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2018 A Comparative Analysis of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Homologs Carolyn Kellie Dang

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The Florida State University Department of Biological Science

Honors in the Major Thesis

A Comparative Analysis of KSHV ORF45 Homologues

Carolyn K. Dang

July 2018

THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS & SCIENCES

A COMPARATIVE ANALYSIS OF KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS ORF45 HOMOLOGS

CAROLYN DANG

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded: [Summer, 2018]

The members of the Defense Committee approve the thesis of Carolyn Dang defended on July 19, 2018. Signatures are on file with the Honors Program office

______Dr. Fanxiu Zhu Thesis Director

______Dr. Hengli Tang Committee Member

______Dr. Mia Lustria Committee Member

Table of Contents

 Abstract………………………………...... 5  Introduction A. Kaposi’s Sarcoma……………………………………………………….6 B. Kaposi Sarcoma Herpesvirus (KSHV) C. Herpesviridae Family Classification ……..……………………….…….7-8 D. Significance of Kaposi Sarcoma-Associated Herpesvirus (KSHV) ORF45 and Mitogen-Activated Protein Kinase Pathway (MAPK)……………..8-11  Research Question/Focus ……………………………………………………...12  Materials and Methods A. Multiple Alignments…………………………………………………….13 B. Plasmid Preparation via Molecular Cloning……………………………14-18 C. Transfection…………………………………………………………….14-20 D. G418 Selection….……………………………………………………....20-22  Results A. Conservation of ERK, RSK, and ORF33 Binding Site Amongst ORF45 Gammaherpesvirus-2 Homologues ……………………………………..26 B. ORF45 Homologues Express Different Levels of Phosphorylated RSK and ERK……………………………………………………...... 26 C. G418 Selection.….……………………………………………………....28  Discussion and Conclusion A. Similar phylogenic relationship amongst gammaherpesvirus-2 proteins….……………………………………………………...... 29 B. Different ORF45 homologues show differing levels of RSK and ERK activation….…………………………………………………………….29 C. Different ORF45 homologues show differing rates of proliferation….…………………………………………………33  Acknowledgements………………………………………………………….…31  Supplementary Material……………………………………………………….34-44  References…………………………………………………………...…….…..45-46

Abstract

Kaposi sarcoma is a type of lymphatic and skin cancer that is caused by Kaposi Sarcoma- associated virus (KSHV). KSHV ORF45 is classified as an immediate early gene that has multifunctional roles. One of its major functions is to activate the extracellular signal-regulated kinase

(ERK) and p90 ribosomal kinase 1 and 2 (RSK1/2) in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. It has been discovered that KSHV ORF45 can activate both the ERK and RSK which promote the reactivation of KSHV. There is limited evidence that observes the behavior of all KSHV ORF45 homologs. Therefore, we are interested in studying the functional conservativeness within gammaherpesvirus ORF45 subfamily, including the ability of ORF45 homologues to activate ERK and RSK, and the colonigenesis of HeLa cells transfected with ORF45 homologues. In this study, we found that amongst all ORF45 homologues, there is strong conservation in the amino-terminus, ERK, RSK and ORF33 binding sites. We examined that there are different levels of phosphorylated RSK and ERK amongst the gammaherpesvirus homologues. In addition, we examined that G418 selection of different ORF45 homologues. The result showed no significant difference between the gammaherpesvirus-2 subgroups (Rhadinovirus, Macavirus, and Percavirus).

Introduction

Kaposi Sarcoma

Kaposi sarcoma (KS) is an aggressive lymphatic and skin cancer that arises from Kaposi

Sarcoma-associated virus (KSHV). Moritz Kaposi was the first to formally describe KS in 1872; after observing abnormal skin lesions in elderly men (Chang and Moore, 2014). By 1994, KSHV was further described. Later, KSHV was deemed responsible for the progression of three human malignancies: (1)

Kaposi sarcoma, primary effusion lymphoma (PEL) and Multicentric Castleman’s disease (Fu et al.,

2016).

Kaposi Sarcoma can be classified into four clinical variants: (1) classic KS, (2) endemic KS, (3) iatrogenic KS, and (4) AIDS-associated KS. Classic KS primarily affects Mediterranean and Eastern

European decent, HIV-negative, elderly men. Iatrogenic KS influences immunosuppressive patients after organ transplants. Meanwhile, endemic KS can develop in all population demographics residing in

Eastern, Equatorial, and Southern Africa. The human immunodeficiency virus (HIV) was initially discovered in 1983; shortly after the AIDs epidemic. It was found that endemic KS increased along with the AIDs epidemic – affecting both HIV-negative children and adults. AIDS-associated KS is most prevalent among gay and bisexual males whom are HIV-positive in North American and European populations (Jha et al., 2016).

Solid tumor masses can be found in the lymph nodes, lungs, and gastrointestinal tract of individuals affected with KSHV. Upon physical examination, dark purple lesions can be observed on the skin’s surface. Closer examination of KS lesions shows characteristics such as (1) erythrocyte extravasation, (2) infiltrated inflammatory cells, and (3) “spindle” cells. “Spindle” cells are KSHV- affected cells that change to an elongated shape (Jha et al., 2016).

Kaposi Sarcoma-Associated Virus (KSHV)

Kaposi Sarcoma-associated virus (KSHV) was further described in 1994 (Chang and Moore,

2014). By 1996, KSHV was sequenced and was genomically mapped showing 81 ORFs and multiple untranslated transcriptions were discovered in the entire genome (Russo et al., 1996). KSHV was classified into the Gammaherpesvirinae subfamily; specifically, the Rhadinovirus genus. Further studies showed that KSHV was a DNA tumor virus that has the ability to cause Kaposi Sarcoma in skin and multiple organs. KSHV, like other herpesviruses, has two different life cycles; lytic and latent cycle.

KSHV is able to switch the life cycle according to host stress and immune response. During the latent life cycle, the virus manipulates the cell’s DNA host machinery to produce circular viral episomes in tandem. Few viral genes are produced. Meanwhile, the lytic life cycle allows for the viral genome to be replicated effectively by utilizing the host’s DNA machinery. The full panel of viral genes is expressed and the virion is assembled and mass released to cause the infection of cells. Therefore, we are interested in the reactivation of KSHV from a latent to lytic life cycle; specifically, the role of KSHV

ORF45 (Zhu and Yuan., 2003).

Herpesviridae Family Classification

The Herpesviridae family consist of three subfamilies; (1) Alphaherpesvirinae, (2)

Betaherpesvirinae, and (3) Gammaherpesvirinae. Examples of viruses included in the

Alphaherpesvirinae subfamily are Herpes Simplex Virus 1 and 2 (HVS-1 and HVS-2) and Varicella zoster virus (VZV). Members of Alphaherpesvirinae can infect a wide range of species and have the ability to establish latency in neuron cells. Meanwhile, viruses within the Betaherpesvirinae subfamily include human Cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6) and human herpesvirus 7

(HHV-7). Viruses defined within the Gammaherpesvirinae subfamily include Epstein-Barr virus (HHV-

4) and human herpesvirus 8 (HHV-8). Members of the Betaherpesvirinae and Gammaherpesvirinae subfamily can infect a narrow range of species. Members of the Betaherpesvirinae can establish latent infection within monocytes, while Gammaherpesvirinae can establish latency in lymphocytes (Davison,

2007).

Significance of Kaposi Sarcoma-Associated Herpesvirus (KSHV) ORF45 and Mitogen-Activated

Protein Kinase Pathway (MAPK)

KSHV ORF45 is a multifunctional, immediate and early protein that can modulate the host cell environment and promote the lytic viral life cycle. Two major functions of KSHV ORF45 have been noted. (1) KSHV ORF45 inhibits the interferon regulatory factor 7 (IRF-7). IRF-7 is the host’s innate immune response that antagonizes DNA and RNA viruses. (2) KSHV ORF45 has the ability to interact with ERK and p90 ribosomal kinase 1 and 2 (RSK1/RSK2) which have major roles in the

Ras/Raf/MAPK/ERK pathway (Li and Zhu, 2009). The MAPK/ERK pathway is a chain of proteins in a cell that communicate via a membrane receptor on the cell’s surface. If the proteins within the pathway are mutated, a potential effect is prolonging of protein transcription. Most human cancers can deregulate the MAPK/ERK pathway causing continuous production of proteins. The MAPK is able to relay extracellular signals to activate intracellular responses. The MAP kinases pathways can be further divided into three families; (1) extracellular-signal-regulated kinase (ERK), (2) Jun amino-terminal kinase (JNKs), and (3) stress-activated protein kinase (p38/SAPKs) (Morrison, 2012). In our study, we will be focusing on the ERK and RSK activation within the MAP kinase pathway.

It has been found that the MAPK pathway consist of three evolutionarily conserved kinases (MAPK,

MAPKK, and MAPKKK). Extracellular cues such as growth factors, stress, and inflammatory cytokines can lead to the Ras/Rho family. The activation of the Ras/Rho family would phosphorylate the Ser/Thr

kinase (MAPKKK). As a result, the MAPKKK activation would lead to the phosphorylation of

MAPKK. The MAPKK kinase is also known as the MAPK/ERK kinase (MEK). The activation of

MAPKK leads to the phosphorylation of MAPK or ERK pathway (Cargnello and Roux, 2011). Once the

ERK pathway (ERK1/2) is activated, RSK can be phosphorylated to induce cell growth and differentiation. Hence, the affect of KSHV ORF45 on RSK1/RSK2 (protein kinases) in the

ERK/MAPK pathway is a major factor for the reactivation of a latent to lytic life cycle.

