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2019 An Analysis of ERK/RSK Activation of Kaposis Sarcoma-Associated Herpesvirus ORF45 Homologues Miranda J Brown

Follow this and additional works at DigiNole: FSU's Digital Repository. For more information, please contact [email protected] THE FLORIDA STATE UNIVERISTY

COLLEGE OF ARTS AND SCIENCES

AN ANALYSIS OF ERK/RSK ACTIVATION OF

KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS

ORF45 HOMOLOGUES

MIRANDA BROWN

A Thesis submitted to the Department of Biology in partial fulfillment of the Requirements for graduation with Honors in the Major

Degree Awarded: [Spring, 2019]

TOPIC KSHV ORF45 is important in ERK/RSK prolonged activation. Further experiments need to be done in order to determine if different homologous sequences of ORF45 show disparity in ERK/RSK activation, and how these correlate to their phylogeny and evolution.

TABLE OF CONTENTS

I. Abstract………………………………………………………………………………………………..4 II. Introduction………………………………………………………………………………………....5 a. Kaposi’s Sarcoma b. Kaposi’s Sarcoma Herpesvirus(KSHV) c. KSHV ORF45 Infection and Signaling Pathways d. Herpesviridae Family Classification and ORF 45 Homologues e. Herpesviridae Family Evolution III. Research Questions……………………………………………………………………………...12 IV. Materials and Methods…………………………………………………………………………13 a. Previous Work b. Alignments and Phylogeny c. Plasmid Preparation by Transformation i. Transformation ii. Inoculation iii. DNA Collection d. Transfection i. Cell Culture ii. Cell Splitting iii. Calcium-Chloride Transfection e. Western Sample Collection f. Analysis by Western Blot V. Results……………………………………………………………………………………………..…20 a. Activation of ERK and RSK by ORF45 Homologues b. pERK and pRSK Relationship to Phylogeny VI. Discussion and Future Studies……………………………………………………………..26 VII. Conclusion……………………………………………………………………………………….…29 VIII. Acknowledgments………………………………………………………………………………30 IX. Supplementary Material……………………………………………………………………...31 X. References………………………………………………………………………………………….32

ABSTRACT

Kaposi’s Sarcoma Herpesvirus (KSHV) is an oncogenic virus that causes human malignancies, including Kaposi Sarcoma, multicentric castleman disease, and primary effusion lymphoma. KSHV is a gammaherpes virus of the genus Rhadinovirus, which all contain tegument proteins. KSHV

ORF45 is one such protein that is critical in production of viral progeny during lytic replication, and is an immediate early gene in viral infection. The ORF45 protein of gammaherpesvirinae has multiple functions, including inhibition of IRF-7 and prolonged activation of the MAPK/ERK pathways. In KSHV, ORF45 can activate extracellular signal-regulated kinase (ERK) and p90 ribosomal kinase (RSK) to form a complex with pERK and pRSK to prolong their phosphorylation and their abilities to transcribe DNA for viral progeny. In order to better understand the functions of ORF45, we are interested in exploring the ability of other gammaherpes homologues of KSHV

ORF45 to activate ERK and RSK. In addition, we wish to better understand how the ability to activate ERK and RSK relates to the amino acid sequence of each homologue, and how expression of pERK and pRSK correlates to phylogeny. By comparing homologues’ amino acid sequences and activation, further insight into the important process of how KSHV replicates in the cell can lead to future studies on how to prevent the formation of viral progeny in live hosts such as humans. We found that activation of ERK and RSK, and therefore levels of pERK and pRSK, varies greatly between different homologues of KSHV ORF45. In addition, levels of pERK and pRSK do have some correlation to differences in the amino acid sequences of ERK and RSK binding sites of

ORF45. Lastly, we observed that closely related homologues on the phylogenetic tree have similar levels of phosphorylated ERK and RSK, demonstrating a pattern between ancestry and ability to form pERK and pRSK complexes.

I. INTRODUCTION

Kaposi’s Sarcoma

Kaposi Sarcoma Herpesvirus (KSHV) received its namesake by first being known to be the cause of Kaposi Sarcoma (KS), a lymphatic and skin cancer first described by Mortiz Kaposi in

1872 (Chang and Moore, 2014). There are four variations of KS including classic KS, endemic KS, iatrogenic or immunosuppression-associated KS, and AIDs-associated KS (Jha et al., 2016). Classic

KS begins with the appearance of purple lesions on the skin, while immunosuppression-associated

KS normally occurs after an organ transplant, and tends to be an aggressive cancer in the lymphnodes and visceral organs, sometimes with the absence of any skin lesions. AIDS-associated

KS is the most present form of KS in the United States and most common AIDS related cancer.

Endemic KS infects a wide range of ages, genders, and patients with and without previous illness in Africa (Antman and Chang, 2000). In all of these cases, dark purple lesions and skin cancer may be present, and tumors may be found in the lungs, gastrointestinal tract, and lymph nodes (Jha et al., 2016).

Kaposi’s Sarcoma Herpesvirus (KSHV)

KSHV is an oncogenic virus that is found to lead to the following three human ailments: Kaposi sarcoma, Multicentric Castleman disease, and Primary Effusion Lymphoma (Chang et al., 1994;

Moore & Chang, 2001). This virus is also known as human herpesvirus-8 (HHV-8), and belongs to the Herpesviridae family. This family can then be further categorized into three subfamilies, including the subfamily of KSHV, Gammaherpesvirinae. The other two subfamilies are

Alphaherpesvirinae and Betaherpesvirinae. With in the subfamily of gammaherpesviruses, KSHV is considered to be of the Rhadinovirus genus (Russo et al., 1996).

So far, KSHV has been studied in various procedures to better determine its structure and modes of replication. It has been found that herpesviruses contain linear, double-stranded DNA

(Gibson, 1966). This DNA is located inside an icosahedral capsid made of proteins, with a lipid membrane envelope surrounding it. This lipid bilayer contains viral glycoproteins produced during lytic replication and added to the host cell membrane before it is used to enclose the new capsids upon release of viral progeny. Between the viral capsid and the envelope is the tegument layer, which is a matrix of proteins containing both proteins unique to each subfamily of

Herpesviridae and some conserved within the entire family (1966). Much remains unknown about tegument proteins, compared to the relatively conserved capsid proteins. For this reason, studying them and their importance in viral infection and replication is a next step in analyzing herpesviruses.

