Cellular and Viral Determinants of Hsv-1 Entry and Transport

CELLULAR AND VIRAL DETERMINANTS OF HSV-1 ENTRY AND TRANSPORT

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Pathobiological Sciences

by Farhana Musarrat M.S. University of Dhaka, Bangladesh, 2010 December 2019 This dissertation is dedicated to my late father Qazi Muzammil Haque and my mother Hasna Haq.

ACKNOWLEDGEMENTS

I came to the United States of America to fulfill my dream of pursuing a career in the

field of Molecular Biology. From the very beginning of my undergraduate studies in

Microbiology at the University of Dhaka, Bangladesh, I knew that I wanted to become a scientist. Growing up in an educated family has always inspired me to learn and explore new things. My late father, who was a great scholar and my eldest brother, were my idols. After the completion of my M.S., I was appointed as a faculty member at the BRAC University for teaching undergraduates. I have spent the best days of my life at that time. I felt independent, free and loved. I thank all my mentors and colleagues at the University of Dhaka and BRAC

University. Especially I was highly inspired by the philosophy of Dr. Naiyyum Chowdhury, Dr.

Zia Uddin, Dr. Sirajul Islam Khan, Dr. Donald James Gomez, Dr. Mozammel Hoq, Dr. Humaira

Akhter and Dr. Chowdhury Rafiqul Ahsan.

My journey as a graduate student in the USA started with Dr. Ya-Ping Tang and Dr. Donna

Neumann in the Louisiana State University, Health Sciences Center at New Orleans. I am

grateful for the training that I received there.

As I joined Dr. Konstantin Gus Kousoulas’s laboratory at Louisiana State University, I began to explore the vast field of herpes virology with an expert group of scientists. My mentor

provided me with the courage to think creatively in the light of science. I highly appreciate his

patience, constant encouragement and valuable critics in my research. The advanced research

facilities and a friendly work environment made my journey a great one in the Kousoulas lab. I

want to express my deep gratitude to my mentor Dr. Kousoulas and all the lab members for

being supportive.

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I want to thank all my graduate committee members, Dr. Rhonda Cardin, Dr. Shafiqul

Chowdhury, Dr. Masami Yushimura and Dr. Jeremy Brown for their time, patience and constant guidance. I want to thank all the faculty members and staff in the Department of Pathobiological

Sciences for their support.

Very special gratitude goes to the love of my life and my best friend, Dr. Rafiq Un Nabi for moral and emotional support throughout this journey. Last but not least, I am grateful to my family for always being there for me.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………………………………iii

ABSTRACT…………………………………………………………………………………vii

CHAPTER 1. INTRODUCTION……………………………………………... 1 1.1 Introduction to herpesviruses………………………………….. 1 1.2 Life cycle of herpesviruses……………………………………. 3 1.3 Intracellular transport………………………………………….. 7 1.4 Regulation of intracellular transport…………………………... 12 1.5 Diseases due to intracellular transport defects………………… 19 1.6 Virus and cellular transport……………………………………. 21 1.7 Conclusions……………………………………………………. 25 1.8 References……………………………………………………... 25

CHAPTER 2. THE AMINO-TERMINUS OF HSV-1 GLYCOPROTEIN K (GK) IS REQUIRED FOR GB BINDING TO AKT, RELEASE OF INTRACELLULAR CALCIUM AND FUSION OF THE VIRAL ENVELOPE WITH PLASMA MEMBRANES 2.1 Introduction……………………………………………………. 48 2.2 Methods and materials………………………………………… 51 2.3 Results………………………………………………………… 56 2.4 Discussion……………………………………………………... 70 2.5 References……………………………………………………... 74

CHAPTER 3. CELLULAR AND VIRAL DETERMINANTS OF HSV-1 TRANSPORT TOWARD NUCLEUS 3.1 Introduction……………………………………………………. 84 3.2 Methods and materials………………………………………… 88 3.3 Results…………………………………………………………. 92 3.4 Discussion……………………………………………………... 108 3.5 References……………………………………………………... 111

CHAPTER 4. CONCLUDING REMARKS 4.1 Introduction …………………………………………………… 119 4.2 Summary of the results ………………………………………... 119 4.3 Future directions ………………………………………………. 120

APPENDIX A. ADDITIONAL WORK A.1 The role of UL37 in HSV-1 retrograde transport in primary neurons………………………………………………………… 122 A.2 Effect of antidepressant anti-psychotic drugs on HSV-1 infection……………………………………………………….. 140 A.3 Transcriptome analysis of human neuroblastoma (SK-N-SH)

cell line following HSV-1 infection…………………………… 147

APPENDIX B. PERMISSION TO REPRINT CHAPTER 2 FROM JOURNAL OF VIROLOGY…………………………………. 163

LIST OF REFERENCES …………………………………………………………………. 164

VITA ……………………………………………………………...... 193

ABSTRACT

Previously, it was shown that the deletion of 38 amino acids from the N terminal end of

glycoprotein K (gK) prevents the Herpes Simplex virus Type-1 (HSV-1McKrae ∆gK31-68) from

entering into the axons of neurons. Herein, we showed for the first time that this modification in gK disrupts the ability of the virus to carry out a fusion of the viral envelope with the cellular plasma membrane and forces the virus to enter via endocytosis in epithelial cells and neuronal cell bodies. We showed that HSV-1 McKrae infection triggers cellular calcium signaling, Akt activation and flipping of Akt to the external membrane surface, which facilitates interaction

between Akt and HSV-1McKrae glycoprotein B (gB). However, the gK mutant virus fails to

trigger calcium signaling and Akt-gB interaction and, therefore, utilizes endocytosis as an

alternative to fusion-mediated entry. Following the entry into cells, the HSV-1tegument and

capsid proteins utilize cellular motor machinery to mediate capsid transport to the nucleus where

the virus replicates its genome. Herein, we showed that the inner tegument protein (UL37) of

HSV-1 McKrae interacts with motor protein dynein, and the capsid ICP5 protein interacts with

motor accessory proteins, for example, dynactin and EB1. Hence, knocking out selected motor

accessory proteins reduced the HSV-1 capsid transport towards the nucleus in human neuroblastoma (SK-N-SH) cells. Interestingly, HSV-1 McKrae altered several cellular regulatory pathways during virus entry and capsid transport. For example, MAP kinases (MAPK) and phosphokinase C (PKC) pathways. Although the virus activates MAPK, this activation does not appear to play important roles in virus entry and intracellular capsid transport. In contrast, inhibition of PKC inhibited virus entry indicating that this kinase is involved in virus entry and possibly in intracellular capsid transport. The results obtained in these studies indicate that HSV-

vii

1 regulates downstream neuronal signaling to facilitate virus entry and intracellular retrograde transport toward the nucleus of infected cells.

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CHAPTER 1 INTRODUCTION

1.1 Introduction to herpesviruses

Herpesvirus falls into the order Herpesvirales and family Herpesvirideae. They form a

large family of enveloped, double-stranded DNA viruses. Herpesviruses have a diverse range of

biological niche including, mammals, birds, reptiles, fish, frogs, and bivalves (1). There are three

subfamilies, namely alpha, beta and gamma herpesvirinea (2-4). Molecular phylogenetic analysis

suggests that the Herpesvirivae subfamilies diverged from a common ancestor around 400

million years ago, and evolution over this time has given rise to at least 135 species (1, 5, 6).

Herpes subfamilies differ greatly in their host range and properties of the viral life cycle. Despite

having all the differences, all herpesvirus share similar morphology: a large double-stranded

DNA genome packaged into an icosahedral capsid, which is approximately 125 nm in diameter

with T=16 symmetry and has a single unique (portal) vertex through which DNA enters and

leaves the capsid. The nucleocapsid is encased in a layer of proteins called tegument surrounded

by an envelope composed of a large number of viral glycoproteins embedded in a lipid bilayer

derived from the host cell (Fig. 1.1) (3, 4, 7-11). All herpesvirus infects their hosts for life. They

primarily infect epithelial cells like the mucosal cell in the oropharynx or genitalia, then spread

to secondary cells. Interestingly herpesviruses establish latency in the host cell and reactivate by

environmental cues (3, 4, 12).

There are eight herpesviruses known to infect humans namely herpes simplex virus type

1 (HSV-1) and type 2 (HSV-2) varicella-zoster virus (VZV) (alpha herpesvirus), cytomegalovirus (CMV), human herpesviruses 6 and 7 (HHV-6 and HHV-7) (beta herpesvirus),

Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) (gammaherpesvirus) (3).

Figure 1.1. Structure of herpes virus. The double-stranded DNA genome is enclosed in the icosahedral capsid. The capsid is around 100 nm in size and is surrounded by tegument proteins and glycoprotein envelope.

Alpha herpesviruses are unique in their ability to infect and establish latency in neurons

(13). The first evidence of neurotropism of these viruses came out from the study of the

Pseudorabies virus (PRV) published by Dr. Albert Sabin in 1938. Since then, it is well

established that infection with alpha-herpesviruses in rodents results in the transmission of the

virus from the peripheral nervous system (PNS) to the central nervous system (CNS) and causes fatal encephalitis (13). Primary infection with HSV-1 or HSV-2 cause only mild orofacial or genital lesion respectively but can cause severe disease in neonates or immunocompromised people. Herpesvirus infects the cornea of the eye where the virus replicates and travels through the optic nerve to reach trigeminal ganglia and eventually the brain. Severe HSV-1 infection in the brain leads to encephalitis. Recurrent eye infection due to HSV-1 is a major cause of infectious blindness in the USA. Varicella-zoster virus, another member of the alpha-herpesvirus subfamily, causes chickenpox or shingles (14). Moreover this subfamily contains veterinary viruses with high economic impact for example Bovineherpesviruses (BoHV-1,-5), equine

herpesviruses (EHV-1,-2,-4,-8,-9), gallid herpesviruses (GaHV-1,-2,-3) and pseudorabies virus

(PRV) (2, 15, 16).

1.2 Life cycle of herpesviruses

Upon entry into the host cells, herpesviruses undergo a lytic life cycle using cellular

machinery to replicate its double-stranded DNA. Herpes DNA forms intranuclear episome but

does not integrate into the host genome. Most of the enveloped virus enters the host cell by

fusion of the viral envelope with the cellular plasma membrane. Although the presence of one

fusion protein is enough for most enveloped viruses to facilitate virus entry, herpesviruses use a

complex entry mechanism involving a ‘fusion complex’ consisting of at least three fusion

proteins gB, gH, and gL. These glycoproteins are conserved among the herpesviruses with gB

exhibiting the highest sequence similarity. For some herpesviruses, gB forms a spike consisting

of a homodimer or homotrimer. The heterodimerization of gH/gL is conserved among the

herpesviruses (2, 12, 17, 18). Herpesviruses use different additional accessory activating proteins to trigger gB-mediated membrane fusion. For example, the HSV alphaherpesvirus uses

gD, PRV uses gp50; the CMV betaherpesvirus uses UL128, UL130, and UL131, and the EBV

gammaherpesviruses use gp41 (17). Recently it was shown that HSV-1 requires glycoprotein K

(gK) for fusion mediated entry (19), and gK is essential for the corneal spread and neuroinvasion

(20, 21). The essential fusion protein gB interacts with the amino-terminal of HSV-1 gK and

UL20 physically and functionally (22).

HSV-1 attaches to the cellular membrane by glycoproteins B (gB) and C (gC) with

glycosaminoglycan (GAG) moieties of cell surface proteoglycans (23, 24). Glycoprotein B can

also interact with cellular receptors including paired immunoglobulin-like type 2 receptor alpha

(PILR), non-muscle myosin heavy chain IIA (NMHC-IIA), and myelin-associated glycoprotein

(MAG). Glycoprotein D binds to cellular receptors like herpesvirus entry mediator (HVEM, also

called HveA), nectin-1 (HveC), and 3-O-sulfated heparan sulfate (25-31). Following binding of gD and gB, a conformational change occurs in gH/gL that alters the conformation of gB

triggering membrane fusion (18, 32-36). Virus-induced cell fusion is thought to be mediated by a mechanism very similar to that occurring during fusion of the viral envelope with cellular membranes, inasmuch as, viral glycoproteins gD, gB, gH and gL and the presence of viral receptors are also required for virus-induced cell fusion (37-40). However, virus-induced cell fusion requires the presence of additional viral glycoproteins and membrane proteins including gE, gI, gM, gK, and the UL20 and UL45 proteins (41-46) (Fig. 1.2).

Figure 1.2. Entry mechanism of HSV-1. A) Fusion. B) Endocytosis. C) Mechanism of fusion mediated entry. (Anthony V. Nicola et al. J. Virol. 2003;77:5324-5332. Adapted from Karasne et al 2011)

Herpesvirus also enters host cells via receptor-mediated endocytosis which can be pH- dependent or independent based on the cell type (47, 48). For example, in CHO cells expressing nectin-1, HeLa and human keratinocytes, HSV-1enters via pH-dependent endocytosis; however,

in mouse melanoma cells (B78C10) HSV-1 prefers pH-independent endocytosis (49-54). The

virus can spread from infected to uninfected cells by causing virus-induced cell fusion allowing

virions to enter into uninfected cells without being exposed to extracellular spaces (12, 49, 55).

Following fusion mediated entry, most of the viral glycoproteins remain on the cell

membrane releasing the viral nucleocapsid with tegument in the cytoplasm. Mass spectrometric analysis of extracellular HSV-1 virions reveals 23 virally encoded tegument proteins and several host cell enzymes, chaperones, and structural proteins, some of which possibly get incorporated

in the tegument layer. The host cell proteins identified were proteins involved in transport and

exocytosis (56). The most abundant tegument proteins are pUL47/VP13-14, pUL48/VP16, and

pUL49/VP22, which are present in 600-1300 copies per virion (57). With the advent of super-

resolution microscopy, it is now possible to locate specific tegument proteins in the viral tegument layer. The outer tegument proteins of herpes viruses are amorphous and quickly

dissociate in the cytoplasm following virus entry. The inner tegument layer is icosahedral due to

its close association with the capsid. Viral capsid travels along with the cellular microtubule

network with three inner tegument proteins UL36, UL37, US3 attached to the capsid (58-64).

Tegument proteins provide structural integrity of virions, transcriptionally activate immediate

early genes by forming transcription complex between host cell factors HCF-1 and Oct-1 and the

HSV-1 pUL48/VP16 or VZV homolog ORF10. For HSV-1 and PRV, the tegument protein

UL41 or VHS (host shutoff protein) suppresses host protein expression (65-74). The HSV-1

tegument proteins UL36 and UL37 mediate capsid anterograde and retrograde transport,

respectively (63).

Figure 1.3. The life cycle of HSV-1. A) Attachment. B) Receptor binding. C) Fusion. D) Release of outer tegument protein and transport of capsid with inner tegument proteins attached to the capsid. E) Capsid docking at the nuclear pore complex. F) Injection of DNA into the nucleus. G) Formation of the DNA episome. (Adapted from Ralph et al., 2017) Initial transport of HSV-1 following entry is mediated by actin and microfilament which

promote short distance motility (75-77). Later on, long-distance travel is facilitated by cellular

motor proteins dynein/dynactin complex and kinesin (63, 77, 78). Additional adaptor proteins and regulatory kinases may have roles in virus transport and replication (79). Although HSV-1 and PRV retrograde transport are bidirectional and saltatory, however, the net movement occurs towards the nucleus following entry (5, 77, 80-84).

After reaching the host nucleus, HSV-1 tegument protein UL36 mediates capsid docking at the nuclear pore complex (NPC) (5, 85) and injects the viral DNA into the nucleoplasm as the capsid (125 nm) is too large to pass across the nuclear pores by diffusion (Fig. 1.3). The herpes

virus genome can be as small as 125 kb (VZV genome) and as large as 235 kb (HCMV genome).

Genomic transcription takes place in the host nucleus immediately to produce early mRNAs,

which get translated into viral proteins required for transcription of other viral genes. Viral DNA

replication is mediated by the viral polymerase and other viral replicative machinery. Newly

synthesized DNA is used as the template for late gene transcription. Late mRNAs encode for

structural proteins for the virus. Part of the newly synthesized DNA is packaged into capsids

under strict viral packaging signals in the nucleus and then the nucleocapsid egress from the

nucleus. The newly formed nucleocapsid undergoes primary envelopment at the inner nuclear

membrane (INM) and reaches the perinuclear space. The glycoprotein embedded enveloped capsids get de-enveloped at the outer nuclear membrane (ONM) and bud off into the cytoplasm

as viral capsids (4). In the cytoplasm, viral capsids incorporate 16-35 tegument proteins and

travel to the trans-Golgi network (TGN) where the tegument coated capsid gets enveloped

acquiring a full constellation of viral glycoproteins.

1.3 Intracellular transport

Cells depend on transport systems for efficient growth and survival. Cytoskeletons

provide the cells with distinct shapes and mediate intracellular transport. Actin and myosin reside

mainly at the periphery of cells and regulate short distance travel of cellular cargo whereas

microtubules confer long-distance transport and spatial organization of organelle, vesicle,

protein, and RNA containing cargos (86-88). Being polarized microtubules have a plus and a

minus end. The minus end originates at the microtubule-organizing center or MTOC which is

often located near the nucleus but can be found at other locations of the cell (89, 90). The minus

end is stabilized by the MTOC. However, the plus end is dynamic and it constantly shrinks and

extends at the periphery. Post-translational modification like acetylation and detyrosination of

tubulin subunit helps to stabilize microtubules (79, 91-93).

Transport along the cytoskeleton can be fast or slow. In neurons, fast transport occurs at a rate of (0.5–10) µM/second. Mitochondria, neurotransmitters, membrane-bound organelle, endosome, and channel proteins travel via fast transport. Although fast transport takes place in both retrograde and anterograde way, slow transport occurs only anterograde at the rate of (0.01-

0.001) µM/second. Cytoskeletal components, for example, neurofilament, tubulin, actin, etc. and proteins like clathrin are transported via slow transport (94-99).

Inside cells, most of the transport occurs in vesicles. Some molecules enter cells by receptor-mediated endocytosis. Once the molecule binds to the receptor, signaling events occur and the molecule gets endocytosed. Vesicles activate signaling cascade, which in turn activates several molecules that are responsible for the transport of the vesicle to the appropriate location.

Transport of molecules can be towards the nucleus or away from the nucleus, depending on the requirement of the cell.

1.3.1 Cellular motors

Mostly cells use motor proteins that carry cargo along microtubules. There are two major motor proteins in eukaryotic cells for long-distance movements, namely, dynein and kinesin.

Dynein mostly mediates transport of cellular cargos to the negative end of MTOC, towards the nucleus. Dynein helps position organelles and spindle during mitosis (96, 100-102). On the other hand, kinesin travels from the minus end to the plus (Fig. 1.4). Several kinesin families give specificity to their actions. The motor dynein often complexes with another smaller molecule called dynactin. Binding of dynactin to dynein activates the complex. Dynactin helps processivity of the overall complex. Overexpression of dynactin disrupts the complex and halts dynein function (103, 104).

Figure 1.4. Cytoskeleton and motor protein-mediated cellular cargo transport. Dynein and kinesin are two major motor proteins that move along the microtubule. Dynein binds cellular cargo directly and moves towards the minus end of the microtubule, whereas, kinesin moves cargo towards the plus end of the microtubule. Microtubule-associated proteins (MAPs), kinases and adaptor proteins aid in this process.

Dynein was first discovered in ciliates (tetrahymena pyriformis) as motor proteins (105).

Dynein is a 1.4 MDa protein complex (96). Two isoforms are identified so far, dynein 1

(cytoplasmic) and dynein 2 (ciliary). Dynein is essential for many organisms like mice and

Drosophila melanogaster (90, 106, 107). The dynein complex has two copies of heavy chains

(cytoplasmic DYNC1H1 or ciliary DYNC2H1), two intermediate chains (DYNC1I1 and

DYNC1I2), two light intermediate chains (DYNC1LI1 and DYNC1LI2) and 3 classes of light

chains (Tctex-1/RP3 (DYNLT1/3, LC7/ roadblock (DYNRB1/2), and LC8 (DYNLL1/2)(96,

108, 109) (Fig. 1.5). The heavy chains contain ATPase activity which is required for movement

along the microtubules. The intermediate chain and light chain regulate motor activity and

mediate interaction with other cofactors and cargos (108-110).

The intermediate chain of dynein (DIC) is a 74-KDa protein and is a necessary part of the

dynein complex (111). Along with cargo recognition and assembly of the complex, DIC helps in

the regulation of the overall process. For example, DIC directly connects huntingtin, adenovirus,

the CIC-2 chloride channel and PLAC 24 to the dynein complex (112-115). DIC also interacts with the light chains (Tctex1, LC8, and LC7) and some regulatory proteins like NudE, dynactin,

NudC and zeste white 10 (116-119). The C terminal of DIC is WD-rich, and the N terminal is unstructured. In case of Drosophila melanogaster, the N terminal residues (3–36) are helical, consistent with the coiled-coil secondary structure predicted for residues (1–40) from sequence analysis, residues (48–60) have a nascent helical propensity, and residues (222–231) constitute a self-association domain. The N-terminal domain binds to all three light chains. For D. melanogaster, the light chain-binding sites of DIC are mapped to residues 110–122 (Tctex1),

126–138 (LC8), and 221–258 (LC7) (120-124).

Figure 1.5. Schematic illustration of the location and approximate structural features of dynein and dynactin subunits. Dynein is composed of heavy chains, intermediate chains, and light chains. The intermediate chain binds cargo and dynactin. Heavy chains mediate ATP dependent movement along the microtubule. Dynactin helps in the processivity of the overall complex. (Adapted from Schroer et al Annu. Rev. Cell Dev. Biol. 2004. 20:759–79).

1.3.2 Cellular adaptors and linkers

After dynein was discovered, it was assumed that other accessory proteins might aid in

dynein function. For accuracy and efficient transport processes, dynein activity is highly regulated by other accessory factors. One major factor is dynactin (DCTN), as mentioned before.