It has been found that KSHV ORF45 can cause sustained activation of p90 ribosomal S6 kinases

(RSK) and extracellular regulated kinase (ERK). Prior studies have identified the amino-terminal between residues 1-115 is sufficient for the binding and activation of RSK. By conducting amino acid deletions, it was further identified as regions aa56-70 as being the range for activation of RSK. When comparing the alignment of ORF45 sequences to other Rhadinovirus, gamma-2 herpesvirus, there was limited homology found within the region of aa56-70.

Figure: Alignments of residues 51-75 of KSHV ORF45 and its respective homologs of RRV, HVS, MVH68a, and MVH68b.

Source: 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. doi:10.3410/f.722183224.793516429

The single, fully conserved residue resides at position 66. By conducting a single point mutation and changing the F66 to alanine, the result was no binding of ORF45 to ERK and RSK. When introducing the mutation to the entire genome of KSHV, there was seen a reduction in RSK activation and decrease in infectious viruses produced by 10-fold less. The sustained phosphorylation of ERK and

RSK may be due to KSHV ORF45 protecting the active kinases and preventing these kinases from dephosphorylating (Fu et al., 2016).

There is further evidence to suggest that the sustained activation of ERK/RSK signaling pathway may cause continuous phosphorylation of eIF4B. Eukaryotic translation initiation factors (eIFs) are involved in initiation; the first step in protein translation. During protein translation, various eIFs (ex. eIF4F, eIF4G, eIF4B) are involved in different roles to prepare the protein for translation. The eIF4B is responsible for ribosome recruitment to the mRNA and initiates the translation of capped and uncapped mRNA by working in collaboration with eIF4A and eIF3. The function of eIF4A is to unwind a protein’s secondary structure through helicase activity. Meanwhile, the function of eIF3 is to stimulate all steps during translation initiation. Hence, the sustained activation of RSK increases phosphorylation of eIF4A, eIF4B, and eIF3 preventing controlled regulation of the ERK/RSK signaling pathway (Kuang et al., 2011).

Of all gamma-2 herpesviruses that are studied, KSHV is the most well-characterized. However, there are few studies observing the behavior of KSHV homologs and KSHV ORF45 itself. Homologs of

KSHV ORF45 that have been studied include: (1) MHV-68, (2) EBV, (3) RRV, and (4) HVS. Amongst the homologs, some similarities that are observed is both the amino- and carboxyl- terminal are strongly conserved across all gamma-2 herpesviruses. In addition, MHV-68 ORF45 is seen as a tegument protein which may play a role in its presence in both the nucleus and cytoplasm (Xing et al., 2016). In other

ORF45 homologs subcellular localization, KSHV ORF45 is found in the nucleus. Meanwhile, RRV and

HVS show evidence of ORF45 localization in the nucleus. The gamma-1 herpesvirus homolog of KSHV

ORF45 – EBV BLRF4 – is localized in both the nucleus and the cytoplasm (Li and Zhu, 2009). These studies suggest that ORF45 homologs react differently in a host cell. Therefore, the activation of the

ERK/RSK pathway may vary between homologs – but the point of activation divergence amongst different viruses is unknown.

Research Question/Focus

The homology for KSHV ORF45 has not been widely studied. ORF45 is found to be conserved among gammaherpesviruses with close relations to Rhesus monkey rhadinovirus (RRV), herpesvirus saimiri (HVS), and murine gammaherpesvirus 68 (MHV-68). Even when evaluating the conservation between the different homologs, it is found the homology is limited amongst the various ORF45 proteins. Using multiple alignment, it is found that all the homologs differ in lengths and the middle portion diverges. However, regardless of host organism, it is seen that the amino- and carboxyl- terminal ends of the sequences are strongly conserved within those regions.

The critical region of aa1-115 in KSHV ORF45 has been shown to be involved in RSK/ERK binding and activation. In addition, there is evidence showing the RSK/ERK pathway activation within

MHV-68 and RRV lytic infection as well. The scope of this thesis will focused on the following questions:

1) Upon multiple alignment analysis, there are patterns of conserved regions amongst the

gammaherpes-2 ORF45 homologs. Do these ORF45 homologs differ in their ability to activate

RSK and ERK?

2) At which point in the phylogenetic tree does the activation of the ERK/RSK pathway diverge?

3) Is there a difference in proliferation of HeLa cells transfected with ORF45 homologs in G418

selection?

Materials and Methods

A. Multiple Alignments The protein and nucleotide sequences of KSHV ORF45 homologues were found using the Basic

Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information

(NCBI). Afterwards, multiple alignment tools from European Bioinformatics Institute (EBI) was used for alignment analysis. Alignment results were yielded from T-Coffee, PRALINE and manually adjustment.

B. Plasmids Preparation via Molecular Cloning

1a. Synthesis of Homologue Genes The following nine (9) homologue genes were synthesized by Genscript: Full ORF45 Virus Name Abbreviation DNA length Protein Length Protein size Accession Number (bp) (aa) (kDa) Alcelaphine ALHV-2 774 258 28.38 NP_065543.1 gammaherpesvirus 2

ORF45 Bovine gammaherpesvirus 4 BoHV-4 726 242 26.62 NP_076537.1 ORF45

Bovine gammaherpesvirus 6 BoHV-6 822 274 30.14 YP_009042022.1 ORF45

Equid gammaherpesvirus 2 EHV-2 966 322 35.42 NP_042642.1

ORF45 Equid gammaherpesvirus 5 EHV-5 936 312 34.32 YP_009118434.1 ORF45

Ovine gammaherpesvirus 2 OvHV-2 786 262 28.82 YP_438167.1 ORF45

Porcine lymphotropic PLHV-1 672 224 24.64 AAM22144.1 herpesvirus 1

ORf45

Porcine lymphotropic PLHV-3 687 229 25.19 AAO12348.1 herpesvirus 3

ORF45 Retroperitoneal RFHV 1089 363 39.93 AGY30726.1 fibromatosis-associated herpesvirus ORF45 Table 1: ORF45 homologue viruses synthesized by Genscript with respective DNA length (bp), protein length (aa), and protein size (kDa).

All gene inserts were synthesized and cloned into a standard pUC57 vector. The pUC57 vector is 2,710 bp and isolated from Escherichia coli strain DH5a. Upon request from Genscript, the BamHI and XhoI digestion cloning sites were added into the vector.

1b. Digestion

To improve visibility and measure gene expression future experiments, each homologue gene- insert was cloned into a pEGFP-C2 vector. The Genscript gene products were provided in a pUC57 vector. To release the gene-insert from the pUC57 vector, 5 ug of the gene-insert-pUC57 vector was digested with New England Biolabs (NEB) restriction enzymes BamHI (1.5 µL) and XhoI (1.5 µL). The pEGFP-C2 vector (5 ug) was digested with NEB BgIII and SalI restriction enzymes. To ensure an optimal digestion environment, 10x NEB Buffer 3.1 (7.5 µL), 100x Bovine Serum Albumin (BSA) (0.75

µL), Shrimp Alkaline Phosphatase (SAP) (1 µL), and ddH2O (used to increase final reaction volume to

75 uL) were included in each digestion reaction.

The gene-insert release digestion system was conducted for two (2) hours in a 37° Celsius waterbath. After two (2) hours, 1 µL of the digestion product was ran through a 50-mL 1.2% agarose electrophoresis gel for 45 minutes at 100V to check for digestion efficiency. Upon the gel-

electrophoresis results, the gene-insert digestion system was heated at 65° Celsius for 10 minutes for restriction enzyme inactivation.

The pEGFP-C2 vector digestion system was placed in a 37° Celsius incubator overnight (12 hours) since the digestion system had lower enzymatic activity. After 12 hours of digestion, 1 µL of the digestion product was ran through an 1.2% agarose electrophoresis gel for 45 minutes at 100 volts (V) to check for proper digestion of the vector. Afterwards, the pEGFP-C2 vector digestion system was heat in-activated at 65° Celsius for 10 minutes.

The remaining gene-inserts and pEGFP-C2 digestion system (74 µL) were ran through 150-mL

1.2% agarose gel at 60 volts (V) for one (1) hour. After one (1) hour, the bands corresponding to the desired gene-inserts and pEGFP-C2 vector were extracted with a razor and placed into separate microcentrifuge tubes.

Vector Digestion Site Sequence

pEGFP-C2 BgIII 5’ . . . A G A T C T . . . 3’

3’ . . . T C T A G A . . . 5’

SalI 5’ . . . G T C G A C . . . 3’

3’ . . . C A G C T G . . . 5’

pUC57 BamHI 5’ . . . G G A T C C . . . 3’

3’ . . . C C T A G G . . . 5’

XhoI 5’ . . . C T C G A G . . . 3’

3’ . . . G A G C T C . . . 5’

Table 2: Restriction enzymes used for digestion of pEGFP-C2 and pUC57 vector.