Herpesviruses have two separate life cycles, which are the latent and the lytic life cycle. During the latent cycle, few viral genomes are produced or expressed; therefore, they are only producing viral episomes for maintenance and immune evasion (Decker et al., 1996). During the lytic life cycle, the virus utilizes the host DNA machinery in order to replicate its viral genome and produce virus replicates. It is this part of the life cycle that needs further analysis in order to determine the mode of reactivation, invasion, and production of new virions. During this time, the complete viral genome is expressed as immediate early genes, early genes, and then late genes. At the end of this life cycle, the new viruses are gathered and then released from the cell (Zhu & Yuan, 2003). The interest is on researching the advantages ORF45 provides to the virus in entering the lytic cycle and producing viral progeny (Zhu & Yuan, 2003).

KSHV ORF45 Infection and Signaling Pathways

Research has uncovered information about KSHV ORF45’s structure, function, and localization in the virus and host cells. In the virion itself, ORF45 is a protein found in the tegument protein matrix of KSHV (Zhu & Yuan, 2003). The localization in the virus tegument makes it possible for early entry into the cell after initial infection. Whether or not it is involved in early or late stages of infection is not known for each homolog, but it has been shown that the disruption of this protein decreases the number infectious virions produced dramatically (Li &

Zhu, 2008). From this information, ORF45 may be necessary in both early and late stages of infection to increase viral replication (Zhu et al., 2006).

KSHV ORF45 research has provided information on what the protein may affect during infection. First, this protein has been found to inhibit the host’s innate immune response, interferon regulatory factor 7 (IRF-7) (Zhu et al., 2002). This decreases the host’s ability to defend against infection. Second, ORF45 has been observed to form complexes and interact with p90 ribosomal s6 kinase (RSK) and extracellular regulated kinase (ERK), leading to sustained phosphorylation and activation of these kinases (Fu et al., 2016;

Kuang et al., 2008). These two kinases play major roles in the

MAPK/ERK pathway, leading to lytic replication success and increased progeny in the presence of ORF45 (Kuang et al., 2008).

In the MAPK pathway, environmental cues first cause the

Ras/Rho family to phosphorylate. An SOS protein removes the

GDP off of Ras and replaces it with a GTP. As a result, a chain of phosphorylation leads to the activation of the ERK pathway.

Once ERK is activated to pERK, RSK is phosphorylated (pRSK), Figure 1. Activation of ERK by MAPK Pathway (Kochanczyk et al., 2017)

7 leading to cell growth and replication (Cargnello and Roux, 2011). After activation, pERK and pRSK have both been shown to activate transcription factors to increase DNA replication. As

ORF45 has the ability to prolong phosphorylation of RSK and ERK, presence of this protein increases activation of these kinases and optimizes virion DNA replication and production during

KSHV lytic replication (Fu et al., 2016). In addition to activating transcription of DNA, activation of

ERK and RSK may phosphorylate eukaryotic translation initiation factors (eIFs) and allow them to continually initiate translation of proteins, furthering the activation of the MAPK signaling pathway (Kuang et al, 2011). In further studies involving amino acid (aa) deletions, it was found that aa1-115, and more specifically aa56-70, are involved in binding to ERK/RSK to prolong their activation. In KSHV, a single point mutation at aa66 showed significantly less activation of pRSK, and therefore little to no creation of the ORF45 RSK complex. Even when expanding the genetic information used to the entire KSHV genome, there was a reduction of RSK activation and virus production (Fu et al., 2016).

Herpesviridae Family Classification and ORF 45 Homologues

In order to better understand ORF45 and its functions, research involving homologs has been conducted. Firstly, it is critical to reiterate that there are three subfamilies in the Herpesviridae family: Gammaherpesvirinae, Alphaherpesvirinae, and Betaherpesvirinae. KSHV is part of the

Gammaherpesvirinae subfamily. In further classification, it is in the genus Rhadinovirus. Other classifications in Gammaherpesvirinae include Macavirus, Lymphocryptovirus, and Percavirus

(Russo et al., 1996).

Knowing this information, it was found that ORF45 is not found just in KSHV, but is conserved among gammaherpesviruses. Although homologues of ORF45 are found in gammaherpesviruses, homology in the ORF45 protein itself is more limited, in that these other gammaherpesviruses

8 have relatively conserved amino- and carboxyl- terminals of ORF45, but diverse interiors and lengths (Fu et al., 2016). In addition, KSHV has a relatively long amino acid sequence (407 aa) compared to other homologs, which have a wide range of sizes starting from MHV-68 (206 aa) (Fu et al., 2016). On the other hand, the localization within host cells varies between homologs.

Restricting whether ORF45 stays in the cytoplasm or the nucleus did affect the number of progeny viruses (Li & Zhu, 2008). Due to the difference in localization, length, and sequence of the protein between homologs, it is possible that ERK/RSK activation in host cells differs between homologs.

Herpesviridae Family Evolution

In order to further understand why these differences may be present, understanding the evolution of the Gammaherpesvirinae is important. To start, the Herpesviridae family is estimated to be around 400 million years old, as is the formation of Pangea. Starting with only the

Alphaherpesvirinae, about 200 million years ago Gondwanaland and Africa were formed and the family continued to speciate into beta and gamma herpesviruses (Grose, 2012). From the ancestral core set of mammalian herpesvirus genes, 42 of those core genes are conserved in the gammaherpesvirinae subfamily. The overall genomic layout is conserved as well, with a long unique sequence bound by terminal end repeats. What varies between Gammaherpesvirinae is the divergence of those core genes through nucleotide mutations such as deletions, additions, and substitutions( McGeoch, 2001).

There are many contributors to viral evolution including geography, co-speciation with hosts, gene duplication, cross-species transmission events, mutation rate, and other factors. Co- speciation is when one species evolves with or in reaction to another species (Hafner & Nadler,

1988). Over all, co-speciation occurs less in gammaherpesviruses than the other two subfamilies, but is still common. With in the Gammaherpesvirinae subfamily, it is thought that Percaviruses

9 originated in perissodactyls and carnivores, Macaviruses in artiodactyls, and Lymphocryptoviruses in primates. The Rhadinovirus genus where KSHV resides has a broader host range and less co- speciation events (Escalera-Zamudio, 2016). One example where co-speciation is not observed is between BoHV-4 and BoHV-6, where they are both bovine viruses but do not share a recent clade, and therefore a recent common ancestor (Figure 6). On the other hand, RFHV and RRV are closely related and both speciated similarly with closely related primates (McGeoch, 2001). In fact, a viral strain of each RFHV and RRV both infect the same African Green Monkey (2001). Co-speciation can sometimes be hard to recognize as cross-species transmission events, where a virus infects a new species, is also seen in gammaherpesvirinae (2001).