It is approximately a 1.2 MDa multi-subunit complex which plays multiple roles in dynein

10 mediated transport. Dynactin (DCTN) is a big molecule consisting of several components namely, DCTN 1 (p150), DCTN 2 (dynamitin), DCTN 3 (p24/22), DCTN 4 (p62), DCTN 5

(p25), DCTN 6 (p27), CapZ and Arp polymer (Fig. 1.5) (Table 1.1). The subunit p150 binds to the IC of dynein, and the other end is associated with microtubule. P62, Arp polymer, and CapZ take part in cargo binding (125-129). Dynactin and kinesin recruit dynein to the plus end of microtubules (130-134). Dynactin also plays a role in cargo recognition, initiation, and processivity of the transport complex. Overexpression of dynactin reduces the motility of the dynein dynactin complex (135).

Table 1.1. The number of subunits of the dynactin molecule.

Dynactin

Subunit P150 P62 P50/ P27 P25 P24 Arp1 Arp11 CapZa CapZB B actin

dynamitin

Number 2 1 4 1 1 2 1 8 1 1 1

There are linker proteins that help dynein to function. End binding protein (EB1) and

CLIP 170 directly recruit dynactin (p150) and dynein sequentially to microtubule plus ends as shown in in vitro reconstitution experiment with purified recombinant proteins (104, 136, 137)

(138). CLIP 170 was identified as a microtubule-binding protein that remains attached to the distal end of microtubules and detaches when microtubule stabilizes. CLIP 170 falls under a group of proteins called +TIPS or plus-end tracking proteins. CLIP 170 also plays a role in vesicle transport in vitro (137, 139-141). Phosphorylated CLIP 170 is essential for dynein recruitment to the plus end of the microtubules and repositioning of the MTOC at the immunological synapse during T cell activation (142). CLIP 170 binds to the dynein-dynactin complex via either the proximal or distal zinc finger motif in the C terminal end. It was shown

that the distal zinc finger motif mediates binding with Lis1, a protein important for neuronal

migration (137). CLIP 170 and Lis1, also known as NudF, a member of the nuclear distribution

(Nud) family, a regulator of dynein motility, mediate the transport of dynein to the plus end of

microtubule by kinesin (143).

EB1 specifically recognizes dynamic microtubule growing ends and recruits other +TIPs

thereby regulates the growth and polymerization of microtubule (144, 145). EB1 is essential for

specific accumulation of other +TIPS and cancer cell migration and proliferation (79, 92, 146-

148). The C terminal linker region of EB1 binds to microtubule and has two phosphorylation

sites, S 155 and Thr 166. Phosphorylation of these sites either activates or inactivates the EB1

respectively.

Activation of the dynein-dynactin complex requires coiled-coil proteins (activating adaptor proteins) like BICD1/2, TRAK1/2, HOOK1/2/3, etc. The presence of the N terminal of

BICD2 (bicaudal D homolog 2) increases the binding between dynein and dynactin in vitro.

Activating adaptors not only mediate the binding of the motor to its cargo but also activate motility of the complex (90).

1.4 Regulation of intracellular transport

Intracellular transport is highly regulated by accessory factors and kinases. Mitogen- activated protein kinases (ERK1/2) regulate cellular motility by activating myosin light chain

(MLC). The activation of MAPK requires growth factor or integrin-mediated receptor activation and signal transduction (149). There is also evidence that p38 MAPK can be activated via phosphokinase C (PKC) activation by G protein-coupled receptors (GPCRs). However, once

MAPK is activated, it induces the activation of intracellular motility machinery (150). PKC also regulates cytoskeleton dynamics. For example, PKC directly activates microtubule-associated

protein tau in vitro and in vivo (151). Tau is highly expressed in the brain and is activated by

glycogen synthase kinase (GSK3β) via PKC (152).

GSK3β activity aids in EB1 phosphorylation and accumulation at the plus end of the

microtubule, however, Akt mediated mitochondrial ROS production inhibits GSK3β induced

EB1 activation by inactivating GSK3β (148). GSK3β targets dynein intermediate chain (DIC)

directly and phosphorylates DIC 1C 1B S87/T88 and DIC 1C 2C S88/T89. Pharmacologic and

genetic inhibition of GSK3β stimulates dynein motility (153).

PI3 kinase is involved in endocytic vesicle transport. For example, phagocytosis, clathrin-

dependent, and independent endocytosis, phagocytosis, glucose-induced endocytosis, and Trk

receptor internalization require PI3 kinase activation (154).

The function of cellular motor dynein can be regulated directly by other proteins. Lis1 directly binds to dynein heavy chain and Model 1 (nuclear distribution protein nudE-like 1) and regulates the distribution of DHC along the microtubule. Lis is a gene located on chromosome

17p13 and is responsible partly for brain malformation due to a defect in neuronal migration

(lissencephaly). Ndel 1 is a mammalian homolog of Aspergillus NUDE protein. Ndel activation is mediated by Cdk5/p35, a necessary complex for neuronal migration (155, 156).

1.4.1 Regulation of axonal transport

Neurons have unique morphology consisting of a cell body, dendrites, and axons. Cell bodies can be a meter away from the axon in mammalian neurons. Therefore, long-distance transport of intracellular organelles, RNA, protein, lipids along the axon and disposal of waste is crucial for the survival and proper function of neurons. Axonal transport plays an essential role

in overall neuronal homeostasis by maintaining long-distance communication between the distal

regions, by maintaining neurotrophic and injury signaling and by regulating axonal length (157-

160).

Axonal transport is highly regulated by direct phosphorylation of motor proteins, adaptors and cargos or by indirect modification of microtubules. Although the overall mechanism is still unknown, mostly axonal transport is regulated by various kinases (Fig. 1.6)

(161).

Figure 1.6. Protein Kinases Implicated in the Regulation of Axonal Transport adapted from Gibbs et al, 2015. (161)

The serine/threonine kinase Akt (also known as protein kinase B) plays a role in the directionality of fast axonal transport. Akt phosphorylates an adaptor protein called htt at Ser

421, which increases the recruitment of kinesin-1. This phosphorylation event ultimately leads to anterograde transport of BDNF-containing secretory vesicles, APP-containing vesicles, and

synaptic vesicles. The inhibition of Akt enhances the retrograde transport of those vesicles. Akt

activation also enhances kinesin binding to microtubules (162). It was shown that the rate of

retrograde axonal transport of BDNF-containing vesicles is decreased by activation of Akt (162).

Akt mediated inactivation of GSK3β expedites fast axonal transport of mitochondria in

hippocampal neurons (163, 164). On the other hand, overexpression of GSK3β inhibits axonal transport of mitochondria and amyloid precursor protein (APP) containing vesicle (164, 165).

However, the inhibitory function of GSK3β in axonal transport of vesicles is not conserved among all species (166). GSK3β directly phosphorylates tau, increases its anterograde movement (167). In contrast, it reduces the anterograde movement of neurofilament by phosphorylating its heavy chain (168). The mitogen-activated protein kinase ERK1/2 directly phosphorylates dynein (DIC1B and DIC2C) and kinesin (KLC1) and regulates fast retrograde cargo transport (169, 170). There is evidence that ERK1/2 facilitates slow anterograde axonal transport of neurofilaments (168). Cyclin-dependent kinase 5 (Cdk 5) is another important kinase that has no role in cell cycle control, unlike other Cdks, but it plays a role in neurodegeneration, neurite growth, migration, and synaptic activity (171, 172). In rodent dorsal root ganglia (DRG), it was shown that Cdk5 phosphorylates Ndel1, an adaptor of dynein motor and activates fast axonal transport (173). Cdk5 also does its effect indirectly on axonal transport by inhibiting

GSK3β (174). Table 1.2 summarizes kinases involved in axonal transport.

Table 1.2. Protein Kinases that Directly Regulate Axonal Transport (161) (rAT, retrograde axonal transport; aAT, anterograde axonal transport; +, enhanced; −, inhibited; NE, no effect.)

Effect on AT Type of Neuron Kinase Target Cargo Affected aAT rAT

GSK3β Membrane-bound KLC2 – – Squid axoplasm organelles

(table

cont’d)

Effect on AT Type of Neuron Kinase Target Cargo Affected aAT rAT

DIC1B Acidic organelles NE – Dorsal root ganglia

(Ser87/Thr88);

DIC2C

(Ser88/Thr89)

Mitochondria,

APP-containing

vesicles, Hippocampal

lipid droplets, neurons, ? – – synaptic vesicles Drosophilasegmental

(?), nerves

BDNF-containing

secretory vesicles

Tau Tau + Cortical neurons

NF-H Neurofilaments – NB2a/d1 cells

DIC1B (Ser80); Signaling ERK1/2 + Hippocampal neurons DIC2C (Ser81) endosomes

Calsyntenin-

KLC1 (Ser460) associated – + Cortical neurons

vesicles (APP)

(table

cont’d)

Effect on AT Type of Neuron Kinase Target Cargo Affected aAT rAT

GSK3β (Ser9) Neurofilaments + NB2a/d1 cells, dorsal

root ganglia

KIF5C Membrane-bound – NE Squid axoplasm (Ser175/176) organelles p38 MAPK ? Mitochondria – – Hippocampal neurons

Drosophilasegmental ? Synaptic vesicles – nerves

NF-M and -H Neurofilaments – Cortical neurons

APP-containing JIP1 (Ser421) + – Dorsal root ganglia vesicles

Membrane-bound KIF5C (Ser176) – – Squid axoplasm organelles JNK Mitochondria, KIF5B – – Hippocampal neurons synaptic vesicles

NB2a/d1 cells, dorsal ? Neurofilaments – root ganglia

Cdk5 Membrane-bound Squid axoplasm, LMTK2/PP1 + + organelles dorsal root ganglia

(table

cont’d)

Effect on AT Type of Neuron Kinase Target Cargo Affected aAT rAT

? Secretory vesicles + Caenorhabditis

elegans motor

neurons

Ndel1 Acidic organelles + + Dorsal root ganglia

MEK Neurofilaments – NB2a/d1 cells

Dorsal root ganglia, NF-M and -H Neurofilaments – SH-SY5Y

BDNF-containing

secretory

vesicles, htt (Ser421) + – Cortical neurons Akt APP-containing vesicles, synaptic

vesicles

GSK3β (Ser9) Mitochondria + + Hippocampal neurons

Small vesicles Crayfish walking leg Kinesin (not – NE PKA giant axon mitochondria)

? Mitochondria + + Dorsal root ganglia

Membrane-bound CK2 KLC1 and 2 – – Squid axoplasm organelles

(table cont’d)

Effect on AT Type of Neuron Kinase Target Cargo Affected aAT rAT

PKC ? Membrane-bound – – Sympathetic and

organelles sensory neurons,

squid axoplasm

PINK1 Miro (Ser156) Mitochondria – – Cortical neurons

1.5 Diseases due to intracellular transport defects

The overall transport inside cells is strictly regulated by motor proteins, adaptor, and

linker proteins and by the function of regulatory molecules and kinases. Disruption in the

number or ratio of motors, linkers, adaptors involved, or aberrant function of the regulatory

molecules and kinases leads to several diseases (175). Misregulation of axonal transport by

protein kinases has a direct role in the pathogenesis of various nervous system disorders (97,

161, 176-182). Many proteins associated with neurodegenerative disease activate p38 MAPK to

exert their toxic effect. For example, amyloid-beta (Aβ) protein associated with Alzheimer’s and

SOD1(G93A) protein associated with Amyotrophic lateral sclerosis (ALS) (183-189). A list of diseases associated with motor defects is described in Table. 1.3.

Table. 1.3. Neurodevelopmental and Neurodegenerative Diseases Caused by Mutations in the Axonal Transport Machinery (97)

Protein(s) Gene(s) with Known Mutation Disease(s) Motor Proteins Dynein DYNC1H1 CMT, SMA-LED, ID, MCD (Epilepsy) Kinesin-1 KIF5A, KIF5C HSP (SPG10), ID, MCD Kinesin-13 KIF2A CDCBM3/MCD Kinesin-3 KIF1A, KIF1B, KIF1C HSP (SPG30), CMT2A, HSN, MR, SPAX Kinesin-4 KIF21A CFEOM Motor Adaptors and Regulators Dynactin DCTN1 Perry syndrome, MND BICD2 BICD2 SMA, HSP Huntingtin HTT HD Lis-1 PAFAH1B1 Lissencephaly NDE1 NDE1 Microcephaly, MHAC Rab7 RAB7A CMT2B

Cytoskeleton and Associated Proteins (e.g., MAPs) CLIP-170 CLIP1 ARID Doublecortin DCX Lissencephaly Microtubules TUBA1A, TUBA8, TUBG1, TUBB3, TUBB2B Lissencephaly, MCD, microcephaly, polymicrogyria, CFEOM Neurofilaments NEFL CMT Spastin SPAST HSP (SPG4) Tau MAPT FTD, Pick disease, AD Abbreviations are as follows: AD, Alzheimer’s disease; ARID, autosomal recessive intellectual disability; CDCBM3, complex cortical dysplasia with other brain malformations-3; CFEOM, congenital fibrosis of the extraocular muscles; CMT, Charcot-Marie-Tooth disease; FTD, frontotemporal dementia; HD, Huntington’s disease; HMN, hereditary motor neuropathy; HSN, hereditary sensory neuropathy; HSP, hereditary spastic paraplegia; ID, intellectual disability; MCD, malformations of cortical development; MHAC, microhydranencephaly; MND, motor neuron disease; MR, mental retardation; SMA, spinal muscular atrophy; SMA-LED, SMA-lower extremity dominant; and SPAX, spastic ataxia

1.6 Virus and cellular transport

Animal viruses enter into host cells by adsorption to the cell membrane receptors,

penetration into the cytoplasm, and eventually uncoating of their genome inside the cytoplasm or

nucleus depending on the type of virus. Some viruses that replicate in the nucleus need to travel a

long distance, especially if they are neurotropic DNA viruses such as herpesviruses (80).

However, the movement of the viral particles through the cytoplasm is not mediated by free

diffusion because of the high viscosity and steric obstacles (190).

The virus exploits cellular machinery for replicating its genome inside the cell. The virus would

likely use cellular motor proteins to travel inside the cells. DNA viruses travel to the nucleus for

replication of their genome. It is assumed that virion capsids would travel along with the

microtubule networks with the help of minus or plus end-directed motor proteins to reach the

nucleus , respectively. Recently a number of viruses showed association with cellular motor

proteins like dynein and kinesin. In many cases it was shown that viruses travel along the

microtubules with or without the presence of motor proteins. The requirement for adaptor proteins and cofactors along with motor proteins in viral transport was also demonstrated.

Several viruses stimulate upstream signaling to change microtubule dynamics and enhance the

infection process. Viral infection triggers many cellular kinases, which may or may not have

specific roles in activating factors responsible for movement inside a cell (78, 191-195).

1.6.1 Herpes simplex virus

HSV-1 enters into host cells via the fusion of its envelope with the cell membrane. Viral

glycoproteins remain at the plasma membrane and most of the outer tegument proteins dissociate

in the cytoplasm except three inner tegument proteins UL36, UL37 and US3 (196). Recently it

was shown that HSV-1 capsid travels across the cytoplasm via microtubules in Vero cells (79).

HSV-1 capsid was shown to be attached to the dynein motor protein (80, 197). The UL34 protein

was thought to be associated with the neuronal dynein intermediate chain 1a (DIC 1a). However,

UL34 is not a structural protein, and the UL34 null virus is found to be able to infect cells (197).

Moreover, the expression of UL34 in baculovirus suggested nuclear membrane localization of

UL34 (112). Later, the HSV-1 capsid protein VP26 was shown to interact with the dynein light

chain component RP3 and Tctex1 by yeast two-hybrid systems and in vitro pull-down assay.

Microinjection of the VP26 null virus in the cell showed the random distribution of viral capsid compared to nuclear-localized wild type HSV-1 capsids 2-4 hours post-infection (197).

Later, the Sodeik laboratory showed that the capsid interactions are mediated by inner tegument proteins, and both plus and minus end-directed motor binds to HSV capsid simultaneously (63, 198) (Fig. 1.7). Recently the role of the microtubule-binding protein in retrograde transport was also elucidated. Following entry, through the plasma membrane, HSV capsid reaches the plus end of microtubules where accessory proteins regulate the efficient interaction with motor proteins and retrograde movement of the viral capsid. End binding protein

1 (EB1) was found to be essential for HSV-1 infection. Also, it was shown that the microtubule plus end associated protein CLIP 170 initiates HSV-1 retrograde transport in primary human cells (79). HSV-1 was also shown to cause stabilization of microtubules (199) (200).

Figure 1.7. Retrograde and anterograde transport of HSV-1 using cellular motors and cytoskeleton. (Adapted from Owen et al, 2015)

1.6.2 Other viruses

Recently it was shown that efficient transport and infection of TZM-bl reporter cells by

HIV requires the dynein-dynactin-BICD2 complex. Depletion of dynein heavy chains

(DYNC1H1) reduced HIV infection significantly, whereas other dynein components have little

to no effects on HIV infection on TZM-bl cells. The depletion of Dynactin components like

DCTN2, DCTN3, and ACTR1A (arp1 rod) resulted in decreased HIV infection in the same cell

line. It was also shown that HIV-1 exploits one or more dynein adaptors for efficient infection

and transport towards the nucleus. From a panel of host adaptor proteins, one possible candidate

for HIV-1 dynein adaptor was found, which is called the bicaudal D homolog 2 (BICD2).

Usually, BICD2 is associated with the cellular transport of mRNA and organelles and helps to

position the nucleus during cell division by docking at the nuclear pore (201). There was a marked reduction in HIV-1 permissiveness in TZM-bl cells in BICD2 knocked out cells (110).

Dynein light chain 2 (DLC-2) transports HIV integrase, and DLC-1 interacts with HIV capsid

23 and affects reverse transcription (202, 203). HIV was found to stabilize microtubule during infection, and plus-end binding protein, EB1, and Kif4 play an important role during the overall process of infection (199).

Interestingly, the virus SV40 directly recruits dynein. Following being endocytosed, the virus enters into the endoplasmic reticulum (ER) and then it travels to the nucleus via cytoplasm.

At this step, the virus requires dynein for the dissociation of the SV40 that enables the virus for successful penetration into the nucleus (204).

Adenoviral capsid (hexon) directly interacts with intermediate dynein chain and dynein light intermediate chain. Dynactin also plays a role in adenovirus transport. However, dynactin was not found to have a role in recruiting dynein. Dynein, dynactin, NudE/NudEL but not Lis1 or ZW10 were found to be colocalized with post endosomal adenoviral particle (112, 205). The phosphoproteins of the Lyssavirus and Rabies virus were also found to be associated with dynein motor (DLC-8) (206).

It was shown by Enquist lab that PRV and HSV-1 infection in the rodent superior cervical ganglion neurons disrupts mitochondrial transport in the cytoplasm. Following gB mediated fusion at the plasma membrane, the neuronal action potential and firing rate increase, which increases the intracellular calcium level. This event activates the calcium-sensitive protein

Miro and modifies kinesin-1 mediated recruitment of mitochondria (207). Other herpesviruses like Kaposi’s sarcoma-associated herpesvirus, can play a role in stabilizing microtubules during early infection via stimulating the Rho-Dia signaling (208).

A tegument protein VP1/2 was shown to be sufficient to move cargo via the dynein- dynactin- microtubule network. The deletion of VP1/2 was found to be lethal. It was shown that

VP1/2 interacts with dynein intermediate chain (DIC) and dynactin (p150) in HEK293 cell lines.

The deletion of the proline-rich sequence from VP1/2 impairs retrograde transport of PRV in neuronal culture and in vivo (209). Pseudorabies, alpha herpes capsid was shown to bind the

dynein light chain (LC8). However, this interaction is not essential for retrograde travel across

the cytoplasm for PRV (210).

1.7 Conclusions

The prevalence of HSV-1 in the USA in persons aged 14 to 49 in 2015-2016 was 47.8%

(211). The virus persists in the body life long once acquired from a source. HSV-1 establishes

latency in the nervous system and occasionally reactivates. Moreover, recurrent eye infection

leads to blindness and HSV-1 is the number one cause of infectious blindness in the USA. The

first step of HSV-1 infection is the entry of the virus into host cells crossing the plasma

membrane and traveling through the complex cytoplasmic barrier to reach the nucleus where the

virus replicates its genome leading to the formation of new infectious particles that spread to

neighboring cells. Unfortunately, it is still unknown how the virus mechanistically enters into

cells and transports its virion capsids to the nucleus where it can establish latency in neurons.

Understanding the overall mechanism of HSV-1 entry and transport through the cytoplasm may provide basic insights that can lead to pharmacologic interventions that can prevent virus entry and intracellular transport.

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CHAPTER 2 THE AMINO-TERMINUS OF HSV-1 GLYCOPROTEINK (GK) IS REQUIRED FOR GB BINDING TO AKT, RELEASE OF INTRACELLULAR CALCIUM AND FUSION OF THE VIRAL ENVELOPE WITH PLASMA MEMBRANES1

2.1 Introduction

2.1.1 HSV-1 entry mechanisms. Herpes simplex virus type-1 (HSV-1) utilizes membrane fusion mediated by viral glycoproteins embedded within the viral envelope and expressed on infected cell-surfaces to infect a variety of cell types and facilitate the spread of infectious virions to adjacent cells via virus-induced cell fusion, respectively (1). Viral entry into cells can occur in a pH-independent manner via fusion of the viral envelope with cellular plasma membranes resulting in the deposition of viral capsids into the cytoplasm of infected cells. This mode of entry is exclusively utilized during virus entry into neuronal axons (2-4). Also, a variety of other cell types, including African monkey kidney cells (Vero), human foreskin fibroblasts,

HaCat, and rat-kangaroo kidney (PtK2) cells, appear to be infected via direct fusion of the viral envelope with cellular plasma membranes (3, 5-8).

Alternatively, enveloped virions can enter a variety of cells by endocytosis into early endosomes followed by pH-dependent and independent fusion of the viral envelope with endosomal membranes resulting in the release of virion capsids into the cytoplasm of infected cells (3, 9). HSV-1 prefers pH-dependent endocytosis for entering into CHO cells expressing nectin-1, HeLa and human keratinocytes and pH-independent endocytosis in mouse melanoma cells (B78C10) (6, 10-14). The virus can spread from infected to uninfected cells by causing

1 This chapter is previously appeared as Musarrat F, Jambunathan N, Rider PJF, Chouljenko VN, Kousoulas KG. 2018. The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes. Journal of Virology 92

virus-induced cell fusion allowing virions to enter into uninfected cells without being exposed to

extracellular spaces.