1c. Gel-Extraction

The gene-inserts and pEGFP-C2 vector were purified following strict protocol from the Omega Bio-Tek

E.Z.N.A. Gel Extraction Kit (D200-02). The pEGFP-C2 vector and gene inserts were separated at 100V for one (1) hour using a 1.2% agarose gel via gel electrophoresis. The fragment of interests were excised from the gel using a sterilized razor for each sample. The gel fragment with gene insert were placed with into a microcentrifuge tube. The weight of the gel fragment with gene insert was weigh in grams using a scale. The conversion for density was 1 gram was equivalent to 1 milliliter. One (1) volume of binding buffer (XP2) was added in respect to the density conversion. The gel fragment was incubated for 7 minutes at 60 degrees Celsius with the binding buffer solution. Every 2-3 minutes, the solution in the tube was vortex to homogenize the mix and aid in melting the gel. Once the gel had completely melted, the solution was filtered through a HiBind DNA mini column by centrifuging at

10,000 g for 1 minute at room temperature. After the entire volume of solution had been filtered through the column, 300 µL of binding buffer (XP2) was added to the HiBind DNA mini column and centrifuged at max speed (>13,000 g) for 1 minute at room temperature. The filtrate was discarded and an additional 700 µL of SPW wash buffer (diluted with 100% ethanol) was added to the DNA mini column and centrifuge at max speed for 1 minute. The SPW wash buffer step was repeated for an additional time. Afterwards, the HiBind DNA mini column was centrifuge at max speed for 2 minutes to remove remaining ethanol solution and to dry the column. The HiBind DNA mini column was transferred to a sterilized micro centrifuge tube. Exactly 41 µL of ddH2O was added directly to the

HiBind DNA mini column. The HiBind DNA mini column was incubated at room temperature for 2 minutes. After 2 minutes, the HiBind DNA mini column and tube was centrifuged at max speed for 1 minute to elute the digested insert or vector DNA. The concentration of the gene-inserts and pEGFP-C2 vector was measured using a Nanodrop.

1d. Ligation

The digested pEGFP-C2 vector (25 ng) and gene-insert (75 ng) were ligated with NEB T4 ligase

(0.5 uL). To ensure an optimal ligation environment, NEB 10x ligase buffer (1 µL) and ddH2O (to increase final reaction volume to 10 µL) were added to each ligation system. Each ligation system was incubated overnight at 16°C.

Figure 1: The molecular cloning construct of ligating pEGFP-C2 with respective gene inserts.

1e. Transformation

The ligation products (3 µL) were added into TOP10 chemically competent Escherichia coli cells (100 µL) and incubated on ice for 25 minutes. After 25 minutes, the ligation and TOP10 cell solution was heatshock for 90 seconds in a 42 degree Celsius waterbath. After 90 seconds, the tube was

reset and incubated on ice for 5 minutes. After 5 minutes of incubation on ice, 200 µL of Luria broth

(LB) was added to the tube using aseptic technique. The tube was incubated in a rotary for 45 minutes at

37 degrees Celsius. After 45 minutes, 300 µL of the ligation, TOP10 cells, and LB broth solution was spread on Luria broth (LB) plates with kanamycin. The LB plates with kanamycin were incubated overnight at 37 degrees Celsius. The next day, the transformants are inoculated into LB broth with kanamycin and shaken at 37°C overnight for further selection. Afterwards, isolation of plasmid DNA is attained by following the strict protocol by Omega-Trek E.Z.N.A Plasmid Mini Kit I. Next, the plasmid

DNA were digested with NEB restriction enzymes NheI and BamHI to confirm the presence of the gene-insert. The digested plasmid DNA were ran on a 1.2% agarose gel at 100V for 45 minutes to check for appropriate insert-size. The sequence of plasmid DNA were verified from sequencing results from the FSU Biology Sequencing Facility.

Figure 2: Summary of general method used for molecular cloning.

C. Transfection

2a. Cell Culture

HEK293T embryonic human kidney cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% L-Glutamine (100x), and 1%

Antibiotic-Antimycotic (100x) at 37°C.

2b. Calcium-Chloride Transfection

One day before transfection, HEK293T cells were seeded into two 12-well plates. By cell- counting, approximately 4.4 x 105 cells were seeded into each well. Transfection of HEK293T cells began when cells were approximately 70-75% confluent. Exactly 2000 ng of plasmid DNA from each representative virus was added into 50 µL of 0.25 M CaCl2 and incubated at room temperature (23°C) for 5 minutes. Afterwards, the plasmid-CaCl2 solution was added drop-wise to 50 µL of HEPES buffer solution (HBS) and incubated at room temperature for an additional 5 minutes. The transfection solution was added drop-wise to the respective well. The cells are returned to the 37°C incubator.

After 6 hours, the old DMEM media was removed and new DMEM media was added to each well.

After 48 hours, the serum-starve process began. The DMEM (10% Fetal Bovine Serum (FBS),

1% L-Glutamine, and 1% Antibiotic-Antimycotic) is removed and replaced with serum-free DMEM

(1% L-Glutamine, and 1% Antibiotic-Antimycotic). The cells were serum-starved overnight at 37 degrees Celsius.

2c. Sample Collection

After overnight incubation, the serum-free DMEM was removed from each well. Cold 1x phosphate- buffered saline (PBS) was added to each well and aspirated after 10-20 seconds. A cell lysis solution consisting of 140 µL whole cell lysis buffer, 1% phenylmethane sulfonyl fluoride (PMSF), 1% NaVO4, and 1% protase inhibitor cocktail (PIC) was added into each well. The cell-dishes were rocked on ice for

15 minutes. After 15 minutes, the lysis solution was collected and placed into the respective microcentrifuge tube. Each sample was sonicated for five seconds. Afterwards, 5x loading dye (36.5 µL) was added to each sample. Before utilizing proteins for western blot analysis, samples were boiled for five minutes.

D. Measuring Protein Expression Using Western Blot

Western blot analysis was used to measure ORF45 homologue protein expression in HEK293T cells.

Each ORF45 homologue protein sample (10 µL) was loaded into a 10% sodium dodecyl sulfate (SDS) gel. The gel ran at 200 volts for 2.5 hours for protein separation. Proteins were transferred onto a nitrocellulose membrane (GE Healthcare Amersham Protran) at 42V for 1.3 hours with ice surrounding the transfer cassette. Afterwards, the membrane was blocked with milk buffer (300 mL 1x PBS, 300 uL

Tween-20, 15g dry milk) for one hour at room temperature (23°C). The membrane was washed for 5 minutes for four times with 1x PBST to remove blocking buffer.

Depending on the desire result, the membrane was submerged with GFP (1:1000), Actin (1:2000) , anti-phosphorylated RSK (1:500), anti-phosphorylated ERK (1:500), anti-total RSK 1/2/3 (1:500), or anti-total ERK1/2 (1:500) primary antibodies overnight at 4°C. The following day, the primary antibody was removed. Nitrocellulose membranes are washed for 5 minutes for four times with 1x PBS.

Secondary antibody (5uL) with milk buffer (10 mL) was added to the nitrocellulose membrane and incubated at room temperature for one hour on the rocker. After one hour, the secondary antibody solution was discarded. The nitrocellulose membrane was washed for 5 minutes for four times with 1x

PBS.

E. G418 Colony Selection

3a. Cell Culture

Human cervix HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% L-Glutamine (100x), and 1% Antibiotic-Antimycotic

(100x) at 37°C.

3b. Lipofectomine Transfection

One day before transfection, HeLa cells were seeded into two 12-well plates. By cell-counting, approximately 1.5 x 105 cells were seeded into each well. Transfection of HeLa cells began when cells were ~70-75% confluent. Before transfection, the DMEM media in the wells were replaced with 1mL of opti-MEM (1x) media. A solution of 2000 ng of DNA plasmids and Invitrogen © Lipofectamine 2000

Reagent (4 µL) were prepared and incubated at room temperature for 30 minutes. After 30 minutes, the

DNA and Lipofectamine solution is added drop-wise to the respective cells. Approximately 6 hours after transfection, the opti-MEM (1x) media was replaced with DMEM media (10% Fetal Bovine Serum

(FBS), 1% L-Glutamine, and 1% Antibiotic-Antimycotic).

3c. Cell Collection

After 48 hours, the DMEM media was removed from the wells. Trypsin (300 µL) was added to each well and the culture plate was incubated for 10 minutes at 37°C. After 10 minutes, DMEM media

(700 µL) was added to each well and the cells were gently resuspended. All resuspended cells were transferred into 1.5mL microcentrifuge tubes.

3d. Seeding Plates

DMEM media (5mL) was allocated to 5-centimeter petri-dishes. Afterwards, 20% (200uL) of the resuspended cells were added to each respective dish.

3e. G418 Selection

One day after transfection, the DMEM media (10% Fetal Bovine Serum (FBS), 1% L-

Glutamine, and 1% Antibiotic-Antimycotic) was replaced with DMEM media containing Geneticin

Selective Antibiotic (G418 Sulfate). DMEM media containing G418 Sulfate was changed every three

(3) days until day 14.

3f. Cell fixation and Staining

DMEM media was removed from each plate and 5mL of 10% formaldehyde was added to each plate. The cell plates were incubated in 10% formaldehyde overnight at room temperature. The next day, the 10% formaldehyde was removed and the cell plates were rinsed with ddH2O. Afterwards, 0.5% crystal violet (5mL) were added to each plate and incubated for 30 minutes at room temperature. After

30 minutes, the plates were thoroughly rinsed with water after discarding the 0.5% crystal violet solution.