A majority of viral evolution occurs through orthologous genes substitution, deletions, and insertions. The average rate of evolution for Gammaherpesvirinae is about 6*10-9 substitutions per site per year. In relationship to the homologues, MHV-4 has a long unique terminal branch and mutates rather quickly, while EBV evolves slowly. Comparing which sites are conserved between homologues, even in the presence of a high mutation rate, can provide insight into which parts of the genome or protein are most important (McGeoch, 2001).

Using the variation in the gamma-herpes coding regions, even tracking the evolution of a single type of virus is possible. For example, it was found that KSHV has variable sequences in different regions of the world. The strains found in East and West Africa are similar, and both phylogenetically different from those found in Europe. Meanwhile, the two main strains found in

Europe are more closely related (Cook et al., 1999). KSHV can be tracked to its human host’s prehistoric spread out of Africa. They first found Europe and Northern Asia. Because of geographical isolation, KSHV was able to form four major clades: Africa, Europe, Europe and N

Asia, and S Asia. For this reason, variability may also be associated with human movement after

10 introduction to this virus (McGeoch, 2001).

In relationship to ORF45, this protein is conserved in all Gammaherpesvirinae, with no presence in alpha or beta herpesviruses (Fu et al., 2016). This could mean that ORF45 was present in the common ancestor of all gammaherpesvirinae, and has since then been conserved.

In regards to KSHV ORF45, the evolutionary history remains incomplete. The length of KSHV

ORF45 (407aa) is a great deal larger than other gammaherpes viruses such as ALHV-2 or PLHV-1

(Table 1). The evolutionary advantage to this could be more available binding sites for different host cell partners, leading to new acquired functions compared to other homologues. Analyzing the amino acid sequence of KSHV ORF45 in conjunction with other homologues may provide insight into why it has expanded in size and if this is significant to its functions (Gillen et al., 2015).

Over all, evolutionary mechanisms including geography, human interaction, host interactions, time, rate of mutation, and co-speciation all effect virus evolutionary history. Analyzing this history is part of understanding the difference in localization, length, mechanisms, activation, and sequence of the ORF45 protein. When homologues are studied in conjunction with evolution, important conserved sites and their purpose can be analyzed.

II. RESEARCH QUESTIONS

Although ORF45 is conserved among gammaherpesviruses, the localization, length, and residue sequence of ORF45 varies between gammaherpesvirus homologs. Also, a limited amount of the ORF45 amino acid sequence is conserved between homologous versions of ORF45 (Li &

Zhu, 2008). Although it has been shown that aa1-115 of KSHV ORF45 are important in ERK/RSK prolonged activation, further experiments need to be done in order to determine differences in

ERK/RSK activation of different homologue sequences of ORF45 and how activation relates to their amino acid sequences and phylogeny.

The following questions will begin to be answered in this paper.

1. What are the differences in patterns of the genetic sequences in ORF45 between

different gammaherpesvirus homologues?

2. Do the differences in amino acid sequences affect the homologues’ ability to affect the

ERK/RSK pathway?

3. Do these differences in ability to alter ERK/RSK correlate to the phylogenetic tree and

homologue history?

III. MATERIALS AND METHODS

The following table contains the nine ORF45 homologues obtained from Genescript. There are five more homologues that were used for experimentation and were supplied by Dr. Fanxiu

Zhu's laboratory at Florida State University: Epstein-Barr virus (EBV, 217aa), Herpesvirus Saimiri

(HVS, 217aa), Kaposi’s Sarcoma herpesvirus (KSHV, 407aa), Murine gammaherpesvirus (MHV-68,

206aa), and Rhesus rhadinovirus (RRV, 353aa).

Virus Name Virus Abbreviation ORF45 DNA ORF45 Protein length (bp) Length (aa) Alcelaphine ALHV-2 774 258 gammaherpesvirus 2

Bovine gammaherpesvirus 4 BoHV-4 726 242

Bovine gammaherpesvirus 6 BoHV-6 822 274

Equid gammaherpesvirus 2 EHV-2 966 322

Equid gammaherpesvirus 5 EHV-5 936 312

Ovine gammaherpesvirus 2 OvHV-2 786 262

Porcine lymphotrophic PLHV-1 672 224 herpesvirus 1

Porcine lymphotrophic PLHV-3 687 229 herpesvirus 3

Retroperitoneal RFHV 1089 363 fibromatosis-associated herpesvirus Table 1. ORF45 homologues utilized in experimentation with their corresponding DNA length (bp) and protein length (aa). Genescript synthesized these ORF45 homologues.

Previous Work

Carolyn Dang did the following experiments with assistance and participation from myself.

Digestion

First, Genescript synthesized ORF45 homologue DNA. The ORF45 protein was isolated and inserted into a pUC57 vector (2,710bp) by Genescript, with BanHI and Xhol digestion sites. The gene-insert was excised out of the pUC57 vector using the restriction enzymes BamHI and Xhol and a digestion solution. This gene insert solution was incubated for two hours in a 37°C for two hours, and then 65°C for ten minutes to inactivate the enzymes. The pEGFP-C2 vector was digested overnight at 37°C by the restriction enzymes BgIII and SalI, and then inactivated by 10 minutes at 65°C. After, both the excised gene-inserts from pUC57 and the pEGFP-62 vector were run through an agarose gel by electrophoresis.

DNA Gel-extraction

The pEGFP-C2 vector and the gene-inserts were extracted from the gel using the Omega Bio-

Tek E.Z.N.A Gel Extraction Kit (D200-02). After, the concentrations of the gene-inserts and the pEGFP-C2 vector were determined via Nanodrop.

Ligation

For ligation, 25ng of the digested pEGFP-C2 vector was ligated over night with 75ng of the gene-insert using NEB T4 ligase.

