filaments that are enriched in SCAMP3 and lack endosomal markers. They noted that upon knocking down SCAMP3, Salmonella replication was not affected, however Salmonella virulence was ablated. This suggests that while SCAMP3 and the SCAMP3-containing Salmonella Induced Filaments aren’t necessary for the survival and replication of Salmonella in vitro they do play some role in virulence, which to this day hasn’t been expanded on112.

An interesting observation that the authors of this study didn’t choose to expand upon was that non-filament-associated SCAMP3 localization changed drastically after Salmonella infection. Before the bacteria was added to the cells, the majority of

SCAMP3 co-localized with TGN46, a trans Golgi marker, in agreement with previous studies regarding SCAMP3’s subcellular distribution. However, after Salmonella

43 infection, this co-localization appears to decrease greatly and a large amount of SCAMP3

(not including that which distributes to Salmonella filaments) appears to localize to a different subcellular compartment. Salmonella bacteria are intracellular pathogens, and therefore Salmonella infection activates the innate immune response upon infection. It is therefore possible that this dynamic relocalization of SCAMP3 in salmonella infected epithelial cells could be caused by the innate immune response. This is an intriguing question, as SCAMP3 has been shown to interact with TSG101, which has been shown to be critically important for the replication of intracellular pathogens such as Influenza and

HIV. Therefore it is possible that SCAMP3 may play some role in TSG101 mediated innate immunity against these viruses.

44 1.17 References

1. Morens, D. M., Taubenberger, J. K., Folkers, G. K., & Fauci, A. S. (2010). Pandemic influenza’s 500th anniversary. Clinical Infectious Diseases, 51(12), 1442– 1444. doi:10.1086/657429

2. THE HISTORY OF INFLUENZA (1919). Science, 49(1261), 216–217. doi:10.1126/science.49.1261.216

3. Morens, D. M., North, M., & Taubenberger, J. K. (2010). Eyewitness accounts of the 1510 influenza pandemic in Europe. The Lancet, 376(9756), 1894–1895. doi:10.1016/s0140-6736(10)62204-0

4. Cannell, J. J., Zasloff, M., Garland, C. F., Scragg, R., & Giovannucci, E. (2008). On the epidemiology of influenza. Virology Journal, 5(1), 29. doi:10.1186/1743-422x- 5-29

5. Fedson, D. S. (2009). Meeting the challenge of influenza pandemic preparedness in developing countries. Emerging Infectious Diseases, 15(3), 365–371. doi:10.3201/eid1503.080857

6. Du Ry van Beest Holle M, Meijer A, Koopmans M, de Jager CM. Human-to-human transmission of avian influenza A/H7N7, The Netherlands, 2003. Euro Surveill. 2005;10(12):pii=584.

7. Uyeki, T. M. (2009). Human infection with highly pathogenic avian influenza A (H5N1) virus: Review of clinical issues. Clinical Infectious Diseases, 49(2), 279– 290. doi:10.1086/600035

8. Avian influenza (H5N1) infection in humans (2006). New England Journal of Medicine, 354(8), 884–884. doi:10.1056/nejmx060010

9. Morens, D. M., Taubenberger, J. K., Folkers, G. K., & Fauci, A. S. (2010). Pandemic influenza’s 500th anniversary. Clinical Infectious Diseases, 51(12), 1442– 1444. doi:10.1086/657429

10. Morens, D. M., North, M., & Taubenberger, J. K. (2010). Eyewitness accounts of the 1510 influenza pandemic in Europe. The Lancet, 376(9756), 1894–1895. doi:10.1016/s0140-6736(10)62204-0

45

11. Hampton, T. (2007). Virulence of 1918 influenza virus linked to inflammatory innate immune response. JAMA, 297(6), 573. doi:10.1001/jama.297.6.580

12. Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med. 2002;76:105–15.

13. Kash JC, Basler CF, García-Sastre A, Carter V, Billharz R, Swayne DE, Przygodzki RM, Taubenberger JK, Palese P, Katze MG, Tumpey TM. The global host immune response: Contribution of HA and NA genes from the 1918 Spanish influenza to viral pathogenesis. Journal of Virology.2004;78:9499–511.

14. Lewis PA and Shope RE. Swine influenza: II. A Hemophilic bacillus from the respiratory tract of infected swine. J Exp Med, 1931, 54:361–371

15. Osterholm, M. T., Kelley, N. S., Sommer, A., & Belongia, E. A. (2012). Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. The Lancet infectious diseases, 12(1), 36-44.

16. Collin, N., de Radiguès, X., & World Health Organization H1N1 Vaccine Task Force. (2009). Vaccine production capacity for seasonal and pandemic (H1N1) 2009 influenza. Vaccine, 27(38), 5184-5186.

17. Henderson, D. A., Courtney, B., Inglesby, T. V., Toner, E., & Nuzzo, J. B. (2009). Public health and medical responses to the 1957-58 influenza pandemic. Biosecurity and bioterrorism: biodefense strategy, practice, and science, 7(3), 265-273.

18. Xu, R., McBride, R., Paulson, J. C., Basler, C. F., & Wilson, I. A. (2010). Structure, receptor binding, and antigenicity of influenza virus from the 1957 H2N2 pandemic. Journal of virology, 84(4), 1715-1721.

19. REF, A. F. (2010). Portrait of a year-old pandemic. Nature, 464, 1112-1113.

20. Earn, D. J., Dushoff, J., & Levin, S. A. (2002). Ecology and evolution of the flu. Trends in ecology & evolution, 17(7), 334-340.

21. Kavet, J. (1977). A perspective on the significance of pandemic influenza. American Journal of Public Health, 67(11), 1063-1070.

46

22. Sencer, D. J., & Millar, J. D. (2006). Reflections on the 1976 swine flu vaccination program. Emerging infectious diseases, 12(1).

23. Gatherer, D. (2009). The 2009 H1N1 influenza outbreak in its historical context. Journal of Clinical Virology, 45(3), 174-178.

24. Su, Y. C., Bahl, J., Joseph, U., Butt, K. M., Peck, H. A., Koay, E. S., ... & Smith, G. J. (2015). Phylodynamics of H1N1/2009 influenza reveals the transition from host adaptation to immune-driven selection. Nature Communications, 6.

25. Kiseleva, I., Larionova, N., Kuznetsov, V., & Rudenko, L. (2010). Phenotypic characteristics of novel swine-origin influenza A/California/07/2009 (H1N1) virus. Influenza and Other Respiratory Viruses, 4(1), 1–5. doi:10.1111/j.1750- 2659.2009.00118.x

26. Palese, PaSML, and Megan L. Shaw. "Orthomyxoviridae: the viruses and their replication." Fields virology 2 (2007): 1647-1689.

27. Arranz, R., Coloma, R., Chichón, F. J., Conesa, J. J., Carrascosa, J. L., Valpuesta, J. M., ... & Martín-Benito, J. (2012). The structure of native influenza virion ribonucleoproteins. Science, 338(6114), 1634-1637.

28. Mitnaul, L. J., Castrucci, M. R., Murti, K. G., & Kawaoka, Y. (1996). The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication. Journal of Virology, 70(2), 873-879.

29. Gómez-Puertas, P., Albo, C., Pérez-Pastrana, E., Vivo, A., & Portela, A. (2000). Influenza virus matrix protein is the major driving force in virus budding. Journal of virology, 74(24), 11538-11547.

30. Couch RB. Orthomyxoviruses. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 58.Available from: https://www.ncbi.nlm.nih.gov/books/NBK8611/

31. A revision of the system of nomenclature for influenza viruses: a WHO memorandum. Bulletin of the World Health Organization, 58 (4), 585-91 PMID: 6969132

47 32. Kamal, R. P., Tosh, C., Pattnaik, B., Behera, P., Nagarajan, S., Gounalan, S., … Pradhan, H. K. (2007). Analysis of the PB2 gene reveals that Indian H5N1 influenza virus belongs to a mixed-migratory bird sub-lineage possessing the amino acid lysine at position 627 of the PB2 protein. Archives of Virology, 152(9), 1637–1644. doi:10.1007/s00705-007-1002-5

33. Zamarin, D., Ortigoza, M. B., & Palese, P. (2006). Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. Journal of virology, 80(16), 7976-7983.

34. Eisfeld, A. J., Neumann, G., & Kawaoka, Y. (2014). At the centre: Influenza A virus ribonucleoproteins. Nature Reviews Microbiology, 13(1), 28–41. doi:10.1038/nrmicro3367

35. Chenavas, S., Crépin, T., Delmas, B., Ruigrok, R. W., & Slama-Schwok, A. (2013). Influenza virus nucleoprotein: Structure, RNA binding, oligomerization and antiviral drug target. Future Microbiology, 8(12), 1537–1545. doi:10.2217/fmb.13.128

36. Jakubova, L., Holly, J., & Vareckova, E. (2016). The role of fusion activity of influenza A viruses in their biological properties. Acta virologica, 60(02), 121–135. doi:10.4149/av_2016_02_121

37. Jagadesh, A., Salam, A. A. A., Mudgal, P. P., & Arunkumar, G. (2016). Influenza virus neuraminidase (NA): A target for antivirals and vaccines. Archives of Virology, 161(8), 2087–2094. doi:10.1007/s00705-016-2907-7

38. Gregoriades, A., & Frangione, B. (1981). Insertion of influenza M protein into the viral lipid bilayer and localization of site of insertion. Journal of virology, 40(1), 323-328.

39. Ritchey, M. B., Palese, P. E. T. E. R., & Schulman, J. L. (1976). Mapping of the influenza virus genome. III. Identification of genes coding for nucleoprotein, membrane protein, and nonstructural protein. Journal of virology, 20(1), 307-313.

40. Shih, S. R., Nemeroff, M. E., & Krug, R. M. (1995). The choice of alternative 5'splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex. Proceedings of the National Academy of Sciences, 92(14), 6324-6328.

41. Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J. J., & Wiley, D. C. (1988). Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature, 333(6172), 426-431.

48 42. Sauter, N. K., Bednarski, M. D., Wurzburg, B. A., Hanson, J. E., Whitesides, G. M., Skehel, J. J., & Wiley, D. C. (1989). Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500- MHz proton nuclear magnetic resonance study. Biochemistry, 28(21), 8388-8396.

43. White, J., Helenius, A., & Gething, M. J. (1982). Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature, 300, 658-659.

44. Rust, M. J., Lakadamyali, M., Zhang, F., & Zhuang, X. (2004). Assembly of endocytic machinery around individual influenza viruses during viral entry. Nature structural & molecular biology, 11(6), 567-573.

45. Compans, R. W., Content, J., & Duesberg, P. H. (1972). Structure of the ribonucleoprotein of influenza virus. Journal of Virology, 10(4), 795-800.

46. Klumpp, K., Ruigrok, R. W., & Baudin, F. (1997). Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. The EMBO Journal, 16(6), 1248-1257.

47. Pons, M. W. (1971). Isolation of influenza virus ribonucleoprotein from infected cells. Demonstration of the presence of negative-stranded RNA in viral RNP. Virology, 46(1), 149-160.

48. Winterling, C., Koch, M., Koeppel, M., Garcia-Alcalde, F., Karlas, A., & Meyer, T. F. (2014). Evidence for a crucial role of a host non-coding RNA in influenza A virus replication. RNA biology, 11(1), 66-75.

49. Stoeckle, M. Y., Shaw, M. W., & Choppin, P. W. (1987). Segment-specific and common nucleotide sequences in the noncoding regions of influenza B virus genome RNAs. Proceedings of the National Academy of Sciences, 84(9), 2703-2707.

50. Fortes, P., Lamond, A. I., & Ortín, J. (1995). Influenza virus NS1 protein alters the subnuclear localization of cellular splicing components. Journal of general virology, 76(4), 1001-1007.

51. Dubois J, Terrier O, Rosa-Calatrava M. 2014. Influenza viruses and mRNA splicing: doing more with less. mBio 5(3):e00070-14 doi:10.1128/mBio.00070-14.

49 52. Flick, R., & Hobom, G. (1999). Transient bicistronic vRNA segments for indirect selection of recombinant influenza viruses. Virology, 262(1), 93-103.

53. Azzeh, M., Flick, R., & Hobom, G. (2001). Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system. Virology, 289(2), 400-410.

54. Digard P, Elton D, Bishop K, Medcalf E, Weeds A, Pope B. Modulation of Nuclear Localization of the Influenza Virus Nucleoprotein through Interaction with Actin Filaments. Journal of Virology. 1999;73(3):2222-2231.

55. Rossman, Jeremy S., and Robert A. Lamb. “Influenza Virus Assembly and Budding.” Virology 411.2 (2011): 229–236. PMC. Web. 1 Dec. 2016.

56. Stevaert A, Naesens L. The Influenza Virus Polymerase Complex: An Update on Its Structure, Functions, and Significance for Antiviral Drug Design. Medicinal Research Reviews. 2016;36(6):1127-1173. doi:10.1002/med.21401.

57. Moreira ÉA, Weber A, Bolte H, et al. A conserved influenza A virus nucleoprotein code controls specific viral genome packaging. Nature Communications. 2016;7:12861. doi:10.1038/ncomms12861.

58. Matsuoka Y, Matsumae H, Katoh M, et al. A comprehensive map of the influenza A virus replication cycle. BMC Systems Biology. 2013;7:97. doi:10.1186/1752-0509-7- 97.

59. Oh DY, Hurt AC. A Review of the Antiviral Susceptibility of Human and Avian Influenza Viruses over the Last Decade. Scientifica. 2014;2014:430629. doi:10.1155/2014/430629.

60. Wilks S, De Graaf M, Smith DJ, Burke DF. A review of Influenza haemagglutinin receptor binding as it relates to pandemic properties. Vaccine. 2012;30(29):4369- 4376. doi:10.1016/j.vaccine.2012.02.076.

61. Wiley, D. (1987). The structure and function of the Hemagglutinin membrane Glycoprotein of influenza virus. Annual Review of Biochemistry, 56(1), 365–394. doi:10.1146/annurev.biochem.56.1.365

50 62. Jiang S, Li R, Du L, Liu S. Roles of the hemagglutinin of influenza A virus in viral entry and development of antiviral therapeutics and vaccines. Protein & Cell. 2010;1(4):342-354. doi:10.1007/s13238-010-0054-6.

63. Horimoto, T., & Kawaoka, Y. (1995). The Hemagglutinin Cleavability of a virulent avian influenza virus by Subtilisin-like Endoproteases is influenced by the amino acid immediately downstream of the cleavage site. Virology, 210(2), 466–470. doi:10.1006/viro.1995.1363

64. Walker JA, Molloy SS, Thomas G, et al. Sequence specificity of furin, a proprotein- processing endoprotease, for the hemagglutinin of a virulent avian influenza virus. Journal of Virology. 1994;68(2):1213-1218.

65. Oshansky CM, Pickens JA, Bradley KC, et al. Avian Influenza Viruses Infect Primary Human Bronchial Epithelial Cells Unconstrained by Sialic Acid α2,3 Residues. Poon LLM, ed. PLoS ONE. 2011;6(6):e21183. doi:10.1371/journal.pone.0021183.