3g. Data Analysis

The ratio of the total area and surviving colonies were measured using ImageJ. To determine the percentage of colonies to area, the area of colonies was divided by total area of plate. A t-test was used to calculate whether there was significant difference between the gamma-2 herpesvirus subgroups.

Results

Figure 3: Conservative motif of ORF45 homologues. (A) Displays the conserved amino-terminus (N- terminal) of subset of ORF45 homologues. (B) Displays the conserved ERK binding site region of

ORF45 homologues. (C) Shows the RSK binding site of ORF45 homologues. (D) Displays the conserved ORF33 binding site of ORF45 homologues.

Figure 4: Conservative motif of in all ORF45 homologues . (A) conserved amino-terminal (N-terminal) region, (B) conserved ERK binding site, (C) conserved RSK binding site, (D) conserved ORF33 binding site of -2 ORF45 homologues.

Figure 5: Phylogeny analysis of ORF45 homologues. Phylogenic tree of ORF45 homologues using neighbor-joining method by T-Coffee program provided by European Bioinformatics Institute (EMBL-

EBI)

Conservation of ERK, RSK, and ORF33 Binding Site Amongst ORF45 -2 Herpesvirus

Homologues

Amongst all of the ORF45 gamma-2 herpesvirus homologues, there is strong conservation in the

ERK, RSK, and ORF33 binding sites. The strongest conservation is the ERK and ORF33 binding site

(Figure 4B and 4D). Residues 65-66 are strongly conserved in all gamma-2 herpesvirus homologues as

RSK binding site (Figure 4C), but the region surrounding the two residues show weaker conservation.

ORF45 Homologues showed different levels of phosphorylated RSK and ERK

Serum-starved HEK293T cells were lysed and analyzed via western blotting. The cell lysates were blotted with anti-phosphorylated-RSK1/RSK2 and anti-phosphorylated-ERK1/ERK2 antibodies. It was found that there are different levels of phosphorylated RSK1/RSK2/RSK3 and ERK1/ERK2 amongst the different homologues (Figure 6B). The same membrane was used to reprobe for total RSK and ERK protein level. The total RSK and total ERK expression levels also varied amongst the ORF45 homologues. Levels of phosphorylated-RSK1/RSK2 were stronger in KSHV, AlHV-2, BoHV-6, PLHV-

3, BoHV-4, and EHV-5 compared to the control. In addition, levels of phosphorylated-ERK1/ERK2 were stronger in KSHV, RFHV, HVS, OvHV-2, AlHV-2, BoHV-6, PLHV-3, PLHV-1, BoHV-4, EHV-

5, EHV-2, and MHV-68 compared with the control. The GFP signal varied amongst the homologues with stronger signals found in JSHV, EHV-5, EHV-2, MHV-68, EBV, RFHV, OvHV-2, and BoHV-6

(Figure 6A).

Figure 6: ORF45 homologues induced ERK and RSK activation. (A) Western blot showing the GFP expression of ORF45 homologues. (B) Western blot showing the phosphorylated-RSK, phosphorylated-

ERK, total RSK, total ERK, and actin level of amongst ORF45 homologue samples.

G418 Selection

G418 selection was performed on HeLa cells transfected by ORF45 homologues. It was found that under G418 selection, HeLa cells transfected with HVS, AlHV-2, MHV-68, and EBV showed the most proliferation compared to other ORF45 homologues after 14 days of G418 selection. The percentage in Figure 7 was determined by dividing the area of surviving colonies to total area of the plate. It was found that RRV, HVS, AlHV-2, MHV-68 and EBV showed the most proliferation compared to the other ORF45 homologues (Figure 7). MHV-68 showed the most proliferation while

EHV-2 showed the least proliferation.

Image 1 G418 selection of HeLa cells transfected by ORF45 homologues on day 14.

Figure 7 G418 selection of HeLA cells transfected by ORF45 homologues. Figure displays the percentage of G418 resistant colonies in relation to the total area of plate. G418 selection experiment was repeated two (2) times.

Discussion and Conclusion

Similar phylogenic relationships amongst gamma-2 herpesvirus proteins

ORF45 homologue phylogenic relationship is similar to other gamma-2 herpesvirus protein

From multiple alignments of ORF45 gamma-2 herpesvirus homologues (Figure 3 and Figure 4), there is evidence to show that it is strongly conservative within the amino-terminus, ERK binding site, RSK binding site, and ORF33 binding sites. Upon phylogenic analysis of those alignments, we found that the phylogenic organization of ORF45 gamma-2 herpesvirus homologues are similar to ORF75 and ORF3

(Skike et al., 2018). When observing the phylogenic relationship of the entire gamma-2 herpesvirus

genome, the phylogenetic tree also shows similar structure (Alba et al., 2001) when compared to the

ORF45, ORF75 and ORF3 phylogenic tree.

Different ORF45 homologues show differing levels of RSK and ERK activation

Different levels of GFP expression (Figure 6A) suggests that different ORF45 homologues show different levels of insert ORF expression. According to our experience, naturally, these ORF45 homologues show different efficacy of expression or stability in cell, consequently, it showed different protein level after deliver the same amount of plasmid by transfection. The GFP expression (Figure 6A) indicated that KSHV, RFHV, OvHv-2, BoHV-6, PLHV-1, EHV-2, EHV-5, and EBV can strongly express effectively naturally. In Figure 6B, there are different expression levels of total RSK and total

ERK. Phosphorylated-RSK is strongly expressed in KSHV, ALHV-2, and PLHV-3. However, there is weak phosphorylated-RSK in RFHV, RRV, HVS, BoHV-6, PLHV-1, BoHV-4, EHV-5, EHV-2, MHV-

68, and EBV. There is strong phosphorylation of ERK in KSHV, RFHV, PLHV-3, BoHV-4, and MHV-

68.

Different ORF45 homologues show differing rates of proliferation

Geneticin 418 (G418) colony selection is the method used to evaluate the colonigenesis of HeLa cell transfected by various ORF45 homologues. Mammalian cells that bearing the Neomycin resistant gene will survive and keep growing throughout the selection process. The Neomycin resistant gene encodes for amino-glycoside 3’-phosphotransferase. When amino-glycoside 3’-phosphotransferase is expressed, the mammalian cell will show resistant to the Geneticin Selective Antibiotic. However, if insert expression would compromise the capability of cell proliferation, the capability of forming

colonies during G418 selection will be undermined. In our study, MHV-68 ORF45 showed the highest proliferation level (17.15%). Meanwhile, EHV-2 showed the least proliferation (0.37%). There is no significant difference in G418 selection between the gamma-2 herpesvirus subfamilies. When performing a t-test, it was found that when comparing the colony proliferation between Rhadinoviruses and Percaviruses, there was no significant difference (p-value = 0.2741). Since KSHV classified within the monkey subfamily, there is an interest in whether there is significant difference between the monkey subfamily (KSHV, RFHV, RRV, HVS, BoHV-4, MHV-68) and Macavirus (AlHV-2, BoHV-6, PLHV-

1, PLHV-3, OvHv-2). When comparing the Rhadinovirus group with the Macavirus, there was deemed to be no significant difference (p-value = 0.406). When comparing G418 selection results between the

Percavirus and Macavirus subgroups, there also showed no significant difference between the two subgroups (p-value = 0.222).

Acknowledgements

I would like to express my sincere gratitude to my thesis advisor Dr. Fanxiu Zhu for his support, patience, enthusiasm while guiding me through my honors thesis. Thank you for allowing me the opportunity to learn in your lab this past year and for being an inspiration. By being involved in research, I have gained a stronger passion for microbiology.

I would also like to extend my sincere gratitude to Dr. Hengli Tang and Dr. Mia Lustria for serving on my HITM thesis committee. Thank you for taking the time out of your busy schedules to guide me with my honors thesis.

I would like to thank Dr. Steve Miller at the Biology Sequencing Facility for his service.

Lastly, I would like to thank all the undergraduate, graduate, and post-doctorate members in the Zhu lab for your constant support and friendship. A special thank you to Hongyu Zhang and Ruiming Hu for guiding me throughout the semester.