Alignments and Phylogeny

Various programs were used in order to create the most accurate alignments. First, the protein and nucleotide sequences for the KSHV ORF45 homologues were found from the National

Center of Biotechnology Information using a Basic Local Alignment Search Tool. After, working together with Caroline Dang, alignment tools from European Bioinformatics Institute (EBI) were

14 used for alignment analysis and yielded results from T-Coffee and Praline. Then, they were manually adjusted with the help of Dr. Scott Steppan and Hongyu Zhang. The phylogenetic tree of homologues was then made based off the nucleotide sequences of ORF45 using the neighbor- joining method in the T-Coffee program of EBI. The phylogenetic tree and alignment results from

Carolyn Dang’s paper were then re-made and can be found in my results section with my pERK and pRSK activation mapped onto them. The original alignments from her paper can be found in the supplementary section.

Plasmid Preparation by Transformation

Transformation was all done aseptically over fire. If using ligation product, 3ul were added into TOP10 chemically competent E. coli cells (30ul). After collection of DNA, further transformation was done by adding about 50ng (0.2ul) of DNA to the thawed TOP10 cells (30ul).

The TOP10 cells and DNA were incubated on ice for 25 minutes for the DNA to associate with the

cell membrane. They were then heat shocked in Ligated gene insert Plasmid taken up by E. coli a 42°C water bath for 90 seconds to open the (Transformed) membranes to the DNA. Immediately after 90 Top10 E.coli Heat Shock seconds had passed, the samples were Amplified Plasmid removed from the water bath and rested on ice Selection + Growth for 5 minutes. Then, 1mL Luria Broth (LB) free was added to each tube and allowed to incubate Inoculation Figure 2. General mechanisms of Transformation from the for one hour in a 37°C rotary to begin ligated gene insert in the pEGFP-C2 vector through inoculation. (Smally & Smalley, 2018). expression of inserted DNA. After one hour, 100ul of each sample consisting of the plasmid DNA,

TOP10 cells, and LB broth were spread onto a LB plate with kanamycin using beads. The gene inserts code for kanamycin resistance. Any colonies that did not express resistance to kanamycin

15 were killed, while the other E. coli colonies with the desired gene inserts were aloud to incubate and grow at 37°C over night (Figure 3).

The following day, inoculation was completed aseptically. A colony from each plate was inoculated into a 500mL flask with 50mL LB broth with kanamycin. These shook vigorously overnight for about 18 hours at 37°C.

The third day, the amplified plasmid DNA was No resistance Kanamycin resistance collected using the protocol and supplies from

Bacterial Omega-Trek E.Z.N.A Plasmid Mini Kit I. After, the Transformation Selection DNA concentration was found using Nanodrop. Figure 3. Selection for kanamycin resistant gene insert in transformation (Young Pearse Lab, Harvard) Transfection

The cells used were HEK293T embryonic human kidney cells. These were cultivated in

Dulbecco’s modified Eagle’s medium (DMEM) with 10% Fetal Bovine Serum (FBS), 1% L-

Glutamine, and 1% Antibiotic-Antimycotic. They were retained in a 37°C incubator. Every two to three days the cells were diluted in DMEM for maintenance.

When the HEK293T cells were approximately 70% confluent, the 12-well dishes were seeded for transfection. First, the DMEM solution was removed from the plate of cells. Then,

1.5mL of trypsin was added to the plate and then aspirated immediately. Following this wash,

1.5mL of trypsin was added to the plate, and then the plate was incubated at 37°C for eight minutes. After, 6.5mL of the DMEM solution was added to the plate, and the cells were suspended using a pipette. Once all cells were individual and no longer clumped, 1mL of the cell solution was added to 11.5mL of DMEM in a conical tube. This was mixed, and then 1mL of this solution was added to each well of the dish. This was incubated at 37°C.

The next day, when the 12-well dish was approximately 70% confluent, transfection of the

HEK293T cells was completed. First, 2000ng of plasmid DNA was added to 50ul of 0.25M CaCl2 and then incubated at room temperature for 10 minutes. After, the CaCl2 and DNA mixture was slowly added drop-wise to 50ul 2xHEPES buffer solution (HBS) while tapping the micro centrifuge tube to mix. It was then briefly vortexed and then incubated at room temperature for 15 minutes.

The DNA, CaCl2, and HBS solution was added drop-wise to the correct well of the 12-well dish and then the dish was swirled to homogenize CaCl2 DNA CaCl2 phosphate co- precipitate the DMEM transfection solution. After, it DNA CaCl2 phosphate co-precipitate DNA returned to 37°C for incubation. After six Phagocytosis hours, the old DMEM media and transfection solution mixture was aspirated and new DMEM Integration of plasmid into cell genome was added to each well. Figure 4. Basic s of CaCl2 transfection (NPTEL)

After 24 hours, the GFP expression was checked and recorded. After 48 hours, the 10%

FBS DMEM media was removed. It was then replaced with DMEM containing no FBS (1% L-

Glutamine, and 1% Antibiotic-Antimycotic). The cells were therefore serum starved in this solution over night at 37°C.

Western Sample Collection

After being serum starved over night, the samples were collected from each well. First, the

DMEM was removed from each well and spun in a micro centrifuge tube at 400ref for 1 minute.

The supernatant was removed, leaving behind a small pellet with a small amount of DMEM on top.

500ul of cold 1x phosphate-buffered saline (PBS) was added to each well and aspirated after 10-

20 seconds. Second, 100ul of the cell lysis solution was added to each well (1% phenylmethane

17 sulfonyl fluoride, 1% NaVO4, 2% protease inhibitor cocktail, 25% 5x Loading dye, and the remaining solution was whole cell lysis buffer). The 12-well dishes were then rocked on ice for 15 minutes before the cells with the cell lysis solution were collected in their respective micro centrifuge tubes with the pellet. Afterwards, all samples were boiled for 5 minutes and then stored in -80°C.

Analysis by Western Blot

Protein expression was measured using a Western Blot. The western samples were boiled for five minutes prior to use. First, 12% SDS Page gels were made by placing two glass plates separately by a spacer on each side into a cassette. Once no leaks in the cassette are confirmed by filling it with ethanol, two gels are made by taking14mL of 12% resolving gel and mixing it with

100ul APS and 10ul TEMED. This solution is then poured between the glass plates, leaving about

15mm of space at the top, and then covered in ethanol. The gel was done polymerizing after 30 minutes, and the ethanol was removed prior to adding the stacking (5mL stacking gel, 50ul APS,

10ul TEMED). Then, a 15 well comb is inserted into the stacking gel and the gel is aloud to harden for about 20 minutes. The cassette was then placed in a running chamber filled with 10x Tris-

Glycine buffer, and fresh buffer was added to the cassette. Lastly, the comb was removed.