66. Chaipan, C., Kobasa, D., Bertram, S., Glowacka, I., Steffen, I., Solomon Tsegaye, T., Takeda, M., Bugge, T., Kim, S., Park, Y., Marzi, A., & Pohlmann, S. (2009). Proteolytic Activation of the 1918 Influenza Virus Hemagglutinin Journal of Virology, 83 (7), 3200-3211 DOI: 10.1128/JVI.02205-08

67. Pulendran B, Maddur MS. Innate Immune Sensing and Response to Influenza. Current topics in microbiology and immunology. 2015;386:23-71. doi:10.1007/82_2014_405.

68. Rajsbaum R, Albrecht RA, Wang MK, et al. Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein. Pekosz A, ed. PLoS Pathogens. 2012;8(11):e1003059. doi:10.1371/journal.ppat.1003059.

69. Van de Sandt CE, Kreijtz JHCM, Rimmelzwaan GF. Evasion of Influenza A Viruses from Innate and Adaptive Immune Responses. Viruses. 2012;4(9):1438-1476. doi:10.3390/v4091438.

70. Yang C-W, Chen S-M. A Comparative Study of Human TLR 7/8 Stimulatory Trimer Compositions in Influenza A Viral Genomes. Park M-S, ed. PLoS ONE. 2012;7(2):e30751. doi:10.1371/journal.pone.0030751

51 71. García-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D. E., Durbin, J. E., ... & Muster, T. (1998). Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology, 252(2), 324-330.

72. Hale, B. G., Randall, R. E., Ortín, J., & Jackson, D. (2008). The multifunctional NS1 protein of influenza A viruses. Journal of General Virology, 89(10), 2359-2376.

73. Staeheli, P., Haller, O., Boll, W., Lindenmann, J., & Weissmann, C. (1986). Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell, 44(1), 147-158.

74. Gregg, M. B., Hinman, A. R., & Craven, R. B. (1978). The Russian flu: its history and implications for this year's influenza season. Jama, 240(21), 2260-2263.

75. Boni, M. F., Gog, J. R., Andreasen, V., & Christiansen, F. B. (2004). Influenza drift and epidemic size: the race between generating and escaping immunity. Theoretical population biology, 65(2), 179-191.

76. Carrat, F., & Flahault, A. (2007). Influenza vaccine: the challenge of antigenic drift. Vaccine, 25(39), 6852-6862.

77. Virelizier, J. L. (1975). Host defenses against influenza virus: the role of anti- hemagglutinin antibody. The Journal of Immunology, 115(2), 434-439.

78. Webster, R. G., & Laver, W. G. (1967). Preparation and properties of antibody directed specifically against the neuraminidase of influenza virus. The Journal of Immunology, 99(1), 49-55.

79. Goodwin, K., Viboud, C., & Simonsen, L. (2006). Antibody response to influenza vaccination in the elderly: a quantitative review. Vaccine, 24(8), 1159-1169.

80. Fu, T. M., Friedman, A., Ulmer, J. B., Liu, M. A., & Donnelly, J. J. (1997). Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. Journal of virology, 71(4), 2715-2721.

81. McMichael, A. J., Gotch, F. M., Noble, G. R., & Beare, P. A. (1983). Cytotoxic T- cell immunity to influenza. New England Journal of Medicine, 309(1), 13-17.

52 82. He, X. S., Holmes, T. H., Zhang, C., Mahmood, K., Kemble, G. W., Lewis, D. B., ... & Arvin, A. M. (2006). Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines. Journal of virology, 80(23), 11756- 11766.

83. Pielak, R. M., Schnell, J. R., & Chou, J. J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel. Proceedings of the National Academy of Sciences, 106(18), 7379-7384.

84. Moscona, Anne. "Neuraminidase inhibitors for influenza." New England Journal of Medicine 353.13 (2005): 1363-1373.

85. Kim, C. U., Lew, W., Williams, M. A., Liu, H., Zhang, L., Swaminathan, S., ... & Laver, W. G. (1997). Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. Journal of the American Chemical Society, 119(4), 681-690.

86. Peters, P. H., Gravenstein, S., Norwood, P., De Bock, V., Van Couter, A., Gibbens, M., ... & Ward, P. (2001). Long‐term use of oseltamivir for the prophylaxis of influenza in a vaccinated frail older population. Journal of the American Geriatrics Society, 49(8), 1025-1031.

87. Brass, A. L., Huang, I. C., Benita, Y., John, S. P., Krishnan, M. N., Feeley, E. M., ... & Adams, D. J. (2009). The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, , and dengue virus. Cell, 139(7), 1243-1254.

88. Diamond, M. S., & Farzan, M. (2013). The broad-spectrum antiviral functions of IFIT and IFITM proteins. Nature reviews Immunology, 13(1), 46-57.

89. Chutiwitoonchai, N., Hiyoshi, M., Hiyoshi-Yoshidomi, Y., Hashimoto, M., Tokunaga, K., & Suzu, S. (2013). Characteristics of IFITM, the newly identified IFN- inducible anti-HIV-1 family proteins. Microbes and Infection, 15(4), 280-290.

90. Diamond, M. S., & Farzan, M. (2013). The broad-spectrum antiviral functions of IFIT and IFITM proteins. Nature reviews Immunology, 13(1), 46-57.

91. Bailey, C. C., Zhong, G., Huang, I. C., & Farzan, M. (2014). IFITM-family proteins: the cell’s first line of antiviral defense. Annual review of virology, 1, 261.

53

92. Bailey, C. C., Huang, I. C., Kam, C., & Farzan, M. (2012). Ifitm3 limits the severity of acute influenza in mice. PLoS Pathog, 8(9), e1002909.

93. Everitt, A. R., Clare, S., McDonald, J. U., Kane, L., Harcourt, K., Ahras, M., ... & Haque, A. (2013). Defining the range of pathogens susceptible to Ifitm3 restriction using a knockout mouse model. PLoS One, 8(11), e80723.

94. Zhang, Y. H., Zhao, Y., Li, N., Peng, Y. C., Giannoulatou, E., Jin, R. H., ... & Wang, D. Y. (2013). Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nature communications, 4, 1418.

95. Markosyan, R., Li, K., Zheng, Y. M., Lin, S. L., & Cohen, F. S. (2013). IFITM Proteins Block the Creation of Hemifusion. Biophysical Journal, 104(2), 89a.

96. Weidner, J. M., Jiang, D., Pan, X. B., Chang, J., Block, T. M., & Guo, J. T. (2010). Interferon-induced cell membrane proteins, IFITM3 and , inhibit vesicular stomatitis virus infection via distinct mechanisms. Journal of virology, 84(24), 12646-12657.

97. Bailey, C. C., Kondur, H. R., Huang, I. C., & Farzan, M. (2013). Interferon-induced transmembrane protein 3 is a type II transmembrane protein. Journal of Biological Chemistry, 288(45), 32184-32193.

98. Yount, J. S., Karssemeijer, R. A., & Hang, H. C. (2012). S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus. Journal of Biological Chemistry, 287(23), 19631-19641.

99. Jia, R., Pan, Q., Ding, S., Rong, L., Liu, S. L., Geng, Y., ... & Liang, C. (2012). The N-terminal region of IFITM3 modulates its antiviral activity by regulating IFITM3 cellular localization. Journal of virology, 86(24), 13697-13707.

100. Desai, T. M., Marin, M., Chin, C. R., Savidis, G., Brass, A. L., & Melikyan, G. B. (2014). The Viral Restriction Factor IFITM3 Promotes Hemifusion but Blocks Full Fusion of Influenza Virus. Biophysical Journal, 106(2), 708a.

54 101. John, S. P., Chin, C. R., Perreira, J. M., Feeley, E. M., Aker, A. M., Savidis, G., ... & Pertel, T. (2013). The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication. Journal of virology, 87(14), 7837-7852.

102. Lin, T. Y., Chin, C. R., Everitt, A. R., Clare, S., Perreira, J. M., Savidis, G., ... & Elledge, S. J. (2013). Amphotericin B increases influenza A virus infection by preventing IFITM3-mediated restriction. Cell reports, 5(4), 895-908.

103. Amini-Bavil-Olyaee, S., Choi, Y. J., Lee, J. H., Shi, M., Huang, I. C., Farzan, M., & Jung, J. U. (2013). The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry. Cell host & microbe, 13(4), 452-464.

104. Liao, Z., Cimakasky, L. M., Hampton, R., Nguyen, D. H., & Hildreth, J. E. (2001). Lipid rafts and HIV pathogenesis: host membrane cholesterol is required for infection by HIV type 1. AIDS research and human , 17(11), 1009-1019.

105. Wrensch, F., Winkler, M., & Pöhlmann, S. (2014). IFITM proteins inhibit entry driven by the MERS- spike protein: evidence for cholesterol-independent mechanisms. Viruses, 6(9), 3683-3698.

106. Hubbard, C. et al. The secretory carrier membrane protein family: structure and membrane topology. Molecular Biology of the Cell. 11:2933-2947 (2000).

107. Aoh, Q., Castle, A., Hubbard, C., Katsumata, O. & Castle, J. SCAMP3 Negatively Regulates Epidermal Growth Factor Receptor Degradation and Promotes Receptor Recycling. Molecular Biology of the Cell 20, 1816-1832 (2009).

108. Wu, TT. & Castle, JD. Tyrosine phosphorylation of selected secretory carrier membrane proteins, SCAMP1 and SCAMP3, and association with the EGF receptor. Molecular Biology of the Cell 9, 1661-1674 (1998).

109. Shaw M. The host interactome of influenza virus presents new potential targets for antiviral drugs. Rev Medical Virology. 21, 358-369 (2011).

110. Dolnik, O. et al. Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in -Infected Cells. PLoS Pathogens 10 e1004463 (2014)

55 111. Wu, TT. & Castle, JD. Tyrosine phosphorylation of selected secretory carrier membrane proteins, SCAMP1 and SCAMP3, and association with the EGF receptor. Molecular Biology of the Cell 9, 1661-1674 (1998).

112. Mota, L. J., Ramsden, A. E., Liu, M., Castle, J. D., & Holden, D. W. (2009). SCAMP3 is a component of the Salmonella‐induced tubular network and reveals an interaction between bacterial effectors and post‐Golgi trafficking. Cellular microbiology, 11(8), 1236-1253.

56

CHAPTER 2

SCAMP3 regulates IFITM3 expression

57 SCAMP3 regulates IFITM3 expression

Vladimir Radica#, Christopher Robert Grotefenda#, Wan-Lin Wua, David Evan Joseph Williamsa, Michael Farzanb, I-Chueh Huanga* aDepartment of Molecular Microbiology and Immunology, Keck School of Medicine,

University of Southern California, Los Angeles, CA 90033 bDepartment of Infectious Diseases, The Scripps Research Institute, Jupiter, FL 33458

#C.R.G. and V.R. contributed equally to this work.

*Address correspondence to: I-Chueh Huang ([email protected])

Running head: SCAMP3 regulates retrograde transport

Abbreviations used in this paper: IFITM, interferon-induced transmembrane; SCAMP, secretory carrier membrane protein; CTXB, cholera toxin subunit B; IAV, influenza A viruses; TSG101, tumor susceptibility gene 101; NEDD4, neural precursor cell expressed, developmentally down-regulated 4 (NEDD4); NEDD4L, NEDD4-like; EGFR, epidermal growth factor receptor; MVB, multivesicular body; IFN, interferon; MLV, murine leukemia virus; GFP, green fluorescence preotin; MACV, machupo virus.

58 Abstract

Interferon-induced transmembrane (IFITM) proteins restrict a broad range of viruses. Despite their critical role in the innate immune control of viral infections, current understanding of IFITM metabolism is lacking. Though immunoprecipitation and mass spectrometry analysis, we found that IFITM3 interacts with secretory carrier membrane protein 3 (SCAMP3) and co-localizes with it in the late endosomes/lysosomes and the

Golgi network. SCAMP3 stabilizes IFITM3 expression by interfering with lysosomal degradation. However, it is not essential for the antiviral activity of IFITM proteins. In contrast to previous research, we observed that the proline-tyrosine (PY) motif, instead of the proline-serine-alanine-proline (PSAP) motif, is the principal determinant for

SCAMP3-mediated IFITM3 stabilization. Furthermore, SCAMP3 regulates early endosome-Golgi retrograde transport of IFITM3 and cholera toxin subunit B (CTXB).

Decrease Golgi network- and increase plasma membrane-associated CTXB and IFITM3 could be observed upon depletion of SCAMP3. Our studies indicate that SCAMP3 regulates IFITM3 expression by increasing its endosome-Golgi retrograde transport and diminishing lysosomal degradation.

59 2.1 INTRODUCTION

The IFITM protein family are viral restriction factors (Brass et al., 2009). In vitro studies have shown that IFITM1, 2, and 3 restrict a broad range of viruses, including influenza A viruses (IAVs), dengue virus, West Nile virus, severe acute respiratory syndrome coronavirus, Ebola virus, and Marburg virus (Brass et al., 2009; Huang et al.,

2011). IFITM3 also plays a central role in limiting IAV replication in vivo (Bailey et al.,

2012; Everitt et al., 2012). The biochemical properties of IFITM proteins have been characterized and several post-translational modifications of IFITM protein have been described. IFITM3 has three cysteines (C71, C72, and C105), which are S-palmitoylated in the hydrophobic domain. These modifications are essential for the membrane association of IFITM3 and its antiviral activity (Yount et al., 2010). Polyubiquitination via the lysine 48 linkage on four lysine residues of IFITM3 has also been reported to regulate proteasomal degradation of IFITM proteins (Yount et al., 2012).

SCAMP3 is a member of the SCAMP family, which contains four ubiquitously expressed members, SCAMP1, SCAMP2, SCAMP3, and SCAMP4, and a predominantly neural SCAMP5 (Singleton et al., 1997; Fernandez-Chacon and Sudhof, 2000; Krebs and

Pfaff, 2001; Aoh et al., 2009; Han et al., 2009). Unlike other members, SCAMP3 has PY and PSAP motifs in its N-terminal region, which allow it to interact with E3 ubiquitin ligases — neural precursor cell expressed, developmentally down-regulated 4 (NEDD4) and NEDD4-like (NEDD4L) — and with tumor susceptibility gene 101 (TSG101), respectively (Aoh et al., 2009; Falguieres et al., 2012). Studies have shown that, different from SCAMP1 and SCAMP2, which mainly localize to post-Golgi vesicles, SCAMP3

60 has a broader subcellular distribution, including the Golgi apparatus, early endosomes, late endosomes/lysosomes, and the plasma membrane (Wu and Castle, 1998; Liu et al.,

2002; Liu et al., 2005; Liao et al., 2007). Functionally, SCAMP3 is involved in the formation of Salmonella-induced tubular filaments in infected cells (Mota et al., 2009;

Schroeder et al., 2011). It regulates epidermal growth factor receptor (EGFR) expression by interfering with vesicular trafficking, multivesicular body (MVB) biogenesis, and lysosomal degradation and the PSAP motif is critical for SCAMP3-mediated EGFR stabilization (Aoh et al., 2009; Falguieres et al., 2012).