Supplemental Material

Full ORF45 Virus Abbreviation Protein Sequence Name

Alcelaphine ALHV-2 MAMFLGKKKDDRLLPIEGAPRRKRTNYFTFPCFKTLKQLSYTGIQSKE KIKENILSGEDVFYPDPVAGPD gammaherpesvirus 2 ALLRDPPLTPGLIFENSDADSDFEDPDISPGGGMESPIKPQRAQPLGE RCSTKFKRSSSSTETAPSGGSG ORF45 TSSGDSDLECSFFEPPPKRPASLVDGPLICKRRCALDESPKIENLSGS SSCGSSTSTDSFEFVNTRDTCS GTSCCQGGNPSANEVLGKLNLQAEDQDHSMRETPPLKGGEDYTWPWN Bovine BoHV-4 MAMFVQKLKGGFSDNDIPRMAPLPGAPRLKLTNYFKFPPEEDENTPSE VQKAELEGIDSDSSSGSDNVFYENLPDPPKTPGFEESNSEESNNGERQ gammaherpesvirus 4 YDYYKDKDFKFKPQDEDTSSDDESAVADFIKCQARGLSSSESDGEPYS RQKMSSPITISDSSSGEEDVVKQQFTGKKKRGISSTCNSDSGPLRRRQ ORF45 RTKHPHEYDSSTSSDDEEPGEHSRDVRRHAMLTQPLMTVNKSYNWPWI D

Bovine BoHV-6 MAMFLKKPFTVSKVPKDDRLLPVEGAPRRKKTQFFKFPPWKSPVQLAE SGCQSPSQFVKQILDEDEVFYP gammaherpesvirus 6 DGIVGPNVMLTDDPIRPLRPISQTHSSSSEESDTESSSEDESEEEQES YVLDEAEEDSEGDTEDSAVDCS ORF45 SDEELHTSPLTPSPLKNLPSRHPLSSSSSSDEERMAVARGSVVSKSRK SVKKCRKLSSSNNESDTDEEFM FLRPPIKEAKRVHTPSPPSSPVQLKRPSKGGAVEDADENYENRVMLDT PPIKNNDESYPWPWL

Equid EHV-2 MSMFLKKQKKTKGGSSEEKRRGRTGSPPRMTSDPGAPRLKRATYFQFP KDGIKETMRKTKDAGDLDDVFF gammaherpesvirus 2 EDCTQCNPPSHVPVFSKPKPRTRAGGAADDSDSESSEDGGEDDEETLH SQDTPPGGSSSDSDDDDQKLPF ORF45 TATGGIKMPGYMSRISDSSSSSSSSSDSESSSSSDSESDGDRSTPEPD ILRQVTSSLARGVSPPRAKPPP AKGEVPVISLLSSEESDSEGEPSPLRAAAAAASQKRKHTSSSSDNDPK HTKVIYISSGESEDEGEGAGAG EGEPLGPEDQVLVVMSQESCEHYMATTPPVAGNPPYNWPWL

Equid EHV-5 MSMFLKKLKKSRGGDEKKREEQQGEQQRQRQQQQRPPKSPTPRMTNDP GAPRLKQATYFQFPSSAIREQI gammaherpesvirus 5 KVTKESGDLDDVFFPDETQCKRSSKVPFVGESDSGSEEEEEEEEDDDE ETLNSEDTTTSESSSSSDSDED ORF45 FELPFTKRGGIKIPDYRSRSNDTISSSSSGSSSDSDSDRSTPEPEVLK AVKRASGRGGSSSSKTAAPSPG KGKIPVINLISSEESEGEGPSSSAGASKKRKRASSGSVSGRKHPKPIV ISSGSESESEDEGEGGYEHRDD ALVILSQESWEHYMATTPPVTGNPEYNWPWM

Ovine OvHV-2 MAMFLKPKGALEDDRMLPLEGAPRRKRTTFFTFPAFKNMKQLTHSGNI SMSQLKREMLDVEEVFYPEALN gammaherpesvirus 2 GPPAIVQGKSYKTLKVLSDSDWSSGSDSSMEDDSPRSPPEQKHCQKTC QPPANGRSQGVKRSSSTDTASS ORF45 PGSASGSESSSDEEVPPAKRAAPDRYSAPICKRQCGLEEPVTIGKQEE IVLSISSSSCGSTTSTDSFDFV STENTTASDGKSGLGPKEMQSLDLNAPAYHDHIMPETPPIKGGAGYPW

PWK

Porcine lymphotropic PLHV-1 MAMFLKKTKKPKHEVPANSDRLFDLEGAPRKKKTTYFKFPPFSSVVQL AYSGSPPSMKDVRSILGDDDVF herpesvirus 1 YPEGFSGPHDILPPRPKFANPEMDIEEQYNYDDSSNDESFSDIECDEE DELGEDEDSSDEGSSYGASMME ORf45 SMVSKSLNIVESTSSSDEEDEFVFIRPPIGQTADVPGKRSRTPNTGAH SDSNKKIKAQTNYSEHNMLDTP PIKEDEYNWPWLN

Porcine lymphotropic PLHV-3 MLKMAMFLKKTQTAKHGKTGNGDRLFDLEGAPRRKKTTYFKFPPFKSL VQLTYSNARNSTDVESMVRDDE herpesvirus 3 VFYPEGFDGPDILHSIPKFSNPESDSSSSEEERFVGYDDTDSSDETFS DVDCENDGKSVDEDTSDDSYSS ORF45 NEMESMVTKSLNIVETDSSSSDEEDEFVYLRPPIGHTADMGSKRSRTP TIGTVPETQKKLKPHVDYSEHE MLDTPPIKDDDYNWPWLN

Retroperitoneal RFHV MAMFLDDSPPPMDDDRLFPYEGAPRRVPPRRFIFPPPRPVQPYGGPPV IDLSAPDDVFAEDDTSPPATPLDLLPSPGPADDRALGINAHHPGAGDW fibromatosis-associated MPAALPPPLAGDRGRPLASTVRRTVPVAVVTGHVRSPLRSETVSSDSF PDWESEGEGFSPGESFSDGETTDTFLSESSVTRDSTSEGAHLEMDPDE herpesvirus GPSWRPLRAEVVATIDLLSDSDSESPTEGTHGHSSEETLSADEGPSTA VAQVRETVIKRKRQSTASSASEASGRPSRRRLWSSGHTSIIIISSDSD ORF45 SEPEPTQRSSFGPPTGQADAAQRLPENLDDESTSATSLSSRSSDSSSG DGDASRALTSTPPLSGNGNYNWPWLD

Table 1 Full protein sequences of ORF45 homologues.

Full ORF45 Virus Abbreviation DNA Sequences Name

Alcelaphine ALHV-2 ATGGCAATGTTTTTGGGTAAAAAAAAGGATGATAGGCTGTTGCCAAT TGAAGGAGCTCCTAGAAGAAAAA gammaherpesvirus 2 GGACTAACTACTTCACTTTTCCCTGCTTTAAAACCTTAAAACAGCTA TCTTACACTGGAATTCAATCTAA ORF45 AGAGAAAATTAAGGAAAACATTTTAAGCGGCGAGGATGTCTTCTACC CAGACCCTGTGGCCGGGCCTGAT GCCCTACTTCGGGATCCACCGTTAACTCCAGGTCTAATTTTTGAGAA CAGTGATGCTGACAGCGACTTTG AAGATCCTGACATCAGTCCAGGGGGAGGTATGGAGTCTCCCATCAAG CCCCAAAGGGCTCAGCCCTTAGG GGAAAGATGCTCCACAAAATTTAAGAGGTCGAGTAGCAGCACGGAAA CAGCGCCTAGCGGTGGCAGTGGT ACCAGCAGTGGGGATAGTGACTTAGAATGTAGCTTCTTTGAGCCACC TCCTAAGCGTCCAGCATCTCTTG TAGATGGACCATTGATCTGCAAGCGCCGATGTGCTTTAGATGAGTCC CCTAAGATTGAAAACCTGAGCGG CTCATCGAGTTGCGGAAGCAGCACTTCTACTGACAGCTTTGAGTTTG TGAACACGAGAGACACCTGCAGC GGTACCAGTTGCTGTCAGGGTGGGAATCCATCAGCTAACGAAGTCCT GGGTAAATTAAACCTGCAAGCTG AAGACCAAGACCATAGCATGCGAGAAACACCACCACTGAAGGGTGGT GAGGATTATACGTGGCCCTGGAA TTAA

Bovine BoHV-4 ATGGCGATGTTTGTGCAGAAGCTCAAGGGGGGATTTTCTGATAATGA TATTCCAAGAATGGCCCCCCTCC gammaherpesvirus 4 CAGGAGCACCAAGACTTAAGTTAACAAACTACTTCAAATTTCCACCA GAGGAGGATGAGAACACCCCTTC ORF45 TGAAGTCCAGAAAGCTGAACTAGAAGGGATAGATTCAGATTCCTCTT CAGGATCTGATAATGTCTTTTAT GAAAATCTTCCTGACCCACCCAAGACGCCCGGCTTTGAGGAATCTAA CAGTGAAGAATCAAATAATGGAG AGAGACAATATGATTATTACAAGGATAAGGACTTCAAATTCAAGCCC CAAGATGAGGATACATCTTCTGA CGATGAAAGTGCAGTGGCAGACTTTATAAAATGCCAGGCGAGGGGCT TATCATCTTCAGAGAGCGACGGG GAACCTTATTCCAGACAGAAAATGTCATCCCCCATTACTATCAGTGA CTCTTCATCCGGTGAGGAAGATG TAGTGAAGCAACAATTTACGGGGAAAAAGAAGAGGGGAATTTCCTCT ACATGTAACTCAGACAGTGGCCC CCTGCGTCGGCGCCAGCGAACTAAACATCCACATGAATATGATTCAT CAACTTCCAGTGATGATGAAGAA CCTGGGGAACATTCCAGGGATGTGAGGAGGCATGCCATGTTAACCCA GCCCCTGATGACAGTAAATAAAA GTTACAATTGGCCATGGATTGACTGA

Bovine BoHV-6 ATGGCTATGTTTTTAAAAAAGCCTTTTACTGTTTCTAAAGTGCCAAA AGACGACCGACTGCTCCCCGTGG gammaherpesvirus 6 AAGGCGCACCAAGGCGCAAAAAAACTCAGTTCTTTAAATTTCCTCCT TGGAAAAGTCCTGTGCAGCTAGC ORF45 GGAGTCTGGTTGCCAGTCGCCCAGTCAATTCGTTAAACAGATTTTGG ACGAAGATGAAGTTTTTTACCCA GATGGCATAGTGGGACCTAACGTTATGTTGACGGACGACCCGATAAG