2.1.2 The role of viral glycoproteins in virus entry. HSV-1 attaches to the cellular

membrane by glycoproteins B (gB) and C (gC) with glycosaminoglycan (GAG) moieties of cell

surface proteoglycans (15, 16). Glycoprotein B can also interact with cellular receptors including

paired immunoglobulin-like type 2 receptor alpha (PILR), non-muscle myosin heavy chain IIA

(NMHC-IIA), and myelin-associated glycoprotein (MAG). Glycoprotein D binds to cellular

receptors like herpesvirus entry mediator (HVEM, also called HveA), nectin-1 (HveC), and 3-O-

sulfated heparan sulfate (17-23). Following binding of gD and gB, a conformational change

occurs in gH/gL that alters the conformation of gB triggering membrane fusion (24-29). Virus- induced cell fusion is thought to be mediated by a mechanism very similar to that occurring during fusion of the viral envelope with cellular membranes, inasmuch as, viral glycoproteins gD, gB, gH and gL and the presence of viral receptors are also required for virus-induced cell fusion (30-33). However, virus-induced cell fusion requires the presence of additional viral glycoproteins and membrane proteins including gE, gI, gM, gK, and the UL20 and UL45 proteins (34-39).

2.1.3 Role of HSV-1 glycoprotein K (gK) in membrane fusion. The UL20 and UL53

(gK) genes are highly conserved in all alphaherpesviruses and encode proteins of 222 and 338 amino acids, respectively, each with four membrane-spanning domains (40-44). HSV-1 gK and

UL20 functionally and physically interact and these interactions are necessary for their coordinate intracellular transport and cell-surface expression and functions in virus-induced cell fusion, virus entry, virion envelopment and egress from infected cells (37, 38, 43, 45-53). The gK/UL20 protein complex interacts with gB and gH and is required for gB-mediated cell fusion

(39, 54). HSV-1 gK is a structural component of virions and functions in virion entry (50, 55,

56). Deletion of amino acids 31-68 within the amino terminus of gK inhibits virus-induced cell- to-cell fusion and virus entry without drastically inhibiting virion envelopment and egress. We have shown that gK is essential for neuronal infection and virulence (57, 58). Besides, we have recently shown that the HSV-1(McKrae) gK∆31-68 virus specifying gK with a deletion of amino acids 31-68 was unable to efficiently infect mouse trigeminal ganglia after ocular infection of scarified mouse eyes (59). These results indicate that the amino terminus of gK plays a pivotal role in corneal infection and neuroinvasiveness (58, 59).

2.1.4 Role of cell signaling in membrane fusion. Akt is a serine/threonine kinase involved in important cellular signaling pathways (60). There are three isoforms of Akt: Akt1,

Akt2, and Akt3 (61-66). Although different genes encode these isoforms, they share an N- terminal pleckstrin homology domain, a kinase domain and a C-terminal regulatory domain (65).

In this work, we are focusing on Akt-1, because it is the predominant isoform of Akt expressed ubiquitously, while Akt-2 and Akt-3 expression are limited to insulin-responsive tissues and brain or testes, respectively (67). Signaling through plasma membrane-embedded receptors activates the phosphoinositide-3-kinase (PI3K) signaling pathway causing recruitment of Akt-1 to plasma membranes where it is activated via phosphorylation at S473 and T308 (68-73).

Herpesviruses express several proteins that alter PI3K/Akt signaling pathway which regulates cell survival, growth, apoptosis, inflammation, cell motility, calcium signaling, etc. (74). These regulatory mechanisms can influence viral entry, replication, latency and reactivation of HSV-1

(75, 76). Recently, it was shown that herpes glycoprotein B activates Akt by phosphorylation and

that gB physically interacts with Akt during virus entry (77). Herpes infection also induces an

intracellular calcium response (78). Binding of glycoprotein H (gH) of equine herpesviruses

(EHV) to cellular integrin (α4β1) triggers intracellular calcium signaling and phosphatidylserine

exposure on the plasma membrane to facilitate EHV-1 entry (79). Furthermore, Akt silencing or

Akt inhibitor miltefosine inhibited calcium signaling and virus entry into cells (77).

In this manuscript, we show that gK is required for entry via fusion of the viral envelope

with cellular membranes. The underlying mechanism involves a role for gK in the regulation of

intracellular calcium release and expression of Akt-1(S473) on cell surfaces required for binding

to gB during virus entry.

2.2 Methods and materials

Cell lines and viruses. African green monkey kidney (Vero) cells and Neuroblastoma cell lines SK-N-SH (HTB11) were obtained from the American Type Culture Collection

(Manassas, VA) and were maintained in either Dulbecco's modified Eagle's medium or Essential modified Eagle's medium (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal calf serum and antibiotics respectively. Mostly HSV-1 strain McKrae and McKrae gKΔ31–68, viruses were used in this study. For some experiments, McKrae VP26 EGFP and McKrae gKΔ31–68 VP26 EGFP viruses were used. HSV-1(McKrae gKΔ31–68) was engineered in our

lab to express gK lacking 38 amino acids immediately after the gK signal sequence, cloned as a

bacterial artificial chromosome, as described previously (59). McKrae VP26 EGFP and McKrae

gKΔ31–68 VP26 EGFP viruses were also constructed in our lab.

Reagents and antibodies. Primary antibodies used are as follows: Mouse anti-gB

(Abcam) antibody, Mouse anti-gD (Abcam) antibody, Rabbit anti-Akt-1 S473 (Abcam)

antibody, Rabbit anti-Nectin-1 (Santa Cruz) antibody, Rabbit anti-UL37 antibody (a gift from

Frank J. Jenkins, University of Pittsburgh Cancer Institute), Mouse anti-Dynein antibody

(Abcam). Pitstop-2 was obtained from Abcam. Fura-2, AM (AS-84015) was obtained from

Anaspec. Miltefosine (sc-203135) was obtained from Santa Cruz biotechnology, Dynasore hydrate was obtained from Sigma. Annexin V-FITC fluorescent microscopy kit was obtained from BD Bioscience. Duolink® In Situ Red Starter Kit Mouse/Rabbit was obtained from Sigma.

Cell mask Deep red plasma membrane stain was obtained from Thermo Fischer and Goat anti- rabbit antibody conjugated with Alexa fluore 594 was used as a secondary antibody.

Virus purification. Both McKrae (WT) and McKrae gKΔ31–68 viruses were purified as

we have described earlier (80). Briefly, supernatants and cells from 6 T-150 flasks of Vero cells infected with McKrae (WT) and McKrae gKΔ31–68, viruses were collected at 36 h postinfection (hpi) and purified by 50 to 20% discontinuous iodixanol gradients twice, followed by a 20% iodixanol cushion. The resulting pellet was resuspended in 250 μl of PBS and used for calcium assay.

Drug assay. Vero cells were treated with drugs for 15 mins at 37°C and then viruses

(100PFU) were adsorbed at 4°C for an hour. Unbound viruses and unabsorbed drug were washed with PBS, and fresh media with methylcellulose were added on the cells. Plates were incubated at 37°C for 48 hours and plaques were counted after being fixed with formalin and stained with crystal violet. In the case of SK-N-SH cells, drug treatment was done for 15 mins, the infection was done at MOI-10 and 1-hour post-infection slides were prepared for respective PLA assay.

Proximity Ligation Assay (PLA). SK-N-SH cells were grown on 8-well chamber slides

(Nunc Lab-Tek II chamber slide system) and infected with McKrae wild type and McK gK∆31-

68 at an MOI of 10. At 1h postinfection, the cells were fixed with ice-cold methanol for 10 min at -20°C. After three washes with PBS, the samples were blocked for two hours at 37°C with

Duolink blocking buffer in a humidity chamber. Primary antibodies that were raised in two

different species were diluted in antibody diluting buffer, added to the samples, and incubated

overnight at 4°C.

Mouse anti-gD antibody (Abcam.) and rabbit anti-nectin-1 antibody (Santa Cruz) were used for gD and Nectin-1 detection, respectively (positive control). Mouse anti-gB (Abcam) and rabbit anti-AKT-1 S473 (Abcam) antibodies were used for the HSV-1 McKRae wild type and

McK gK∆31-68 infected cells to detect gB-AKT1 S473 interaction. Mouse anti-dynein antibody

against intermediate-chain I (Abcam) and rabbit anti-UL37 antibody (a gift from Frank J.

Jenkins, University of Pittsburgh Cancer Institute) were used for dynein/UL37 detection as a measure of entry in the endocytosis assay.

Unbound primary antibodies were removed by washing with 1X Tris-buffered saline

(TBS)– Tween 20 (0.05%) three times for 5 min each. Duolink Anti-Rabbit Plus and Anti-Mouse

Minus in situ proximity ligation assay (PLA) probes were added to the samples (1:5 dilution) and incubated at 37°C for one h. After the incubation step, washes were done with 1XTBST, twice for 5 min each. The ligation stock was diluted 1:5 in high-purity water and added to the wells

(40µl), and the slides were incubated at 37°C for 30 min. The slides were washed with buffer A three times for 5 min each, amplification solution (40µl) was added, and the slides were incubated for 1.5 h at 37°C. The slides were then washed with wash buffer B twice for 10 min each, and once with 0.01% buffer B. The slides were mounted with mounting medium (Duolink

II), stored at -20°C, and protected from light until confocal images were taken. The confocal images were taken using a 60X objective on an Olympus Fluoview FV10i confocal laser scanning microscope.

Immunoprecipitation and immunoblot assays. SK-N-SH cells were infected with

McKrae wild type and McK gK∆31-68 at an MOI of 10. At 1 hpi, the infected cells were lysed with NP-40 cell lysis buffer (Life Technologies) supplemented with protease inhibitor tablets

(Roche). The samples were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were then used for immunoprecipitation. The proteins from virus-infected cells were

immunoprecipitated using protein G magnetic Dynabeads according to the manufacturer’s

instructions (Invitrogen). Briefly, the beads were bound to their respective antibodies and left on

a nutator for 10 min, followed by the addition of cell lysates. The lysate-bead mixture was kept on the nutator for 10 min at room temperature and subsequently washed three times with phosphate-buffered saline (PBS). The protein was eluted from the magnetic beads in 40 µl of elution buffer and used for immunoblot assays. Sample buffer containing 5%β-mercaptoethanol was added to the protein and heated at 55°C for 15 min. Proteins were resolved in a 4 to 20%

SDS-PAGE gel and immobilized on nitrocellulose membranes. For Western immunoblots, subconfluent SK-N-SH cells were infected with the indicated viruses at an MOI of 10 for different time points as mentioned elsewhere. The cells were lysed with NP-40 lysis buffer and the lysate was used for Western immunoblots. Immunoblot assays were carried out using monoclonal mouse anti-gB antibody (Abcam), rabbit anti-Akt-1 S473 antibody (Millipore), horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies against the light chain

(Fab) and heavy chain (Fc) (Abcam, Inc., Cambridge, MA), and HRP-conjugated goat anti-rabbit antibody (Abcam, Inc., Cambridge, MA).

Calcium signaling assay. Vero and neuroblastoma (SK-N-SH) cells were grown in 96 well black clear bottom plates overnight. The cells were washed twice with PBS and incubated with a calcium indicator Fura-2AM dye solution (25uM) at 37°C for 30mins. Cells were washed

54 twice with PBS and kept on ice for 5 minutes. Then purified viruses and PBS were added on the cells and incubated at 4°C for an hour for adsorption. Then the fluorescence was measured every minute for an hour by spectramax M2 which was set at 37°C. The excitation and emission wavelengths for Fura-2AM were 340nm and 380nm and intracellular calcium concentration was measured by the ratio of bound to unbound dye following the equation mentioned elsewhere

(15).

PS detection on the cell surface. Vero cells were grown on 8 well chamber slides and adsorbed with both McKrae and gK∆31-68 viruses at 4°C for 1 hour and shifted to temperature

37°C for 5 minutes. Cells were then washed twice with 1X PBS and once with 1X Annexin

Binding Buffer. Then the cells were stained with FITC-Annexin-V for 5 minutes at room temperature. Unbound stain was washed off, and the slides were observed under a fluorescent microscope and counted.

Akt-1 S473 detection on the cell surface. Vero cells were grown on 8 well chamber slides and infected with both McKrae VP26 EGFP and gK∆31-68 VP26 EGFP viruses at 37°C, and after 30 minutes, rabbit anti-Akt-1 S473 antibody (1:100) was added under the live condition and incubated for 30 minutes at 37°C. The cells were then washed with 1X PBS and fixed with formalin and reacted with goat anti-rabbit IgG conjugated with Alexa fluor 594 (red) for 1 hour at 37°C. Cell mask deep red plasma membrane stain was added to the cells (8 µg/ml) and incubated at 37°C for a minute and washed with 1X PBS. The slides were then mounted with

DAPI containing mounting media and observed under a confocal microscope at 60X with oil.

Uninfected cells were stained for intracellular protein dynein the same way (mouse anti dynein antibody under live condition then fixed) as a negative control. Infected cells were permeabilized with methanol and reacted with rabbit anti-Akt-1 S473 and goat anti-rabbit IgG conjugated with

Alexa fluor 594 and mounted the same way with DAPI to show the intracellular Akt-1 S473.

Uninfected cells were permeabilized and stained for intracellular protein dynein the same way to show cytoplasmic dynein.

Endocytosis inhibition Assay. To asses the endocytosis inhibition, two inhibitors were used. pitstop-2 is a clathrin inhibitor, and dynasore Hydrate is a dynamin inhibitor. SK-N-SH

cells were treated with the drugs for 30 min at 37°C. Drugs had been used alone either 30µM

pitstop-2 or 80µM dynasore hydrate and in combination (30µM pitstop-2 + 80µM dynasore hydrate) . The cells were then infected with the McKrae and McKrae gKΔ31–68 at MOI 10 for 1 hour at 37°C with the drugs on. In the case of Vero cells, a series of dilution of pitstop-2 plus dynasore hydrate (80µM) was added combinedly, and then the cells were infected with McKrae and gK∆31-68 for 48 hours and plaques were counted.

Statistical analyses. Statistical analyses were performed by using ANOVA and Student's t-tests; values of P < 0.05 were considered significant. Bonferroni adjustments were applied for multiple comparisons between each treatment group and the control. All analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

2.3 Results

The gK∆31-68 mutation causes resistance to miltefosine-mediated inhibition of virus entry. Previous work has shown that Akt inhibitor miltefosine, prevents HSV entry into cells

(77). Miltefosine mimics the PH domain of Akt and blocks the recruitment of Akt to the inner leaflet of the plasma membrane (81). We tested whether Akt is important for HSV-1 entry using miltefosine. Nearly confluent monolayers of Vero cells were pre-treated for 15 min with increasing doses of miltefosine, and virus entry experiments were performed as described in

Materials and Methods. As expected, the McKrae wild-type strain was inhibited in a dose- dependent manner, with more than 90% inhibition achieved at 100 µM miltefosine. Surprisingly, the gK∆31-68 mutant entry was not inhibited (Fig. 2.1 A). The effect of miltefosine on virus entry was also tested in SK-N-SH cells using the proximity ligation assay (PLA) for virus entry

(see Materials and Methods). Briefly, PLA assay detects the deposition of capsids into the cytoplasm of infected cells by detecting the interaction of the HSV-1 UL37 protein with dynein, while cell-surface bound virions are detected via the interaction of gD with nectin-1 (82). Both

viruses attached to cell surfaces equally well, however, at the maximum concentration of

miltefosine (30 µM, since 100 µM miltefosine was toxic to SK-N-SH cells), the McKrae wild-

type virus entry was drastically inhibited, while gK∆31-68 entry was unaffected (Fig. 2.1 B).

Figure 2.1. Effect of Akt inhibitor on HSV-1 growth/entry. (A) Vero cells were treated with a series of dilutions of Akt inhibitor Miltefosine for 15 minutes and then adsorbed with McKrae and McKrae gK∆31-68 (100 PFU) for 1 hour at 4°C. After incubation at 37°C for 48 hours, plaques (PFU) were counted using crystal violet staining. * P<0.05 and ***P<0.001 vs. no drug-treated control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent 95% confidence interval about the

mean. (B) SK-N-SH cells were treated with miltefosine for 15 minutes and infected with McKrae or McKrae gK∆31-68 (MOI-10) for 1 hour at 37°C and proximity ligation assay (PLA) was performed. Confocal microscopy was used to detect bright red spots that indicate an interaction between two proteins after drug treatment. Interaction between UL37 (capsid protein)-Dynein (cellular protein) was used as a measure of entry of the virus. Interaction between gD and Nectin-1 was used as a positive control in this experiment. DAPI was used to stain the nuclei of the cells.

The gK∆31-68 mutation inhibits gB binding to Akt-1(S473). Previous work has shown that gB binds to Akt and Akt is required for virus entry (77). The ability of HSV-1 gB to bind

Akt-1(S473) specified by the gK∆31-68 mutant virus was tested using the proximity ligation assay (PLA) and two-way co-immunoprecipitation assay. We focused on the detection of Akt-

1(S473) due to the availability of highly reactive-Akt-1(S473) antibody (see Materials and

Methods). PLA using specific antibodies against Akt-1 (S473) and gB, detected close association of gB and Akt-1 (473) in McKrae-infected, but not in gK∆31-68-infected SK-N-SH cells at 1 hour post-infection (hpi) at MOI-10 (Fig 2.2 A).

Figure 2.2. Interaction between gB and Akt-1(S473). (A) Proximity ligation assay showing the interaction between gB and Akt-1(S473) in McKrae and gK∆31-68 infected SK-N- SH cells 1-hour post-infection at MOI-10. gD-Nectin-1 interaction was used as positive control and gD-Akt-1 S473 interaction was used as a negative control. Confocal microscopy was used to detect the bright red spots that suggest the interaction between two proteins. DAPI was used to stain the nuclei of the cells. (B) Two-way immunoprecipitation showing gB-Akt-1 S473 interaction in McKrae and gK∆31-68 infected SK-N-SH lysates.

(figure cont’d)

Two-way co-immunoprecipitation assays were performed using anti-gB and anti-Akt-1 monoclonal antibodies and the presence of gB and Akt-1 was detected in immunoblots of infected SK-N-SH lysates and in immunoprecipitates with either anti-gB or anti-Akt-1 antibody.

Similar amounts of gB were detected in either McKrae or gK∆31-68 infected lysates with gB appearing as two major protein species migrating with apparent molecular masses of 130 and

120 kDa representing most likely the high mannose precursor and fully glycosylated species, as we have reported previously (54, 82, 83). Similar amounts of Akt-1(S473) were detected in both

McKrae and gK∆31-68 infected SK-N-SH lysates; however, the overall levels of Akt-1 (S473) were substantially higher in infected than uninfected cells indicating that both viruses induced

Akt-1 phosphorylation equally well. Anti-gB antibody detected the presence of gB in anti-Akt-

1(S473) immunoprecipitates of McKrae-infected SK-N-SH cellular lysates at 1 hpi; however, a substantially lower amount of gB was detected in the immunoprecipitate from gK∆31-68- infected cellular lysates. Similarly, the anti-Akt-1 antibody detected Akt-1 in anti-gB immunoprecipitates of McKrae, but not gK∆31-68 infected SK-N-SH lysates (Fig. 2.2 B).

The gK Δ31-68 mutation fails to induce the release of intracellular calcium. The

concentration of intracellular free calcium regulates a variety of cellular processes, including

sperm-egg fusion, contraction, secretion, cell growth, and apoptosis (84). Previous work

indicated that HSV-1 infection triggers the release of intracellular calcium, which is required for

virus entry (77, 85). The inhibition of Akt prevents calcium release and HSV-1 entry (77). To

test whether McKrae and gK∆31-68 and viruses triggered intracellular calcium release, we

utilized the indicator Fura 2 AM that binds free calcium in the cytosol immediately following

infection. The relative amount of calcium was calculated every minute over the 1 hour of virus

entry at 37°C (see Materials and Methods). The McKrae wild-type virus triggered almost 14 and

20 times higher calcium release than the gK∆31-68 mutant virus in Vero and SK-N-SH cells, respectively (Fig. 2.3 A and 2.3 B).

Figure 2.3. HSV-1 triggers intracellular calcium release. (A) Vero cells and (B) SK-N-SH cells were grown on 8-wells chamber slides and treated with Fura-2AM for 30 minutes at 37°C. After washing, the cells were then adsorbed with purified McKrae and gK∆31-68 at 4°C for one hour and then shifted to 37°C to measure absorbance. The ratio of bound to unbound dye

(340/380nm) was measured, and intracellular calcium concentration was calculated. Ionomycin was used as a positive control, and 1X PBS was used as a negative control. ***P<0.001 vs PBS control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent the 95% confidence interval about the mean. ns= non-significant.

The gK∆31-68 mutation fails to cause phosphatidylserine (PS) and Akt-1 (S473) expression on infected Vero cell-surfaces. Intracellular calcium activates flippase (scramblase) enzymes, which function in flipping the inner leaflet to the outer leaflet of the plasma membrane

(PM). Phosphatidylserine (PS) resides on the inner leaflet of the PM (86). Therefore, the presence of PS on cell surfaces is indicative of membrane flipping (86). Although Akt is a cytosolic protein, Akt is recruited to the inner leaflet of the membrane by PIP3/PH domain binding. The PH domain of Akt is responsible for anchoring (81). Previous work suggested that the Equine Herpes Virus (EHV-1) infection of epithelial cells triggers exposure of PS from the inner leaflet of the plasma membrane to the outer surface (79). We investigated whether HSV-1

infection causes expression of PS on infected Vero cell-surfaces. Vero cells were adsorbed with

both McKrae and gK∆31-68 viruses at 4°C for 1 hour, and then infected cell cultures were

shifted to 37°C for 5 minutes. FITC conjugated Annexin V staining of PS showed that the

McKrae wild type virus infection caused expression of PS on cell-surfaces (Fig. 2.4 A).

Specifically, there was at least a 3-fold higher PS expression in McKrae infected cells compared

to gK∆31-68, as revealed by quantitative analysis of the fluorescent signals (Fig. 2.4 B).