The samples were all boiled for five minutes and then placed on ice. From there, 8ul of each sample was loaded into the wells. The gel was run at 20AMPs/gel with a max voltage of 180V for 2.5 hours.

After the gel was finished running, the proteins were transferred to a nitrocellulose membrane. In order to do this, the gel is flipped left to right and placed into a transfer cassette.

The membrane was placed on top of the gel and then squeezed together by adding pads and closing the transfer cassette. The cassette was placed in the chamber and filled with transfer

18 buffer. The chamber was filled with ice and ice water. The transfer ran for 1.3 hours at 42V.

After, the membrane was removed, any necessary cuts were made, and the membrane was blocked for one hour on a rocker with milk blocking buffer (15g dry milk, 300mL 1x PBS, and

300ul Tween-20). After, the membranes were washed four times for five minutes each time in

1xPBS plus 0.1% Tween-20 (PBST).

The desired primary antibody was then added to the membrane and incubated over night in 4°C. Potential primary antibodies included GFP, Actin, total ERK, total RSK, phosphorylated

ERK, and phosphorylated RSK. The next day, the primary antibody was returned to its tube and the membrane was washed four times for 5 minutes each with PBST.

In order to visualize the protein, a secondary antibody was applied. The secondary antibody (1.6ul) was added to 10mL of milk buffer and aloud to incubate on the membrane in room temperature for an hour and a half. After, the solution was removed and the membrane was again washed four times for 5 minutes each time with PBST.

For visualization of the secondary antibody, and therefore protein expression, we used chemiluminescence. The solutions provided in the azure biosystems radiance kit were mixed and applied to the membranes, which were then exposed in the azure biosystems imaging machine for various amounts of time.

Data Analysis

The pERK and pRSK activation levels were analyzed on a scale of 0 to 5 based on observation of the Western Blot band shape and intensity and organized into tables. They were then mapped onto Carolyn Dang’s phylogenetic tree using the results from this paper.

IV. RESULTS

Activation of RSK and ERK by ORF45 Homologues

Figure 5. Western blot (WB) results of the ORF45 homologues. The WB nitrocellulose membrane obtained from transfer from an SDS Page gel was first probed for pRSK, pERK, and GFP in order to check the activation of ERK and RSK by ORF45 homologues. Then, they were stripped and re-probed for Total RSK, Total ERK, and Actin. From top to bottom of the figure: WB of phosphorylated RSK, WB of total RSK, WB of phosphorylated ERK, WB of total ERK, WB of GFP expression, and WB of Actin.

The DNA of each ORF45 homologue was inserted into HEK293T cells. These cells then expressed this DNA and made proteins accordingly. The proteins present in the cells were then collected and used in a western blot. The western blot membrane was then tested for specific proteins. For each image in Figure 5, the membrane was probed with an antibody specific to that

20 protein and then imaged in order to check for its presence. Based on Figure 5, the membranes probed for phosphorylated ERK and phosphorylated RSK show that there are different levels of pERK and pRSK amid the homologues. The blots of phosphorylated RSK that were more intense than the control Mock (-) were KSHV, RFHV, RRV, HVS, OVHV-2, AIHV-2, BOHV-6, PLHV-3, PLHV-1,

BoHV-4, EHV-5, and EHV-2. The strongest signals were for RFHV, HVS, BoHV-6, PLHV-1 and

BoHV-4. The weakest signals were for RRV, OVHV-2, and AlHV-2 (Table 2). Comparably, levels of phosphorylated ERK also varied, with KSHV, RFHV, RRV, OVHV-2, BOHV-6, PLHV-3, PLHV-1,

BoHV-4, EHV-5, EHV-2, and MHV-68 having higher levels of pERK than the control. The highest levels of pERK were seen in KSHV, RFHV, BoHV-4, and MHV-68 (Figure 5). The levels of pRSK and pERK activation given to each homologue can be seen in Table 3. The pERK and pRSK scores as well as intensity of the western blot mark for each homologue in Table 3 show that there is a difference between ERK and RSK activation between homologues of ORF45. Lastly, the standards

GFP and Actin were fairly analogous between the ORF45 homologues, but with RRV having a low

GFP expression, and pEGFP, KSHV, RFHV, EHV-5 and EHV-2 having stronger signals. Presence of total ERK and total RSK also showed similar levels between the samples (Figure 5). pERK and pRSK Relationship to Phylogeny

With in the ORF45 homologues, there is a difference between pERK and pRSK activation

(Table 3). The intensity of the signal for pRSK and pERK was observed and recorded on a scale from 0 to 5 based on the size of the protein marker for that homologue as well as the intensity and darkness of the mark (Table 2). These results were then mapped on a phylogenetic tree produced by Carolyn Dang. The phylogenetic tree is based on the genetic sequence of ORF45 for each homologue.

Looking at pERK mapped on the phylogenetic tree of Figure 6, there seem to be some relationship between close relatives and pERK expression. KSHV and RFHV are sister taxa and share similar levels of pERK activation at 4 and 5 respectively. Another clade with high ERK activation is seen between BoHV-4, EHV-5 and EHV-2 with scores of 3, 2, and 2 respectively.

Meanwhile, there are similar low levels of pERK among the larger clade of Macavirus (Figure 6).

Table 2. Level of pERK and pRSK in the western blots of ORF45 homologues as they induce the activation of ERK and RSK. The size and intensity (darkness) of the blot produced by each homologue was judged by observation on a scale from 0 to 5.

A. B.

Table 3. The ability of ORF45 homologues to induce the sustained activation of pRSK (Table A) and pERK (Table B) based on the size and intensity of the blots from Figure 5. The score for observed intensity of each blot goes from 0 to 5.

Similar trends are seen for pRSK. According to the levels of pRSK superimposed on the

neighbor-joining tree, KSHV and RFHV share similar levels of pRSK activation at 3 and 4.

Similarly, BoHV-6, PLHV-1 and PLHV-3 have comparable levels of pRSK activation of 5, 4, and 3

respectively. On the other spectrum, ALHV-2 and OvHV-2 are in the same clade and show

analogous low expression of pRSK, thus splitting the Macavirus genus in two in relationship to

pRSK. Similar to pERK, pRSK activation is again similar in the clade with BoHV-4, EHV-2, and EHV-

5 (Figure 6).