ACCACTCAGGCCCATTTCACAAA CACATAGCTCCAGCAGCGAAGAGAGTGACACAGAAAGCAGCAGCGAA GACGAGTCCGAAGAGGAGCAGGA AAGCTACGTGTTAGACGAAGCAGAGGAAGACTCTGAAGGCGACACCG AAGACAGCGCCGTAGACTGCAGC AGCGACGAAGAGCTCCACACGTCCCCGCTCACCCCATCTCCGCTTAA AAACTTGCCTTCCAGGCACCCGC TAAGCAGCAGTAGCAGCAGCGACGAAGAACGCATGGCTGTTGCTAGG GGCAGCGTCGTTTCCAAATCGCG GAAAAGCGTGAAAAAGTGTAGAAAGCTTTCTAGCTCTAACAACGAAA GCGACACGGATGAAGAGTTTATG TTTTTGAGGCCCCCCATTAAAGAAGCTAAGCGGGTTCATACGCCATC TCCCCCCAGCTCGCCTGTACAGT TGAAAAGGCCTTCAAAGGGTGGCGCCGTAGAAGACGCGGATGAGAAT TATGAAAACAGAGTCATGCTTGA CACTCCCCCTATTAAAAACAATGATGAATCATACCCCTGGCCTTGGC TGTAA

Equid EHV-2 ATGTCTATGTTTCTGAAAAAGCAGAAAAAGACTAAGGGGGGGTCCTC GGAGGAGAAGCGGCGGGGCCGGA gammaherpesvirus 2 CCGGGTCGCCACCCAGGATGACCAGCGACCCGGGGGCGCCCAGGCTA AAGCGGGCCACCTACTTTCAGTT ORF45 TCCCAAAGATGGGATTAAGGAAACTATGAGGAAGACTAAAGACGCGG GGGACCTGGATGATGTCTTTTTT GAGGACTGCACCCAGTGCAACCCCCCTTCCCACGTTCCAGTTTTTAG TAAGCCCAAACCGCGGACGCGTG CCGGGGGCGCTGCCGATGACAGCGACAGCGAGTCCTCCGAGGACGGG GGGGAGGACGATGAGGAGACGCT GCACTCCCAGGACACTCCCCCGGGGGGCTCTAGCAGCGACAGCGATG ACGATGACCAAAAACTTCCCTTT ACGGCCACCGGGGGGATAAAGATGCCCGGCTACATGTCCAGGATCTC GGACAGCTCCTCCTCTTCCTCCT CTTCTTCGGATAGCGAAAGCAGCAGCAGCAGCGACAGCGAGAGCGAT GGCGATCGCAGCACTCCCGAGCC AGATATCCTGAGGCAGGTCACCAGCTCGCTGGCGCGGGGAGTTTCCC CGCCCCGGGCCAAACCCCCTCCG GCCAAAGGGGAAGTCCCTGTCATCAGCCTGCTCTCCTCGGAGGAGTC AGACAGCGAGGGCGAGCCTTCCC CTTTGCGCGCCGCTGCCGCCGCCGCTTCTCAAAAGAGAAAGCACACC TCCAGCAGCTCGGACAATGACCC CAAACACACCAAGGTGATCTATATCTCCTCCGGTGAGTCCGAGGACG AGGGAGAGGGTGCGGGCGCCGGG GAGGGGGAGCCTCTTGGGCCCGAGGATCAGGTCCTGGTGGTCATGTC CCAGGAGTCATGCGAACACTATA TGGCGACGACGCCCCCTGTGGCCGGGAACCCACCCTACAACTGGCCT TGGCTGTAA

Equid EHV-5 ATGTCTATGTTTTTGAAAAAACTAAAGAAATCTAGGGGGGGCGATGA GAAAAAAAGGGAGGAACAACAGGGGGAGCAGCAGCGGCAGCGGCAGC gammaherpesvirus 5 AGCAGCAGCGGCCGCCCAAGTCGCCCACCCCACGCATGACCAACGAC CCCGGGGCCCCAAGGTTAAAGCAGGCCACCTATTTTCAGTTCCCCAG ORF45 CTCGGCCATCAGAGAGCAGATTAAGGTGACCAAAGAGTCCGGGGATT TAGATGATGTTTTTTTTCCGGACGAGACGCAGTGCAAGCGGTCGTCC AAGGTTCCTTTTGTGGGCGAGTCGGACAGCGGCTCGGAGGAGGAAGA GGAAGAGGAAGAGGACGATGACGAGGAGACGCTCAACTCTGAGGACA CAACCACCTCAGAGAGCAGCTCGAGCAGCGATAGCGATGAGGATTTT GAATTGCCCTTTACAAAAAGGGGTGGGATAAAAATTCCAGATTACAG GTCGCGTTCCAACGACACCATCTCCTCCTCGAGCAGCGGCAGCAGCA

GCGACAGCGACAGCGACCGCAGCACCCCGGAGCCAGAGGTTTTAAAA GCGGTTAAGAGGGCCTCTGGCCGCGGCGGTTCCTCTTCCTCTAAAAC CGCAGCGCCCTCGCCGGGCAAGGGGAAAATCCCCGTCATTAATTTAA TTTCTTCTGAAGAGTCTGAGGGGGAGGGCCCCTCTTCTAGCGCCGGC GCCTCTAAAAAGAGAAAGCGCGCCTCTAGCGGTTCTGTTTCTGGGCG CAAACACCCCAAGCCTATAGTCATATCCAGCGGGTCTGAAAGCGAGA GCGAGGATGAGGGTGAAGGCGGGTATGAGCATAGAGATGATGCGTTG GTTATACTGTCGCAAGAGTCTTGGGAGCACTATATGGCGACGACTCC CCCCGTGACCGGCAACCCAGAATACAACTGGCCTTGGATGTGA

Ovine OvHV-2 ATGGCCATGTTTCTCAAACCAAAGGGGGCCCTTGAAGATGACAGAAT GCTGCCCTTGGAGGGGGCGCCTC gammaherpesvirus 2 GGCGAAAGAGGACAACTTTTTTCACCTTCCCCGCCTTCAAAAACATG AAACAGCTCACGCACAGCGGCAA ORF45 TATTTCCATGTCCCAGCTTAAAAGAGAGATGCTGGACGTGGAAGAAG TGTTTTACCCCGAAGCGCTTAAT GGACCACCCGCCATCGTGCAGGGCAAGAGTTACAAAACCCTGAAAGT ACTCTCTGACAGCGACTGGAGCA GCGGCTCGGACAGCAGTATGGAGGATGACTCCCCACGCAGCCCCCCA GAGCAAAAACATTGCCAGAAGAC CTGCCAGCCGCCTGCAAATGGCCGCAGCCAGGGGGTCAAGCGCAGCA GCAGCACCGACACTGCCAGCAGC CCTGGTAGCGCAAGCGGCTCGGAGAGTAGCAGCGACGAGGAGGTGCC GCCAGCAAAGCGGGCAGCACCGG ATAGGTACTCTGCGCCCATCTGTAAGCGGCAGTGCGGTCTAGAGGAG CCCGTAACAATAGGAAAGCAGGA AGAGATTGTCCTGAGCATATCGTCCTCCAGCTGCGGCAGCACTACCT CCACAGACAGCTTTGACTTCGTG TCCACTGAAAACACTACGGCCAGTGATGGCAAAAGCGGACTGGGGCC CAAAGAGATGCAGTCCCTGGACC TAAATGCGCCGGCCTACCACGACCACATTATGCCAGAGACGCCTCCC ATCAAGGGGGGTGCTGGCTACCC ATGGCCCTGGAAATAA

Porcine lymphotropic PLHV-1 ATGGCTATGTTTTTAAAGAAGACTAAGAAACCTAAACATGAAGTCCC TGCAAACTCAGATAGATTGTTCG herpesvirus 1 ACCTGGAAGGTGCACCTCGCAAAAAGAAAACTACCTATTTCAAATTT CCACCATTTTCATCTGTCGTGCA ORf45 GCTTGCTTATTCTGGTTCTCCTCCATCAATGAAGGATGTAAGATCAA TATTGGGAGATGATGATGTGTTT TATCCAGAAGGATTTTCGGGTCCTCATGATATTCTTCCACCTAGACC AAAATTTGCAAACCCTGAGATGG ATATTGAAGAACAGTATAACTATGATGATTCCTCAAACGACGAAAGC TTTTCTGACATAGAATGTGATGA GGAGGATGAATTAGGAGAGGATGAAGATTCTTCCGACGAAGGAAGCT CATATGGTGCGAGCATGATGGAA TCTATGGTGTCAAAGTCCTTAAACATTGTGGAGAGTACCAGTTCATC TGATGAAGAAGATGAGTTTGTGT TTATACGCCCCCCTATAGGACAAACTGCAGATGTCCCTGGGAAAAGA TCTAGAACTCCAAATACTGGAGC ACATTCAGACAGTAACAAGAAAATTAAAGCACAAACAAATTATTCTG AACATAACATGCTGGATACACCC CCTATAAAGGAGGATGAATACAACTGGCCCTGGCTAAACTGA