Figure 2.4. Phosphatidylserine exposure on the cell surface. (A) Vero cells were adsorbed with either McKrae or gK∆31-68 at MOI-10 for 1 hour at 4°C and then slides were shifted to 37°C for 5 minutes. Cells were live stained with FITC conjugated Annexin-V to detect phosphatidyl serine on the surface. Fluorescent (FITC), phase contrast, and merged images of the infected and uninfected control Vero cells were presented. (B) The cells exposing FITC conjugated Annexin-V were counted per area of 100 square millimeters. Values were normalized with uninfected control. Statistical comparison was conducted by graph pad prism using students’ t-test. Bars represent the 95% confidence interval about the mean.

Both McKrae and gK∆31-68 viruses caused efficient phosphorylation of AKt-1 (S473)

(Fig. 2B), indicating that Akt-1 was efficiently associated with the inner leaflet of the plasma

membrane since Akt-1 phosphorylation occurs after Akt-1 is bound to plasma membranes (81).

The previous PS results indicated that gK∆31-68 failed to flip inner leaflet lipids to the outer membrane leaflet. We tested whether the inability of gK∆31-68 to flip PS from inner to outer membrane leaflets was also true for Akt-1. Vero cells were infected with either McKrae VP26

EGFP or gK∆31-68 VP26 EGFP at 37°C for 30 minutes. To detect the cell-surface expression of

Akt-1, infected Vero cells were reacted with rabbit anti-Akt-1 antibody under live conditions for

30 minutes at 37°C. Subsequently, cells were washed and fixed with formalin and reacted with goat anti-rabbit antibody conjugated to Alexa fluor-594. To validate the experimental conditions used to detect cell-surface expressed Akt-1, the anti-dynein antibody was reacted with Vero cells under live conditions for 30 min at 37C immediately followed by formalin fixation. As expected, the anti-dynein antibody failed to react with dynein under these conditions indicating that the anti-dynein antibody did not enter into cells during the 30 min incubation at 37C.

In contrast, the anti-dynein antibody reacted efficiently with dynein when cells were fixed with methanol. The presence of virions was detected by fluorescence emitted by the VP26-

EGFP viral protein. Whole-cell Akt-1 expression was similarly detected with anti-Akt-1 antibody after cellular permeabilization and fixing with methanol (Fig. 2.5 A-C) (see Materials and Methods). These experiments revealed that gK∆31-68 failed to translocate Akt-1 to infected cell surfaces.

Figure 2.5. Akt-1 S473 exposure on the cell surface. (A) Vero cells were infected with either McKrae VP26 EGFP or gK∆31-68 VP26 EGFP for 30 minutes at 37°C and were reacted with rabbit Anti-Akt-1 S473 antibody (1:100) under the live condition for 30 minutes at 37°C. After washing, cells were formalin-fixed and stained with Anti-rabbit antibody conjugated with Alexa fluor 594 (red) and plasma membrane stain (purple). Slides were mounted with mounting media with DAPI (cyan). (B) Vero cells were either reacted with rabbit Anti Akt-1 S473

antibody and formalin-fixed or methanol treated and reacted with rabbit Anti Akt-1 S473 antibody for 30 minutes at 37°C. Cells were stained with Anti-rabbit antibody conjugated with Alexa fluor 594 (red) and were mounted with mounting media with DAPI. (C) Vero cells were infected with either McKrae VP26 EGFP or gK∆31-68 VP26 EGFP for an hour at 37°C and treated with methanol. After washing cells were reacted with rabbit Anti Akt-1 S473 antibody for 30 minutes at 37°C and stained with Anti-rabbit antibody conjugated with Alexa fluor 594 (red). Slides were mounted with mounting media with DAPI.

The gK∆31-68 mutation forces virus entry via endocytosis. Recently, it was shown that lack of intracellular calcium release and scramblase activity caused EHV-1 to enter into cells via endocytosis instead of fusion of the viral envelope with cellular plasma membranes (79).

Therefore, we tested the ability of the endocytosis inhibitors pitstop-2 and dynasore hydrate (79,

87, 88) to inhibit McKrae and gK∆31-68 entry into SK-N-SH and Vero cells. Pitstop-2 blocks clathrin that coats the endocytic vesicle and dynasore hydrate is an inhibitor of dynamin, that creates a nick to release the endosome (87, 88). SK-N-SH or Vero cells were treated with both drugs simultaneously for 15 minutes at 37°C and infected with either McKrae or gK∆31-68 viruses at MOI of 10 or with 100 PFU for 1 hour, respectively. Virus entry was detected using

PLA in the case of SK-N-SH, as we have described previously or by counting plaque-forming units (PFU) in the case of Vero cells (82). Briefly, PLA assay detects the deposition of capsids into the cytoplasm of infected cells by detecting the interaction of the HSV-1 UL37 protein with

Dynein, while cell-surface bound virions are detected via the interaction of gD with nectin-1

(82). A similar number of McKrae and gK∆31-68 virions was attached to SK-N-SH cells as detected by PLA (Fig. 2.6 A). Treatment of cells with either pitstop-2 (30µM) or dynasore- hydrate (80µM) reduced the number of virions detected within the cytoplasm of SK-N-SH cells, while the combined use of both inhibitors (pitstop-2 (30µM)+dynasore-hydrate (80µM)) substantially reduced the number of gK∆31-68 capsids detected within SK-N-SH, but not

McKrae-infected SK-N-SH cells (Fig. 2.6 A). Similarly, when Vero cells were treated with the

same drugs and infected with both McKrae and gK∆31-68 viruses for 48 hours at 37°C, a dose- dependent reduction was observed in the number of plaques in case of gK∆31-68 but not in case of the McKrae wild type virus (Fig. 2.6 B).

Figure 2.6. Endocytosis assay. (A) SK-N-SH cells were treated with no drug or 30µM pitstop-2 or 80µM dynasore hydrate or combined (30µM pitstop-2+ 80µM dynasore hydrate) for 30 minutes at 37°C. Cells were infected with either McKrae or gK∆31-68 for 1 hour at MOI-10 at 37°C and then prepared for PLA. UL37-Dynein interaction was used as a measure of entry of

69 viral capsid into the cell, and gD-Nectin-1 interaction was used as a positive control. Confocal microscopy was used to detect the bright red spots that suggest the interaction between two proteins. DAPI was used to stain the nuclei of the cells. (B) Vero cells were treated with combined doses of pitstop-2 (10-50 µM) and dynasore hydrate (80µM) for 30 mins at 37°C and then infected with 100 PFU McKrae or gK∆31-68 for 1 hour at 37°C (w/o washing the drug off). After infection wells were washed with PBS, and methylcellulose was added. Plaques (PFU) were counted using crystal violet staining after 48 hours. *P<0.05 and ***P<0.001 vs. no drug- treated control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent the 95% confidence interval about the mean.

2.4 Discussion

HSV-1 has been shown to enter into neuronal axons exclusively via fusion of the viral envelope with the synaptic axonal membrane mediated by viral glycoprotein B (gB). This fusion event leads to the deposition of virion capsids that subsequently are transported in a retrograde manner to the neuronal cell body (2). However, HSV-1 can enter into different cells either via fusion of viral envelope with cellular plasma membranes or endocytosis of the enveloped particle into early endosomes where they can be released presumably via fusion of the viral envelope with endosomal membranes. This fusion event is facilitated by the low pH environment within endosomes that triggers gB-mediated fusion. We have recently shown that the amino terminus of

HSV-1 glycoprotein K (gK) interacts with gB and regulates gB-mediated entry into neuronal axons and subsequent infection of ganglionic neurons required for the establishment of latency

(39). Herein, we show for the first time, that virions unable to enter into neuronal axons can enter into Vero and the SK-N-SH neuronal cells via endocytosis. Moreover, we show that binding of gB to plasma membrane phosphorylated Akt-1 (S473) is associated with the ability of the virus to enter via fusion at the plasma membrane. These results identify gK as a regulator of entry via fusion of the viral envelope with cellular plasma membranes.

HSV-1 gK is required for Akt binding, intracellular calcium release,

phosphatidylserine and Akt translocation to the outer plasma membrane leaflet, and

fusion of the viral envelope with cellular plasma membranes. The mechanism by which gB mediates membrane fusion during virus entry, as well as in cell-to-cell fusion, remains enigmatic. The prevalent hypothesis is that the initial binding of gD to cellular receptors nectin-1

or HVEM triggers a conformational change in gH/gL that ultimately triggers gB’s fusogenicity

(28). Conventional fusion models call for the juxtaposition of hydrophobic segments of the

fusion protein within the apposed membrane that leads to the creation of a membrane pore,

which subsequently expands and merges the apposed membranes (29). Previous work by the

Herold laboratory showed that gB bind to phosphorylated Akt-1 (S473) during virus entry (77).

Furthermore, phosphorylated Akt was hypothesized to be flipped to the outside membrane leaflet

by scramblase that can flip the inner membrane leaflet to the outer surface. The observed

inability of the gK∆31-68 virus to induce PS and Akt-1 translocation to the outer plasma

membrane leaflets suggests that scramblase may not be activated. Presumably, scramblase is

triggered via intracellular calcium release (85). Previously, we have shown that gK binds to the amino terminus of gB and gH (54).

Furthermore, previous results with EHV-1 have indicated that gH/gL binding to integrin

αV is responsible for intracellular calcium release (79). Therefore, it is conceivable that destabilization of the gB/gH/gL/gK fusion complex may cause a lack of gH/gL triggering

intracellular calcium release. Alternatively, the amino terminus of gK may also bind cell-surface

expressed integrins causing intracellular calcium release. Additional experimentation is needed

to clarify the role of gK and other viral glycoproteins in intracellular calcium release through the

engagement of cellular receptors.

It is widely accepted that HSV-1 enters into a variety of cells either by endocytosis of

enveloped capsids and/or fusion of the viral envelope with cellular membranes. We show here

that endocytosis of enveloped virions utilizes a clathrin-mediated mechanism, while the fusion of viral envelopes with cellular plasma membranes is associated with binding to phosphorylated

Akt and intracellular calcium release (Fig. 7). It is apparent that the inability of gK∆31-68 to induce intracellular calcium release leads to inhibition of Akt-1 translocation from the inner leaflet to the outer leaflet of plasma membranes resulting in the observed lack of interaction with gB.

Figure 2.7. Model of HSV-1 entry into host cells. Schematic shows the molecules involved in HSV-1 entry via either endocytosis or fusion between the viral envelope and the cellular plasma membrane as well as the intracellular signaling that may be involved with these two entry pathways.

HSV-1 gK’s role has remained controversial over the years, largely due to the fact that gK-mutant viruses can enter into cells and replicate, albeit at highly reduced efficiencies (39).

However, there is a plethora of evidence that gK and its interacting protein UL20 play important roles in membrane fusion exemplified by the fact that the gK gene is the locus of multiple

mutations (40, 89-91) that cause extensive virus-induced cell fusion and that gK has been shown

in our laboratory to be required for virus entry into neuronal axons (59). The fact that gK and

UL20 are highly conserved among all alphaherpesviruses that have the predilection of infecting

neurons via their axonal termini suggests that gK plays a crucial role in gB-mediated fusion events.

We have hypothesized, and herein provide evidence that gK functions exclusively in mediating fusion between the viral envelope and cellular membranes, as well as in cell-to-cell

fusion. The gK∆31-68 and gK-null viruses can enter into other than neuronal axonal

compartments via endocytosis, where the low pH of endosomes can trigger fusion of the viral

envelope with endosomal membranes releasing the virion into the cytoplasm without the need of

the regulation provided by the presence of gK. HSV-1 entry into neuronal axons via fusion of the

viral envelope with synaptic axonal membranes appears to be a highly-regulated process in

which gK plays a crucial role. An obvious explanation is that this exquisite mechanism of highly

regulated virus entry is important not only for ensuring successful entry of virions into the

axoplasm of neurons, but also to induce downstream signaling events that ensure the successful

movement of virions in a retrograde manner and preparation of the neuronal cell as an optimum

host for the establishment of viral latency.

Virus-cell fusion is known to trigger innate antiviral immune responses. Specifically, it

has been reported that virus-cell fusion stimulated a type I interferon (IFN) response

characterized by expression of IFN-stimulated genes (ISGs), engagement of Toll-like receptors 7

and 9 signalings and in vivo recruitment of leukocytes (92). Apparently, intracellular calcium is induced during virus-cell fusion, as well as by fusogenic liposomes. Inhibition of calcium signaling abrogated IFN responses to HSV-1 entry (93, 94). These results suggest that virus-cell

73 fusion and subsequent calcium signaling causes a substantially different innate immune response in comparison to virus entry via endocytosis. We have recently shown that intracellular vaccination of mice with the HSV-1(VC2) attenuated virus that contains the gKD31-68 mutation elicits strong humoral and cellular immune responses that protect mice against both HSV-1 and

HSV-2 lethal, intravaginal challenge (95, 96), as well as ocular challenge with the human ocular clinical strain HSV-1(McKrae) (Naidu and Kousoulas, unpublished). It is unclear at this point, whether endocytotic entry abrogates the IFN response attributed to virus-cell fusion and the mechanisms by which this entry pathway leads to enhanced anti-HSV humoral and cellular immune responses. These topics are subject to ongoing investigations.

2.5 References

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CHAPTER 3 CELLULAR AND VIRAL DETERMINANTS OF HSV-1 TRANSPORT TOWARD NUCLEUS

3.1 Introduction Following entry, the herpes simplex virus type 1 (HSV-1) capsid travels along the cellular microtubule to reach the host nucleus where the virus delivers its genome for replication (1). The

virus uses active transport for this process since simple diffusion is not possible due to the viscosity and steric hindrance (2). The virus hijacks and modifies cellular machinery involved in cellular cargo transport and may utilize other cellular mechanisms to facilitate the transport of capsids to the nucleus (3-8). Although most of the outer tegument proteins of HSV-1 dissociate in the cytoplasm, three inner tegument proteins namely, UL36, UL37, and US3 remain attached to the capsid intracellular transport to the nucleus. Therefore, it is assumed that HSV-1 tegument

proteins along with the capsid, play significant roles in viral capsid transport (9-18).

The cell moves a variety of different cargo along with the cytoskeleton network. Although

the short-distance movement of cargo is mediated by actin and myosin, long-distance transport

(>1µM) of organelles and other targets requires ATP dependent motor proteins and accessory

factors traveling along with the microtubule networks (19-22). Recently it has been shown that

actin is capable of mediating long-distance transport of vesicles carrying different types of cargos as well (21). Microtubules are the major component of the cytoskeleton. The plus end or the growing end of the microtubule is located towards the periphery (cell membrane) of the cell and helps in cell growth and migration. Several end binding proteins (MAPs, +TIPs) aid in this process. For example, EB1 specifically recognizes the growing end of dynamic microtubule and recruits other MAPs. These molecules help binding microtubules with specific targets like cargos, cell cortex and organelles (1, 23, 24). The minus end of the microtubule is close to the

microtubule organizing center (MTOC) and pointed towards the nucleus of the host cell. Motor proteins move along the microtubule depending on the type of cargo they are carrying and the directionality of the specific motor that is utilized. For example, dynein moves along the microtubule towards the minus end (MTOC/nucleus), whereas kinesin moves towards the plus end (away from the nucleus) of the microtubules. Dynein is a multi-subunit complex having two copies of heavy chains (cytoplasmic DYNC1H1 or ciliary DYNC2H1), two intermediate chains

(DYNC1I1 and DYNC1I2), two light intermediate chains (DYNC1LI1 and DYNC1LI2) and 3 classes of light chains (Tctex-1/RP3 (DYNLT1/3, LC7/ roadblock (DYNRB1/2), and LC8

(DYNLL1/2)(25-27). The heavy chains contain ATPase activity which is required for movement along the microtubules. The intermediate chain and light chain regulate motor activity and mediate interaction with other cofactors and cargos (26-30).

Recently several viruses were shown to utilize the cellular motor protein dynein to facilitate intracellular transport. It was shown that HSV-1 capsid travels along with the microtubule network in host cells (1). During infection, HSV-1 utilizes the cellular motor protein dynein for

its capsid transport. Although it was initially evident that HSV-1 capsid directly interacts with

dynein, recent findings suggest that the HSV-1 inner tegument proteins UL36 and UL37 interact

simultaneously with the cellular motor proteins kinesin and dynein respectively and that HSV-1

capsid exhibits bidirectional or saltatory movement along microtubules (10, 14, 31, 32).

However, the kinesin or dynein domains that interact with the HSV-1 capsids are not known.

Sometimes virus transport occurs at the expense of cellular transport. For example, HSV-1

infection disrupts mitochondrial transport by altering kinesin-mediated motility in the cell (6).

For pseudorabies virus (PRV), VP1/2 (UL36 homolog), interacts with the dynein intermediate

chain (DIC) and dynactin (p150) in HEK293 cell lines, and VP1/2 alone was shown to be able to

move cargo via the microtubule network. The proline-rich sequence of VP1/2 seems to play an

essential role in the retrograde transport of PRV in neuronal cultures and in vivo (33). A

phosphoprotein specified by pseudorabies and lyssavirus binds with the dynein light chain 8

(LC8) to perform similar roles. HIV transport is also mediated by cellular motors. The depletion

of dynein heavy chains (DYNC1H1) by siRNA reduced HIV infection significantly since the

dynein heavy chain provides the actual motor function of the complex.

In contrast, depletion of other dynein components by siRNA has little to no effect on HIV infection in TZM-bl cells (34). The dynein light chain 2 (DLC-2) transports HIV integrase and

DLC-1 interacts with HIV capsid and affects reverse transcription (35). In the case of adenovirus, the hexon protein directly recruits the intermediate dynein chain (DIC) for capsid transport (36). Interestingly, the SV40 virus uniquely utilizes dynein. Upon entry via endocytosis virus ends up in the endoplasmic reticulum (ER). From the ER, the virus enters the cytosol to reach the nucleus where it replicates. During this step, SV40 recruits dynein to disassemble the particle and change the conformation of the virus. This change in conformation is essential for the virus to travel to the nucleus (8).

Several accessory proteins work along with the motor proteins to activate or inactivate and to increase or decrease the processivity of the motor complex. One major accessory protein complex is dynactin (DCTN). It is a multi-subunit complex that plays multiple roles in dynein mediated transport. Dynactin (DCTN) is a big molecule consisting of several components namely, DCTN 1 (p150), DCTN 2 (dynamitin), DCTN 3 (p24/22), DCTN 4 (p62), DCTN 5

(p25), DCTN 6 (p27), CapZ and Arp polymer. The subunit p150 binds to IC of dynein and the other end is associated with microtubules. P62, Arp polymer, and CapZ take part in cargo binding (37-40). Bicaudal D homolog 2 (BICD2) is another accessory protein that mediates

binding of cargo to dynein motor. BICD2 is associated with the cellular transport of mRNA and

organelles and helps to position the nucleus during cell division by docking at the nuclear pore. It

was also shown that HIV-1 exploits one or more dynein adaptors for efficient infection and

transport towards the nucleus. Dynein adaptor BICD2, dynactin components DCTN2, DCTN3

and ACTR1A (arp1 rod) are shown to be essential for HIV permissiveness in TZM-bl cells (34).

End binding protein 1 (EB1) was found to be essential for HSV-1 infection. Also, it was shown

that the microtubule plus end associated protein CLIP 170 initiates HSV-1 retrograde transport in primary human cells (1).

Protein kinases play an essential role in receptor signal transduction, cell cycle control, vesicle transport, cell proliferation, and survival (41-48). Some kinases directly modify motor proteins. For example, inhibition of glycogen synthase kinase (GSK3β) enhances retrograde motility, by direct phosphorylation of dynein intermediate chain (DIC)(49). GSK3β also recruits

EB1 to the plus end of microtubule which is important for the loading of cargos and viruses on the microtubule for transport (50). Another kinase, nuclear distribution protein nudE-like 1

(Ndel1), directly binds to dynein after getting activated by cyclin-dependent kinase (Cdk 5) and alters axonal transport (51). Viruses are known to alter kinase functions and activities. For example, it was shown that HSV-1 activates serine-threonine kinase (Akt) (52) and mitogen activating protein kinases (MAPK) later time post entry (53). The PI3K/ Akt pathway gets activated following HIV env-CD4 mediated fusion. HIV requires PI3K p110α/PTEN signaling during entry into T cells, PM1 cells, and TZM-b1 cells (54). Inhibition of certain cellular kinases prevents HSV entry and transport towards the nucleus. For example, Akt inhibition by miltefosine inhibits HSV-1 McKrae entry into Vero and SK-N-SH cells (55). PI3K inhibition resists HSV containing endosome trafficking (56). Some viruses modify the motor proteins via

87 activating kinases for their transport in the cytoplasm. For example, adenovirus activates protein kinase A (PKA) and p38 MAPK and is involved in phosphorylation of dynein light intermediate chain 1 (LIC-1). Adenovirus hexon then binds to the phosphorylated dynein (57). Adenoviral activation of kinases enhances the nuclear targeting possibly through the integrins (58).

In this report we investigated the role of kinases and motor accessory proteins in HSV-1 intracellular transport. We focused on the cellular kinases- MAPK, Akt, PKC, NDEL1, and motor protein dynein, accessory proteins- dynactin, EB1, BICD2, and HSV-1 capsid protein VP5 and tegument protein UL37. We found that HSV-1 triggers MAPK activation, although virus entry or capsid transport does not require this kinase activity. However, the Akt signaling pathway is required for viral capsid transport to the nucleus. PKC inhibition restricted HSV-1 entry into Vero cells. During transport, HSV-1 utilized both cellular motor proteins and accessory proteins. Specifically, HSV-1 tegument protein UL37 binds DIC early post-infection and major capsid protein ICP5 interacts with dynactin and +TIPs. Therefore, shRNA mediated silencing of the individual accessory motor proteins reduced capsid transport. Interestingly,

HSV-1 McKrae infection activates DIC early post-infection; but the activation of DIC was neither by Akt nor by adaptor protein Ndel1.

3.2 Methods and materials Cells and viruses. African green monkey kidney cells (Vero) and human neuroblastoma cells (SKNSH) were used for most of the experiments. Vero cells were grown in filter-sterilized

DMEM media supplemented with 10% FBS and primocin. SKNSH cells were grown in filter- sterilized EMEM media supplemented with 10% FBS and primocin. All cells were incubated at

37°C with 5% CO2. The pathogenic strain of HSV-1 (McKrae GFP) virus was used throughout the experiments.