Through immunoprecipitation and mass spectrometry, we found that IFITM3 interacts with SCAMP3. IFITM3 colocalizes with SCAMP3 and is distributed in the late endosomal/lysosomal compartments and the Golgi network. SCAMP3 is not essential to the antiviral activity of IFITM proteins but it stabilizes IFITM3 expression by interfering with lysosomal degradation. In contrast to previous studies, our research demonstrated that the PY, instead of the PSAP, motif is the principal determinant contributing to

SCAMP3-mediated IFITM3 stabilization. We also showed that SCAMP3 plays a critical role retrograde transport. Early endosome to Golgi trafficking of CTXB and IFITM3 is impaired when SCAMP3 expression is depleted. These observations indicate that enhanced endosome-Golgi transport by SCAMP3 leads to the accumulation of IFITM3 at the Golgi network and decreases lysosomal degradation. As IFITM proteins are important innate immune factors against a broad range of viral infections, this work deepens the knowledge of the regulation of IFITM metabolism, which in turn may inspire new approaches to stabilizing IFITM expression as novel antiviral therapies.

61

2.2 RESULTS

IFITM3 interacts with SCAMP3

To identify host factors interacting with IFITM3 proteins, immunoprecipitation and mass spectrometry were performed. Among the three cell lines analyzed, SCAMP3 was isolated (Supplemental Figure S1A). SCAMP3 is a member of the tetraspanin protein family (Aoh et al., 2009). To examine whether the interaction between SCAMP3 and

IFITM3 is specific, coprecipitation and immunoblotting were conducted. We included two additional tetraspanin members, CD9 and CD81, which have been shown to interact with IFITM1 (Bradbury et al., 1992), as our controls. We found that HA-tagged

SCAMP3 (HA-SCAMP3), as well as HA-CD9, interacted with IFITM3 (Figure 1A and

Supplemental Figure S1B). However, the direct interaction between HA-CD81 and

IFITM3 could not be detected. Similarly, IFITM2, but not IFITM1, also interacted with

SCAMP3 (Figure 1B). Confocal microscopic analyses revealed that IFITM3 and

SCAMP3 colocalized in late endosomes/lysosomes and the trans- and cis-Golgi apparatus (Figure 1C and Supplemental Figure S1C).

SCAMP3 is not essential for IFITM-mediated viral restriction.

Because SCAMP3 interacts with IFITM3, we sought to examine the effect of

SCAMP3 on the antiviral activities of IFITM proteins. Viral entry and replication assays in cells expressing scrambled shRNA or shRNA targeting SCAMP3 were performed. We found that the entry of murine leukemia virus carrying the green fluorescent protein

62 reporter (MLV-GFP) and pseudotyped with various IAV hemagglutinin (HA) proteins, and the replication of infectious influenza A/PR/8/34 (H1N1) and A/Udorn/72 (H3N2) viruses were both increased upon SCAMP3 depletion (Figure 2, A and B). However, endogenous expression of IFITM3 was downregulated in cells expressing SCAMP3 shRNA (Figure 2C). To further clarify the role of SCAMP3 in IFITM-mediated viral restriction, we hyperexpressed IFITM1, 2, or 3 in control cells and SCAMP3-depleted cells (Figure 2D). Enhanced IAV entry resulted from the attenuated expression of IFITM proteins in cells expressing SCAMP3 shRNA could be observed (Supplemental Figure

S1D). Nevertheless, the effect of IFITM proteins on viral entry restriction in these cells was comparable to that in scrambled shRNA-expressing cells (Figure 2, E and F). Our data indicate that SCAMP3 may play an important role in regulating IFITM protein expression but is not an essential host factor for IFITM-mediated restriction of IAVs.

SCAMP3 stabilizes IFITM protein expression.

To evaluate whether SCAMP3 expression is critical to IFITM stabilization, we depleted SCAMP3 and found that the endogenous and interferon (IFN)-induced expression of IFITM1 and IFITM2/3 was downregulated (Figure 3, A and B). Using

A549 cells expressing tet-inducible IFITM3, we further demonstrated that SCAMP3 prolonged IFITM3 turnover (Figure 3C). Proteasomal or lysosomal degradation are two major pathways of protein metabolism (Hirsch and Ploegh, 2000; Hislop et al., 2011;

Tofaris et al., 2011). To determine the mechanism of IFITM3 metabolism and whether

SCAMP3 regulates this mechanism, we analyzed IFITM3 expression in cells treated with

63 a proteasomal inhibitor, MG132, or with a lysosomal inhibitor, bafilomycin (BAF) A1

(Umata et al., 1990; Yoshimori et al., 1991). In contrast to previous studies, our research showed that IFITM3 expression was strongly enhanced by BAF A1, whereas MG132 had a marginal effect (Figure 3D). When SCAMP3 was hyper-expressed, IFITM3 could be upregulated. However, this enhancement was diminished when cells were treated with

BAF A1 (Figure 3E). Our data indicated that lysosomal degradation contributes to the metabolism of IFITM3 and that SCAMP3 stabilizes IFITM3 expression by interfering with this pathway.

The PY motif of SCAMP3 is essential for SCAMP3-mediated stabilization of IFITM proteins.

Among all members of the SCAMP family, SCAMP3 has unique PY and PSAP motifs in the N terminus (Figure 3F) (Aoh et al., 2009). The PY motif has been shown to interact with NEDD4 and NEDD4L, whereas the PSAP motif may associate with

TSG101 (Aoh et al., 2009). To determine whether these motifs play a role in the stabilization of IFITM3, ΔPY and ΔPSAP SCAMP3 variants were generated by replacing proline residues 52 and 53 or the serine residue 65 with alanines. We coexpressed

IFITM3 and SCAMP3 variants and observed that IFITM3 expression was enhanced by wildtype and ΔPSAP, but to a lesser extent by ΔPY, SCAMP3 (Figure 3G). Unlike previous studies, our data indicate that the PY, instead of PSAP, motif is the determinant of SCAMP3-mediated IFITM3 stabilization.

64 The interaction between the PY motif of SCAMP3 and E3 ubiquitin ligases raises the possibility that SCAMP3 may alter the ubiquitination of IFITM3 and, therefore, interfere with its trafficking and degradation (Bradbury et al., 1992). To clarify this issue, a set of

IFITM3 variants was generated by replacing all lysine residues with arginines or maintain a single ubiquitination site: IFITM3-4R, IFITM3 24(K), 83(K), or 104(K) (Yount et al.,

2012). We found that IFITM3 24(K) had a longer half-life suggesting that ubiquitination of this site may contribute to stabilization of IFITM3 (Supplemental Figure S2A).

However, when we coexpressed SCAMP3, expression of all IFITM3 variants was enhanced (Supplemental Figure S2B). Furthermore, the ubiquitination pattern of IFITM3 was not altered by either WT or ΔPY SCAMP3 (Supplemental Figure S2, C-E). These data indicated that although the PY motif is essential for SCAMP3-mediated IFITM3 stabilization, SCAMP3 does not regulate IFITM3 by interfering with its ubiquitination.

SCAMP3 regulates early endosome-Golgi retrograde transport.

It is also possible that IFITM3 trafficking and subcellular localization may be regulated by SCAMP3. Previous studies as well as our research have shown that IFITM3 is mainly distributed in late endosomes/lysosomes and the Golgi network. We investigated whether subcellular localization of IFITM3 is altered when we disturb SCAMP3 expression. We observed that IFITM3 colocalized with a trans- Golgi marker and a cis-Golgi marker in control cells. However, this distribution was strongly diminished upon depletion of

SCAMP3 (Figure 4, A and B). The attenuated accumulation of IFITM3 in the Golgi network raises the possibility that SCAMP3 may promote retrograde trafficking of

65 IFITM3. To clarify this possibility, two model molecules, CTXB and mannose-6- phosphate receptor (M6PR) were used to assay early endosome-Golgi and late endosome/lysosome-Golgi retrograde transport respectively (Johannes and Popoff, 2008).

We found that SCAMP3 did not alter the steady-state distribution of M6PR (Figure 4, C and D). In contrast, retrograde trafficking of CTXB from the plasma membrane to the

Golgi network was impeded upon SCAMP3 depletion despite the total amount of CTXB remaining constant (Figure 5, A-C). Similarly, accumulation of IFITM3 at the plasma membrane could be observed up depletion of SCAMP3 (Figure 5 D and E). These observations indicate that SCAMP3 plays a critical role in regulating retrograde transport from early endosomes, rather than late endosomes, to the Golgi network. They also imply that SCAMP3 may increase steady-state IFITM3 expression by promoting its retrograde trafficking.

2.3 DISCUSSION

In our studies, we identified SCAMP3 as one of the cellular factors regulating the expression of IFITM proteins. SCAMP3 stabilized IFITM3 by interfering with its lysosomal degradation and contributed to distribution of IFITM3 in the Golgi network by promoting early endosome-Golgi retrograde trafficking. Based on our findings, we propose a mechanistic model for IFITM3 metabolism. At the steady state, IFITM3 distributes in late endosomes/lysosomes, the Golgi network, and the plasma membrane.

The majority of IFITM3 at the plasma membrane is recycled via early endosome-Golgi retrograde transport. The rest traffics to late endosomes/lysosomes and ultimately

66 undergoes lysosomal degradation. In the presence of SCAMP3, the balance between endosome-Golgi recycling and lysosomal degradation maintains the steady-state expression of IFITM3 (Figure 5F). Upon depletion of SCAMP3, retrograde transport of

IFITM3 to the Golgi network diminishes resulting in enhanced lysosomal degradation and decreased IFITM3 expression (Figure 5G).

A different functional model for SCAMP3 in the stabilization of EGFR has been proposed by Aoh et al (Aoh et al., 2009; Falguieres et al., 2012). They observed that

EGFR expression could be stabilized by SCAMP3 and its PSAP motif, which was reported to interact with TSG101, was essential for SCAMP3-mediated EGFR stabilization. Because Hrs and TSG101 are members of endosomal-sorting complex required for transport (ESCRT) complexes, they proposed that SCAMP3 inhibits MVB formation and, therefore, suppresses EGFR degradation (Falguieres et al., 2012). In contrast to previous work, our studies showed that the PY motif, which interacts with

NEDD4 and NEDD4L (Aoh et al., 2009), plays a critical role in IFITM3 stabilization.

Although these two models have the same downstream event (reduced lysosomal protein degradation), the regulatory mechanisms are quite different. Our research is the first to show that SCAMP3 regulates protein expression via retrograde transport.

The expression of IFITM proteins is induced by IFNs and several cytokines. We could not exclude the possibility that the function of SCAMP3 on IFITM3 stabilization might be regulated by IFNs or by other pathogen patterns. Mota et al. have demonstrated that the subcellular localization of SCAMP3 is altered during the course of Salmonella infection (Mota et al., 2009). Wu et al. have also shown that phosphorylation of

67 SCAMP1 and SCAMP3 changes their distribution (Wu and Castle, 1998). These observations suggest that pathogens may affect the localization of SCAMP3, and possibility also its activity. The antiviral activities of IFITM proteins have been well characterized. Our research sheds light on the metabolic mechanism of IFITM proteins.

Because the expression of IFITM proteins is critical for controlling the entry of viruses, interfering with the degradation of these proteins may provide novel approaches for treatment and prevention of viral infections.

2.4 MATERIALS AND METHODS

Cells, Chemicals, and Reagents

Human embryonic kidney 293T, human epithelial HeLa, and African green monkey kidney Vero E6 cells were maintained in Dulbecco's modified eagle medium (DMEM;

Corning). Human lung epithelial A549 and chronic myelogenous leukemia K562 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium or Iscove's

Modified Dulbecco's Medium (IMDM; ThermoFisher) respectively. All media were supplemented with 10% fetal bovine serum (FBS; Corning), 100 U/ ml penicillin, and

100 μg/ml streptomycin (Corning). A549 cells transduced with an empty vector or to express tetracycline (tet)-inducible IFITM3 variants were selected with 3 µg/ml puromycin (ThermoFisher) and 400 µg/ml gentamicin (ThermoFisher). HeLa and K562 cells expressing scrambled shRNA or shRNA targeting IFITM1, IFITM2, IFITM3, or

SCAMP3 were selected with 4 µg/ml puromycin (ThermoFisher). DMSO, BAF A1, and

68 MG132 were purchased from Sigma-Aldrich whereas human IFN-β 1A was obtained from R&D Systems.

Plasmids and constructs

DNA fragments encoding native and c-myc-tagged IFITM1, IFITM2, or IFITM3 were cloned into the pQCXIP vector (Clontech) using restriction enzyme cutting sites, NotI and BamHI. N-terminal hemagglutinin (HA)-tagged SCAMP3, HA-CD9, and HA-CD81 and N-terminal flag-tagged ubiquitin were also cloned into the pQCXIP vector

(Clontech) using the same restriction enzyme cutting sites. IFITM3 variants (24(K),

83(K), 88(K), 104(K), and IFITM3-4R) and SCAMP3 mutants (ΔPY, and ΔPSAP) were generated using the QuikChange method (Agilent Technologies). To generate plasmids encoding tet-inducible IFITM3 variants, open-reading frames of IFITM3 variants were introduced into the pRetroX-Tight-Pur vector (Clontech) using restriction enzyme cutting sites, BamHI and EcoRI. Retroviral pRS vector-based shRNA plasmids (the targeting sequence for scrambled shRNA is GCACTACCAGAGCTAACTCAGATAGTACT and the targeting sequence for IFITM1/IFITM3 shRNA is

TCATAGCATTCGCCTACTCCGTGAAGTCT) were purchased from OriGene.

Lentiviral pLKO.1 vector-based shRNA plasmids (the targeting sequence for scrambled shRNA is GCGGTTGCCAAGAGGTTCCAT and the targeting sequences for SCAMP3 shRNA are CAGCTACTCGACAGAACAATT [SCAMP3-1 shRNA] and

CGGAGTGACAGTTCATTCAAT [SCAMP3-2 shRNA]) were obtained from GE

Healthcare Life Sciences.

69

Pseudotyped virus transduction and infection assays

Plasmids and procedures used to generate pseudotyped MLV-GFP and transducing viruses have previously been described (Li et al., 2003; Huang et al., 2008; Huang et al.,

2011). Viral entry glycoproteins used to generate MLV-GFP included IAV HA proteins from A/PR/8/34 (H1N1) (H1[PR]), A/Udorn/72 (H3N2) (H3[Ud]), A/Thailand/2(SP-

33)/2004(H5N1) (H5[Thai]), and A/FPV/Rostock/34 (H7N1) (H7[FPV]), the protein from amphotropic MLV, and the glycoprotein (GPC) from Machupo virus (MACV,

Carvallo strain) (Li et al., 2003; Kuhn et al., 2006; Radoshitzky et al., 2007; Huang et al.,

2008). Viral entry glycoprotein used to generate transducing viruses included the G protein from vesicular stomatitis virus (VSV, Indiana strain), GPC proteins from MACV or lymphocytic choriomeningitis virus (Radoshitzky et al., 2007). The transduction assay and the pseudotyped virus entry assay were performed following procedures described in previous publications (Huang et al., 2008; Williams et al., 2014).