Porcine lymphotropic PLHV-3 ATGCTTAAAATGGCTATGTTTTTAAAGAAGACACAGACAGCTAAACA TGGGAAAACTGGCAATGGTGACA herpesvirus 3 GATTGTTTGATTTGGAAGGTGCTCCACGGAGAAAGAAAACAACATAC TTTAAATTTCCACCATTTAAATC ORF45 TCTGGTACAGCTGACCTACTCCAATGCTCGAAATTCTACGGATGTCG AATCAATGGTGCGTGATGATGAA GTATTTTATCCAGAAGGATTTGATGGCCCTGATATACTTCACTCCAT TCCAAAGTTTTCAAACCCAGAAA GTGATAGTAGTAGTAGTGAAGAAGAAAGGTTTGTGGGATATGATGAT ACAGATTCGTCTGATGAAACCTT CTCAGATGTGGACTGTGAAAATGATGGGAAGTCTGTGGATGAGGATA CATCCGATGACTCTTACAGCTCC AATGAAATGGAGTCAATGGTGACAAAATCTCTAAATATTGTTGAAAC GGACAGTTCATCATCAGATGAAG AAGATGAGTTTGTCTACTTGCGTCCTCCTATTGGACATACGGCTGAC ATGGGCAGTAAGAGATCCAGGAC GCCGACCATTGGCACAGTTCCAGAAACACAGAAGAAACTGAAGCCAC ATGTGGATTATTCTGAACATGAA ATGCTGGATACCCCACCGATAAAGGATGATGACTACAATTGGCCATG GTTAAATTAA

Retroperitoneal RFHV ATGGCCATGTTTTTGGACGACTCCCCGCCCCCCATGGACGACGACCG ACTATTTCCGTACGAGGGCGCAC fibromatosis-associated CGCGTCGCGTCCCCCCAAGGAGGTTTATATTTCCTCCCCCACGACCG GTACAACCATACGGCGGCCCTCC herpesvirus CGTCATCGATCTTTCTGCCCCAGACGATGTGTTTGCAGAGGACGACA CGTCGCCACCGGCAACGCCACTG ORF45 GACCTCTTACCATCCCCGGGACCGGCCGATGACAGGGCACTAGGTAT AAATGCCCATCACCCTGGCGCAG GAGACTGGATGCCCGCGGCGTTACCCCCTCCTCTCGCTGGAGACAGG GGGCGCCCTCTGGCGTCCACGGT GCGCAGAACAGTTCCAGTTGCCGTTGTTACGGGCCACGTTCGGAGTC CCCTTCGCTCGGAGACCGTAAGT TCTGATAGCTTTCCGGACTGGGAAAGCGAGGGGGAAGGATTTTCTCC CGGCGAGTCTTTCTCTGACGGCG AAACGACAGATACTTTTCTGAGCGAATCGTCAGTCACCCGTGACTCC ACCTCGGAAGGGGCCCATTTAGA AATGGACCCGGATGAGGGTCCCAGCTGGCGTCCCTTACGCGCAGAAG TAGTCGCAACCATCGACCTATTG TCGGACTCAGACAGCGAATCGCCAACCGAGGGGACTCACGGACACTC ATCGGAAGAGACACTATCGGCCG ACGAAGGGCCGTCAACAGCGGTAGCGCAGGTGCGGGAGACGGTAATT AAACGCAAAAGACAAAGCACGGC ATCGTCGGCCAGCGAGGCATCGGGGCGACCCAGTCGCCGCCGTCTGT GGTCATCAGGTCATACGTCCATA ATCATCATATCGTCAGACAGTGATTCCGAACCCGAACCGACGCAGAG GTCCTCATTCGGCCCCCCGACTG GTCAGGCCGACGCCGCTCAGCGCCTACCGGAGAATTTAGACGATGAA AGTACGTCCGCTACGTCCCTCTC GTCCAGAAGCAGCGACTCATCCTCAGGCGACGGAGATGCGAGTCGTG CGCTTACCTCCACGCCGCCCCTT AGTGGCAACGGGAATTACAACTGGCCCTGGCTTGATTAA Table 2 Full DNA sequences of ORF45 homologues.

Full ORF45 Virus Abbreviation Optimized DNA Sequences Synthesized Name

Alcelaphine ALHV-2 GGATCCAAAT GGCCATGTTC CTGGGCAAGA AGAAGGACGA TAGGCTGCTG CCTATCGAGG gammaherpesvirus 2 GAGCACCAAG GAGAAAGCGG ACCAACTACT TCACATTTCC ATGCTTCAAG ACCCTGAAGC ORF45 AGCTGAGCTA TACAGGCATC CAGTCCAAGG AGAAGATCAA GGAGAATATC CTGTCCGGCG AGGACGTGTT CTACCCTGAC CCAGTGGCAG GACCAGATGC ACTGCTGCGC GACCCCCCTC TGACCCCAGG CCTGATCTTC GAGAACTCTG ACGCCGATAG CGACTTTGAG GACCCCGACA TCTCTCCAGG AGGAGGAATG GAGAGCCCCA TCAAGCCTCA GAGGGCACAG CCTCTGGGAG AGAGATGCTC TACCAAGTTT AAGAGGAGCT CCTCTAGCAC CGAGACAGCA CCATCTGGAG GCAGCGGCAC ATCCTCTGGC GATTCCGACC TGGAGTGTTC TTTCTTTGAG CCACCACCTA AGAGGCCAGC AAGCCTGGTG GATGGCCCTC TGATCTGCAA GAGGCGCTGT GCCCTGGACG AGTCCCCCAA GATCGAGAAC CTGTCTGGCA GCTCCTCTTG CGGCAGCTCC ACCTCCACAG ATTCTTTCGA GTTTGTGAAT ACCAGAGACA CATGTAGCGG CACCTCCTGC TGTCAGGGAG GAAACCCAAG CGCCAATGAG GTGCTGGGCA AGCTGAACCT GCAGGCCGAG GATCAGGACC ACTCCATGAG GGAGACCCCA CCCCTGAAGG GAGGAGAGGA TTATACATGG CCTTGGAATT GACTCGAG Bovine BoHV-4 GGATCCAAAT GGCCATGTTC GTGCAGAAGC TGAAGGGCGG CTTTTCCGAC AACGATATCC gammaherpesvirus 4 CAAGAATGGC ACCTCTGCCA GGAGCACCAA GGCTGAAGCT GACCAACTAC TTCAAGTTTC ORF45 CCCCTGAGGA GGACGAGAAT ACACCTAGCG AGGTGCAGAA GGCCGAGCTG GAGGGCATCG ACTCTGATAG CTCCTCTGGC AGCGACAACG TGTTCTACGA GAATCTGCCC GATCCACCCA AGACCCCTGG CTTTGAGGAG TCCAATTCTG AGGAGTCTAA CAATGGCGAG CGCCAGTATG ATTACTACAA GGACAAGGAT TTCAAGTTTA AGCCACAGGA CGAGGATACA AGCTCCGACG ATGAGAGCGC CGTGGCCGAC TTCATCAAGT GCCAGGCCAG AGGCCTGTCT AGCTCCGAGT CCGATGGCGA GCCTTATTCT CGGCAGAAGA TGTCTAGCCC AATCACCATC TCCGACTCCT CTAGCGGCGA GGAGGATGTG GTGAAGCAGC AGTTTACCGG CAAGAAGAAG CGGGGCATCT CCTCTACATG TAACAGCGAC TCCGGACCAC TGCGGAGAAG GCAGAGGACC AAGCACCCTC ACGAGTACGA CAGCTCCACA TCTAGCGACG ATGAGGAGCC AGGAGAGCAC AGCCGGGATG TGCGCCGGCA CGCAATGCTG ACCCAGCCAC TGATGACAGT GAACAAGTCC TATAATTGGC CCTGGATCGA TTGACTCGAG