Antibodies and reagents. Antibodies against alpha-tubulin, HSV-1 ICP5, HSV-1 gD,

HSV-1 gB, intermediate dynein chain (DIC), dynein heavy chain (DHC), Dynactin 1/p150

(DCTN 1), dynactin 2/dynamitin (DCTN 2), dynactin 3 (DCTN 3) were bought from Abcam biotechnology. Antibody against end binding protein 1 (EB1), HSV-1 VP5, was bought from

Santa Cruz biotechnology. Antibody against total NDEL 1 and phospho NDEL 1 was bought from Antibodies.com. HSV-1 UL37 antibody was a gift from Frank J. Jenkins, University of

Pittsburgh Cancer Institute. MAPK family antibody sampler kit was collected from cell

signaling. Akt inhibitor miltefosine (sc-203135) was bought from Santa Cruz biotechnology.

MAPK inhibitor PD 98059 was bought from Santa Cruz, and PKC inhibitors STS and Goml

were bought from Abcam biotechnology.

MAP kinase activation assay. Vero cells were grown in a 12 well plate and starved overnight. HSV-1 McKrae was adsorbed at 4°C for 1 hour at an MOI of 10 and then transferred

to 37°C. Lysates were collected at 0 min, 15 min, 30 min, 1hr, 2hr and 3 hours using NP40 lysis

buffer + protease inhibitor. The lysates were analyzed by western blot and were detected for

MAPK activation using antibody against phosphorylated ERK1/2, SAPK/JNK and p38 MAPK

(cell signaling #9910 phospho- MAPK family antibody sampler kit). For control, total ERK 1/2,

total SAPK/JNK, and total p38 were measured the same way.

Entry Assay. Vero cells were starved overnight and then treated with MAPK inhibitor PD

98059 (Santa Cruz) for 30 min at 37°C. Cells were adsorbed with HSV-1 McKrae at 4°C at 100

PFU for 1hr. After that, media was removed, and methylcellulose was added on top before incubation at 37°C. The number of plaques was counted after 48 hours.

Retrograde transport Assay. SKNSH cells were grown in eight well chamber slides and

starved overnight. Cells were then adsorbed with HSV-1 McKrae at 4°C at MOI of 20. Then the

slides were shifted to 37°C for 15 minutes and then washed with a low pH buffer. The cells were

treated with inhibitors for 5 hours at 37°C. After that, the slides were fixed in formalin and

permeabilized with ice-cold methanol followed by blocking with goat serum. Antibody against

ICP5 was used to detect viral capsid transport to the nucleus, which was stained with DAPI.

Images were taken using a 60x objective on a fluorescent microscope.

Immunoprecipitation and immunoblot Assays. SK-N-SH cells were infected with

McKrae wild type and McK gK∆31-68 at an MOI of 10. At one-hour post infection, the infected cells were lysed with NP-40 cell lysis buffer (Life Technologies) supplemented with protease inhibitor tablets (Roche). The samples were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were then used for immunoprecipitation. The proteins from virus-infected cells were immunoprecipitated using protein G magnetic Dynabeads according to the manufacturer’s instructions (Invitrogen). Briefly, the beads were bound to their respective antibodies and left on a nutator for 10 min, followed by the addition of cell lysates. The lysate-bead mixture was kept on the nutator for 10 min at room temperature and subsequently washed three times with phosphate-buffered saline (PBS). The protein was eluted from the magnetic beads in 40 µl of elution buffer and used for immunoblot assays. Sample buffer containing five percent β- mercaptoethanol was added to the protein and heated at 55°C for 15 min. Proteins were resolved in a 4 to 20% SDS-PAGE gel and immobilized on either nitrocellulose or PVDF membranes. For

Western immunoblots, subconfluent SK-N-SH cells were infected with the indicated viruses at an MOI of 10 for different time points, as mentioned elsewhere. The cells were lysed with NP-40 lysis buffer, and the lysate was used for Western immunoblots. Immunoblot assays were carried

90 out using rabbit anti-Akt-1 S473 antibody (Millipore), rabbit anti-Akt 1/2/3 antibody (Millipore), rabbit anti-MAPK family antibodies (cell signaling), rabbit anti-dynein intermediate chain phosphorylation (DIC 1B S80A) antibody (from Dr. Kevin Pfister laboratory), mouse anti DIC antibody (Abcam), horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies against the light chain (Fab) and heavy chain (Fc) (Abcam, Inc., Cambridge, MA), and HRP-conjugated goat anti-rabbit antibody (Abcam, Inc., Cambridge, MA).

Lentivirus shRNA mediated gene silencing. shRNA against dynein heavy chain (DHC),

BICD2, dynactin 2 (DCTN 2) dynactin 3 (DCTN 3), and end binding protein 1 (EB1) were used and delivered in lentiviral vectors to knockdown these proteins in SKNSH cells. The level of expression of these proteins was checked simply by western blot analysis of transduced cell lysates. Gene silencing by shRNA was done by following the protocol provided by Santa Cruz biotechnology. Briefly, cells were grown in complete media (serum and antibiotic) in 12 well plates. When they are approximately 50% confluent, they are ready for lentiviral transduction.

To increase the binding between pseudo viral capsid and the cellular membrane, a compound called polybrene (sc-134220) was used at a final concentration of 5µg/ml of complete medium.

The media was removed from the plate and replaced with 1ml of polybrene media per well.

Lentiviral particles were thawed at room temperature and mixed gently before use. The cells were infected by adding shRNA lentiviral particles to the culture. Following overnight incubation, at 37°C, the medium was removed and replaced with 1 ml of complete medium

(without polybrene). To select stable clones expressing the shRNA, cells were split from 1:3 to

1:5 depending on the cell type, and puromycin hydrochloride was used in the media as a selection agent. A puromycin titration was done before to determine the sufficient amount to kill

the non-transduced cells. For Vero cells 15µg/ml and for SKNSH cells, 10µg/ml puromycin was used to kill non-transduced cells.

Drug toxicity test. Vero cells were grown in 12 well plates and starved overnight before the drug test. Drugs were dissolved in DMSO, and the dilutions were made in DMEM without serum. Cells were treated with different dilutions of drugs and incubated at 37°C for 24 hours.

Each dilution was tested in triplicates. The percentage of live cells was quantified by using trypan blue. The counts from treated wells were compared with the no drug-treated wells.

3.3 Results

3.3.1 Herpes simplex virus type 1 (HSV-1 McKrae) activates MAP kinases (MAPK).

Previously it was shown that HSV-1 triggers MAPK pathway late post-infection (after 3

hours)(53). However, it was unclear if HSV-1 activates MAPK soon after entry or during capsid

transport to the nucleus. Recently, we showed that HSV-1 activates the Akt pathway as early as 5 minutes post-infection and pharmacologic inhibition of Akt blocks HSV-1 entry in epithelial

cells (52, 59). Similarly, PI3K inhibition blocks HSV-1 capsid transport to the nucleus (60).

Both PI3K and Akt are upstream of MAPK. Therefore, we investigated the role of MAPK in

HSV-1 infection during early times post-infection. Following HSV-1 McKrae infection of Vero

cells, we observed an increase in phosphorylated MAPK indicating that the MAPK pathway was

activated by viral entry into cells. MAPK activation occurs as early as 15 minutes post-infection

by HSV-1 McKrae (Fig. 3.1). Since binding of the virus to the cell did not trigger the pathway,

indicating that HSV-1 entry (membrane fusion) is required for the activation of the pathway (Fig

3.1).

Figure 3.1. MAPK activation by HSV-1. HAV-` (McKrae) was adsorbed onto starved Vero cells at 4°C for 1 hour at MOI 5 and then infected cell cultures were shifted to 37°C for 15 m, 30 m, 1 hr, 2 hrs, and 3 hrs. Cells were lysed with NP40 lysis buffer in the presence of protease inhibitor cocktail and analyzed for the presence of phosphorylated ERK1/2, phosphorylated SAPK/JNK and phosphorylated p38 MAPK by Western blot. Total ERK, Total SAPK/JNK, and Total p38 MAPK were used as a loading control.

3.3.2 MAP kinases (MAPK) are not required for herpes simplex virus type 1 (HSV-1

McKrae) entry and transport towards the nucleus. Recently it was shown that adenovirus

activates PKA and MAPK pathways that facilitate intracellular transport toward the nucleus (58).

To determine if HSV-1 McKrae requires MAPK for successful infection, we tested whether the

MAPK inhibitor PD 98059 had any effect in HSV-1 McKrae entry and capsid transport towards

the nucleus. Starved Vero cells were treated with the drug at different dilutions (10 µM – 90 µM)

for 30 minutes. HSV-1 McKrae was added at 100PFU per well, and the efficiency of viral entry

was quantified by plaque assay. PD98059 failed to exhibit any effect on virus entry (Fig. 3.2).

Figure 3.2. Effect of MAPK inhibitor PD 98059 on HSV-1 entry. Vero cells were treated with a series of dilutions of MAPK inhibitor (PD 98059) for 30 minutes and then adsorbed with McKrae (100 PFU) for 1 hour at 4°C. After incubation at 37°C for 48 hours, plaques (PFU) were counted using crystal violet staining. Ns vs. no drug-treated control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent 95% confidence interval about the mean.

Next, we tested whether the MAPK inhibitor PD 98059 could inhibit intracellular virus transport in SK-N-SH cells. The virus was adsorbed to the cell for an hour and shifted to 37°C for 15 minutes to let the virus enter inside the cell before treatment with the drug at different concentrations (10 µM – 50 µM) was conducted. Cells were washed with low pH buffer to remove the attached virus particle, which did not enter the cell in 15 minutes. Both untreated control and treated wells show replication of HSV-1 McKrae wild type virus in the nucleus following 5 hours post-infection. The major capsid protein ICP5 was stained with Alexa fluore

647 (red), and the nucleus was stained with DAPI (blue), as shown in Figure 3.3. Our results suggest that MAPK is not required for HSV-1 McKrae entry or transport in epithelial or neuroblastoma cell lines. However, activation of MAPK by HSV-1 may have a role in the regulation of motor proteins and other kinases that is needed for optimal viral replication.

Figure 3.3. Effect of MAPK inhibitor on HSV-1 transport. SK-N-SH cells were adsorbed with HSV-1 McKrae at MOI 20 for 1 hour at 4°C then shifted to 37°C for 15 minutes and then washed with low pH buffer. MAPK inhibitor PD 98059 was added and incubated for 5 hours at 37°C. The eight well chamber slide was then fixed with formalin, permeabilized with methanol and prepared for fluorescent microscopy. Antibody against ICP5 (red) was used and nuclei were stained with DAPI (blue). Capsids colocalized in the nuclei are purple (red+blue).

3.3.3 Akt is required for herpes simplex virus type 1 (HSV-1 McKRae) transport

towards the nucleus. Previously, we showed that Akt inhibition blocks HSV-1 McRae entry into epithelial and neuroblastoma cell lines (59). To determine if Akt has a role in HSV-1

intracellular transport, SK-N-SH cells were treated with Akt inhibitor miltefosine at different

concentrations (10µM, 30µM, 50µM, and 70µM) at 37°C and intracellular transport was

monitored by immunofluorescence microscopy. Before the drug treatment, cells were adsorbed with HSV-1 McKRae (MOI 20) for 1 hour at 4°C and then shifted to 37°C for 15 minutes to let

95 the virus get into the cells. Following a low pH buffer wash, the cells were incubated with the drugs for 5 hours and prepared for immunofluorescence. As shown in Figure 3.4, after 5 hours post-infection, HSV-1 McRae travels into the nucleus and starts replication in the untreated control well. The HSV-1 major capsid protein ICP5 (red) was visible inside the nucleus (blue).

However, viral capsids were stuck in the cytoplasm in Akt inhibitor-treated wells. This result suggests that Akt is required for both HSV-1 McRae entry and efficient transport towards the nucleus.

Figure 3.4. Effect of Akt inhibitor on HSV-1 transport. SK-N-SH cells were adsorbed at 4°C with HSV-1 McKRae at MOI 20 for 1 hour then shifted to 37°C for 15 minutes and then washed with low pH buffer. Akt inhibitor miltefosine was added at 30µM and incubated for 5 hours at 37°C. The 8 well chamber slide is then fixed with formalin, permeabilized with methanol, and prepared for fluorescent microscopy. Antibody against ICP5 (red) was used, and nuclei were stained with DAPI (blue). Capsids colocalized in the nuclei are purple.

3.3.5 Protein kinase C (PKC) is required for herpes simplex virus type 1 (HSV-1

McKRae) entry. Protein kinases play multiple roles in cellular transport. Protein kinase C has specific roles in retrograde axonal transport of cargoes. Our previous results indicated that Akt, which is a serine-threonine kinase, is required for HSV-1 entry and transport towards the nucleus. Miltefosine blocks both Akt and PKC enzymatic activities. Therefore, we investigated the role of PKC in HSV-1 entry and transport into Vero cells to determine whether the inhibitory effect was due to Akt or PKC. There was a dose-dependent reduction in the entry of HSV-1

McKrae in Vero cells following treatment with either (a highly specific PKC inhibitor) or staurosporine (STS, abroad spectrum PKC inhibitor). Maximum inhibition was observed at

90µM (gouml) and 0.5µM (STS) concentrations respectively (Fig.3.5).

Figure 3.5. Effect of protein kinase C in HSV-1 McRae entry in Vero cells. Vero cells were treated with a series of dilutions of PKC inhibitor (Gouml) or the protein kinase inhibitor (staurosporine STS) for 4 hours and then adsorbed with McKrae (100 PFU) for 1 hour at 4°C. Cells were washed, and methylcellulose was added. After incubation at 37°C for 48 hours, plaques (PFU) were counted using crystal violet staining. ***P<0.001 vs. no drug-treated control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent 95% confidence interval about the mean.

3.3.6 HSV-1 McRae activates dynein. Dynein is the major cellular motor protein that carries cargo towards the nucleus (28). Activation of the motor complex, as well as activation of regulatory molecules, is essential for any cellular cargo to be transported to the correct location

(40, 61). Many viruses were shown to activate motors directly (7, 8, 10, 12, 32, 33, 36, 62-64). It was shown that HSV-1 tegument proteins bind both retrograde and anterograde motors simultaneously while moving along the microtubules (9, 10, 65). It was unclear how HSV-1 recruits cellular motors and exploit them to carry its capsid to the nucleus where the virus replicates its genome. We observed that HSV-1 activates dynein by phosphorylating the intermediate chain 1B as early as 15 minutes post-infection (Fig. 3.6). The phosphorylation pattern is 15 minutes post-infection to 3 hours post-infection which is approximately the time for

HSV-1 to travel from the membrane after entry to the nucleus (56).

Figure 3.6: Dynein activation by HSV-1. Starved Vero cells were adsorbed with McKrae at 4°C for 1 hour at MOI 5 and then shifted to 37°C for 15 m, 30 m, 1 hr, 2 hrs, and 3 hrs. Cells were lysed with NP40 lysis buffer with protease inhibitor cocktail and analyzed for phosphorylated dynein intermediate chain (DIC 1BS80A) by Western blot. The total dynein intermediate chain (DIC) was used as the loading control.

3.3.7 Effect of Akt in dynein activation by HSV-1 McRae. Previously we showed that

HSV-1 activates Akt and MAPK pathways at early times post-infection. We assume that the Akt pathway is upstream of dynein activation and plays a role in activating dynein for the capsid motility. To investigate if HSV-1 McRae activates dynein via the Akt pathway, miltefosine was used to treat cells before and after virus entry (Fig. 3.7) and the resultant dynein phosphorylation pattern was monitored. Miltefosine-mediated inhibition of virus entry also blocked dynein phosphorylation (at 75 uM, 100 uM miltefosine). However, once the virus entered the cell, blocking Akt did not inhibit the activation of dynein by HSV-1 McKrae (Fig. 3.7).

Figure 3.7: Role of Akt in dynein activation by HSV-1. McRkae virus was adsorbed onto Starved Vero cells at 4°C for 1 hour at MOI 5 and then shifted to 37°C for 1 hour. Cells were lysed with NP40 lysis buffer with protease inhibitor cocktail and analyzed for phosphorylated dynein intermediate chain (DIC 1BS80A) by Western blot. (A). Cells were pre-treated with miltefosine before the virus was added. (B) Following adsorption cells were incubated at 37°C for 15 minutes, then miltefosine was added and incubated for 1 hour. (C) Effect of miltefosine on Akt phosphorylation

3.3.8 Ndel1 activation by HSV-1 McRae. Kinases play important role in cargo movement along microtubules (66, 67). Some motors have specific adaptors which regulate the activity and processivity of the motor complex (61). Lis1 and Ndel1 are adaptors that directly regulate dynein

(68). Activation of Ndel1 by Cdk5 regulates dynein activity (69-71). We investigated whether

HSV-1 McRae infection activates dynein by activating Ndel1. SK-N-SH cells were infected with HSV-1 McRae at MOI-5 for 5m, 15m, 30m, 1hour (data not shown) and 2 hours at 37°C and the lysates were analysed by western blot. There was no detectable increase in the phosphorylation of Ndel following HSV-1 McRae infection (Fig. 3.8).

Figure 3.8: Activation of Ndel1 by HSV-1. Vero cells were adsorbed with McKrae at MOI 5 at 4°C for 1 hour and then shifted to 37°C for 2 hours. Cells were lysed with NP40 lysis buffer with protease inhibitor cocktail and analyzed for phosphorylated Ndel1 by Western blot.

3.3.9 RNA interference by shRNA. To determine the role of motor proteins and adaptors in HSV-1 McRae intracellular transport towards the nucleus, we generated several knockout cell lines using the lentivirus-based expression of specific shRNAs (Santa Cruz, Inc). We used shRNA against dynein heavy chain (DHC), dynactin 2 (DCTN2), dynactin 3 (DCTN3), end binding protein 1 (EB1), and BICD2 and made the knockouts in Vero and SK-N-SH cells. GFP control shRNA was used to determine the efficiency of lentiviral infection in both cell lines (Fig.

3.9A). The vector also contains the puromycin resistance gene for positive clone selection. A

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toxicity test for puromycin was done for both Vero and SK-N-SH cell lines and 10 µg/ml and 15

µg/ml were efficient for selecting knocked out cells in SK-N-SH and Vero cell lines respectively

(Fig. 3.9B).

Figure 3.9: shRNA mediated knock-out cell lines. (A) Vero and SK-N-SH cells were infected with lentivirus carrying control-GFP. (B) Puromycin toxicity assay on Vero and SK-N- SH cells. (C) Lentivirus shRNA (human) mediated knockouts in SK-N-SH cells.

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3.3.10 Role of motor adaptors in HSV-1 McRae intracellular transport. Previously, it was shown that HSV-1 capsid transport requires EB1 (1). Recently, it was shown that BICD2 is

essential for HIV infection (34). Dynein heavy chain is required for the motor function of the

dynein complex (28, 72). We investigated HSV-1 McKrae intracellular transport in the EB1,

BICD2, DCTN2, DCTN3, and DHC knockout Vero and SK-N-SH cells. Since the shRNA we

used were of human origin, the production of the targeted proteins was lower in SK-N-SH cells

(human neuroblastoma cell line) (Fig. 3.9C) than in Vero (African green monkey cell line) (data

not shown). Dynein heavy chain knockout SK-N-SH cells did not survive. However, there was a marked reduction in the number of the virus particles in the nucleus visible in EB1, BICD2,

DCTN2, and DCTN3 knockout SK-N-SH cells 5 hours post-infection with HSV-1 McKrae compared to the control shRNA treated SK-N-SH cells as shown in the immunofluorescent images in Figure 3.10. HSV-1 McRae travels into the nucleus and starts replication in the untreated control well. The HSV-1 major capsid protein ICP5 (red) was visible inside the nucleus (blue). However, viral capsids were stuck in the cytoplasm in the shRNA treated wells.

For further confirmation, transmission electron microscopy was performed on EB1, BICD2 and

DCTN3 knockout cells post HSV-1 McKrae infection, and similar results were observed (Fig.

3.11-3.14).

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Figure 3.10. Role of motor adaptors in HSV-1 transport. shRNA treated SK-N-SH cells were infected with HSV-1 McKrae at MOI 20 for 5 hours at 37°C. The 8 well chamber slide is then fixed with formalin, permeabilized with methanol, and prepared for fluorescent microscopy. Antibody against ICP5 (red) was used, and nuclei were stained with DAPI (blue). Capsids colocalized in the nuclei are purple.

Figure 3.11. Transmission Electron micrograph (TEM) of HSV-1 McKrae infected control shRNA treated SK-N-SH cells. Following 5 hours post-infection virus traveled to the nucleus, and new viral capsids were seen in the nucleus (cytoplasm = C, nucleus = N)

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Figure 3.12. Transmission Electron micrograph (TEM) of HSV-1 McKrae infected EB1 shRNA treated SK-N-SH cells. Following 5 hours post-infection enveloped HSV-1 virion was seen on the plasma membrane, HSV-1 capsid with tegument was seen in the cytoplasm, but no new viral capsids were seen in the nucleus (cytoplasm = C, nucleus = N)

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Figure 3.13. Transmission Electron micrograph (TEM) of HSV-1 McKrae infected BICD2 shRNA treated SK-N-SH cells. Following 5 hours post-infection HSV-1 capsid with tegument was seen in the cytoplasm close to the membrane (A, C), in endosome (B), in the cytoplasm away from the membrane (D) and new viral capsids were seen in the nucleus (in one cell only). (cytoplasm = C, nucleus = N)

Figure 3.14. Transmission Electron micrograph (TEM) of HSV-1 McKrae infected DCTN3 shRNA treated SK-N-SH cells. Following 5 hours post-infection HSV-1 capsid with tegument was seen in the cytoplasm close to the membrane (cytoplasm = C, nucleus = N)

105

3.3.10 Role of HSV-1 tegument and capsid in motor-adaptors mediated transport. It was shown that the viral particle directly interacts with motor proteins for intracellular transport.