IAV infection assay

Influenza A/PR/8/34 (H1N1) and A/England/42/72 (H3N2) viruses purchased from

American type culture collection (ATCC) were propagated and titered as described previously (Huang et al., 2008). HeLa cells expressing scrambled shRNA or shRNA targeting SCAMP3 were incubated with influenza A/PR/8/34 (H1N1) or

A/England/42/72 (H3N2) virus at a multiplicity of infection (M.O.I.) of 1 for 3 hours.

After infection, cells were maintained in regular growth medium and harvested 16 hours

70 after viral inoculation. Infected cells were labeled with 0.5 μg/ml murine anti-influenza

H1 IgG2a (C179; Clontech) or with 1 μg/ml murine anti-influenza viral H3 IgG1 (F49;

Clontech), followed by an PE-conjugated secondary antibody (Thermo scientific)

(Williams et al., 2014). Fixed cells were analyzed by flow cytometry.

Immunoprecipitation and immunoblotting

For protein analysis, cells were lysed with 2% Triton X-100 (Sigma-Aldrich) or 2% lubrol (MP Biomedicals). Lysates were incubated with 25 μl of protein G sepharose (4 fast flow; GE Healthcare Life Sciences) overnight before immunoprecipitation.

Immunoprecipitation was performed using a goat anti-IFITM2/3 antibody (1 μg/mL;

R&D Systems [Cat. AF4834]), a murine anti-HA antibody (1 μg/mL, clone 16B12;

Biolegend), or a murine anti-flag antibody (1 μg/mL, clone M2; Sigma-Aldrich). Samples for western blotting were prepared in the reducing buffer, heated at 75 °C for 10 minutes, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), and transferred to the polyvinylidene difluoride (PVDF) membrane (ThermoFisher). The expression of native IFITM1, IFITM2, IFITM3, and IFITM3 variants was detected by goat anti-IFITM1 antibody (1 μg/mL; R&D Systems [Cat. AF4827]) and by goat anti-

IFITM2/3 antibodies (1 μg/mL; R&D Systems [Cat. AF4834]). The expression of HA-

CD9, HA-CD81, and HA-SCAMP3 variants was recognized by a Horseradish peroxidase

(HRP)-conjugated anti-HA antibody (Clone 3F10; Roche Life Science). The expression of endogenous SCAMP3 was detected by a rabbit anti-SCAMP3 (0.5 μg/mL, N1N3;

GeneTex [Cat. GTX102216]) or by a sheep anti-SCAMP3 (1 μg/mL; R&D Systems [Cat.

71 AF5344]) antibody. β-tubulin recognized by 1 µg/ml murine monoclonal anti-β-tubulin antibody (clone SAP.4G5, Sigma-Aldrich [Cat. T-7816]) was used as loading controls.

Secondary antibodies used for western blotting included HRP-conjugated rabbit anti-goat

IgG (1:20,000 dilution; Sigma-Aldrich [Cat. A-5420]), HRP-conjugated donkey anti- sheep IgG (1:20,000 dilution; R&D Systems [Cat. HAF016]), HRP-conjugated goat anti- mouse IgG (1:10,000 dilution; Santa Cruz Biotechnology [Cat. sc-2005]), and HRP- conjugated mouse anti-rabbit IgG (1:5,000 dilution; Santa Cruz Biotechnology [Cat. sc-

2357]) antibodies.

Confocal microscopy

Subcellular localizations of M6PR, IFITM3, and HA-tagged SCAMP3 were analyzed by confocal microscopy. Cells in chamber slides were washed twice with phosphate buffered saline (PBS) and fixed with 4% formaldehyde (Polysciences) at 25 °C for 20 minutes. Fixed cells were then washed with PBS twice, permeabilized with 0.2% Triton

X-100 (Sigma) for 10 min. Slides were than blocked in 3% bovine serum albumin (BSA;

Sigma) at 25 °C for 1 hour, and labeled with primary antibodies overnight at 4 °C.

Primary antibodies used in our research included rabbit anti-M6RP (1:1000 dilution,

ThermoFisher [Cat. PA5-24608]), goat anti-IFITM2/3 antibody (1 μg/mL; R&D Systems

[Cat. AF4834]), sheep anti-SCAMP3 (1:500 dilution; R&D Systems [Cat. AF5344]), rabbit anti-IFITM3 (1:1000 dilution; Proteintech [Cat. 11714-1-AP]), rabbit anti-early endosome antigen 1 (EEA1; 1:50 dilution; Thermo Scientific [Cat. MA5-14794]), murine anti-protein disulfide (PDI, 1:100 dilution; Enzo Life Sciences [Cat. ADI-SPA-

72 891-D]), goat anti-Golgi matrix protein GM130 (1:50 dilution; Santa Cruz Biotechnology

[Cat. sc-16268]), goat anti-trans-Golgi network integral membrane protein TGN38 (1:50 dilution; Santa Cruz Biotechnology [Cat. sc-27680]), and murine Alexa Fluor 488- conjugated anti-lysosomal-associated membrane protein 2 (LAMP2; 1:50 dilution, Santa

Cruz Biotechnology [Cat. sc-18822]) antibodies. After primary antibody staining, cells were washed three time with PBS and labeled with fluorophore-conjugated secondary antibodies for 1 hour at 25°C. Antibodies used included Alexa Fluor 555-conjugated donkey anti-sheep IgG (1:2000 dilution; ThermoFisher [Cat. A21436]), Alexa Fluor 647- conjugated chicken anti-rabbit IgG (1:2000 dilution; ThermoFisher [Cat. A21443]),

Alexa Fluor 488-conjugated donkey anti-goat IgG (1:2000 dilution; ThermoFisher [Cat.

A11055]), and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2000 dilution;

ThermoFisher [Cat. A10684]) antibodies. Cells were then mounted in prolong gold anti- fade reagent with 4',6-diamidino-2-phenylindole (DAPI, ThermoFisher). Finally, cells were imaged using a Zeiss LSM 510 confocal microscopy system (objective 63X).

CTXB trafficking assay

A549 cells expressing either scrambled shRNA or shRNA targeting SCAMP3 were incubated on ice with 500ng/mL Biotin- conjugated CTXB (1:1000 dilution,

ThermoFisher [Cat. C34779]), or Alexa-488 conjugated CTXB (1:1000 dilution,

ThermoFisher [Cat. C34775]) for 30 minutes. Cells were then washed with PBS 3 times and maintained in a 37°C incubator. Thirty minutes later, labeled cells were detached using a non-enzymatic cell dissociation reagent (Cellstripper; Corning), washed with PBS

73 for 3 times, and fixed with 2% formaldehyde (Polysciences). Cells were then analyzed by flow cytometry. For biotin-conjugated CTXB labeling, cells were incubated with a

1:1000 dilution of PE conjugated streptavidin (ThermoFisher [Cat. S866]) for 1 hour at

4°C before fixing and flow cytometry analysis. For confocal analysis, cells labeled with

Alexa-488 conjugated CTXB were fixed, permeabilized, and stained with the organelle marker following the procedures mentioned.

Surface staining

A549 cells expressing scrambled shRNA and SCAMP3 shRNA were transduced to express IFITM3. Two days later, cells were detached using a non-enzymatic cell dissociation reagent (Cellstripper, Corning), washed once with PBS, and then incubated with a rabbit anti-IFITM3 antibody (1:500 dilution; Proteintech [Cat. 11714-1-AP]) for 1 hour at 4°C. After primary antibody labeling, cells were washed three times with PBS and then incubated with an Alexa-488-conjugated anti-rabbit secondary antibody (1:2000 dilution, ThermoFisher [Cat. A21206]) for 1 hour at 4°C in the dark. Labeled cells were washed three times with PBS, fixed in 2% formaldehyde (Polysciences), and analyzed by flow cytometry.

Statistical analyses

Values are presented as relative infectivity or fluorescence intensity. Errors bars denote one s.d. (n=3). Every experiments were performed at least twice or three times. Sample

74 numbers were determined based on the preliminary studies. We used a paired two-tailed

Student's t-test for statistical analyses, and a P <0.05 was considered significant.

75 2.5 FIGURES

76

Figure 2.1. IFITM3 interacts with SCAMP3. (A) IFITM3-expressing 293T cells were transfected with an empty vector or to express the indicated HA-tagged tetraspanins. Two days later, cells were lysed. Supernatants were immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting using the indicated antibodies (WCL: whole cell lysates, IP: immunoprecipitation). (B) Experiments similar to that in (A) except that SCAMP3-expressing 293T cells were transfected with an empty vector or to express the indicated IFITM proteins. (C) A549 cells expressing IFITM3 were fixed, permeabilized, and labeled with anti-IFITM3, anti-SCAMP3, and the indicated organelle antibodies. Labeled cells were analyzed by confocal microscopy. Experiments were performed at least twice with similar results.

77

78 Figure 2.2. SCAMP3 is not essential for IFITM-mediated viral restriction. (A) Naïve HeLa cells or HeLa cells expressing the scrambled or SCAMP3 shRNA were incubated with MLV-GFP pseudotyped with the indicated viral glycoproteins. Infected cells were analyzed by flow cytometry 48 hours after infection. The relative infectivity was determined as the percentage of GFP positive cells normalized to that of vector-transduced cells. (B) HeLa cells expressing the indicated shRNA were infected with replicating influenza A/PR/8/34 (H1N1) or A/Udorn/72 (H3N2) virus at a multiplicity of infection (M.O.I.) of 1. One day later, cells were harvested, labeled with anti-H1 or anti-H3 primary and phycoerythin (PE)-conjugated secondary antibodies. Labeled cells were analyzed by flow cytometry. The relative infectivity was determined as the percentage of PE positive cells normalized to that of scrambled shRNA-expressing cells. (C) Aliquots of cells used in (A) and (B) were analyzed by western blotting. (D) Scrambled or SCAMP3 shRNA- expressing HeLa cells were transduced with an empty vector or to express the indicated IFITM proteins. Cells were analyzed by western blotting. (E) Experiments similar to that in (A) except that scrambled shRNA-expressing HeLa cells transduced with an empty vector or to express the indicated IFITM proteins as described in (D) were used for the viral entry assay. (F) Experiments similar to that in (A) and (E) except that SCAMP3 shRNA-expressing cells were used. Experiments were performed at least three times with similar results. Errors bars denote one standard deviation (s.d.; n=3). * P <0.05; paired two- tailed Student's t-test.

79

80 Figure 2.3. SCAMP3 stabilizes expression of IFITM3. (A) K562 cells expressing scrambled or shRNA or shRNA targeting the indicated genes were lysed and analyzed by western blotting using the indicated antibodies. (B) HeLa cells stably expressing scrambled shRNA or shRNA targeting the indicated genes were treated with 1000U/ml IFN-β. Two days later, cells were analyzed by western blotting. (C) Tet-inducible IFITM3-expressing A549 cells were transduced with an empty vector or to express HA-tagged SCAMP3 and then treated with 1 μg/ml doxycycline. Two days later, cells were washed and maintained in doxycycline free medium. At the indicated time points, cells were lysed. The time course of IFITM3 metabolism was determined by western blotting. (D) 293T cells transduced to express IFITM3 were treated with dimethyl sulfoxide (DMSO), 50 nM BAF A1, or 10 μM MG-132. Sixteen hours later, cells were analyzed by western blotting. (E) 293T cells expressing IFITM3 were transfected with an empty vector or to express HA-tagged SCAMP3. Cells were treated with DMSO or with 50 nM BAF A1. Sixteen hours later, cells were analyzed by western blotting. (F) A representation of SCAMP3. Note that the PY motif (PPAY) and the PSAP motif are in the N-terminal region (TM: transmembrane). (G) 293T cells expressing IFITM3 were transfected with an empty vector or to express the indicated SCAMP3 variants. Two days later, cells were analyzed by western blotting. Experiments were performed at least twice with similar results.

81

82 Figure 2.4. SCAMP3 contributes the accumulation of IFITM3 in the Golgi network. (A) Scrambled or SCAMP3 shRNA-expressing A549 cells were transduced to express IFITM3. Two days later, cells were fixed, permeabilized, and labeled with anti-IFITM3 and anti-GM130 antibodies. Labeled cells were analyzed by confocal microscopy. (B) Experiments similar to that in (A) except that an anti-TGN38 antibody was used. (C) Scrambled or SCAMP3 shRNA-expressing A549 cells were fixed, permeabilized, and labeled with anti-M6PR antibody and anti-GM130 antibodies. Labeled cells were then imaged by confocal microscopy. (D) Experiments similar to that in (C) except that an anti- TGN38 antibody was used. Experiments were performed at least twice with similar results.

83

84 Figure 2.5. SCAMP3 regulates early endosome-Golgi retrograde transport. (A) Scrambled or SCAMP3 shRNA-expressing A549 cells were incubated with Alexa488-conjugated CTXB (green) on ice for 30 minutes and then maintained at 37C. Thirty minutes later, cells were fixed, permeabilized, and labeled with the indicated organelle marker (red). Labeled cells were imaged by confocal microscopy. (B) Experiments similar to that in (A) expect that biotin-conjugated CTXB was used and plasma membrane-associated CTXB was labeled with PE-conjugated streptavidin. Cells were then fixed without permeabilization and analyzed by flow cytometry. The relative fluorescence intensity was determined as the mean fluorescence intensity of PE normalized to that of scrambled shRNA-expressing cells. (C) Experiments similar to that in (A) and (B) except that Alexa488-conjugated CTXB was used. Cells were fixed without permeabilization and analyzed by flow cytometry. (D) Scrambled or SCAMP3 shRNA-expressing A549 cells were transduced to express IFITM3. Two days later, cells were labeled with anti-IFITM2/3 and PE-conjugated antibodies. Surface expression of IFITM3 was determined by flow cytometry. The relative fluorescence intensity was determined as the mean fluorescence intensity of PE normalized to that of scrambled shRNA-expressing cells. (E) Same aliquots of cells used in (D) were analyzed by western blotting. Experiments were performed at least three times with similar results. Errors bars denote one s.d. (n=3). * P <0.05; paired two-tailed Student's t-test. A representation of IFITM3 trafficking model in the presence (F) or upon depletion (G) of SCAMP3.

85

86 Figure 2.6. IFITM3 interacts with SCAMP3. (A) 293T, A549, or Vero E6 cells transduced to express c-myc-tagged IFITM3 were lysed, immunoprecipitated with an anti-c-myc antibody, and analyzed by SDS-PAGE and mass spectrometry. Fragmented amino acid sequences of SCAMP3 isolated by mass spectrometry were listed. (B) IFITM3-expressing 293T cells were transfected with an empty vector or to express the indicated HA-tagged SCAMP3. Two days later, cells were lysed. Supernatants were immunoprecipitated with an anti-IFITM2/3 antibody and analyzed by immunoblotting with the indicated antibodies. (C) A549 cells were transduced to express IFITM3. Two days later, cells were fixed, permeabilized, and labeled with the indicated antibodies (anti-IFITM3 (red), the indicated organelle markers (Li et al.), and anti-SCAMP3 (blue) antibodies). Labeled cells were analyzed by confocal microscopy. (A) Same experiments as described in Figs. 2E and F. The relative infectivity was determined as the percentage of GFP positive cells normalized to that of scrambled shRNA-expressing and vector-transduced HeLa cells. Experiments were performed at least three times with similar results.