BoSHV-1 0,346 Bottlenose dolphin gammaherpesvirus-3

ovirus Lymphnocrypt

Neighbor-Joining pERK pRSK

Phylogeny of CalHV-3 0.2885 Callitrichine gammaherpesvirus 3

ORF45 Homologues 0 0 EBV 0.1634 Human gammaherpesbirus 4 Mapped with pERK and pRSK LCHV 0.05784 Lymphocryptovirus macaca RhEBV 0.04721 Macacine gammahperesvirus 4

AIHV-1 0.23792 Alcelaphine gammaherpesvirus 1

0 1 AIHV-2 0.2426 Alcelaphine gammaherpesvirus 2

1 1 OvHV-2 0.28606 Ovine gammaherpesvirus 2 Macavirus

2 5 BoHV-6 0.29178 Bovine gammaeherpesvirus 6

2 4 PLHV-1 0.05082 Porcine lymphotropic herpesvirus 1

PLHV-2 0.05232 Porcine lymphotrophic herpesvirus 2

1 3 PLHV-3 0.19728 Porcine lymphotrophic herpesvirus 3

AtHV-3 0.20348 Ateline gammaherpesvirus 3 0 4 HVS Saimiriine gammaherpesvirus 2 Rhadinovirus SaHV-2 0.02081 Saimiriine gammaherpesvirus 2

LHV-1 0.36802 Leporid herpesvirus 1

FCHV 0.35803 Felis catus gammaherpesvirus 1

EFHV 0.39739 Eptesicus fuscus gammaherpesvirus 3 4 BoHV-4 0.33315 Bovine gammaherpesvirus 4 Percavirus 2 3 EHV-2 0.0886 Equid gammaherpesvirus 2

2 3 EHV-5 0.20695 Equid gammaherpesvirus 5

HSHV 0.35083 Harp seal herpesvirus

4 0 MHV-68 0.09946 Murid gammaherpesvirus 4

WoMHV 0.0245 Wood mouse herpesvirus

WoMHV_1 0.024 Wood mouse herpesvirus Rhadinovirus RHVP 0.31858 Cricetid gammaherpesvirus 2

4 3 KSHV 0.2804 Humman Gammaherpesvirus 8

5 4 RFHV 0.2742 Retroperitoneal herpesvirus

MFHV 0.05097 Macaca fuscata gammaherpesvirus

1 1 RRV 0.04846 Rehus Monkey gammaherpesvirus

MNHV-2 0.13953 Macaca nemestrina rhadinovirus 2

My-HV8 0.40801 Myotis gammaherpesvirus 8

Figure 6. The score for pERK and pRSK activation by ORF45 homologues mapped onto the neighbor-joining phylogenetic tree re- produced based on the results of Carolyn Dang. This tree was produced based on the nucleotide sequence that encodes for ORF45 for each homologue. 24

RSK Binding site Score 54 69 KSHV 3 P - T V I D M S A P D D - V F A E D

RFHV 4 P - P V I D L S A P D D - V F A E D RRV 1 P - P V I D M T A P E D - V F D Q D

Rhadino HVS 4 E D G L L T S S G S D S - V F N S T MHV-68 0 - - D S D G S S T P D S - V F E A E OvHV-2 1 Q - L K R E M L D V E E - V F Y P E AlHV-2 1 K - I K E N I L S G E D - V F Y P D

BoHV-6 5 Q - F V K Q I L D E D E - V F Y P D Maca PLHV-3 3 T - D V E S M V R D D E - V F Y P E

PLHV-1 4 K - D V R S I L C D D D - V F Y P E EHV-5 3 - - - T K E S G D L D D - V F F P D

Perca EHV-2 3 - - - T K D A G D L D D - V F F E D

CONS * * * Figure 7. Conservation of the amino acid sequences for the RSK binding sites in ORF45 homologues listed by genus. These alignments are reproductions of the alignments in the honors thesis of Carolyn Dang seen in Figures 9 and 10.

ERK Binding Site Score 15 69

KSHV 4 E R M L P I E G A P R R R P P V K F I F P P P P L S RFHV 5 D R L F P Y E G A P R R V P P R R F I F P P P - - R

Rhadino RRV 1 P R M L P I P G A P R K K R T R R F L F - - A G S R HVS 0 R K L W P L P G A P R E K Q T N V F K F P T D G N D MHV-68 4 - R M L P I K G A P I S R P I S V F T F D V - F - N OvHV-2 1 D R M I P L E G A P R R K R T T F F T F P A F K N M

AlHV-2 0 D R L L P I E G A P R R K R T N Y F T F P C F K T L

Maca BoHV-6 2 D R L L P V E G A P R R K K T Q F F K F P P W K S P PLHV-3 1 D R L F D L E G A P R R K K T T Y F K F P P F K S L PLHV-1 2 D R L F D L E G A P R K K K T T Y F K F P P F S S V EHV-5 2 P R M T N D P G A P R L K Q A R Y F Q F P S S A I R

Perca EHV-2 2 P R M T S D P G A P R L K R A R Y F Q F P K D G I K

CONS * * * * * * * Figure 8. . Conservation of the amino acid sequences for the ERK binding sites in ORF45 homologues listed by genus. These alignments are reproductions of the alignments in the honors thesis of Carolyn Dang seen in Figures 9 and 10.

V. DISCUSSION AND FUTURE STUDIES Discussion

Multiple alignments of the ORF45 protein sequence of gammaherpesviruses show strong conservation in some regions. In this case, the binding sites of ORF45 that bind to ERK and RSK in order to promote prolonged phosphorylation and activation are conserved compared to the rest of the protein (Figures 7 and 8). Looking Figure 7, There is an over all strong conservation of amino acid 66 in the RSK binding site. According to Figure 8, there is strong conservation of a larger area for ERK binding. This could be due to multiple reasons. First, these regions could be conserved due to the high level of importance of sustained phosphorylation of ERK and RSK in the production of viral progeny. Another reason is due to convergent evolution, which is when two species have a similar trait due to evolving it for a common reason, not due to a shared ancestor.

It is possible that these viruses adapted to host cell mechanisms separately and evolved to form these complexes with ERK and RSK due to their independent needs instead of the protein being conserved by a common ancestor. As it is present in all gammaherpesvirinae and not other viruses, it is more likely that this is an example of conservation of ORF45 from an ancestor than convergent evolution of the protein.