Adenovirus directly binds to dynein via hexons, rabies virus, lyssavirus directly interact with dynein light chain with viral phosphoprotein, HSV-1 capsid was shown to interact with EB1 and dynein, HSV-1 inner tegument protein UL37/UL36 was shown to be associated with dynein and kinesin motors (8-10, 12, 32, 36, 63). However, during HSV-1 transport towards the nucleus which motor proteins and viral proteins take part is still unclear. We demonstrate here that viral tegument protein UL37 and the major capsid protein VP5 interact with dynein intermediate chain and dynactin (p150 and p62), and the microtubule-binding protein EB1. Two-way immunoprecipitation and immunofluorescence data suggest that HSV-1 UL37 interacts with dynein intermediate chain (DIC) following 2-hour infection at MOI 10 in SK-N-SH cells (Fig.

3.15 A, 3.15 C). Immunoprecipitation results also suggest a possible interaction between HSV-1 capsid and dynactin and EB, but not between HSV-1 capsid and HSV-1 tegument proteins following 2-hour infection at MOI 10 in SK-N-SH cells (Fig. 3.15 B).

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Figure 3.15. Two way immunoprecipitation showing (A) UL37-dynein intermediate chain (DIC) interaction (B) VP5-p150, VP5-p62 and VP5-EB1 interaction in HSV-1 McKrae infected SK-N-SH lysates. (C) UL37-DIC colocalization in McKrae infected SK-N-SH cells. The 8 well chamber slide was fixed with formalin, permeabilized with methanol, and prepared for fluorescent microscopy. Antibody against DIC (red) and UL37 (green) were used, and nuclei were stained with DAPI (blue).

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3.4 Discussion

We have previously shown that the N terminal 38 amino acids in glycoprotein K (gK) play an important role in virus entry via fusion of the viral envelope with cellular membranes. During entry into Vero or SK-N-SH cells, Viral glycoprotein B (gB) interacts with Akt and triggers intracellular calcium mobilization. HSV-1 McKrae entry via fusion of the viral envelope with cellular membranes induced flipping of the inner leaflet of the plasma membrane to the outer side, exposing Akt on cell surfaces. We showed that the presence of HSV-1 McKrae gK was necessary for these events. The salient features presented herein is that the Akt signaling pathway is involved in both virus entry and intracellular transport of virion capsids to the nucleus. Our results suggest that dynein phosphorylation via an Akt independent pathway, as well as direct interaction of capsid and tegument proteins with the dynein motor machinery, are required for efficient intracellular transport toward the nucleus (Fig. 3.16).

HSV-1 McKrae infection activates several signaling pathways, for example, calcium signaling, MAPK, and PI3K/Akt pathways. HSV-1 McKrae activated MAPK pathways as early as 15 minutes post entry. However, virus entry or capsid transport along microtubules were not dependent on the MAPK pathways, since a specific MAPK inhibitor did not prevent HSV-1

McKrae entry and replication. In contrast, the Akt pathway was found to be essential for HSV-1

McKrae entry into Vero and SK-N-SH cells as well as intracellular capsid transport in both types of cells. Our results also suggest that HSV-1 McKrae entry and replication significantly depended on PKC activity. The fact that both Akt and PKC inhibitors prevented both virus entry and intracellular transport suggest that both enzymes are essential in modulating both events.

Besides, it is possible that phosphorylation of the same or different viral and cellular proteins by

these two enzymes are differentially involved in virus entry and intracellular transport.

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The role of dynein, kinesin and additional proteins in intracellular virus transport is well

known. Recently it was shown that herpes virus uses dynein, dynactin and the EB1 cofactor for transport towards the nucleus along microtubules following the entry into host cells. Herein, we show for the first time that the HSV-1 McKrae major capsid protein ICP5 (VP5) physically interacts with dynactin (p150 and p62) and EB1 and HSV-1 McKrae tegument protein UL37 interacts with DIC early post-entry by two-way immunoprecipitation assay and colocalization assays. Absence of dynactin (DCTN 2 and DCTN3), EB1, and BICD2 adaptors significantly reduced virus transport following entry compared to the mock infection. We also showed that

HSV-1 McKrae activates DIC, but activation of DIC is not dependent on Akt or Ndel 1 activation by HSV-1.

HSV-1 infection causes rapid dynein phosphorylation that is not mediated by either Akt or

PKC since miltefosine which inhibits both enzymes allowed dynein phosphorylation. Therefore,

dynein must be phosphorylated by a different cellular or viral kinase. It is possible that the viral

kinase encoded by the US3 gene, which is a structural component of the virus tegument may be

directly involved in dynein phosphorylation. US3 is one of three tegument proteins that remain

attached to the capsid during intracellular capsids transport toward the nucleus in close proximity

to the dynein motor machinery. Future work will deal with whether the US3 protein is involved

in dynein phosphorylation and the facilitation of intracellular virion transport.

In this work, we show for the first time that the major capsid protein directly interacts with

dynactin and the EB1 cofactor which has been shown to facilitate HSV-1 transport by helping to

load viral capsids onto microtubules, The involvement of dynactin in intracellular transport is further supported by the fact that the dynactin cofactors BICD2 is required for efficient intracellular transport. The tegument protein UL37 appears to interact with the dynein

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intermediate chain (DIC), which is phosphorylated after HSV-1 infection. It is not clear at this

point whether DIC phosphorylation is required for interaction with the UL37 protein.

Overall, these results show that virus entry and intracellular capsid transport may be

functionally linked through the action of cellular and viral kinases that potentially regulate

interactions of viral tegument and capsid proteins with the dynein motor machinery including

dynactin and its accessory cofactors. Elucidation of the nature of these kinases and the specific

interactions that are involved may provide new ways to block both virus entry and intracellular

transport.

Figure 3.16. Model of HSV-1 transport into host cells. Schematic shows the molecules involved in HSV-1 transport, as well as the intracellular signaling that may be involved with this process. (Adapted from Ralph et al., 2017)

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CHAPTER 4 CONCLUDING REMARKS

4.1 Introduction

In this dissertation, we presented possible mechanisms by which Herpes Simplex Virus

Type-1enters into the host cells. In chapter 2, we showed that modification in one of the glycoproteins on the surface of the HSV-1 modifies the entry mechanism of the virus and forces the virus to enter into host cells via an alternate route. In chapter 3, we investigated how HSV-1

travels along the microtubules to reach the nucleus where the virus replicates. We focused on the

cellular motors, adaptor proteins, some regulatory kinases and determined their possible roles in

HSV-1 transport towards the nucleus. The following are the results of my current research and

future research plans.

4.2 Summary of the results

In chapter 2, we have elucidated the role of glycoprotein K (gK) in Herpes Simplex Virus

Type-1 entry. Previously, we have shown that the deletion of the 38 amino acids from the N- terminal end of the gK made the virus unable to enter through the axon of the neurons. However, the mechanism behind this phenomenon was unknown. In this project, we showed for the first time that modification in gK force the virus to enter into host cells via endocytosis instead of fusion of the envelope with the host plasma membrane. Here we showed that HSV-1 infection triggers the release of intracellular calcium and express Akt, a serine-threonine kinase, on the surface of the host cell. However, the deletion of 38 amino acids from the N-terminal end of the gK modifies the gB mediated fusion machinery in such a way that the mutant virus can not trigger the release of calcium from the intracellular source and cannot express Akt on the surface.

These two phenomena probably play an important role in fusion mediated entry and hence, the

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mutant virus is unable to enter the axonal terminals of neurons, as the dogma is, HSV-1 enters into axons only by the fusion of the envelope with the cell membrane.

In chapter 3, we further analyze the transport mechanism that how HSV-1 travels in the cytoplasm to enter into the nucleus of the host cell, where the virus replicates its genome. We showed that both tegument and capsid proteins interact with cellular motor and adaptor proteins during this process. HSV-1 recruits dynein, a major minus end-directed motor protein, either directly or by other unknown proteins and HSV-1 phosphorylates dynein intermediate chain

(DIC) early time post-infection. We showed that the inner tegument protein UL37 physically interacts with the DIC. It is still unclear how HSV-1 phosphorylates DIC. Our project also shows a possibility that the major capsid protein ICP5 may interact with components of the adaptor protein dynactin. Following silencing several motor adaptor proteins, namely, BICD2, EB1,

DCTN2, and DCTN 3 in the neuroblastoma cell line, we observed a reduction in viral capsid transport towards the nucleus of the host cell, compared to the control cell line. In this project, we also investigated the roles of kinases during HSV-1 transport. We showed that HSV-1 activates the MAP kinase pathway early post-infection and transport of HSV-1 towards the nucleus is dependent on the Akt and PKC pathway.

4.3 Future direction

We have used epithelial cell lines and neuroblastoma cell lines for most of our experiments. We believe that the effect of the treatments would be greater and more profound if we have used primary neurons or in vivo animal models. It will be difficult to use in vivo model for most of our experiments; however, we plan to use human primary neurons and replicate our experiments. For this, we plan to use human induced pluripotent stem cells (iPSC) and differentiate them into neurons, which will grow long exons as shown by other laboratories. We

120 will be determining the effect of several kinase inhibitors along with the ones we have already used. Pharmacological inhibitors have non-specific targets. That is why other methods like RNA interference should confirm the effect of the inhibitors. We are trying to develop CRISPR- Cas9 based methods to knock-out cellular transcripts to determine the role of cellular motors and adaptor proteins in HSV-1 transport. We are also interested in the inner tegument proteins of the virus, particularly, UL37 and US3. It is known that US3 is a virus-encoded kinase, and US3 resides close to the capsid during the transport toward the nucleus. We believe that the HSV-1 capsid carries this kinase along with the capsid during transport for its benefit. In the future, we plan to make a US3 knockout HSV-1 to see the effect of the kinase in retrograde transport of the virus in neurons.

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APPENDIX A. ADDITIONAL WORK

A.1 The role of UL37 in HSV-1 retrograde transport in primary neurons

UL37 is conserved among all herpesviruses and it is an essential tegument protein

required for virus growth and virion envelopment (1-3). It is a 120 KDa protein consisting of

1123 amino acids. UL37 forms a complex with UL36, another tegument protein of herpesviruses

(Fig.A.1) (4-6). The protein complex between UL37 and UL36 homologs is conserved among all

three subfamilies of herpesviruses and it is documented for herpes simplex virus (HSV-1), pseudorabies virus (PRV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and

Kaposi’s sarcoma-associated herpes viruses (KSHV) (7-9). Following the entry into host cells herpesviruses lose their outer tegument proteins in the cytoplasm, while three inner tegument proteins including UL36, UL37 and US3 remain attached to the capsid during capsid transport towards the nucleus where the virus replicates its DNA (2, 10). Each of these three inner tegument proteins has enzymatic activities. UL37 has deamidase activity in its C-terminal end, but this activity is dispensable for retrograde transport (11, 12). UL36 has deubiquitinase activity in its N-terminal end (13, 14). US3 is a serine-threonine kinase and involved in virus envelopment and glycoprotein K (gK) distribution (15-17). According to the current literature, the UL36/UL37 complex or its homologs are involved in herpesvirus transport (2, 18).

Specifically, it was shown that PRV utilizes cellular proteins dynein and kinesin during retrograde transport, whereas it utilizes the only kinesin during anterograde transport in peripheral nervous system (PNS) neurons (19). In the case of both HSV-1 and PRV, it was shown that a surface region of UL37 (R2) is essential for neuroinvasion and retrograde transport

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in both cell culture and in vivo (1). UL37 is phosphorylated by cellular kinases, as it was shown that if recombinant UL37 is expressed in the vaccinia virus system it still gets phosphorylated.

Figure A.1. Schematic of HSV-1 structure showing the respective location of tegument proteins.

The schematic in Figure A.2 A describes the UL37 protein structure. The C terminal end of UL37 has tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding domain

(aa 1099-1104) (4) responsible for NFκβ activation (20) and dystonin interaction domain (aa

578-899) involved in microtubule stabilization and transport (21). UL37 interacts with UL36 via the C terminal domain as well; specifically, D631 residue mediates this binding (4, 22). The N terminal of UL37 has self-association domains (aa 1-300, 568-1123), leucine-rich nuclear export signal (NES) (aa 263-273), alanine-rich region (ARR) (aa 44-80) and a leucine zipper motif (aa

203-224) (18, 23). The central portion of the UL37 is not very well defined. The sequence alignment of UL37 among alphaherpesviruses revealed some conserved regions in the central portion. Our laboratory recently showed that replacement of tyrosine residues from positions 476 and 477 by alanine does complement for UL37 null virus but does not complement for the same if the tyrosine at positions 474 and 480 are replaced by alanine. The quadruple mutant virus, having the replacement of all four tyrosine by alanine at positions 474, 476, 477, and 480,

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showed gK null phenotype, as evident by smaller plaque size and accumulation of capsids in the

nuclear periphery in the infected cells. These mutations also reduced the interaction between gK

and UL37 in infected cells (24).

In this report, we aimed to determine the role of UL37 in HSV-1 entry and retrograde transport in epithelial and primary neurons. The HSV-1 (F) UL37 ∆TRAF6 mutant virus has a deletion of six amino acids in the TRAF6 binding domain of UL37. Deletion of these six amino acids did not affect the growth rate or morphology of the plaque formation compared to the wild type HSV-1 (F). The entry characteristics for different mutant viruses were compared utilizing mutant viruses having a deletion of 38 amino acid from N terminal end of gK in HSV-1 F (VC1) or tyrosine replaced by alanine in UL37 position 476 (DC 476) or 477 (DC 477). We found that

VC1, DC 476 and DC 477 were affected similarly when endocytosis was inhibited. Live imaging suggested that alanine replacement of tyrosine at position 476 or 477 of UL37 had no effect on

HSV-1 retrograde transport in primary rat dorsal root ganglia (DRG) neurons. However, the quadruple mutation at positions 474, 476, 477 and 480 was detrimental for the virus and it severely affected viral growth.

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Figure A.2. (A) Schematic of the structure of HSV-1 UL37 protein showing different functional domains and tentative position of the conserved tyrosine residues. (B) Schematic representation of the UL37 mutant viruses constructed on the viral genomic background on the HSV-1(F) and VC1 genomes cloned as a bacterial artificial chromosome. The approximate locations of the constructed mutations are shown.

Methods and materials

Cells and viruses. African green monkey kidney (Vero) cells were obtained from the

American Type Culture Collection (Rockville, MD). The Vero cell-based UL37-complementing cell line BD45 was a gift from Prashant Desai (Johns Hopkins University, Baltimore, MD). All cells were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal calf serum and antibiotics. HSV-1 (F) and all the other mutants were grown in Vero cells except DC 474-480, which required BD45 cells.

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Construction of mutant HSV-1 (F). UL37 mutagenesis was done in our laboratory following the instructions of the GeneTailor site-directed mutagenesis system kit from Invitrogen as published previously (3, 24, 25). Construction of viral mutants with specific amino acid changes was accomplished in Escherichia coli by using the markerless two-step Red recombination mutagenesis system and synthetic oligonucleotides implemented on the bacterial artificial chromosome (BAC) plasmid pYEbac102 carrying the HSV-1(F) genome (a gift from

Y. Kawaguchi, University of Tokyo, Japan). Double Red recombination was also used to construct the mutant DC474-480, which has the four conserved tyrosines at positions 474, 476,

477, and 480 of HSV-1 UL37 changed to alanine, as well as mutant viruses DC474 and DC476 with single (Y-to-A) amino acid changes at positions 476 and 477 (Fog. 2B). We also have generated a rescuing virus from the quadruple mutant. All mutated DNA regions were sequenced to verify the presence of the desired mutations in BACs and the absence of any other spurious mutations on the viral genome (3). The UL37 ∆TRAF6 mutant was constructed similarly by deleting six amino acids (1099 P-V-E-D-D-E-1104) from the C terminal end (20).

Purification of HSV-1. DC 474-480 and the rescued viruses were purified as described previously. Briefly, DC 474-480 was grown in BD45 complementing cell line and then passaged in Vero cells. The inoculum virus was able to replicate in Vero but the progeny capsids accumulate in the cytoplasm. Vero cells were infected with the rescued virus as well and cells from 6 T-150 flasks of Vero cells infected with either virus were collected at 24 hours post- infection (hpi) and cells were broke open by NP-40 lysis buffer (Invitrogen). The virus capsids were purified by 50 to 20% discontinuous iodixanol gradients twice, followed by a 20% iodixanol cushion. The resulting pellet was resuspended in PBS (25, 26).

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Plaque assay. A confluent monolayer of Vero cells was infected with HSV-1 (F) or

UL37 TRAF6 mutant. Following shaking at room temperature for 1 hour, the 12 well plates were washed with 1X PBS and methylcellulose was added to each well. The plates were then incubated at 37°C for 48 hours, fixed with formalin and the plaques were stained with antibody against HSV-1 for bright field microscopy.

Replication kinetics. Viral growth kinetics was done as previously described (24).

Briefly, Vero cells were absorbed with the virus at 4°C for 1 h in 12 well plates. Then the plates

were transferred to 37°C with 5% CO2 for 1 h for virus entry. Unbound viruses were washed off

with low pH buffer and then the plates were incubated for 0, 6, 12, 24 and 48 hours. Virus titer

was measured, averaged and the standard deviation was calculated for each time point.

Endocytosis inhibition Assay. To asses the endocytosis inhibition, two inhibitors were

used. Pitstop-2 (Abcam) is a clathrin inhibitor, and dynasore Hydrate (sigma) is a dynamin

inhibitor. A series of dilution of pitstop-2 (10-50) µM and dynasore hydrate (80µM) was added combinedly at 37°C for 30 minutes. Then the Vero cells were infected with VC1, DC 476 or DC

477 at MOI 10 without washing the drug off. After 1 hour the cells were washed with 1X PBS and methylcellulose was added. Following 48 hours the cells were fixed and plaques were counted.

Transport Assay. Vero cells were grown in eight well chamber slides and starved overnight. Cells were then adsorbed with either purified Rescue (F) or purified UL37 Y(474-

480)A and at 4°Cat MOI of 20. Then the slides were shifted to 37°C for 3, 5 and 24 hours. After that, the slides were fixed in formalin and permeabilized with ice-cold methanol followed by blocking with goat serum. Antibody against UL37 was used to detect viral capsid transport to the

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nucleus, which was stained with DAPI. Images were taken using a 60x objective on a fluorescent microscope.

Primary rat dorsal root ganglia neuron. Pregnant rat (E16) was euthanized and dissected under sterile conditions. Dorsal root ganglia were collected from the pups under a bright field microscope. The ganglia were dissociated using dissociation media (Brains Bit) and spun to get rid of the undissociated ganglia. The microfluidic chamber was placed on glass- bottom dishes which were coated with poly-D-lysin. The neurons were grown in the microfluidic chamber with complete media from Brains bit supplemented with primocin (antibiotic).

Following dissociation of the ganglia, neurons were added to only one side of the groove (cell body side) and the other side (axon side) was loaded with complete media with neuronal growth factor (NGF). The next day an anti-mitotic agent (Ara C) was added to the microfluidic device to get rid of the non-neuronal dividing cells. The following day the media was removed and fresh media was added to both side and NGF was added to the axon side. NGF will attract the growth of the axonal terminals to grow through the groove from the cell body side to the axon side. The media volume of the cell body side was kept higher than the axon side to create a gradient.

However, the narrow grooves only allow axons to pass through but not cell bodies. The microfluidic device was observed every day for optimal axonal growth. Once a good number of axons were visible on the axon side, the device was considered ready for the experiment.

Results

Characterization of UL37 ∆TRAF6 mutant. Previously it was shown that HSV-1

UL37 has a binding site for cellular TRAF6 at the C terminal end. HSV-1 UL37 activates the

NFκβ pathway via TLR2. Activation of this pathway requires UL37- TRAF6 interaction.

Particularly the C terminal 123 amino acid of UL37 is required for NFκβ activation (3, 4, 15).

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Single amino acid replacement (UL37 E1101A) in the TRAF6 binding domain significantly reduced NFKB activation (20). Here we generated a UL37 mutant virus deleting six amino acids from position 1099 to 1104 (P-V-E-D-D-E) in the TRAF6 binding domain. This sequence is identical to the TRAF6 binding motif in IRAK-M (27, 28). Our initial characterization of the mutant virus showed similar plaque morphology and growth kinetics as the wild type HSV-1 (F) as shown in figure A.3 , Future experiments should be focused on the role of this motif in retrograde transport of the virus as UL37 is an essential tegument protein and plays pivotal role in virus transport and secondary envelopment.

Figure A.3. Characterization of UL37 ∆TRAF6 mutant. Vero cells were infected with either (A) HSV-1 (F) or (B) UL37 ∆TRAF6 for 48 hours at 37°C and plaques were stained with rabbit anti-HSV-1 polyclonal antibody. (C) Replication kinetics of wild-type and UL37 ∆TRAF6 mutant on Vero cells. HSV-1 (F) and UL37 ∆TRAF6 viruses were adsorbed on nearly confluent

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monolayers of Vero cells. Cells were infected with each virus at an MOI of 0.1 and 5. Virus stocks were collected at the indicated times, and titers were determined on Vero cells. Virus titers from three independent cultures were averaged, and the standard deviation was calculated for each time point.

Mutation in gK and UL37 in HSV-1 (F) exhibits similar features as the mutation in

gK in HSV-1 (McKrae) in terms of entry. Previously we have shown that the deletion of 38

amino acid from the N terminal end of McKrae gK modifies the viral entry process from fusion

to endocytosis while compared to the wild type McKrae which was resistant to endocytosis

inhibitors (29). Here we wanted to determine if the similar phenomenon can be observed in case of VC1 which has a 38 amino acid deletion from the N terminal end of HSV-1 (F) gK or in case

of UL37 mutations at 476 or 477 positions. Our preliminary data suggests that both gK and

UL37 mutations showed a similar pattern in virus entry in HSV-1 F strain (Fig.A.4).

Figure A. 4. Endocytosis assay. Vero cells were treated with combined doses of pitstop-2 (10-50 µM) and dynasore hydrate (80µM) for 30 mins at 37°C and then infected with 100 PFU VC1, DC 476 or DC 477 for 1 hour at 37°C (w/o washing the drug off). After infection wells were washed with PBS and methylcellulose was added. Plaques (PFU) were counted using crystal violet staining after 48 hours. ***P<0.001 vs no drug-treated control. Statistical

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comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent the 95% confidence interval about the mean.