87

88 Figure 2.7. SCAMP3 does not alter the ubiquitination pattern of IFITM3. (A) A549 cells expressing the indicated tet-inducible IFITM3 variants were treated with 1 μg/ml doxycycline. Two days later, cells were washed and maintained in doxycycline free medium. At the indicated time points, cells were harvested and lysed. The time courses of the metabolism of IFITM3 variants were determined by western blotting using the indicated antibodies. (B) 293T cells expressing the indicated IFITM3 variants were transfected with an empty vector or to express SCAMP3. Two days after transfection, cells were lysed and analyzed by western blotting using the indicated antibodies. (C) and (D) 293T cells stably expressing flag-tagged ubiquitin were transfected with an empty vector or to express the indicated SCAMP3 variants followed by transduction to express the indicated IFITM3 variants. Cells were then treated with 50 nM BAF A1 to maintain all ubiquitinated IFITM3 species. Sixteen hours later, cells were lysed, supernatants were immunoprecipitated with an anti-flag antibody, and analyzed by immunoblotting using an IFITM2/3 antibody. (E) Experiments similar to that in (C) except that the expression of the indicated proteins in the whole cell lysates were detected by the indicated antibodies. Experiments were performed at least twice with similar results.

89 2.6 References

1. Aoh, Q.L., A.M. Castle, C.H. Hubbard, O. Katsumata, and J.D. Castle. 2009. SCAMP3 negatively regulates epidermal growth factor receptor degradation and promotes receptor recycling. Mol Biol Cell. 20:1816-1832.

2. Bailey, C.C., I.C. Huang, C. Kam, and M. Farzan. 2012. Ifitm3 limits the severity of acute influenza in mice. PLoS Pathog. 8:e1002909.

3. Bradbury, L.E., G.S. Kansas, S. Levy, R.L. Evans, and T.F. Tedder. 1992. The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. J Immunol. 149:2841-2850.

4. Brass, A.L., I.C. Huang, Y. Benita, S.P. John, M.N. Krishnan, E.M. Feeley, B.J. Ryan, J.L. Weyer, L. van der Weyden, E. Fikrig, D.J. Adams, R.J. Xavier, M. Farzan, and S.J. Elledge. 2009. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell. 139:1243-1254.

5. Everitt, A.R., S. Clare, T. Pertel, S.P. John, R.S. Wash, S.E. Smith, C.R. Chin, E.M. Feeley, J.S. Sims, D.J. Adams, H.M. Wise, L. Kane, D. Goulding, P. Digard, V. Anttila, J.K. Baillie, T.S. Walsh, D.A. Hume, A. Palotie, Y. Xue, V. Colonna, C. Tyler-Smith, J. Dunning, S.B. Gordon, R.L. Smyth, P.J. Openshaw, G. Dougan, A.L. Brass, and P. Kellam. 2012. IFITM3 restricts the morbidity and mortality associated with influenza. Nature. 484:519-523.

6. Falguieres, T., D. Castle, and J. Gruenberg. 2012. Regulation of the MVB pathway by SCAMP3. Traffic. 13:131-142.

7. Fernandez-Chacon, R., and T.C. Sudhof. 2000. Novel SCAMPs lacking NPF repeats: ubiquitous and synaptic vesicle-specific forms implicate SCAMPs in multiple membrane-trafficking functions. J Neurosci. 20:7941-7950.

8. Han, C., T. Chen, M. Yang, N. Li, H. Liu, and X. Cao. 2009. Human SCAMP5, a novel secretory carrier membrane protein, facilitates calcium-triggered cytokine secretion by interaction with SNARE machinery. J Immunol. 182:2986-2996.

9. Hirsch, C., and H.L. Ploegh. 2000. Intracellular targeting of the proteasome. Trends Cell Biol. 10:268-272.

90

10. Hislop, J.N., A.G. Henry, and M. von Zastrow. 2011. Ubiquitination in the first cytoplasmic loop of mu-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation. J Biol Chem. 286:40193-40204.

11. Huang, I.C., C.C. Bailey, J.L. Weyer, S.R. Radoshitzky, M.M. Becker, J.J. Chiang, A.L. Brass, A.A. Ahmed, X. Chi, L. Dong, L.E. Longobardi, D. Boltz, J.H. Kuhn, S.J. Elledge, S. Bavari, M.R. Denison, H. Choe, and M. Farzan. 2011. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus. PLoS Pathog. 7:e1001258.

12. Huang, I.C., W. Li, J. Sui, W. Marasco, H. Choe, and M. Farzan. 2008. Influenza A virus neuraminidase limits viral superinfection. J Virol. 82:4834-4843.

13. Johannes, L., and V. Popoff. 2008. Tracing the retrograde route in protein trafficking. Cell. 135:1175-1187.

14. Krebs, C.J., and D.W. Pfaff. 2001. Expression of the SCAMP-4 gene, a new member of the secretory carrier membrane protein family, is repressed by progesterone in brain regions associated with female sexual behavior. Brain research. Molecular brain research. 88:144-154.

15. Kuhn, J.H., S.R. Radoshitzky, A.C. Guth, K.L. Warfield, W. Li, M.J. Vincent, J.S. Towner, S.T. Nichol, S. Bavari, H. Choe, M.J. Aman, and M. Farzan. 2006. Conserved receptor-binding domains of Lake Victoria marburgvirus and bind a common receptor. J Biol Chem. 281:15951-15958.

16. Li, W., M.J. Moore, N. Vasilieva, J. Sui, S.K. Wong, M.A. Berne, M. Somasundaran, J.L. Sullivan, K. Luzuriaga, T.C. Greenough, H. Choe, and M. Farzan. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 426:450-454.

17. Liao, H., J. Ellena, L. Liu, G. Szabo, D. Cafiso, and D. Castle. 2007. Secretory carrier membrane protein SCAMP2 and phosphatidylinositol 4,5-bisphosphate interactions in the regulation of dense core vesicle exocytosis. Biochemistry. 46:10909-10920.

18. Liu, L., Z. Guo, Q. Tieu, A. Castle, and D. Castle. 2002. Role of secretory carrier membrane protein SCAMP2 in granule exocytosis. Mol Biol Cell. 13:4266-4278.

91 19. Liu, L., H. Liao, A. Castle, J. Zhang, J. Casanova, G. Szabo, and D. Castle. 2005. SCAMP2 interacts with Arf6 and phospholipase D1 and links their function to exocytotic fusion pore formation in PC12 cells. Mol Biol Cell. 16:4463-4472.

20. Mota, L.J., A.E. Ramsden, M. Liu, J.D. Castle, and D.W. Holden. 2009. SCAMP3 is a component of the Salmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking. Cellular microbiology. 11:1236-1253.

21. Radoshitzky, S.R., J. Abraham, C.F. Spiropoulou, J.H. Kuhn, D. Nguyen, W. Li, J. Nagel, P.J. Schmidt, J.H. Nunberg, N.C. Andrews, M. Farzan, and H. Choe. 2007. 1 is a cellular receptor for New World haemorrhagic fever . Nature. 446:92-96.

22. Riederer, M.A., T. Soldati, A.D. Shapiro, J. Lin, and S.R. Pfeffer. 1994. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Cell Biol. 125:573-582.

23. Schroeder, N., L.J. Mota, and S. Meresse. 2011. Salmonella-induced tubular networks. Trends in microbiology. 19:268-277.

24. Singleton, D.R., T.T. Wu, and J.D. Castle. 1997. Three mammalian SCAMPs (secretory carrier membrane proteins) are highly related products of distinct genes having similar subcellular distributions. J Cell Sci. 110 ( Pt 17):2099-2107.

25. Tofaris, G.K., H.T. Kim, R. Hourez, J.W. Jung, K.P. Kim, and A.L. Goldberg. 2011. Ubiquitin Nedd4 promotes alpha-synuclein degradation by the endosomal-lysosomal pathway. Proceedings of the National Academy of Sciences of the United States of America. 108:17004-17009.

26. Umata, T., Y. Moriyama, M. Futai, and E. Mekada. 1990. The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase. J Biol Chem. 265:21940-21945.

27. Utskarpen, A., H.H. Slagsvold, T.G. Iversen, S. Walchli, and K. Sandvig. 2006. Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'. Traffic. 7:663-672.

92 28. White, J., L. Johannes, F. Mallard, A. Girod, S. Grill, S. Reinsch, P. Keller, B. Tzschaschel, A. Echard, B. Goud, and E.H. Stelzer. 1999. Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. J Cell Biol. 147:743- 760.

29. Williams, D.E., W.L. Wu, C.R. Grotefend, V. Radic, C. Chung, Y.H. Chung, M. Farzan, and I.C. Huang. 2014. IFITM3 polymorphism rs12252-C restricts influenza A viruses. PloS one. 9:e110096.

30. Wu, T.T., and J.D. Castle. 1998. Tyrosine phosphorylation of selected secretory carrier membrane proteins, SCAMP1 and SCAMP3, and association with the EGF receptor. Mol Biol Cell. 9:1661-1674.

31. Yoshimori, T., A. Yamamoto, Y. Moriyama, M. Futai, and Y. Tashiro. 1991. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 266:17707-17712.

32. Yount, J.S., R.A. Karssemeijer, and H.C. Hang. 2012. S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus. J Biol Chem. 287:19631-19641.

33. Yount, J.S., B. Moltedo, Y.Y. Yang, G. Charron, T.M. Moran, C.B. Lopez, and H.C. Hang. 2010. Palmitoylome profiling reveals S-palmitoylation-dependent antiviral activity of IFITM3. Nat Chem Biol. 6:610-614.

93 CHAPTER 3

SCAMP3 Is and Innate Immune Factor which Inhibits

IAV infection by altering Hemagglutinin Trafficking.

94 3.1 Introduction

Influenza A Viruses.

Influenza A Viruses (IAVs) are members of the family Orthomyxoviridae1. They are enveloped viruses containing an eight-segmented, negative-sense single-stranded RNA genome. The eight genomic RNA total about 13Kb and encode 11 different proteins2.

The viral life cycle can roughly be broken down into four stages: Entry, replication, assembly and egress. During entry, the viral Hemagglutinin (HA) binds to sialic acid residues on cell membrane glycoproteins resulting in the virion undergoing receptor- mediated endocytosis. Fusion between the viral and cell membrane occurs in the late endosome allowing for entry of viral ribonucleoprotein complex (RNP) into the cytosol.

The RNP is then translocated into the nucleus, where it hijacks cellular transcription and translation machinery in order to replicate the viral genome and begin producing viral proteins. Once the viral genome has been replicated and viral proteins translated, the virus utilizes the host secretory pathway to transport viral proteins and genomic material to the apical plasma membrane of the infected cell. Assembly of progeny virions is completed at the plasma membrane, where the RNP interacts with envelope glycoproteins and nascent virions bud from the host cell.3

Influenza A Viruses are a Major Public Health Concern

Influenza has long been an important human pathogen. Influenza-like syndromes have been described through antiquity and the virus continues to cause seasonal epidemics and

95 pandemics4. On average, seasonal influenza pandemics cause 200,000 hospitalizations and 25,000 deaths per year in the United States alone 4,5 This is despite the great strides which have been made in understanding influenza biology and how to treat the disease.

Furthermore, with the emergence of Highly Pathogenic Avian Influenza Virus (HPAIV) in humans, influenza viruses now pose an even greater global health threat.

Hemagglutinin Cleavage is a Major Determinant for Influenza Pathogenicity

Hemagglutinin (HA) is responsible for binding to host cell receptors at the cell membrane. This results in the virus being internalized on via receptor-mediated endocytosis. As the endosome matures, it acidifies, resulting in a conformational change in HA. This allows the fusion domain of HA to insert into the endosomal membrane and facilitate creation of a fusion pore through which the viral ribonucleoprotein complex can enter the cytoplasm. HA cleavage is the main determinant of IAV infectivity due to the fact that cleavage of the HA0 proprotein into disulfide linked HA1 and HA2 exposes the fusion peptide6. Uncleaved HA cannot mediate membrane fusion and the contents of the virion cannot enter the cytoplasm, and without fusion occurring the virion ends up being degraded in the lysosome7. In low pathogenicity influenza viruses, HA0 is cleaved at a terminal arginine residue by trypsin-like proteases. In mammals, expression of these proteases is limited to the upper respiratory tract, therefore efficient viral replication can only occur in these specific tissues8. In contrast, HPAIV HA contains a polybasic cleavage site and thus is cleaved by the ubiquitously expressed protease Furin9. This allows HPAIVs to replicate efficiently in most tissues allowing these viruses to establish

96 systemic infection and leading to a much more lethal disease syndrome than seasonal pandemic viruses. Mutation of the polybasic site in HPAIVs leads to a highly attenuated phenotype and loss of ability to cause systemic infection10.

The Innate Immune Response to IAV Infection.

The innate immune system provides a critical barrier against influenza virus infection11.

The first step in induction of innate immunity against IAV happens when the virus is sensed by various pathogen recognition receptors. In the case of influenza, the virus is sensed by either TLR3, TLR7 or RIG-I. These pathogen recognition receptors are activated by viral RNA (double stranded in the case of TLR3 and RIG-I and single stranded for TLR7) and initiate a signaling cascade, which culminates in the expression of type I interferon. Type I interferon can then signal in an autocrine and paracrine manner, causing infected cells, as well as any cells stimulated by secreted interferon to express thousands of interferon stimulated genes which inhibit viral replication. The innate immune response also results in production of cytokines and chemokines which aid in the recruitment of cellular immunity12,13. This pathway is critical to surviving influenza challenge, as is evidenced by the fact that knocking out the interferon receptor in mice leads to much higher susceptibility to IAV infection14. Despite the importance that the interferon response plays in controlling infections, many interferon effectors are still poorly characterized and the full scope of the antiviral state induced by IFN has yet to be fully deciphered.

97 SCAMP3

Secretory Carrier Membrane Protein 3(SCAMP3) is a ubiquitously expressed protein of the SCAMP family. It is a tetraspanning integral membrane protein with cytoplasmic C and N terminal domains15. It has been found to play roles in post-Golgi transport,

Epidermal Growth Factor Receptor degradation and recycling, as well as Salmonella pathogenesis16,17,18. SCAMP 3 also contains various glycosylation, phosphorylation and ubiquitination sites15. Previous studies have shown that altering SCAMP3 phosphorylation can change the protein’s localization. Phosphorylated SCAMP3 seems to migrate from the trans-Golgi network to patches on the plasma membrane19. The purpose of this phenomenon is still unclear. Furthermore, it has been postulated that ubiquitination of SCAMP3 can alter its function from promoting recycling of cell surface receptors to promoting their degradation. In addition to these sites there are many other putative post-translational modifications on SCAMP3 which are yet to be examined.

SCAMP3 has also been shown to have a conserved PSAP motif in an N terminal proline rich domain of the protein. This motif allows SCAMP3 to interact with TSG101, which has been shown to an important factor in Influenza, HIV and Ebola live cycles20-21.

ISGylation of TSG101 Can Prevent Egress of Influenza Virions From Infected Cells.