Next, looking at the western blot results, there is variation in pERK, pRSK, and GFP between the ORF45 homologues. First, differences in GFP expression shows variability in the ability of

ORF45 homologues to express their genome in the host cell since the same amount of plasmid was delivered during transfection. For instance, pEGFP, KSHV, RFHV, EHV-5 and EHV-2 showed higher

GFP expression and therefore get taken into the cell thus expressing their genomes more efficiently (Figure 5). The ORF45 protein of KSHV, RFHV, HVS, BoHV-6, PLHV-1 and BoHV-4 is seen to cause strong levels of pRSK (Table 3, A). Meanwhile, ORF45 of KSHV, RFHV, BoHV-4, and

MHV-68 causes more activation of pERK than other homologues (Table 3, B). This could be due to 26 convergent evolution in the gammaherpesvirinae, conservation and differences in the amino acid sequences between one another and from a common ancestor, or different modes of viral progeny production decreasing or increasing the importance of sustained ERK/RSK.

Differences in amino acid sequences of ORF45 may be related to this difference in ERK/RSK activation. As the RSK activation site is less conserved, differences between homologues are more obvious. For example, KSHV and RFHV have very similar genetic sequences, and share an S at aa61 and a D at aa64 with HVS, which also shows strong activation. Meanwhile, RRV is genetically closely related but does not have that serine or aspartate, which could effect binding and explain its lower activation level (Figure 7). Comparatively, AIHV-2 an OvHV-2 are missing the glutamate or aspartate at aa3, which could be a reason they activate RSK much less than their close relatives

BoHV-6, PLLHV-1, and PLHV-3 (Figure 7). From only looking at the ERK/RSK conserved regions, it seems as though differences in amino acid sequences do have some effect on ERK/RSK activation in ORF45 homologues, but internal deletions and other experiments studying the effects of those differing amino acids would be necessary to make further conclusions.

According to Figure 6, the differences in ability to alter ERK/RSK appear to correlate to the phylogenetic tree. There is a possibility that convergent evolution is responsible for the patterns seen by forming a phylogenetic tree based off ORF45 sequences. As this pathway is very important for virus progeny production, it cannot be for certain that the coding regions did not evolve separately in some of the viruses instead of through a common ancestor. Additionally, as the sequences surrounding the binding regions tend to vary, there is a high possibility that these regions are being purposefully conserved. Assuming that the DNA sequence of ORF45 is due to homology and not convergent evolution, there is a correlation between common ancestry of

ORF45 and the ability to activate these pathways. KSHV and RFHV are sister taxa and share

27 similar levels of pERK and pRSK activation. Also, BoHV-4, EHV-2, and EHV-5 form their own clade and share similar intensities for pRSK and pERK. There are also differences between pERK and pRSK activation that can be related to phylogeny. For example, viruses in the Macavirus genus all have similar pERK activation levels and share a common ancestor. When looking at more recent relationships, this genus splits into two main clades. The clade with ALHV-2 and OvHV-2 has low pRSK activation, while the clade with BoHV-4, PLHV-3, and PLHV-1 have higher levels of pRSK

(Figure 6). Meanwhile, MHV-68 has high pERK activation with a score of 4, and no pRSK activation. Comparatively, HVS has a high pRSK activation of 4 and no pERK activation (Table 2).

Future Studies

Based on this data, we can predict which amino acids are important to ERK and RSK binding, such as amino acid 66 and the larger region of pERK. We can also observe how differences in these sequences are changing activation of ERK and RSK between homologues.

Lastly, we have mapped the activation of ERK and RSK onto the phylogeny and can from there predict that level of function of ORF45 is more consistent in the clades that share the most recent common ancestor than clades that branch off three or four common ancestors back.

Understanding why this is would be the next step.

There are many factors that play a role in ORF45 function, such as length, other regions not involved in ERK and RSK binding, and subcellular localization. For this reason, more studies must be completed to test these predictions. These studies may test the conservation of the ORF45 protein in gammaherpesvirinae, why some activate ERK and RSK and others do not, how they activate ERK/RSK, which regions are important in activation, and when and why the divergence happened between homologues that do and do not activate these signaling pathways. Some research that could be completed to further the knowledge of this subject are internal deletions or

28 substitutions to determine which amino acids are most important in ERK and RSK activation in which homologues and if this relates to the homology and evolution of the protein. In addition, the same experiment completed could be done using amino acids 1-115 as they have been found important in ERK and RSK activation. Deletions or substitutions could also be done using aa1-115 instead of the full length of ORF45 homologues. Other tests would also need to be completed and compared, such as sub cellular localization and how it affects ERK/RSK binding, localization in terms of phylogeny, and a comparison of other functions present in different ORF45 homologues.

VI. CONCLUSION

ORF45 homologues have various similarities and differences based on these results. First, there is a strong conservation of ERK and RSK binding sites of ORF45. It has been concluded that the region from aa1-115 is very important in ERK/RSK binding. Differences in the conserved regions between homologues are likely an effector of ERK/RSK binding and therefore sustained activation of pERK/pRSK. From there, the varying levels of pERK and pRSK between homologues show a strong correlation to close relatives on the phylogenetic tree. By using homologues to study activation of certain pathways, the amino acid sequences responsible for that particular protein function can be determined. Over all, evolutionary mechanisms all effect virus evolutionary history, which is important in understanding the difference in localization, length, mechanisms, activation, and sequence of the ORF45 protein. When homologues are studied in conjunction with evolution, comparing which sites are conserved and the differences between them can provide insight into their purpose and importance to viral efficiency.

VII. ACKNOWLEDGEMENTS I would like to sincerely thank my thesis director Dr. Fanxiu Zhu for his guidance, patience, and support during this project. Thank you for taking a chance and inviting me into your lab even as we believed I would be graduating that semester. When I asked to stay for another year and continue to procure results on this topic through an honors thesis, you supported and encouraged me. I greatly appreciate the opportunity that you’ve given me to learn about lab procedure, microbiology, scientific writing, and the scientific community over the past year. My discovery of research started with your lab, and my love for microbiology and research grew into a future career path because of this lab and your inspiration, advice, and guidance.

I would also like to thank the rest of my honors thesis committee Dr. Scott Steppan and Dr.