Single tyrosine mutation in UL37 does not impair retrograde capsid transport.

Previously our lab showed that single tyrosine replacement by alanine in the UL37 central region

(Y476A or Y477A) does not change the morphology or growth kinetics of the virus compared to

the wild type virus, however quadruple mutant virus DC 474-480 failed to propagate in Vero

cells (3). However, it was not clear if this defect was due to the inability of the capsid to

transport or due to spreading defect. Here we used all three mutant viruses along with the

rescued virus to see if these mutants were able to travel to the nucleus following infection in

Vero cells and in primary rat dorsal root ganglia neurons. Before purifying DC 474-480, we

grew the virus in BD45 cells and ran two passages in Vero cells. For other mutants and the

rescued virus, the viral stock was made in Vero cells. Our preliminary data suggests that the single mutants were able to travel and replicate in the nucleus like the rescued virus whereas the purified DC 474-480 was stuck at the cytoplasm. Figure A.5 shows the location of major capsid protein ICP5 in Vero cells infected with DC 474-480 and rescued virus following 3 hours, 5 hours and 24 hours post-infection. Due to limited volume and low titer of the DC 474-480, this experiment was not repeated to make it statistically significant.

Similar experiments were done with GFP tagged DC 476, DC 477 and the rescued HSV-

1 (F) viruses in primary rat DRG neurons. The virus was added to the axon side of the microfluidic chamber and the movement of the GFP virus observed under the live condition at

37°C under a fluorescent microscope (Zeiss). Within a few minutes following the addition of the virus, green capsid movement was observed in the grooves of the microfluidic chamber from the axon side to the cell bodyside (Fig. A.6). Live recordings and images were taken during the

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infection process. Due to limited volume and low titer of the DC 474-480, this virus could not be used for this experiment.

Figure A.5. Vero cells were adsorbed with either purified Rescue (F) or purified DC 474- 480 at 4°Cat MOI of 20 and then shifted to 37°C for 3, 5 and 24 hours. The 8 well chamber slide is then fixed with formalin, permeabilized with methanol and prepared for fluorescent microscopy. Antibody against ICP5 (red) was used and nuclei were stained with DAPI (blue).

(figure cont’d)

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Figure A.6. Transport of HSV-1 in rat dorsal root ganglia neuron. (A) Pregnant rat (E16) was dissected, and dorsal root ganglia (DRG) neurons were grown in the cell body side of the Xona microfluidic chamber. (B) Schematic shows the principle of Xona microfluidic chamber to physically separate cell bodies from the axons of neurons. Higher volume in the cell body side

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creates pressure that pushes the cells close to the grooves. Added neuronal growth factor (NGF) on the other side attracts axons to go through the grooves. Width of the grooves prevents the cell body to pass through the grooves. (C) Growth of DRG neurons in the chamber four days post- seeding. The white arrow indicates cell body and the black arrow indicates axons of the dorsal root ganglia neurons. (D) Transport of GFP tagged rescued HSV-1 in Xona microfluidic chamber.

Discussion

Published results implicated that UL37 as a crucial determinant in intracellular virion transport (2). Specifically, it was shown that the R2 domain of UL37 played an important role in virion capsid transport in neurons (1). We aimed to take advantage of a cadre of different UL37 mutant viruses created in our laboratory to investigate whether any of these mutations also affected virion transport. Our results suggested that none of these mutations affected capsids transport, although they appeared to differentially affect virus replication and plaque formation

(3). Unfortunately, mutations that caused drastic inhibition of viral replication could not be tested because these mutations produced unenveloped capsids that could not be tested for their ability to be transported intracellularly toward the nucleus. Preliminary work suggested that perhaps lipofectamine or liposome enclosed capsids may be able to insert these capsids into the cytoplasm of cells to test for their ability to bind the dynein motor machinery and be transported in a retrograde manner. This work showed promise but was not completed rendering this work to be included in the thesis appendix instead of the main body of this thesis.

References

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2. Luxton GW, Lee JI, Haverlock-Moyns S, Schober JM, Smith GA. 2006. The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport. J Virol 80:201-209.

3. Chouljenko DV, Jambunathan N, Chouljenko VN, Naderi M, Brylinski M, Caskey JR, Kousoulas KG. 2016. Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production. Journal of Virology 90:10351-10361.

4. Bucks MA, Murphy MA, Kevin JOC, Courtney RJ. 2011. Identification of interaction domains within the UL37 tegument protein of herpes simplex virus type 1. Virology 416:42-53.

5. Kelly BJ, Fraefel C, Cunningham AL, Diefenbach RJ. 2009. Functional roles of the tegument proteins of herpes simplex virus type 1. Virus Research 145:173-186.

6. Maurer UE, Sodeik B, Grunewald K. 2008. Native 3D intermediates of membrane fusion in herpes simplex virus 1 entry. Proceedings of the National Academy of Sciences of the United States of America 105:10559-10564.

7. Lee JH, Vittone V, Diefenbach E, Cunningham AL, Diefenbach RJ. 2008. Identification of structural protein-protein interactions of herpes simplex virus type 1. Virology 378:347-354.

8. Klupp BG, Fuchs W, Granzow H, Nixdorf R, Mettenleiter TC. 2002. Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein. Journal of Virology 76:3065-3071.

9. Mijatov B, Cunningham AL, Diefenbach RJ. 2007. Residues F593 and E596 of HSV-1 tegument protein pUL36 (VP1/2) mediate binding of tegument protein pUL37. Virology 368:26-31.

10. Kramer T, Enquist LW. 2013. Directional spread of alphaherpesviruses in the nervous system. Viruses 5:678-707.

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11. Stults AM, Smith GA. 2019. The herpes simplex virus type I deamidase enhances propagation but is dispensable for retrograde axonal transport into the nervous system. J Virol doi:10.1128/JVI.01172-19.

12. Zhao J, Zeng Y, Xu S, Chen J, Shen G, Yu C, Knipe D, Yuan W, Peng J, Xu W, Zhang C, Xia Z, Feng P. 2016. A Viral Deamidase Targets the Helicase Domain of RIG-I to Block RNA-Induced Activation. Cell Host Microbe 20:770-784.

13. Kattenhorn LM, Korbel GA, Kessler BM, Spooner E, Ploegh HL. 2005. A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Molecular Cell 19:547-557.

14. Schlieker C, Korbel GA, Kattenhorn LM, Ploegh HL. 2005. A deubiquitinating activity is conserved in the large tegument protein of the Herpesviridae. Journal of Virology 79:15582-15585. 15. Kato A, Kawaguchi Y. 2018. Us3 Protein Kinase Encoded by HSV: The Precise Function and Mechanism on Viral Life Cycle. Human Herpesviruses 1045:45-62.

16. McGeoch DJ, Davison AJ. 1986. Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses. Nucleic Acids Res 14:1765- 1777.

17. Tobler K, Senn C, Schraner EM, Ackermann M, Fraefel C, Wild P. 2019. The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K. F1000Res 8:727.

18. Pitts JD, Klabis J, Richards AL, Smith GA, Heldwein EE. 2014. Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking. J Virol 88:5462-5473.

19. Scherer J, Yaffe ZA, Vershinin M, Enquist LW. 2016. Dual-Color Herpesvirus Capsids Discriminate Inoculum from Progeny and Reveal Axonal Transport Dynamics. J Virol 90:9997-10006.

20. Liu X, Fitzgerald K, Kurt-Jones E, Finberg R, Knipe DM. 2008. Herpesvirus tegument protein activates NF-κB signaling through the TRAF6 adaptor protein. Proceedings of the National Academy of Sciences 105:11335-11339.

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21. Pasdeloup D, McElwee M, Beilstein F, Labetoulle M, Rixon FJ. 2013. Herpesvirus Tegument Protein pUL37 Interacts with Dystonin/BPAG1 To Promote Capsid Transport on Microtubules during Egress. Journal of Virology 87:2857-2867.

22. Kelly BJ, Mijatov B, Fraefel C, Cunningham AL, Diefenbach RJ. 2012. Identification of a single amino acid residue which is critical for the interaction between HSV-1 inner tegument proteins pUL36 and pUL37. Virology 422:308-316.

23. Watanabe D, Ushijima Y, Goshima F, Takakuwa H, Tomita Y, Nishiyama Y. 2000. Identification of nuclear export signal in UL37 protein of herpes simplex virus type 2. Biochemical and Biophysical Research Communications 276:1248-1254.

24. Jambunathan N, Chouljenko D, Desai P, Charles A-S, Subramanian R, Chouljenko VN, Kousoulas KG. 2014. Herpes Simplex Virus 1 Protein UL37 Interacts with Viral Glycoprotein gK and Membrane Protein UL20 and Functions in Cytoplasmic Virion Envelopment. Journal of Virology 88:5927-5935.

25. Jambunathan N, Chowdhury S, Subramanian R, Chouljenko VN, Walker JD, Kousoulas KG. 2011. Site-Specific Proteolytic Cleavage of the Amino Terminus of Herpes Simplex Virus Glycoprotein K on Virion Particles Inhibits Virus Entry. Journal of Virology 85:12910-12918.

26. Jambunathan N, Charles AS, Subramanian R, Saied AA, Naderi M, Rider P, Brylinski M, Chouljenko VN, Kousoulas KG. 2015. Deletion of a Predicted beta-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons. J Virol 90:2230-2239.

27. Ye H, Arron JR, Lamothe B, Cirilli M, Kobayashi T, Shevde NK, Segal D, Dzivenu OK, Vologodskaia M, Yim M, Du K, Singh S, Pike JW, Darnay BG, Choi Y, Wu H. 2002. Distinct molecular mechanism for initiating TRAF6 signalling. Nature 418:443-447.

28. Wesche H, Gao X, Li XX, Kirschning CJ, Stark GR, Cao ZD. 1999. IRAK-M is a novel member of the pelle/interleukin-1 receptor-associated kinase (IRAK) family. Journal of Biological Chemistry 274:19403-19410.

29. Musarrat F, Jambunathan N, Rider PJF, Chouljenko VN, Kousoulas KG. 2018. The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB

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Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes. Journal of Virology 92.

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A.2 Effect of antidepressant anti-psychotic drugs on HSV-1 infection

According to the world health organization (WHO), any disorder or disease of the brain, spinal cord or peripheral nervous system is known as a neurological disorder. It includes dementia (Alzheimer’s disease, AD), brain tumors, Parkinson’s disease, seizures (epilepsy), cerebrovascular diseases (stroke), multiple sclerosis, Amyotrophic lateral sclerosis (ALS), migraine and other headache disorders, any other neurologic disorder due to head or nerve damage or malnutrition. Every year millions of people die due to neurological disorders.

Sometimes the causative agents of these disorders are pathogens; sometimes, it is immune response itself, and sometimes it is genetic or unknown. It is estimated that around 6 million people die each year due to stroke, at least 48 million people are affected with dementia of which a major part is contributed by Alzheimer’s disease, and around 50 million people suffer from epilepsy worldwide (WHO, 2016). With the advent of modern technology and advanced research, several drugs are now available to treat neurological disorders. Unfortunately, most of the neurological disorders are treated based on the symptoms since there is not much research done on the actual cause of the diseases.

A number of chronic or long-term neurological disorders such as multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and autistic spectrum disorders are thought to be associated with pathogens but the direct linkage is still elusive (1).

Although most pathogens cannot cross the blood-brain barrier (BBB) to affect the brain, a few bacteria, fungi, amoebae and viruses are well equipped to do that (2). The virus infects peripheral nerves and uses axonal transport to reach CNS. On the other hand, most bacteria reach CNS via

BBB or the cerebrospinal fluid (CSF) from the systemic circulation. Bacteria are mostly responsible for inflammation of the meninges (meningitis) (2). Around 77% of cases are caused by Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumonia, according to

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the National Surveillance of Bacterial Meningitis (3). Encephalitis is characterized by

inflammation of the brain parenchyma and neurologic dysfunction. The causative agents of most

CNS encephalitis are a virus (68.5%) and the most common virus leads to encephalitis in western

countries is herpes simplex type 1 (HSV-1) (4-6). Other viruses that might be associated with

encephalitis are Enteroviruses, varicella-zoster virus, Epstein-Barr virus, measles virus, and

arboviruses, such as Japanese encephalitis virus, West Nile virus, and Murray Valley

encephalitis virus (2, 7). Focal infection of CNS is caused by mostly bacteria such as

Streptococcus and Staphylococcus spp and some Gram-negative enteric bacteria (1).

Neurotropic herpesviruses establish lifelong latency in the nervous system, and

occasionally they reactivate. Although, stress, immunosuppression, radiation, fever, etc. are

thought to be causative agents for herpes reactivation, the actual mechanism of herpes

reactivation in the brain or trigeminal ganglia is still unclear. Recently it was shown that there is

an association between neurodegeneration and HSV-1 (8). For example, in 1997, it was first

shown that 60% of Alzheimer’s patients carrying the apolipoprotein E gene (APOE-Ɛ4) had

HSV-1 in their brains (9, 10). However, the population who was not apolipoprotein E gene

carriers did not develop AD, although they were positive for HSV-1. It was suggested that

recurrent reactivation of HSV-1 in the brain of the carriers accumulated damage that leads to AD

(10). Later on, it was shown that an antibody against HSV-1 was present in the CSF fluid of AD patients (11). HSV-1 DNA was also detected in AD plaques, and specifically, the characteristic beta-amyloid and P-tau were found in HSV-1 infected mice brain and in HSV-1 infected cell culture respectively (11-14). Recently it was found that there might be a role of HSV-1 in cognitive dysfunction in schizophrenia (15). It was shown that herpes encephalitis (HSE) related cognitive impairment is improved following antipsychotic drug treatment in some patients (16).

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Recurrent HSV-1 infection and reactivation lead to encephalitis and psychosis in one reported

patient who was mannose-binding lecithin (MBL) deficient and exposed to Epstein-Barr virus

(EBV) in childhood (17). It was reported that many viruses including HSV-1, get reactivated following immunotherapy (Table A.2.1).

Table A.2.1. List of drugs that reactivate viruses.

Most of the central nervous system (CNS) drugs are either CNS stimulants or CNS depressants. They function directly on the pre- or postsynaptic neurons and affect the release of neurotransmitters like norepinephrine (NE), epinephrine (EPI), dopamine (DA), serotonin (5-

HT), acetyl choline (ACh), etc or their receptors. Therefore, treatments with these agents sometimes change the environment of the nervous system to some extent that it exacerbates the overall pathology (1, 18-21).

In this report, we investigated on some commonly used CNS drugs on HSV-1

(Table.A.2.2). We hypothesize that antipsychotic drugs either enhance or inhibit HSV-1 infection. The latent HSV-1 in the neuron may reactivate at the exposure of drugs. The drug may enhance HSV-1 replication. On the other hand, some antipsychotic or CNS drugs may prevent

HSV-1 reactivation and replication thereby, reduce chances of CNS disease. Although it would be relevant to use neurons or in vivo experiment to see the effect of CNS drugs on HSV-1, we have used Vero cells for a number of reasons- 1) most of these drugs are not highly specific, 2)

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the mode of actions of these drugs are not essentially known, 3) it is easy to perform plaque assay on Vero cells.

Table A.2.2.Common CNS drugs, their mode of action, usage and side effects. (22-24)

Drug name Compound Mode of action Uses Side effects Valproate Sodium valproate Affects GABA Epilepsy, Nausea, levels, blocking seizure, vomiting, voltage-gated sodium bipolar sleepiness, channels, and disorder, dry mouth, inhibiting histone migraine suicidal deacetylases headache tendency, liver problems. Amphetamine/ Alpha- CNS stimulant, Attention Euphoria, methylphenethyla Increases the activity deficit suppresses Methylphenidate mine of a number of hyperactivity appetite, neurotransmitters, disorder weight loss, especially (ADHD), acne, blurred norepinephrine and non-medical, vision, dopamine. recreational seizure, heart problems, psychosis, paranoia, erectile dysfunction, reduction in cognitive ability addiction. Fluoxetine N-methyl-3- Selective serotonin Depression, Insomnia, phenyl-3-4- reuptake inhibitor eating tremor, (trifluoromethyl)p (SSRI) disorder, weakness, henoxy propan-1- Antidepressant obsessive- tunnel vision, amine compulsive dry mouth, disorder decreased sex (OCD), panic drive, disorder, and headache, a premenstrual suicidal dysphoric tendency in disorder young people (PMDD).

(table cont’d)

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Lamotrigine Phenyltriazine Increase GABA, Seizure, Vomiting decrease voltage- bipolar sleepiness, sensitive sodium disorder trouble with channels coordination, and rash, Anticonvulsant headache, lack of red blood cells, increased risk of suicide Venlafaxine 1-2- Selective serotonin Major Diarrhea, (dimethylamino)- and norepinephrine depressive nausea, 1-(4 reuptake inhibitors disorder, Fatigue, dry methoxyphenyl)et (SSNRIs) anxiety and mouth, hyl cyclohexanol, panic dizziness, Antidepressant disorder. insomnia, reduced sex drive

Analysis

Vero cells were grown in 12 well clear bottom plates. Once the cells were confluent, they

were starved overnight. Drugs (tablets) were crushed with the help of mortar and pestle and

dissolved in DMSO at appropriate concentrations. Dilutions were made in DMEM without FBS.

Starved Vero cells were treated with different dilutions of drugs to determine the toxicity of

cells. A non-toxic range of drug concentrations was selected for the plaque assay. Briefly, 300 µl

of drug solution was added to the 12 well plates and incubated at 37°C for 30 minutes.

Approximately 100 PFU of HSV-1 McRae was added to the plate without washing the drug off

and incubated at 4°C on a shaker for 1 hour. Then the plate was washed with ice-cold PBS to remove the drug and unbound virus. Methylcellulose was added on top of the cell and incubated at 37°C for 48 hours. The number of plaques was counted after staining with antibodies against

HSV-1.

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According to our data, overall, there was no significant reduction or increase in HSV-1

McKrae plaque numbers in the drug-treated wells vs. the untreated control well. However, fluoxetine and venlafaxine were promising as there is a reduction of HSV-1 plaque number in venlafaxine (10µg/ml) treated cells and fluoxetine treated cells (10µg/ml and 10 pg/ml) at certain dilutions (Fig. A.2.1). Interestingly both of these drugs are anti-depressant (selective serotonin reuptake blockers). Further analysis of these drugs in primary neurons or in vivo is required to ensure the effect on HSV-1 replication, latency, and reactivation.

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Figure A.2.1. Vero cells were treated with a series of dilutions of drugs for 30 minutes and then adsorbed with HSV-1 McKrae (100 PFU) for 1 hour at 4°C. After incubation at 37°C

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for 48 hours, plaques (PFU) were counted using an antibody against HSV-1. Ns vs. no drug- treated control. Statistical comparison conducted by graph pad prism using ANOVA with post hoc t-test with Bonferroni adjustment. Bars represent 95% confidence interval about the mean

A.3 Transcriptome analysis of human neuroblastoma (SK-N-SH) cell line following HSV-1

infection

With the advent of modern technology and advanced molecular biology, it is now

possible to identify cellular transcripts and quantify gene expression. RNA-seq is a single high-

throughput sequencing assay that combines both identification and quantification of cellular

transcripts. For efficiency, convenience, and quick performance, this technology has become a standard part of biological science research. In the case of pathobiological sciences and cancer biology, this technique plays very important roles. For example, RNA samples from infected cells or cancer cells can be analyzed to check which signaling pathways are activated or inhibited following infection or cancer. These data will provide insights into the therapeutic intervention.

In this report, we analyzed RNA samples from human neuroblastoma (SK-N-SH) cell line infected with HSV-1 McKrae for 24 hours at 37°C. Here we tried to detect and analyze the cellular transcripts which were affected following the infection.

Analysis

RNA nucleic acid was extracted from samples, and sequencing libraries were prepared following previously established techniques. RNA Sequencing (RNASeq) reads from the samples were sequenced using the Ion Torrent Suite with Ion Proton and Ion Personal Genome

Machine (PGM) sequencing machines. The sequenced reads were then mapped to the hg38 human reference genome using the STAR mapping software. This software calculates the

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position within the human reference genome of where RNASeq reads match gene transcripts by using the information on splice junctions and the nucleotide sequences of genes. The mapped and unmapped RNASeq data was then copied to the IBM Delta Power8 High-Performance

Computing (HPC) cluster for subsequent steps. First, we performed the two-step mapping protocol from Life Technologies, which performed a second mapping step with Bowtie2, and then merged the mapped files from both steps using Picard tools . Next, the abundance of RNA transcripts from the RNASeq data was quantified using the Cufflinks software package. The software package Cuffdiff then calculated the gene expression on a log2 fold change scale, and then performed statistical calculations to determine the genes that were statistically significantly expressed in the dataset. To prevent undefined log2 fold change values in cases where 0 reads mapped to a gene, the value 1 x 10^-5 was used instead of 0 for these calculations. The code for these workflows is available upon request.

We generated heat maps that showed gene expression of the top 50 significantly differentially expressed genes for the HSV Control versus HSV wild type groups using R. Genes, and gene expression were selected based on their relevance to HSV infection, and then a heat map was generated using the seaborn visualization software.

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Figure A.3.1. Heat map generated from high expression genes. From a pool of 6000 genes, 350 genes were selected on the basis of relevance to our study. Top 50 significantly differentially expressed genes for the HSV-1 McKare vs control were grouped into high expression and low expression genes. HSV-1 induced a 5 fold high GAS5 expression than control.

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Figure A.3.2. Heat map generated from low expression genes. From a pool of 6000 genes, 350 genes were selected on the basis of relevance to our study. Top 50 significantly differentially expressed genes for the HSV-1 McKare vs control were grouped into high expression and low expression genes. HSV-1 McKrae induced higher expression of VCAM1, AMY2B, DCAF6, ACTN2, and TNFRSF9 compared to control.

In the high expression gene list, we observed at least a 5 fold higher expression of GAS5

in McKrae infected SK-N-SH than the mock-infected cells (Fig. A.3.1). GAS5 is a growth arrest-

specific transcript and it has tumor-suppressive role. Both in lung and breast carcinoma, GAS5

was found to play a role (25). In the low expression gene list the expression of vascular cell

adhesion molecule (VCAM1), amylase alpha (AMY2B), DDB1 and CUL4 associated factor 6

(DCAF6), actinin (ACTN2) and TNF receptor superfamily (TNFRSF9) were higher in McKrae

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infected SK-N-SH than the mock-infected cells (Fig.A.3.2). ACTN2 is a cytoskeleton (actin) binding protein (26). Higher expression of ACTN2 might have a role in HSV-1 transport.