TSG101 is part of the endosomal sorting complex required for transport 1(ESCRT-1). It functions in the biogenesis of the multi-vesicular body, and is thus utilized by certain viruses to gain access to multi-vesicular budding pathway20,21. TSG101 was first implicated to play a role in the life cycle of retroviruses. Specifically, HIV gag protein

98 was shown to have a highly conserved PSAP motif, which when mutated was shown to abrogate viral budding. This was demonstrated to be due to HIV needing to hijack the host ESCRT pathway through Tsg101 to facilitate trafficking of viral proteins to the cell membrane and buddying of progeny virions20. More recently, TSG101 was shown to have an interferon-dependent antiviral effect on IAV. Tsg101 is necessary for trafficking of viral HA from the trans Golgi network to the plasma membrane. The induction of the interferon response results in production of Interferon Stimulated Gene 15(ISG15), a ubiquitin-like molecule which can be ligated to TSG101. This addition of ISG15 (termed

ISGylation) prevents transport of viral HA to the plasma membrane, and in turn prevents formation of infectious progeny virions21.

Overall Significance

Given the preceding information, we believe there is a possibility that SCAMP3 may have some role in innate immunity against influenza. Since SCAMP3 can interact with

TSG101, we believe that it may have some role to play in TSG101 mediated abrogation of hemagglutinin trafficking. Since the mechanism of how ISGylated TSG101 prevents

HA trafficking to the plasma membrane is poorly understood, we believe that by studying

SCAMP3 in the context of IAV infection we can elucidate some step in this phenomenon.

99 3.2 RESULTS

SCAMP3 Interacts with Interferon Induced Transmembrane (IFITM) Family of

Proteins

We first identified SCAMP3 as a putative interferon effector while studying IFITM- interacting proteins. The IFITM family has been shown to be a potent inhibitor of viral entry23,24. Using mass spectrometry, we determined which proteins were interacting with the different IFITM isoforms in HEK 293T, A549 and HeLa cells. We found that

SCAMP3 co-precipitated with the IFITM proteins in all of these cell lines. Given that the

IFITM proteins an instrumental part of the interferon response against IAV, we postulated that this interaction with SCAMP3 could indicate SCAMP3 had some role in the interferon response.

SCAMP3 Efficiently Inhibits Viral Entry of Progeny HPAIV Pseudotype Virions

To test if SCAMP3 had any antiviral activity, we assayed how well pseudotype HPAIV

H7N1 and H5N1 viruses infected SCAMP3 expressing cells. We also tested how well virus produced in SCAMP3 expressing cells was able to infect target cells. As we did not have access to a select agent BSL-3 facility, we used an HIV pseudotype model to assay infectivity in a BSL-2 environment. We transfected 293T cells with either a SCAMP3 expression vector or a control vector, as well as PNL4.3dE (A plasmid encoding the structural elements of HIV, but lacking the glycoprotein), the HA protein from either

H5N1 (A/Thailand/1(KAN-1)/2004) or H7N1 (A/FPV/Rostock/34) and the neuraminidase gene from H1N1 (A/PR/8/1934). This resulted in virions which contained

100 the structural elements of HIV and the HA proteins of two HPAIV strains. To assay infectivity, we used GHOST X4 cells as our target cells. These cells contain an HIV driven promoter, which when activated by HIV genome integration and tat protein production drives the expression of GFP. This causes infected cells to emit green fluorescence, which can be quantified by flow cytometry. We also transduced a group of these target cells to express high levels of SCAMP3. We found that virus produced in normal cells was able to infect SCAMP3 expressing cells and control vector expressing cells at the same rate (Fig 1B). However, when we generated pseudotype virus in

SCAMP3 expressing cells, we found entry of these viruses into target cells was highly attenuated (Fig1A). Remarkably, SCAMP3 was not able to restrict entry of viruses pseudotyped with the VSV glycoprotein (VSV-G). This was not due to higher levels of virus being produced by control cells, as the levels of P24, an HIV structural protein in our viral samples did not differ between viruses produced in SCAMP3 hyper-expressing cells compared to control cells (data not shown).

SCAMP3 Hyperexpression Prevents HPAIV HA Cleavage

Since the only influenza proteins we used in the pseudotype entry assay were

Hemagglutinin (HA) and Neuraminidase (NA), we postulated that SCAMP3 was somehow able to inhibit the functions of one or both of these proteins. The role of the viral neuraminidase is to cleave sialic acids on the host cells, as well as on other viral proteins. Inhibition of neuraminidase activity has been shown to prevent the virus from detaching from the cell surface after budding due to HA staying bound to sialic acids on

101 the plasma membrane25. NA inhibition can also cause aggregation of IAV particles, due to the HA binding to sialic acids on adjacent virions. Since we were able to detect virus in the supernatant of our producing cells, we hypothesized that SCAMP3’s antiviral activity not related to NA activity. We therefore focused the viral HA protein. We then analyzed

HA from cell lysate and progeny virions from our previous experiments by western blot.

We discovered that even though both the SCAMP3 expressing cells and the control cells were able to produce similar amounts of virus, the HA of virions produced by SCAMP3 expressing cells was not cleaved (Fig 1C). This phenomenon was also observed in the lysate of virus producing cells. After HPAIV HA is synthesized in the ER, it is trafficked through the Trans-Golgi Network, where it is cleaved by Furin into HA1 and HA2. In contrast, VSV-G does not contain a polybasic cleavage site and therefore does not need to be cleaved by Furin to be active. We verified these findings by co-expressing

SCAMP3 and HPAIV HA in 293T cells (Fig 1D). We found that during SCAMP3 hyper- expression, HPAIV HA cleavage is ablated. These observations suggest that SCAMP3 restricts HA cleavage by affecting trafficking of the HA though the Golgi network.

SCAMP3 Hyper-Expression Causes a Decrease in Intracellular HA in Influenza

Infected Cells

To discern if SCAMP3 was able to have this effect on a replicating Influenza Virus, we first generated stable SCAMP3 hyper-expressing A549 cells. We accomplished this by transducing naïve A459 cells with a SCAMP3 expression vector containing a puromycin resistance gene. We used puromycin-containing media to obtain a pure culture of

102 SCAMP3 hyper-expressing cells. We used cells transduced with an empty vector as controls. We infected these cells with A/PR 8/34 (H1N1) and A/England/42/72 (H3N2) influenza viruses and assayed HA expression by western blot (Fig 2A). We found that hyperexpressing SCAMP3 led to a decreased level of HA being produced in the infected cells when compared to a control cell line.

Knocking Down SCAMP3 Results in Higher Levels of Intracellular HA In Infected

Cells

To further investigate whether SCAMP3 can play a role in restricting IAV, we used shRNA to knock down SCAMP3 in A549 cells. We generated the knockdown cells by transducing naïve A549 cells with a vector containing an shRNA against SCAMP3 mRNA as well as a puromycin resistance gene. We selected for knockdown cells by plating the transduced cells in puromycin containing media. Cells transduced with the same vector expressing a non-targeting siRNA were used as a control (Fig 2B). We found that depleting SCAMP3 resulted in higher levels of intracellular HA being produced during infection with H1N1 and H3N2 viruses. To test if this was due to reduced entry of viruses into control cells, we assayed the intracellular levels of viral nucleoprotein (NP), a structural protein of IAV. We found that the levels of viral NP present in the cell were similar in the knockdown as well as the control cells, indicating that this knockdown affects only hemagglutinin. Furthermore we found that virion associated Hemagglutinin levels were also increased in the knockdown cells compared to the luciferase shRNA controls (Fig 2C)

103 SCAMP3 Localization Changes During Influenza Challenge.

To further elucidate SCAMP3’s role in viral restriction, we used confocal microscopy to determine if SCAMP3 distribution can change during viral infection. We used A549 cells infected with either H1N1 or H3N2, as well as uninfected cells, cells treated with

2000u/mL IFN-B and interferon treated cells, which were also infected. Cells were incubated for 24 or 48 hours, then fixed, permeabilized and stained for SCAMP3. We analyzed SCAMP3 distribution using confocal microscopy (Fig 3A). Under normal conditions, SCAMP3 has a punctate localization around the cytoplasm, with some protein localizing the perinuclear space. Strikingly, upon influenza infection, we were only able to detect SCAMP3 strongly localizing to the perinuclear space. Interestingly, treating cells with interferon before IAV infection seemed to have an additive effect on SCAMP3 re-localization.

SCAMP3 Is Phosphorylated During Influenza Infection.

Previous studies have shown that SCAMP3 localization can change as a result of phosphorylation of tyrosine residues on the protein19. Since influenza infection appears to alter SCAMP3 localization, we decided to assay if SCAMP3 phosphorylation is also altered during viral infection. To test this, we decided to use Phos-Tag agarose to assay

SCAMP3 phosphorylation. Phos-tag agarose contains an alkoxide-bridged dinuclear zinc(II) complex, which can transiently bind phosphate groups on proteins which are migrating through the gel during electrophoresis. This results in phosphorylated species of a protein having a higher apparent molecular weight on the resulting western blot. To

104 assay SCAMP3 phosphorylation, we compared immunoblots of SCAMP3 from

SCAMP3 hyper-expressing A549 cells infected with either H1N1 or H3N2 viruses to

SCAMP3 from naïve cells(Fig 3B). We found that SCAMP3 from infected cells was enriched for a band of high molecular weight, which was much less pronounced in naïve cells. This indicates that SCAMP3 is being phosphorylated during viral infection.

SCAMP3 Expression Can be Induced by TNFα

Our earlier data suggested that high levels of SCAMP3 could be sufficient to restrict entry of daughter virions. However, at the time of the writing of this dissertation, no research has been done to determine whether any cellular or pathogen factors can induce SCAMP3 expression. We decided to investigate this by searching promoter element databases to determine if perhaps SCAMP3 had any proximal regulatory elements. Interestingly, we found that there are several NFkB binding sites upstream and downstream of the SCAMP3 sequence (Fig 3C). We decided to test if NFkB Induction could induce SCAMP3 expression. To do this, we stimulated cells with three compounds which are known to induce NFkB activation, Lipopolysaccharide (LPS) Interleukin 1 beta (IL1-β) and Tumor Necrosis Factor Alpha (TNFα). We found that LPS and IL1-β treatment had no apparent effect on SCAMP3 expression, however TNFα caused a dose dependent upregulation of SCAMP3 expression (Fig 3D). Influenza infection has been shown to induce TNFα expression, therefore providing a putative link between our observation that high levels of SCAMP3 can alter HA trafficking and the innate immune system.

105 3.3 DISCUSSION

The innate immune response against Influenza is critical in controlling the virus and limiting its pathogenesis. Despite this, many of the thousands of Interferon

Stimulated Genes have not been explored and the full extent of the innate immune response against these viruses is still not well understood. Further confounding the understanding of innate immunity is cases of proteins such as TSG101, which themselves are not stimulated by the interferon response but instead are modified by other interferon stimulated proteins and thus are able to have some kind of antiviral effect. Here we describe a novel antiviral function for SCAMP3. Current research has described

SCAMP3 as a regulator of receptor recycling from the cell surface, however we have shown that SCAMP3 can alter Influenza Virus Hemagglutinin trafficking, thus resulting in reduced levels of HA on nascent virions, as well as ablated cleavage of HPAIV HA.

Hemagglutinin is critically important to influenza infection. This protein is responsible not only for binding to host cell receptors but also for mediating fusion between the virion and host cell membranes. The latter of these phenomenon is dependent on the HA being cleaved, either by extracellular, trypsin-like proteases in the case of low pathogenicity influenza viruses or by intracellular Furin in the case of highly pathogenic influenza viruses. Our research shows that high levels of intracellular

SCAMP3 correlate with a decrease in HPAIV Hemagglutinin cleavage. Since Furin cleavage of HPAIV HA occurs predominantly in the trans Golgi network, we can assume that this is due to incorrect trafficking of HA, as SCAMP3 has been previously shown to be able to alter trafficking of membrane bound vesicles. Another possibility is that

106 SCAMP3 alters Furin function or localization, however we don’t believe that this is likely, as Furin is critical for the processing of many cellular factors and cells that stably express high levels of SCAMP3 don’t appear to have any kind of growth dysfunction.

We have shown that in SCAMP3 hyperexpressing cells, pseudotyped HPAIV’s containing H5 or H7 HA’s are strongly restricted. This restriction is due to the HA not being cleaved in the presence of high levels of SCAMP3, thus nascent virions are unable to fuse in the late endosome and simply undergo lysosomal degradation. Furthermore we have shown that SCAMP3 also reduces intracellular and virion associated HA levels during infection with replicating influenza. Overexpressing SCAMP3 can reduce HA levels and knocking down SCAMP3 via siRNA results in increased levels of intracellular

HA. Furthermore we have shown that during viral infection, SCAMP3 becomes phosphorylated and the subcellular distribution of SCAMP3 changes. Previous research has shown that SCAMP3 phosphorylation results in alteration of the subcellular distribution of the protein, and seeing as how we observed both SCAMP3 phosphorylation and re-localization during IAV infection we can infer that this plays some role in its ability to restrict viruses. Furthermore, we show that SCAMP3 expression can be increased in a dose dependent manner upon treatment with TNFα.

Therefore, we suggest a model where upon either infection with Influenza Virus or stimulation with TNFα, SCAMP3 is upregulated, phosphorylated and re-localizes in order to alter trafficking of viral hemagglutinin and ablate the infectivity of daughter virions.

107 The question remains which of these phenomena is more important to SCAMP3 mediated viral restriction. Influenza infection begins when cells of the upper airway epithelium become infected. Once the virus enters the cell, viral PAMP’s are sensed by intracellular pathogen recognition receptors, which induce expression of type 1 interferons. Based on our findings, at this point SCAMP3 relocalization can begin, thus allowing it to be able to alter HA trafficking as the protein is being produced. We were however only able to test this in low-pathogenicity strains as we didn’t have access to a

BSL-3 select agent facility at the time of this writing.

In our research we have shown that high SCAMP3 expression is sufficient to prevent viral entry of HPAIV strains produced in SCAMP3 hyperexpressing cells.

However, in our work with replicating influenza, we never observed SCAMP3 upregulation as a consequence of influenza infection. Also at the time of this writing there has been no literature published describing a condition in which SCAMP3 expression is upregulated. We did however observe that TNFα treatment caused a dose dependent upregulation of SCAMP3 in A549 cells. This is interesting as in vivo studies have demonstrated that TNFα is mainly produced by macrophages, which have either been infected with a pathogen or stimulated by cells secreting interferon and other cytokines. This provides and intriguing scenario where there are multiple factors that control SCAMP3’s antiviral activity: the intracellular pathogen activating innate immunity and causing re-distribution of SCAMP3 and extracellular factors being secreted by other cells which cause SCAMP3 expression to increase. Further research will need to

108 be done to examine how cytokine signaling as well as innate immunity functions in regulating SCAMP3’s antiviral activity.

At the time of this writing there are only two drugs that are approved to treat influenza infection: Ion Channel Inhibitors and Neuraminidase Inhibitors. Widespread resistance among circulating viruses has rendered Ion Channel Inhibitors useless in most cases of the flu and they are no longer used to treat the disease. Since these viruses pose such a significant health threat, it is imperative that new therapies be discovered and implemented as soon as possible. SCAMP3 presents an interesting therapeutic target. It is constitutively expressed therefore if a drug could be designed to selectively activate

SCAMP3’s antiviral activity it would be able to provide efficient restriction of viral replication in infected individuals, or efficient prophylaxis for uninfected individuals.