Jeff Chanton for supporting me in my endeavors. Your commitment to helping your students and myself is an inspiration. Thank you to you both for your advice and time this past year even though you both have busy schedules. It has been a gift to have you both on my committee.

I want to give a special thanks to Carolyn Dang and Hongyu Zhang for initiating this project.

You both taught me so much about microbiology and lab procedure, and have continued to support and assist me this last year after graduating. Carolyn, your guidance, friendship, and willingness to answer all my questions has made this honors thesis that much better of an experience. Lastly, a thank you to Randula Fonseka, Michael Taylor, and John Henriquez for the hours spent in lab with me teaching me proper techniques and procedures to obtain my results and for the making this lab feel like home. I am honored to be friends with and share this experience with you all.

VIII. Supplementary Material

A. B.

Figure 9. Conservation of ORF45 homologues used in these experiments in their amino acid sequence. (A) displays the conserved RSK binding site. (B) Displays the conserved ERK Binding site. These alignments were produced the honors thesis of Carolyn Dang.

A. B.

Figure 10. Conservation of all ORF45 homologues in terms of genus. (A) displays the conserved RSK binding site. (B) displays the conserved ERK binding site. These alignments were produced the honors thesis of Carolyn Dang.

IX. REFERENCES Antman, Karen and Yuan Chang. 2000. Kaposi Sarcoma. The New Englad Journal of Medicine 342: 1027-1038.

Cargnello, M., & Roux, P. P. (2011). Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases. Microbiology and Molecular Biology Reviews: MMBR, 75(1):50-83. Chang, Y., & Moore, P. 2014. Twenty Years of KSHV. Viruses,6(12): 4258-4264. Chang, Y., Cesarman, E., Pessin, M., Lee, F., Culpepper, J., Knowles, D., et al. 1994. Identification of herpesvirus-like DNA sequences in AIDS-associated kaposi's sarcoma. Science 266(5192):1865-1869. Cook, Pamela M. et al. 1999. Variability and evolution of Kaposi’s sarcoma-associated herpesvirus in Eurpoe and Africa. AIDS, 13: 1165-1176

Decker, L. L., Shankar, P., Khan, G., Freeman, R. B., Dezube, B. J., Lieberman, J., et al. 1996. The kaposi sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients. The Journal of Experimental Medicine, 184(1):283-288.

Escalera-Zamudio, Marinae et al. 2016. Bats, Primates, and the Evolutionary Origins and Diversification of Mammalian Gammaherpesviruses. American Society for Microbio. 7(6).

Fu, B., Kuang, E., Li, W., Avey, D., Li, X., Turpin, Z., Valdes, A., Brulois, K., Myoung, J., & Zhu, F. 2016. Activation of p90 ribosomal S6 kinases by ORF45 of Kaposis sarcoma-associated herpesvirus is critical for optimal production of infectious viruses. Journal of Virology ,89(1):195-207. Gibson, W. 1996. Structure and assembly of the virion. Intervirology, 39(5-6):389-400.

Gillen, Joseph et al. 2015. A Survey of the Interactome of Kaposi’s Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses. Journal of Virology 89(9): 4918-4931.

Gross, Charles. 2012. Pangaea and the Out-of-Africa Model of Varicella-Zoster Virus Evolution and Phylogeography. Journal of Virology, 86(18): 9558-9565.

Hafner, M.S. & Nadler, S.A. 1988. "Phylogenetic trees support the coevolution of parasites and their hosts." Nature 332: 258-259

Jha , H. C., Banerjee, S., & Robertson, E. S. 2016. The role of Gammaherpesviruses in Cancer Pathogenesis [Review]. Pathogens 5(18):1-43. Kochanczyk, Marek et al. 2017. Relaxation oscillations and hierarchy of feedbacks in MAPK signaling. Scientific Reports 7(38244).

Kuang, E., Q. Tang, G. G. Maul, F. Zhu. 2008. Activation of p90 Ribosomal S6 Kinase by ORF45 of Kaposi’s Sarcoma-associated Herpesvirus and its role in viral lytic replication. Journal of Virology 82(4):1838-1850

Kuang, E., Fu, B., Liang, Q., Myoung, J., & Zhu, F. 2011. Phosphorylation of Eukaryotic Translation Initiation Factor 4B (EIF4B) by Open Reading Frame 45/p90 Ribosomal S6 Kinase (ORF45/RSK) Signaling Axis Facilitates Protein Translation during Kaposi Sarcoma- associated Herpesvirus (KSHV) Lytic Replication . Journal of Biological Chemistry, 286(48):41171-41182.

Li, X., & Zhu, F. (2008). Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposis Sarcoma-Associated Herpesvirus. Journal of Virology 83(6):2531-2539. McGeoch, Duncan J. 2001. Molecular Evolution of the y-Herpesvirinae. Phil. Trans. R. Soc. Lond. 356:421-435

Moore, P. S., and Y. Chang. 2001. Kaposi’s sarcoma-associated herpesvirus, p. 2803-2833. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Staus, Fields virology. Lippincott Williams and Wilkins, Philadelphia, Pa Russo, J. J., R. A. Bohenzky, M. C. Chien, J. Chen, M. Uan, D. Maddalena, J. P. Parry, D. Peruzzi, I. S. Edelman, Y. Chang, and P. S. Moore. 1966. Nucleotide sequence of the Kaposi sarcoma- associated herpesvirus (HHV8). Proc. Natl. Acad. Sci. USA 93:14862-14867 Smalley, Inna and Keiran S.M. Smalley. 2016. ERK Inhibiton: A New Front in the War against MAPK Pathway-Driven Cancers? American Association for Cancer Research.

Zhu, F. X., & Yuan, Y. 2003. The ORF45 Protein of Kaposis Sarcoma-Associated Herpesvirus Is Associated with Purified Virions. Journal of Virology 77(7): 4221-4230. Zhu, F. X., S. M. King, E. J. Smith, D. E. Levy and Y. Yuan. 2002. A Kaposi’s sarcoma-associated herpesviral protein inhibits virus-mediated induction of type I interferon by blocking IRF-7 phosphorylation and nuclear accumulation. Proc. Natl. Acad. Sci. USA 99:5573-5578. Zhu, F. X., X. Li, F. Zhou, S. J. Gao, and Y. Yuan. 2006. Functional characterization of Kaposi’s sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis. J. Birol. 80:12187-12196