From the RNA-seq analysis, we also made lists of genes that were affected during HSV-1

McKrae infection in SK-N-SH cells following 24 hours of infection. Table A.3.1 listed genes in different cellular processes and table A.3.2 listed genes involved in calcium flux following HSV-

1 McKrae infection versus mock infection. Further analysis with the help of bioinformatics tools is imperative to come to a relevant conclusion of the study. The future study involves pathway analysis (IPA) from these RNA-Seq inputs to determine the HSV-1 induced activation or inhibition of significant pathways, for example, Akt, MAPK, PKC, calcium signaling, immune pathways (STING-cGAS) and pathways involved in cellular transport in neurons.

Table A.3.1. List of molecules increased in different cellular processes (HSV-1 McRae Vs. control)

Diseases or Functions Annotation Molecules ADCYAP1,ADRA1A,ADRB2,AVP,BLNK, BTK,C3,C5AR1,CALCR,CAMP,CASR,CCL 11,CCL18,CCL19,CCL28,CCL3,CCL4,CCR 7,CCR8,CCR9,CD244,CD247,CD33,CD4,C D40,CD48,CD5,CX3CR1,CXCL1,CXCL10, CXCL11,CXCL3,CXCL8,CXCR1,CXCR2,C XCR3,CXCR5,EGF,F2,F2RL3,FAS,FCER1 G,FCGR2A,FCRL4,FFAR4,FPR1,FPR2,GH SR,GNA15,GNRH1,GPR132,GRM1,GRM5, GRM8,INSL5,KDR,KISS1,KISS1R,LAT,LC K,LILRB2,MLNR,MS4A2,NPSR1,NPY1R, NTSR2,P2RY1,PROKR1,PTAFR,PTPN6,PY Y,RXFP4,SYK,TAC3,TACR1,TAS1R1,TAS mobilization of Ca2+ 1R3,TRPV2,UTS2 ABCB4,ABCC2,ABCG2,ABCG5,CETP,NR excretion of lipid 1H4,SCARB1

(table cont’d)

151

transport of acidic amino acid ABCC11,ABCC2,ABCC5,ABCG2,SLC17A6 ,SLC17A7,SLC17A8,SLC1A2,SLC1A3,SLC 1A6,SLC1A7,SLC22A13,SLC9A1,TNF SLC17A6,SLC17A7,SLC17A8,SLC1A2,SL transport of L-glutamic acid C1A3,SLC1A6,SLC1A7,SLC9A1,TNF ADRA2A,AHR,ALB,APOD,AVP,C3,CASR, CD4,CD40,CLEC7A,CNR2,CSF3,F2,FAS,F CGR2A,GRM1,KISS1,MAS1,NTSR2,PLA2 G1B,PLA2G2D,PLA2G3,PLA2G5,PRKCG, release of arachidonic acid TAC1,TAC3,TNF ABCC11,ABCC2,ABCC9,ADRA2A,ANO1, ANO9,ATP1A2,ATP2A1,ATP6V0C,AVP,C A2,CA4,CASR,CCL3,CDH23,CFTR,CNGA 3,CNGB1,CXCL10,CXCL3,DRD3,EGF,F2,F GF23,GLRA2,HCN4,KCNA1,KCNA2,KCN D3,KCNF1,KCNG2,KCNJ15,KCNJ3,KCNJ4 ,KCNJ6,KCNK4,KCNK5,KCNK6,KCNQ4, KCNS1,KCNS3,KCNV1,KL,NKX2- 5,NR3C2,P2RX1,PDZK1,RHBG,RYR2,RYR 3,SCNN1B,SCNN1G,SGK1,SLC11A1,SLC1 7A1,SLC17A2,SLC17A4,SLC17A6,SLC17A 7,SLC1A2,SLC22A1,SLC22A11,SLC22A25, SLC22A3,SLC22A8,SLC24A2,SLC26A3,SL C26A4,SLC26A7,SLC26A8,SLC26A9,SLC3 4A1,SLC34A2,SLC34A3,SLC39A2,SLC4A1 ,SLC4A4,SLC5A5,SLC9A1,STC1,TCIRG1, TRPC4,TRPC6,TRPM2,TRPM3,TRPV6,TT transport of ion YH1,UCP1,VDR,ZACN ABCC5,ABCG2,AQP9,PDZK1,SLC17A1,S transport of purine base LC22A12,SLC22A13,SLC22A8

(table cont’d)

152

transport of molecule A2M,ABCB4,ABCC11,ABCC2,ABCC5,AB CC9,ABCG2,ABCG5,ABHD5,ADCYAP1,A DIPOQ,ADRA2A,ADRA2B,ADRB2,ALB,A MELX,AMN,ANGPT2,ANO1,ANO9,AOC3, APOA1,APOA2,APOA4,APOB,APOC1,AP OC2,AQP2,AQP3,AQP4,AQP8,AQP9,AR,A TF3,ATP1A2,ATP1B4,ATP2A1,ATP4A,AT P6V0C,ATXN2,AVP,BAIAP3,BCL11B,BC L2L11,BMP4,BMP7,C3,C5AR2,CA2,CA4,C ABP5,CACNA1E,CACNA1H,CALCR,CAM K2A,CAMP,CASR,CCL3,CCR7,CD14,CD3 6,CD40,CD74,CDH23,CETP,CFTR,CGA,C HP2,CHRNA1,CHRNA6,CHRNB3,CIDEA, CIDEB,CLCA1,CLCA2,CNGA3,CNGB1,C RTAM,CXCL10,CXCL3,CXCL8,CYP3A4,C YP7A1,DIO3,DRD3,EGF,ENPP1,ENTPD1, EPO,ERBB3,EXOC3L1,F2,FABP2,FAS,FC GR2A,FFAR4,FGF20,FGF23,FOLR2,FOLR 3,FOXP3,FPR1,FPR2,GABRA1,GABRA2,G ABRA4,GABRA6,GABRB2,GABRD,GABR G2,GABRR1,GABRR2,GALR1,GAST,GC, GCG,GH1,GJA8,GLMN,GLP2R,GLRA2,G NRH1,GPBAR1,GRID2,GRIN2A,GRIN2B, GRIN2C,GRM4,GUCA2A,HBB,HCN4,HDC ,HFE,HFE2,HNF1A,HPX,HSD17B2,HTN3,I L11,IL6,KCNA1,KCNA2,KCND3,KCNF1,K CNG2,KCNJ15,KCNJ3,KCNJ4,KCNJ6,KCN K10,KCNK4,KCNK5,KCNK6,KCNQ4,KCN S1,KCNS3,KCNV1,KL,KRT71,LCN2,LPL, MLANA,MS4A1,MSR1,MT3,MTTP,NAP NFKBIA,NGB,NGFR,NKX2- 5,NLRP12,NPC1L1,NPPC,NPTX1,NR1H3, NR1H4,NR1I2,NR3C2,NXF3,OLR1,OSM,P 2RX1,P2RX3,P2RY12,PDZK1,PECAM1,PI GR,PLA2G1B,POMC,PPBP,PRICKLE1,PR KG2,PROK1,PTK2,RETN,RHBG,RIMS1,R YR2,RYR3,SCARB1,SCNN1B,SCNN1G,SG K1,SHBG,SLC11A1,SLC15A1,SLC15A2,SL C16A10,SLC16A6,SLC16A7,SLC16A8,SLC 17A1,SLC17A2,SLC17A4,SLC17A6,SLC17 A7,SLC17A8,SLC18A2,SLC1A2,SLC1A3,S LC1A6,SLC1A7,SLC22A1,SLC22A11,SLC2 2A12,SLC22A13,SLC22A25,SLC22A3,SLC 22A8,SLC24A2,SLC26A3,SLC26A4,SLC26

(table cont’d)

153

A7,SLC26A8,SLC26A9,SLC28A2,SLC2A2, SLC2A4,SLC2A5,SLC30A3,SLC34A1,SLC 34A2,SLC34A3,SLC39A2,SLC44A4,SLC4A 1,SLC4A4,SLC51A,SLC5A1,SLC5A10,SLC 5A5,SLC6A1,SLC6A12,SLC6A13,SLC6A14 ,SLC6A20,SLC6A3,SLC6A4,SLC6A5,SLC7 A10,SLC9A1,SORBS1,SRSF7,SST,STAR,S TC1,TAC1,TCIRG1,TF,TGFA,TNF,TRPC4, TRPC6,TRPM2,TRPM3,TRPV2,TRPV6,TT PA,TTYH1,TULP1,UCN2,UCP1,VDR,ZAC N ABCG2,PDZK1,SLC17A1,SLC22A12,SLC2 transport of uric acid 2A13 ANO1,CASR,CCR7,CFTR,EGF,F2,FAS,GL secretion of anion P2R,GPBAR1,PROK1,SLC4A4,STC1 ABCC11,ABCC2,ABCC5,ABCG2,AQP9,PD ZK1,SLC17A1,SLC22A1,SLC22A12,SLC22 transport of nitrogenous base A13,SLC22A8,SLC28A2

ABCB4,ABHD5,ADIPOQ,ADRA2A,AHR,A LB,APOA1,APOA2,APOD,AVP,C3,CAMP, CASR,CCL11,CD14,CD4,CD40,CD7,CLEC 7A,CNR2,CSF3,CXCL8,CYP27B1,DRD3,F2 ,FAS,FCER1G,FCGR2A,GAST,GRM1,IL16, KISS1,MAS1,NR5A1,NTSR2,PLA2G1B,PL (table cont’d) A2G2D,PLA2G3,PLA2G5,PLG,POMC,PRK release of lipid CG,PYY,QRFP,SST,SYK,TAC1,TAC3,TNF SLC17A6,SLC17A7,SLC17A8,SLC1A2,SL C1A3,SLC1A6,SLC1A7,SLC22A13,SLC4A transport of alpha-amino acid 1,SLC6A20,SLC6A5,SLC9A1,TNF ADIPOQ,ADRA2A,AHR,ALB,APOD,AVP, C3,CAMP,CASR,CCL11,CD4,CD40,CD7,C LEC7A,CNR2,CSF3,CXCL8,F2,FAS,FCER1 G,FCGR2A,GAST,GRM1,IL16,KISS1,MAS 1,NTSR2,PLA2G1B,PLA2G2D,PLA2G3,PL release of eicosanoid A2G5,PRKCG,SYK,TAC1,TAC3,TNF ABCC9,ADRA2A,ATP1A2,ATP6V0C,AVP, CA2,DRD3,EGF,KCNA1,KCNA2,KCND3, KCNF1,KCNG2,KCNJ15,KCNJ3,KCNJ4,K CNJ6,KCNK4,KCNK5,KCNK6,KCNQ4,KC NS1,KCNS3,KCNV1,NKX2- 5,NR3C2,SCNN1B,SCNN1G,SGK1,SLC17 A2,SLC17A4,SLC1A2,SLC34A3,SLC4A4,S transport of monovalent inorganic cation LC9A1,TCIRG1,UCP1 (table cont’d)

154

flux of Ca2+ A2M,ADIPOQ,ADRB2,APOA1,AVP,BTK, CALB2,CAMP,CASR,CCL11,CCL19,CCL2 0,CCL26,CCL3,CCL4,CCR8,CD247,CD28, CD38,CHRM5,CRACR2B,CXCL10,CXCL3, CXCL8,CXCR3,CXCR5,EGF,EPO,F2,FCG R2A,FCGR3A/FCGR3B,FPR2,GH1,GPBAR 1,GRIN2B,HOXA7,HTR3A,HTR3B,HTR3D ,IL18,LAT,MS4A1,NPPC,NR3C2,NTF3,P2R X3,P2RY1,PECAM1,PLA2G1B,POU5F1,PR KCG,RAPSN,RYR1,RYR2,TRPC4,TRPC6, TRPC7,TRPM2,TRPV6,UCN2 ABCG5,ACSL5,ADIPOQ,AHR,APOA1,AP OA2,APOD,CCKBR,CD14,CD33,CD36,CE TP,CFTR,ESR1,ESR2,FAAH,FABP4,FOXA 1,GHSR,IAPP,IL6,LPL,MSR1,MTTP,NPC1 L1,NR1H4,NRIP1,PGR,PNLIPRP2,PPBP,Q uptake of lipid RFP,SCARB1,STAR,TLR6,TNF

ABCC11,ABCC2,ANO1,ANO9,CA2,CA4,C FTR,FGF23,KL,SLC17A1,SLC17A6,SLC17 A7,SLC22A11,SLC22A25,SLC22A8,SLC26 A3,SLC26A4,SLC26A7,SLC26A8,SLC26A9 ,SLC34A1,SLC34A2,SLC34A3,SLC4A1,SL transport of anion C5A5,STC1,TTYH1 depletion of Ca2+ ATP2A1,AVP,BTK,SYK A2M,ADIPOQ,ADRB2,APOA1,AVP,BTK, CALB2,CAMP,CASP1,CASR,CCL11,CCL1 9,CCL20,CCL26,CCL3,CCL4,CCR8,CD247, CD28,CD38,CHRM5,CRACR2B,CXCL10,C XCL3,CXCL8,CXCR3,CXCR5,EGF,EPO,F2 ,FCGR2A,FCGR3A/FCGR3B,FPR2,GH1,GP BAR1,GRIN2B,HOXA7,HTR3A,HTR3B,H TR3D,IL18,KCNN2,LAT,MS4A1,NPPC,NR 3C2,NTF3,P2RX3,P2RY1,PECAM1,PLA2G 1B,POU5F1,PRKCG,RAPSN,RYR1,RYR2,S LC26A4,SLC4A1,TNF,TRPC4,TRPC6,TRP flux of ion C7,TRPM2,TRPV6,UCN2,UCP1,ZACN release of hydrogen peroxide AQP8,CXCL3,CXCL8,NOX4,PPBP,TNF secretion of cholesterol ABCG5,APOA1,APOB,MTTP,NR1H3

(table cont’d)

155

flux of inorganic cation A2M,ADIPOQ,ADRB2,APOA1,AVP,BTK, CALB2,CAMP,CASR,CCL11,CCL19,CCL2 0,CCL26,CCL3,CCL4,CCR8,CD247,CD28, CD38,CHRM5,CRACR2B,CXCL10,CXCL3, CXCL8,CXCR3,CXCR5,EGF,EPO,F2,FCG R2A,FCGR3A/FCGR3B,FPR2,GH1,GPBAR 1,GRIN2B,HOXA7,HTR3A,HTR3B,HTR3D ,IL18,KCNN2,LAT,MS4A1,NPPC,NR3C2,N TF3,P2RX3,P2RY1,PECAM1,PLA2G1B,PO U5F1,PRKCG,RAPSN,RYR1,RYR2,TRPC4, TRPC6,TRPC7,TRPM2,TRPV6,UCN2,UCP 1 SLC17A6,SLC17A7,SLC17A8,SLC1A2,SL C1A3,SLC1A6,SLC1A7,SLC22A13,SLC6A transport of L-amino acid 20,SLC9A1,TNF ABHD5,ADIPOQ,ADRA2A,AHR,ALB,AP OD,AVP,C3,CAMP,CASR,CCL11,CD4,CD 40,CD7,CLEC7A,CNR2,CSF3,CXCL8,DRD 3,F2,FAS,FCER1G,FCGR2A,GAST,GRM1,I L16,KISS1,MAS1,NTSR2,PLA2G1B,PLA2 G2D,PLA2G3,PLA2G5,PRKCG,SST,SYK,T release of fatty acid AC1,TAC3,TNF A2M,ADCYAP1,ADRA1A,ADRA2A,ADR B1,ATP2A1,AVP,CABP1,CALCR,CASR,C CKBR,CCL11,CCL3,CCL3L3,CCL4,CCL7, CD247,CD38,CXCL10,CXCL11,CXCL3,CX CR3,EDNRB,EGF,ENTPD1,ENTPD2,EPO,F 2,FCGR3A/FCGR3B,FGF3,FGF6,FGF7,GA ST,GCG,GCGR,GH1,GHRH,GNA15,GNRH 1,GNRHR,GP6,GRIN2A,GRM1,HRH4,IAPP ,IGF2,IL6,KDR,LAIR1,LCK,LTA,MCHR2, NPHS1,NTF3,PDGFB,PLA2G5,POMC,PTP RC,PYY,RYR2,SHH,SLC8A1,SST,TAC1,T ACR2,TAS2R4,TFEC,TNF,TNFSF11,TRPC quantity of Ca2+ 6

(table cont’d)

156

flux of cation A2M,ADIPOQ,ADRB2,APOA1,AVP,BTK, CALB2,CAMP,CASR,CCL11,CCL19,CCL2 0,CCL26,CCL3,CCL4,CCR8,CD247,CD28, CD38,CHRM5,CRACR2B,CXCL10,CXCL3, CXCL8,CXCR3,CXCR5,EGF,EPO,F2,FCG R2A,FCGR3A/FCGR3B,FPR2,GH1,GPBAR 1,GRIN2B,HOXA7,HTR3A,HTR3B,HTR3D ,IL18,KCNN2,LAT,MS4A1,NPPC,NR3C2,N TF3,P2RX3,P2RY1,PECAM1,PLA2G1B,PO U5F1,PRKCG,RAPSN,RYR1,RYR2,TNF,T RPC4,TRPC6,TRPC7,TRPM2,TRPV6,UCN 2,UCP1 SLC17A6,SLC17A7,SLC17A8,SLC1A2,SL C1A3,SLC1A6,SLC1A7,SLC6A14,SLC6A2 transport of glutamine family amino acid 0,SLC9A1,TNF CASR,CCR7,CFTR,EGF,FAS,GPBAR1,PR secretion of chloride OK1 absorption of Na+ AVP,CCR7,CFTR,EGF,SLC9A3,SST ABCB4,APOA1,F2,PLA2G1B,PLA2G2D,P release of phosphatidic acid LA2G5 ABCC9,ADRA2A,ANO1,ATP1A2,ATP2A1, ATP6V0C,AVP,CA2,CASR,CCL3,CDH23,C NGA3,CNGB1,CXCL10,CXCL3,DRD3,EG F,F2,FGF23,HCN4,KCNA1,KCNA2,KCND 3,KCNF1,KCNG2,KCNJ15,KCNJ3,KCNJ4, KCNJ6,KCNK4,KCNK5,KCNK6,KCNQ4,K CNS1,KCNS3,KCNV1,NKX2- 5,NR3C2,PDZK1,RHBG,RYR2,RYR3,SCN N1B,SCNN1G,SGK1,SLC11A1,SLC17A2,S LC17A4,SLC1A2,SLC22A1,SLC22A3,SLC3 4A3,SLC39A2,SLC4A4,SLC9A1,TCIRG1,T RPC4,TRPC6,TRPM2,TRPM3,TRPV6,UCP transport of cation 1,VDR FGF23,KL,SLC17A1,SLC17A6,SLC17A7,S transport of phosphate LC34A1,SLC34A2,SLC34A3 ABCC2,ANO1,ANO9,CFTR,FGF23,KL,SL C17A1,SLC17A6,SLC17A7,SLC26A3,SLC2 6A4,SLC26A7,SLC26A8,SLC26A9,SLC34A 1,SLC34A2,SLC34A3,SLC4A1,SLC5A5,TT transport of inorganic anion YH1 secretion of triacylglycerol ABHD5,APOA4,ATF3,CIDEB,HSD17B2 uptake of cholesterol ester APOA1,APOA2,CETP,LPL,SCARB1 absorption of water AVP,PYY,SLC9A3

157

Table A.3.2. List of genes involved in calcium flux HSV-1 McRae Vs. control

Genes in dataset Prediction (based on measurement direction) P2RX3 Increased CCL11 Increased FPR2 Increased EPO Increased EGF Increased CD247 Increased CXCL3 Increased TRPV6 Increased CASR Increased HTR3A Increased APOA1 Increased CCL19 Increased F2 Increased BTK Increased IL18 Increased TRPC6 Increased HTR3B Increased CCL4 Increased CXCR3 Increased CAMP Increased FCGR2A Increased TRPC4 Increased AVP Increased P2RY1 Increased CD38 Increased CCL20 Increased LAT Increased NTF3 Increased CCL26 Increased RYR1 Increased CCR8 Increased TRPC7 Increased PLA2G1B Increased CXCR5 Increased TRPM2 Increased A2M Increased CCL3 Increased (table cont’d)

158

RYR2 Increased PECAM1 Increased GPBAR1 Increased HTR3D Increased FCGR3A/FCGR3B Increased HOXA7 Decreased MS4A1 Decreased CALB2 Decreased RAPSN Decreased NPPC Decreased GH1 Decreased GRIN2B Decreased PRKCG Decreased CXCL10 Decreased UCN2 Decreased CRACR2B Affected CXCL8 Affected ADIPOQ Affected POU5F1 Affected CHRM5 Affected NR3C2 Affected ADRB2 Affected CD28 Affected

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APPENDIX B. PERMISSION TO REPRINT CHAPTER TWO FROM JOURNAL OF VIROLOGY

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VITA

Farhana Musarrat was born in Dhaka, Bangladesh. She received her B.S. in Microbiology

from the University of Dhaka, Bangladesh, in 2008. She completed her M.S. in Microbiology

from the same institute in 2010. She received the prestigious Dean’s award for excellent

performance in undergraduate studies. Following the completion of her studies, she was

immediately appointed as a faculty member in the BRAC University, Dhaka, where she taught

several undergraduate-level courses, including General Microbiology, Basic Biology, and

Enzyme kinetics. To fulfill her dream to pursue a research career, she came to the United States

of America as a Ph.D. student. She joined the laboratory of Dr. Konstantin Gus Kousoulas in the

department of Pathobiological Sciences in the School of Veterinary Medicine of Louisiana State

University, Baton Rouge, in 2015. Her major research is on the molecular determinants of

Herpes Simplex Type 1 entry and transport which is the focus of this dissertation. Farhana plans to graduate in December 2019 and will continue in Dr. Kousoulas’s laboratory as a post-doctoral

researcher.

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