Such a drug would be invaluable in treating high risk influenza patients and it would also provide a much needed therapy for a potenitial HPAIV pandemic.

3.4 Materials and Methods

Cells, Chemicals, and Reagents

Human embryonic kidney 293T, and Human Osteosarcoma (Ghost) cells were maintained in Dulbecco's modified eagle medium (DMEM; Corning). Human lung epithelial A549 were grown in Roswell Park Memorial Institute (RPMI) 1640 medium.

All media were supplemented with 10% fetal bovine serum (FBS; Corning), 100 U/ ml penicillin, and 100 μg/ml streptomycin (Corning). A549 cells transduced with an empty vector or to express SCAMP3 were selected with 3 µg/ml puromycin (ThermoFisher) and

109 400 µg/ml gentamicin (ThermoFisher). HeLa and K562 cells expressing scrambled shRNA or shRNA targeting SCAMP3 were selected with 4 µg/ml puromycin

(ThermoFisher). Human IFN-β 1A was obtained from R&D Systems, while LPS, IL1-β and TNFα were obtained from Proteintech.

Plasmids and constructs

DNA fragments encoding native N-terminal hemagglutinin (HA)-tagged SCAMP3, were cloned into the pQCXIP vector (Clontech) using NotI and BspeI cutting sites.

SCAMP3 mutants (ΔPY, and ΔPSAP) were generated using the QuikChange method

(Agilent Technologies). To generate plasmids encoding tet-inducible SCAMP3, open- reading frames of IFITM3 variants were introduced into the pRetroX-Tight-Pur vector

(Clontech) using restriction enzyme cutting sites, BamHI and EcoRI. Retroviral pRS vector-based shRNA plasmids (the targeting sequence for scrambled shRNA is

GCACTACCAGAGCTAACTCAGATAGTACT and the targeting sequence for

SCAMP3 shRNA are CAGCTACTCGACAGAACAATT [SCAMP3-1 shRNA] and

CGGAGTGACAGTTCATTCAAT [SCAMP3-2 shRNA]) were obtained from GE

Healthcare Life Sciences.

Pseudotyped virus transduction and infection assays

Plasmids and procedures used to generate pseudotyped MLV-GFP and transducing viruses have previously been described (Li et al., 2003; Huang et al., 2008; Huang et al.,

2011). Viral entry glycoproteins used to generate MLV-GFP included IAV HA proteins

110 from A/PR/8/34 (H1N1) (H1[PR]), A/Udorn/72 (H3N2) (H3[Ud]), A/Thailand/2(SP-

33)/2004(H5N1) (H5[Thai]), and A/FPV/Rostock/34 (H7N1) (H7[FPV]), the env protein from amphotropic MLV, and the glycoprotein (GPC) from Machupo virus (MACV,

Carvallo strain) (Li et al., 2003; Kuhn et al., 2006; Radoshitzky et al., 2007; Huang et al.,

2008). HIV Pseudotyped viruses were generated using the PNL4.3ΔEnv HIV backbone obtained from the NIH AIDS reagent program. Viral entry glycoproteins used to generate transducing viruses included the G protein from vesicular stomatitis virus (VSV, Indiana strain), GPC proteins from MACV or lymphocytic choriomeningitis virus (Radoshitzky et al., 2007). The transduction assay and the pseudotyped virus entry assay were performed following procedures described in previous publications (Huang et al., 2008;

Williams et al., 2014).

Immunoblotting

For protein analysis, cells were lysed with 2% Triton X-100 (Sigma-Aldrich) or 2% lubrol (MP Biomedicals). Samples for western blotting were prepared in the reducing buffer, heated at 75 °C for 10 minutes, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), and transferred to the polyvinylidene difluoride (PVDF) or nitrocellulose membrane (ThermoFisher). The expression of HA-

SCAMP3 was recognized by a Horseradish peroxidase (HRP)-conjugated anti-HA antibody (Clone 3F10; Roche Life Science). The expression of endogenous SCAMP3 was detected by a rabbit anti-SCAMP3 (0.5 μg/mL, N1N3; GeneTex [Cat. GTX102216])

111 or by a sheep anti-SCAMP3 (1 μg/mL; R&D Systems [Cat. AF5344]) antibody. H5HA was detected using a goat anti H5HA (1 μg/mL; Santa Cruz Biotech [Cat. sc-54958])

or a Mouse anti H5HA antibody (1 μg/mL; BEI Resources [Cat. NR2729]), H7HA was detected using a goat anti H7HA antiserum (2 μg/mL; BEI Resources [Cat. V304-501-

157]) , H1HA was recognized by a goat anti H1HA antibody(BEI Resources [Cat. V301-

511-552]), H3HA was recognized by a goat anti H3HA antibody (BEI Resources [Cat.

34586]), β-tubulin recognized by 1 µg/ml murine monoclonal anti-β-tubulin antibody

(clone SAP.4G5, Sigma-Aldrich [Cat. T-7816]) was used as loading controls. Secondary antibodies used for western blotting included HRP-conjugated rabbit anti-goat IgG

(1:20,000 dilution; Sigma-Aldrich [Cat. A-5420]), HRP-conjugated donkey anti-sheep

IgG (1:20,000 dilution; R&D Systems [Cat. HAF016]), HRP-conjugated goat anti-mouse

IgG (1:10,000 dilution; Santa Cruz Biotechnology [Cat. sc-2005]), and HRP-conjugated mouse anti-rabbit IgG (1:5,000 dilution; Santa Cruz Biotechnology [Cat. sc-2357]) antibodies.

Phos-Tag Electrophoresis

Phos tag gels were cast using established protocols then supplemented with 10μg

Phos-tag reagent (Waco Industries JP [Cat. AAL-107]) and 100μL of 10mM MnCl2 solution. Gels were allowed to solidify and were used immediately. Electrophoresis was performed at 150V for 1 hour. Gels were then removed from cassettes, incubated with transfer buffer for 30 minutes at room temperature, before being transferred to a wet- blotting tank. Blotting was performed overnight in a 4°C cold room at a constant

112 amperage of 150mA under constant stirring from a stir plate. Proteins were transferred to a PVDF membrane. The next day the gels were removed from the tank and the tank and protein detection was completed in the manner described earlier.

Confocal microscopy

Subcellular localizations of SCAMP3 were analyzed by confocal microscopy. Cells in chamber slides were washed twice with phosphate buffered saline (PBS) and fixed with

4% formaldehyde (Polysciences) at 25 °C for 20 minutes. Fixed cells were then washed with PBS twice, permeabilized with 0.2% Triton X-100 (Sigma) for 10 min. Slides were than blocked in 3% bovine serum albumin (BSA; Sigma) at 25 °C for 1 hour, and labeled with primary antibodies overnight at 4 °C. Primary antibodies used in our research included sheep anti-SCAMP3 (1:500 dilution; R&D Systems [Cat. AF5344]), goat anti-

Golgi matrix protein GM130 (1:50 dilution; Santa Cruz Biotechnology [Cat. sc-16268]), goat anti-trans-Golgi network integral membrane protein TGN38 (1:50 dilution; Santa

Cruz Biotechnology [Cat. sc-27680]), and murine Alexa Fluor 488-conjugated anti- lysosomal-associated membrane protein 2 (LAMP2; 1:50 dilution, Santa Cruz

Biotechnology [Cat. sc-18822]) antibodies. After primary antibody staining, cells were washed three time with PBS and labeled with fluorophore-conjugated secondary antibodies for 1 hour at 25°C. Antibodies used included Alexa Fluor 555-conjugated donkey anti-sheep IgG (1:2000 dilution; ThermoFisher [Cat. A21436]), Alexa Fluor 647- conjugated chicken anti-rabbit IgG (1:2000 dilution; ThermoFisher [Cat. A21443]),

113 Alexa Fluor 488-conjugated donkey anti-goat IgG (1:2000 dilution; ThermoFisher [Cat.

A11055]), and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:2000 dilution;

ThermoFisher [Cat. A10684]) antibodies. Cells were then mounted in prolong gold anti- fade reagent with 4',6-diamidino-2-phenylindole (DAPI, ThermoFisher). Finally, cells were imaged using a Zeiss LSM 510 confocal microscopy system (objective 63X).

Statistical analyses

Values are presented as relative infectivity or fluorescence intensity. Errors bars denote one s.d. (n=3). Every experiments were performed at least twice or three times. Sample numbers were determined based on the preliminary studies. We used a paired two-tailed

Student's t-test for statistical analyses, and a P <0.05 was considered significant.

114 3.5 Figures

115 Figure 1: SCAMP3 Expression in IAV Pseudotype virus producing cells results in decreased viral entry due to a lack of Hemagglutinin Cleavage. (A) Ghost cells were infected Pseudotype Influenza Viruses containing the hemagglutinin protein from either H5N1 (A/Thailand/1(KAN-1)/2004) or H7N1 (A/FPV/Rostock/34) and the neuraminidase gene from H1N1 (A/PR/8/1934) were produced using the PNL4.3ΔEnv HIV backbone. These viruses were produced in 293T SCAMP3 Tet on cells with or without doxycline stimulation. Cells were analyzed by flow cytometry 48 hours after infection. (B) Pseudotyped viruses were used to infect either normal Ghost cells or Ghost cells stably expressing SCAMP3. (C) Cells from (A) were lysed and analyzed via western blotting for virion associated P24, as well as intracellular and virion associated Hemagglutinin. (D) HEK 293T cells were transfected with H5 and H7HA, then co- transfected with either SCAMP3 or an empty vector. Cells were lysed 48 hours after transfection and analyzed by western blotting.

116

117 Figure 2: SCAMP3 Alters intracellular and virion-associated hemagglutinin levels: (A) Stably SCAMP3 expressing A549 cells were infected with Influenza A/England/42/72 (H3N2) at an MOI of 1. 48 hours later the cells were lysed and analyzed by western blot. (B) A549 cells expressing shRNA against luciferase or SCAMP3 were infected with Influenza A/PR 8/34 (H1N1) and A/England/42/72 (H3N2). 48 hours later the cells were lysed and analyzed by western blot. (C) The supernatant from the cells in (B) was harvested and centrifuged at 21000g for 2 hours to pellet free virions. The virions were lysed in 2% lubrol with PBS and then resuspended in reducing buffer and analyzed by SDS-PAGE.

118

119 Figure 3: SCAMP3 and Innate Immunity. (A) A549 cells were incubated either with normal media, or media containing A/England/42/72 (H3N2) Influenza. Cells were also treated with Interferon at the indicated concentration for the indicated amount of time. 48hours later these cells were fixed, permiabilized, stained and mounted for confocal microscopy. (B) 293T cells were infected with the indicated strains of Influenza. 48hours later there cells were lysed and the lysate was used for SDS PAGE in a gel containing phos-tag acrylamide. The gel was then transferred to a PVDF membrane and SCAMP3 was analyzed by imminoblotting. (C) NFkB sites upstream of SCAMP3 in the . (D) A549 Cells were treated with either LPS or the indicated cytokines for 48 hours before being lysed and analyzed by SDS PAGE and immunoblotting.

120 3.5 References

1.Fields, B., Knipe, D. & Howley, P. Fields virology. (Wolters Kluwer Health/Lippincott Williams & Wilkins, 2007).

2.Taubenberger, J. & Morens, D. The Pathology of Influenza Virus Infections. Annu. Rev. Pathol. Mech. Dis. 3, 499-522 (2008).

3.Shi, Y., Wu, Y., Zhang, W., Qi, J. & Gao, G. Enabling the 'host jump': structural determinants of receptor-binding specificity in influenza A viruses. Nature Reviews Microbiology 12, 822-831 (2014).

4.Horimoto, T. & Kawaoka, Y. Influenza: lessons from past pandemics, warnings from current incidents. Nature Reviews Microbiology 3, 591-600 (2005).

5.Cdc.gov,. Seasonal Influenza-Associated Hospitalizations in the United States| Seasonal Influenza (Flu) | CDC. (2015). at http://www.cdc.gov/flu/about/qa/hospital.htm

6.Steinhauer, DA. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 25, 1-20 (1999).

7.Schoch, C & Blumenthal, R. Role of the Fusion Peptide Sequence in Initial Stages of Influenza Hemagglutinin-induced Cell Fusion. Journal of Biological Chemistry 13, 9267- 9274 (1992).

8.Schrauwen, E. & Fouchier, R. Host adaptation and transmission of influenza A viruses in mammals. Emerg Microbes Infect 3, e9 (2014).

9.Kawaoka, Y. & Webster, R. Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. Proceedings of the National Academy of Sciences 85, 324-328 (1988).

10.Horimoto, T. & Kawaoka, Y. Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. Virology 68, 3120-3128 (1994)

11.Iwasaki, A. & Pillai, P. Innate immunity to influenza virus infection. Nature Reviews Immunology 14, 315-328 (2014).

12.Le Goffic, R. et al. Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells. The Journal of Immunology 178, 3368-3372 (2007).

121 13.Diebold, S. Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA. Science 303, 1529-1531 (2004).

14.Shaw M. The host interactome of influenza virus presents new potential targets for antiviral drugs. Rev Medical Virology. 21, 358-369 (2011).

15.Hubbard, C. et al. The secretory carrier membrane protein family: structure and membrane topology. Molecular Biology of the Cell. 11:2933-2947 (2000).

16.Castle, A. and Castle, D. Ubiquitously expressed secretory carrier membrane proteins (SCAMPs) 1-4 mark different pathways and exhibit limited constitutive trafficking to and from the cell surface. J Cell Sci 115(3769-3780) 2005

17. Mota LJ., Ramsden AE., Liu M., Castle JD. & Holden DW. SCAMP3 is a component of the Salmonella-induced tubular network and reveals an interaction between bacterial effectors and post-Golgi trafficking. Cell Microbiology 8:1236-1253 (2009)

18.Aoh, Q., Castle, A., Hubbard, C., Katsumata, O. & Castle, J. SCAMP3 Negatively Regulates Epidermal Growth Factor Receptor Degradation and Promotes Receptor Recycling. Molecular Biology of the Cell 20, 1816-1832 (2009).

19. Wu, TT. & Castle, JD. Tyrosine phosphorylation of selected secretory carrier membrane proteins, SCAMP1 and SCAMP3, and association with the EGF receptor. Molecular Biology of the Cell 9, 1661-1674 (1998).

20. Martin-Serrano, J. Zhang, T. & Bieniasz, PD. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nature Medicine 12, 1313-1319 (2001)

21. Saynal, S. et al. Type I interferon imposes a TSG101/ISG15 checkpoint at the Golgi for glycoprotein trafficking during influenza virus infection. Cell Host Microbe 14, 510- 521 (2013)

22. Dolnik, O. et al. Interaction with Tsg101 Is Necessary for the Efficient Transport and Release of Nucleocapsids in Marburg Virus-Infected Cells. PLoS Pathogens 10 e1004463 (2014)

23. Huang, I. et al. Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus. PLoS Pathogens 7, e1001258 (2011).

24. Brass, A. et al. The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus. Cell 139, 1243-1254 (2009).

122 25. Moscona, A. Neuraminidase Inhibitors for Influenza. New England Journal of Medicine 353, 1363-1373 (2005).

123 CHAPTER 4 DISCUSSION

124 Influenza viruses are a significant threat to global health. The World Health

Organization estimates that each year Influenza kills between 250,000 and 500,000 people worldwide. The 1918 Spanish Flu pandemic was likely the worst natural disaster in human history, claiming between 50 and 100 million lives in the span of a few years.

Furthermore, with the emergence of Highly Pathogenic Influenza Viruses in humans, the threat caused by Influenza continues to be prevalent in modern society. Innate immunity to this disease constitutes a critical barrier against infection, and research has shown that disrupting innate immunity greatly increases susceptibility to this Influenza Viruses. In this dissertation, we have expanded on the knowledge of the regulation of IFITM family proteins and we have also demonstrated a new role for SCAMP3 as an innate immune factor. This knowledge broadens our understanding of the innate immune response to influenza viruses and provides new targets for prophylaxis and treatment of influenza infections.

We have previously shown that the IFITM family of proteins can potently restrict influenza viruses. However, the exact mechanism of how IFITM’s function in viral restriction, as well as the underlying mechanisms of IFITM maintenance and turnover in the cell are largely unknown. In this dissertation, we described the interaction of

SCAMP3 and IFITM3. We showed that these proteins interact in several different cell lines and can co-precipitate together. Furthermore, we show that these proteins localize to similar subcellular compartments. Initially, we believed that SCAMP3 may regulate

IFITM activity, however this hypothesis was wrong, as IFITM3 function did not appear to be affected by knocking down SCAMP3. We did however observe that knocking down

125 SCAMP3 appears to decrease intracellular IFITM levels. We postulated that this could be due to SCAMP3 preventing IFITM turnover. This appeared to be the case as when we knocked down SCAMP3 we observed lower levels of IFITM3 in hyperexpressing cells, interferon stimulated cells and cells that natively express high levels of IFITM3. We also observed that this down regulation of IFITM3 was ablated when we treated cells with a lysosome inhibitor. Since it appeared that SCAMP3 appeared to prevent lysosomal degradation of IFITM3, the next step was to try to clarify the mechanism by which this happens. Past studies have shown that IFITM3 ubquitination contributes to its stability.

When we mutated the PY motif in SCAMP3 (Which allows it to interact with NEDD4, a ubiquitin ligase) we found that SCAMP3’s ability to stabilize IFITM3 was reduced. We therefore decided to investigate whether SCAMP3 can contribute to IFITM3 ubiquitination. Upon analyzing IFITM3 ubiquitination site mutants as well as studying

IFITM ubiquitination patterns in the presence and absence of SCAMP3 we came to the conclusion that SCAMP3 does not have any effect on IFITM3 ubquitination. Since

SCAMP3’s ability to stabilize IFITM did not appear to involve ubiquitination, we assayed whether the subcellular localization of IFITM3 changed in the presence or absence of SCAMP3. Interestingly, we found that there was a marked decrease in cis- golgi and trans-golgi associated IFITM3 in cells which had been depleted of SCAMP3.

This suggested that instead decreasing IFITM3 turnover by modulating ubiquitination,

SCAMP3 could instead affect IFITM3 recycling. To further investigate this, we used two model molecules, Mannose-6-phosphatase receptor (M6PR) and the B subunit of Cholera

Toxin(CTX-B). Both of these proteins undergo recycling from the plasma membrane via

126 retrograde transport. M6PR undergoes recycling through the late endosome while CXTB recycles to the golgi through the early endosome. We found that there was no difference in M6PR distribution between scrambled shRNA and SCAMP3 shRNA expressing cells, however when we treated cells with cholera toxin we found that CTXB was enriched on the plasma membrane in SCAMP3 knockdown cells. When we assayed surface CTXB levels we found that they were increased in SCAMP3 knockdown cells, even though the total CTXB amount remained constant. These observations were consistent when we assayed IFITM3, allowing us to come to the conclusion that SCAMP3 facilitates recycling of IFITM3 between the cell surface and the Golgi network via early endosomes. Our findings are novel as SCAMP3 has only been investigated with regard to recycling of the Epidermal Growth Factor Receptor, and it has never been implicated in contributing to innate immunity. Furthermore, unlike previous studies, which have shown that the PSAP motif of SCAMP3, which allows it to interact with ESCRT complex proteins was the most important factor in allowing SCAMP3 to alter protein metabolism, we have demonstrated that the PY motif is more important in the case of IFITM3.

Furthermore, our research broadens the understanding of how IFITM proteins are maintained in the cell. Further research on this subject is warranted, but this provides an attractive therapeutic target against a wide range of viruses. If a drug could be designed to selectively activate SCAMP3-mediated IFITM recycling, the IFITM proteins would be maintained in the cell longer, likely leading to a decrease in viral entry into affected cells.

Once we established that SCAMP3 could interact with and stabilize IFITM3, we decided to investigate whether SCAMP3 itself has any antiviral effect. SCAMP3 contains

127 a PSAP motif, which has been shown to allow it to interact with TSG101. Previous research has shown that TSG101 plays a role in innate immunity against influenza.

TSG101 is necessary for trafficking of Hemagglutinin to the cell surface in viral infected cells, via its interaction with other ESCRT proteins. Upon interferon stimulation, TSG101 is conjugated to ISG15, a ubiquitin like interferon effector. This ISG-ylation event ablates

TSG101-mediated trafficking of Hemagglutinin from the trans golgi network to the cell surface. Since the regulatory mechanisms behind TSG101 mediated influenza restriction are not well understood we decided to investigate if SCAMP3 had any role in this phenomenon.

The first step in investigating SCAMP3 as an innate immune factor was to determine if it could restrict the entry of various strains of influenza viruses. Since we didn’t have access to a select agent BSL-3 facility, we decided to first test SCAMP3’s activity against HPAIV using the pseudotype viral system in a BSL-2 environment. We initially attempted to infect SCAMP3 hyperexpressing cells with virions produced in

HEK-293T cells and found that there was no difference in infectivity between cells expressing large amounts of SCAMP3 compared to those expressing an empty vector.

However, when we generated virus in SCAMP3 hyperexpressing cells, we found that it was defective in viral entry compared to cells expressing only endogenous SCAMP3.

This was despite there being similar levels of virus produced in both of these cell types.

This suggested that SCAMP3 was able to restrict viral entry, most likely during some step of viral assembly. Since the only influenza proteins expressed in the pseudotype system were Hemagglutinin and Neuraminidase, this allowed us to further focus on the

128 assembly steps of these two proteins. Strikingly, when we performed immunoblotting on these cells and the resulting virus, we found that HPAIV cleavage in SCAMP3 hyperexpressing cells was almost completely ablated. We were able to confirm this finding by coexpressing SCAMP3 and HA from two HPAIV strains: A/Thailand/2(SP-

33)/2004(H5N1) (H5[Thai]), and A/FPV/Rostock/34 (H7N1) (H7[FPV]). Influenza infection is dependent on HA being cleaved to expose the fusion peptide and allow the virus to fuse with the host cell in the late endosome, facilitating release of the RNP complex into the cytosol. HPAIV HA is cleaved in the trans-golgi network by furin, s ubiquitously expressed protease. Since SCAMP3 hyperexpression was preventing HA cleavage we postulated that it accomplishes this either by altering trafficking of HA so it bypasses the trans-golgi network or by altering Furin activity. Furin activity is critical to cell survival, as it is responsible for processing growth factors and other proteins necessary for cell survival and proliferation. As we were able to generate A549 cells that expressed high levels of SCAMP3 and didn’t display and defects in growth or morphology, we were mostly able to rule out Furin inhibition as a mechanism of

SCAMP3 action.

We next focused on whether SCAMP3 can affect HA on replicating viruses. As we didn’t have access to a BSL-3 Select Agent facility, we used two low-pathogenicity strains (A/PR/8/34 (H1N1) (H1[PR]), and A/Udorn/72 (H3N2) (H3[Ud]) to test this. We found that in SCAMP3 hyperexpressing cells, there was less intracellular HA than in control cells. Next, when we knocked down SCAMP3, we saw the opposite phenotype.

When SCAMP3 knockdown cells were infected with influenza. In these cells intracellular

129 and virion-associated HA levels were increased compared to control cells. In conjunction with our previous observations that HPAIV HA is not cleaved in SCAMP3 overexpressing cells suggests that in the presence of SCAMP3, Hemagglutinin trafficking is altered and instead of trafficking through the trans-golgi network, it is trafficked via another route where it is ultimately degraded. Therefore we can assume that SCAMP3 plays some role in viral restriction, however we did not have evidence at this point that this function was tied to the innate immune response.

Previous studies have shown that SCAMP3 contains several post translational modifications, including phosphorylation and ubiquitination sites. Studies have shown that SCAMP3 phosphorylation alters its subcellular distribution, and others have suggested that SCAMP3’s ubiquitination state can alter its function. In order to determine if SCAMP3’s cellular function could be affected by the innate immune response, we first decided to observe how its distribution changes during influenza infection or interferon treatment. Using confocal microscopy, we observed that SCAMP3’s distribution is altered during viral infection, from a punctate, cytoplasmic distribution to a much more condensed perinuclear distribution. We also found that during influenza infection,

SCAMP3 becomes phosphorylated, as evidenced by different banding patterns that appeared when we ran lysates from non-infected and infected cells on a Phos-Tag gel.

Previous studies have shown that SCAMP3’s phosphorylation state alters its intracellular distribution, but we are the first to show that SCAMP3 phosphorylation can be altered as a response to viral infection. While these are intriguing observations and suggest that

SCAMP3 function may be altered by the innate immune response, our previous results

130 suggested that expressing high levels of SCAMP3 is necessary for its ability to restrict progeny virions. We therefor decided to investigate whether SCAMP3 can be upregulated during the innate immune response.

We first decided to search a database of regulatory elements (Motifmap) to see if there were any innate immune transcription factor binding sites proximal to the SCAMP3 sequence in the human genome. We found that there were several putative NFkB binding sites both upstream and downstream of the SCAMP3 gene, which was seemingly never mentioned in any SCAMP3 related literature. We then tested several compounds which have been shown to activate NFkB: Lipopolysaccharide (LPS) Interleukin 1 beta (IL1-β) and Tumor Necrosis Factor Alpha (TNFα). When treated A549 cells with each of these proteins and assayed SCAMP3 levels, we found that LPS and IL1-β had no noticeable effect on intracellular SCAMP3 levels, however when we treated cells with TNFα , we found that there was a dose-dependent increase in intracellular SCAMP3 expression.

Since our previous results showed that infectivity of progeny virus is ablated during

SCAMP3 hyper expression. We can therefore hypothesize that during the innate immune response to Influenza infection, TNFα induces SCAMP3 expression, which prevents infected cells from producing viable virions, restricting viral replication. In vivo studies have shown that the main source of TNFα is activated macrophages. This suggests an interesting dynamic between the innate immune response and SCAMP3 activity. We propose a multi-step model for SCAMP3 activation and antiviral activity. Intracellular

SCAMP3 is activated via Interferon induction, either by viral infection itself or through paracrine Interferon signaling from nearby infected cells. SCAMP3 is phosphorylated

131 during the innate immune response and relocalizes, this corresponds to a switch in

SCAMP3 function, from its normal activity to altering the trafficking of newly synthesized HA proteins, preventing them from trafficking through the trans-golgi network and ultimately leading to their degradation. Concurrently, macrophages in the infected tissue are activated either by influenza infection or chemokine/cytokine signaling from infected cells. These macrophages begin secreting TNFα, which causes cells in the infected tissue to upregulate SCAMP3, increasing viral restriction. More research is necessary to determine the exact timing of these events, as well as which is more critical to SCAMP3-mediated viral restriction, however we believe that we have enough evidence to suggest that SCAMP3 is an antiviral effector capable of restricting a broad range of Influenza viruses.

These two studies seemingly show that SCAMP3 has opposite function. In the case of IFITM3, SCAMP3 contributes to recycling of the protein from the early endosome to the golgi. In the case of Hemagglutinin, SCAMP3 appears to alter its normal trafficking through the golgi to the plasma membrane and instead facilitates aberrant trafficking of this protein and ultimately leads to its degradation. While these are seemingly contradictory, the literature provides a possible explanation for how this is possible. In 2009, Aoh et al showed that SCAMP3 expression correlated with increased

EGFR recycling to the cell surface. When they knocked down SCAMP3, they found that the rate of EGFR turnover was vastly increased a upon EGF treatment compared to their control cells. Two years later, Falguières et al reported the opposite phenotype. In their study, SCAMP3 appeared to increase EGFR degradation, and knocking down the protein

132 correlated with decreased EGFR turnover and higher levels of EGFR recycling compared to control cells. They didn’t investigate this difference further but mentioned that the differences in results were due to different experimental conditions. They also stated that the conditions utilized in the Wu paper, specifically their method of culturing HeLa cells promoted SCAMP3 ubiquitination. They went on to postulate that the discrepancy in their results was due to SCAMP3 function changing as a result of its ubiquitination. This provides a potential explanation to the seemingly contradictory data that we have observed. The model we propose is as follows. At the early stage of influenza infection, the first infected cells begin producing interferon. This interferon signals to nearby cells, which begin producing IFITM and in these non-infected cells SCAMP3 is modified such that it promotes the recycling of IFITM. This renders non-infected cells in this tissue refractory to viral infection. Once a cell is infected, however, IFITM proteins are no longer useful to restricting viral replication. Therefore, we propose that viral infection and subsequent sensing innate immune sensing of the virus and signaling by innate immune factors induces a switch in SCAMP3 activity, which likely involves differential phosphorylation or ubiquitination by Interferon Stimulated Genes. This change in post- translational modifications of SCAMP3 alters its function. Instead of recycling

IFITMs(and likely other proteins) from the early endosome to the golgi, SCAMP3 then begins to alter trafficking of Hemagglutinin and promotes its degradation. Previous studies have suggested that post translational modifications of SCAMP3 can alter its subcellular distribution and likely its function. Furthermore, there have been studies published that implicate SCAMP3 in Salmonella pathogenesis, and present data showing

133 that SCAMP3 re-localizes upon Salmonella infection. While these studies have provided a putative role for SCAMP3 in the life cycle of intracellular pathogens, we are the first group to provide direct evidence that SCAMP3 itself is an innate immune factor.

Further research is warranted to fully examine SCAMP3’s role in the innate immune system. While we showed that in a pseudotype system SCAMP3 was able to potently restrict HPAIV entry, we have yet to assay it’s ability to restrict live HPAIV.

Furthermore, it would be prudent to investigate whether SCAMP3 can restrict other viruses that require Furin mediated activation of their entry glycoproteins. Further studies also need to be done into the exact molecular mechanism of SCAMP3 activation and function. SCAMP3 provides an attractive therapeutic target that can potentially be used for treatment and prophylaxis of various viral pathogens. If a drug could be designed to selectively activate SCAMP3 it would provide a critical tool in limiting viral infection.

Out work has broadened the understanding of the innate immune response and paves the way to a new therapeutic target for protecting against influenza viruses.

134