Bovine BoHV-6 ATGGCTATGT TTTTAAAAAA GCCTTTTACT GTTTCTAAAG TGCCAAAAGA CGACCGACTG gammaherpesvirus 6 CTCCCCGTGG AAGGCGCACC AAGGCGCAAA AAAACTCAGT TCTTTAAATT TCCTCCTTGG ORF45 AAAAGTCCTG TGCAGCTAGC GGAGTCTGGT TGCCAGTCGC CCAGTCAATT CGTTAAACAG ATTTTGGACG AAGATGAAGT TTTTTACCCA GATGGCATAG TGGGACCTAA CGTTATGTTG ACGGACGACC CGATAAGACC ACTCAGGCCC ATTTCACAAA CACATAGCTC CAGCAGCGAA GAGAGTGACA CAGAAAGCAG CAGCGAAGAC GAGTCCGAAG AGGAGCAGGA AAGCTACGTG TTAGACGAAG CAGAGGAAGA CTCTGAAGGC GACACCGAAG ACAGCGCCGT AGACTGCAGC AGCGACGAAG AGCTCCACAC GTCCCCGCTC ACCCCATCTC CGCTTAAAAA CTTGCCTTCC AGGCACCCGC TAAGCAGCAG TAGCAGCAGC GACGAAGAAC GCATGGCTGT TGCTAGGGGC AGCGTCGTTT CCAAATCGCG GAAAAGCGTG AAAAAGTGTA GAAAGCTTTC TAGCTCTAAC AACGAAAGCG ACACGGATGA AGAGTTTATG TTTTTGAGGC CCCCCATTAA AGAAGCTAAG CGGGTTCATA CGCCATCTCC CCCCAGCTCG CCTGTACAGT TGAAAAGGCC TTCAAAGGGT GGCGCCGTAG AAGACGCGGA TGAGAATTAT GAAAACAGAG TCATGCTTGA CACTCCCCCT ATTAAAAACA ATGATGAATC ATACCCCTGG CCTTGGCTGT AA Equid EHV-2 GGATCCAAAT GTCCATGTTC CTGAAGAAGC AGAAGAAGAC CAAGGGAGGC AGCTCCGAGG gammaherpesvirus 2 AGAAGAGGAG AGGAAGGACC GGCTCTCCAC CTAGGATGAC AAGCGACCCC GGAGCACCTC ORF45 GGCTGAAGAG AGCCACCTAC TTCCAGTTTC CCAAGGATGG CATCAAGGAG ACCATGAGGA AGACAAAGGA CGCCGGCGAT CTGGACGACG TGTTCTTCGA GGACTGCACC CAGTGTAACC CACCCTCCCA CGTGCCCGTG TTCAGCAAGC CCAAGCCTAG GACACGCGCC GGAGGAGCAG CAGACGATTC CGACTCTGAG TCTAGCGAGG ATGGCGGCGA GGACGATGAG GAGACCCTGC ACTCTCAGGA CACACCTCCA GGCGGCTCCT CTAGCGATAG CGACGATGAC GATCAGAAGC TGCCTTTTAC CGCCACAGGC GGCATCAAGA TGCCAGGCTA TATGTCCCGG ATCTCTGACT CCTCTAGCTC CTCTAGCTCC TCTAGCGACT CCGAGTCCTC TAGCTCCTCT GATAGCGAGT CCGACGGCGA TAGGAGCACC CCAGAGCCAG ATATCCTGAG GCAGGTGACA AGCTCCCTGG CCCGGGGCGT GTCCCCACCT AGAGCCAAGC CACCACCTGC AAAGGGAGAG GTGCCAGTGA TCAGCCTGCT GTCTAGCGAG GAGTCTGACA GCGAGGGCGA GCCTAGCCCA CTGAGGGCAG CAGCAGCAGC AGCATCCCAG AAGAGAAAGC ACACCTCCTC TAGCTCCGAC AATGATCCCA AGCACACAAA AGTGATCTAC ATCTCTAGCG GCGAGTCCGA AGATGAAGGA GAGGGAGCAG GAGCAGGAGA GGGCGAGCCA CTGGGCCCCG

AGGATCAGGT GCTGGTGGTC ATGAGCCAGG AGTCCTGCGA GCACTACATG GCAACCACAC CACCAGTGGC AGGAAACCCT CCATATAATT GGCCTTGGCT GTGACTCGAG Equid EHV-5 GGATCCAAAT GTCCATGTTC CTGAAGAAGC TGAAGAAGTC TCGGGGCGGC GATGAGAAGA gammaherpesvirus 5 AGCGGGAGGA GCAGCAGGGA GAGCAGCAGA GGCAGAGACA GCAGCAGCAG AGGCCACCTA ORF45 AGTCCCCTAC ACCACGCATG ACCAACGACC CAGGAGCACC TCGGCTGAAG CAGGCCACAT ACTTCCAGTT TCCTAGCTCC GCCATCAGAG AGCAGATCAA GGTGACAAAG GAGAGCGGCG ATCTGGACGA CGTGTTCTTC CCCGACGAGA CCCAGTGCAA GCGGTCTAGC AAGGTGCCTT TTGTGGGCGA GAGCGATTCC GGCTCTGAGG AAGAAGAGGA GGAGGAGGAG GACGATGACG AGGAGACCCT GAACTCTGAG GACACCACAA CCAGCGAGTC CTCTAGCTCC TCTGATAGCG ACGAGGATTT CGAGCTGCCT TTTACAAAGC GGGGCGGCAT CAAGATCCCA GATTATAGGA GCCGCTCCAA TGACACCATC AGCTCCTCTA GCTCCGGCTC TAGCTCCGAC TCTGATAGCG ACAGGAGCAC ACCCGAGCCT GAGGTGCTGA AGGCAGTGAA GAGGGCATCC GGAAGGGGAG GCTCTAGCTC CTCTAAGACC GCAGCACCAA GCCCAGGCAA GGGCAAGATC CCAGTGATCA ATCTGATCAG CTCCGAGGAG AGCGAGGGAG AGGGACCCTC TAGCTCCGCC GGAGCCTCCA AGAAGCGGAA GAGAGCCTCT AGCGGCTCCG TGTCTGGCAG GAAGCACCCT AAGCCAATCG TGATCTCCTC TGGCAGCGAG TCCGAGTCTG AGGATGAAGG AGAGGGAGGA TACGAGCACC GCGATGACGC CCTGGTCATC CTGAGCCAGG AGTCCTGGGA GCACTACATG GCAACAACCC CACCAGTGAC CGGAAACCCA GAGTATAATT GGCCCTGGAT GTGACTCGAG Ovine OvHV-2 GGATCCAAAT GGCCATGTTC CTGAAGCCCA AGGGCGCCCT GGAGGACGAT AGGATGCTGC gammaherpesvirus 2 CACTGGAGGG AGCACCTAGG AGAAAGAGGA CCACATTCTT TACCTTCCCT GCCTTTAAGA ORF45 ACATGAAGCA GCTGACACAC AGCGGCAATA TCTCCATGTC TCAGCTGAAG CGGGAGATGC TGGACGTGGA GGAGGTGTTT TACCCAGAGG CCCTGAACGG ACCACCTGCA ATCGTGCAGG GCAAGAGCTA TAAGACCCTG AAGGTGCTGA GCGACTCCGA TTGGAGCTCC GGCTCCGATT CTAGCATGGA GGACGATTCT CCACGGAGCC CACCCGAGCA GAAGCACTGC CAGAAGACAT GTCAGCCTCC AGCAAATGGC CGGAGCCAGG GAGTGAAGAG ATCCTCTAGC ACCGACACAG CCTCCTCTCC AGGCTCTGCC AGCGGCTCCG AGAGCTCCTC TGACGAGGAG GTGCCACCTG CAAAGAGGGC AGCACCAGAT AGGTACTCCG CCCCCATCTG CAAGAGACAG TGTGGCCTGG AGGAGCCTGT GACCATCGGC AAGCAGGAGG AGATCGTGCT GTCTATCAGC TCCTCTAGCT GCGGCTCCAC CACATCTACC GACAGCTTCG

ATTTCGTGAG CACCGAGAAC ACCACAGCCT CCGATGGCAA GTCTGGCCTG GGCCCAAAGG AGATGCAGAG CCTGGACCTG AATGCCCCTG CCTACCACGA TCACATCATG CCAGAGACCC CACCCATCAA GGGAGGAGCA GGATATCCTT GGCCATGGAA GTGACTCGAG Porcine lymphotropic PLHV-1 GGATCCAAAT GGCCATGTTC CTGAAGAAGA CCAAGAAGCC TAAGCACGAG GTGCCAGCCA herpesvirus 1 ACTCCGATAG GCTGTTTGAC CTGGAGGGAG CACCACGGAA GAAGAAGACC ACATACTTCA ORf45 AGTTTCCCCC TTTCAGCTCC GTGGTGCAGC TGGCCTATTC TGGCAGCCCA CCCAGCATGA AGGATGTGCG GTCCATCCTG GGCGACGATG ACGTGTTCTA CCCAGAGGGC TTTAGCGGCC CCCACGATAT CCTGCCTCCA AGGCCCAAGT TTGCCAACCC TGAGATGGAC ATCGAGGAGC AGTACAACTA TGATGACTCT AGCAATGATG AGTCCTTCTC TGACATCGAG TGCGATGAGG AGGACGAGCT GGGCGAGGAT GAGGACTCCT CTGACGAGGG CAGCTCCTAT GGCGCCTCCA TGATGGAGTC TATGGTGAGC AAGTCCCTGA ATATCGTGGA GTCCACCTCT AGCTCCGATG AGGAGGACGA GTTCGTGTTT ATCCGGCCCC CTATCGGCCA GACAGCCGAT GTGCCTGGCA AGCGGTCTAG AACCCCAAAC ACAGGCGCCC ACTCTGACAG CAATAAGAAG ATCAAGGCCC AGACCAACTA CAGCGAGCAC AATATGCTGG ATACACCACC CATCAAGGAG GACGAGTATA ACTGGCCTTG GCTGAATTGA CTCGAG Porcine lymphotropic PLHV-3 GGATCCAAAT GCTGAAGATG GCCATGTTCC TGAAGAAGAC CCAGACAGCC AAGCACGGCA herpesvirus 3 AGACCGGCAA CGGCGATAGG CTGTTTGACC TGGAGGGAGC ACCAAGGAGA AAGAAGACCA ORF45 CATACTTCAA GTTTCCCCCT TTCAAGTCCC TGGTGCAGCT GACCTATAGC AACGCCCGGA ATTCCACAGA CGTGGAGTCT ATGGTGAGAG ACGATGAGGT GTTCTATCCA GAGGGCTTTG ATGGCCCCGA CATCCTGCAC AGCATCCCCA AGTTTTCCAA CCCTGAGTCT GATAGCTCCT CTAGCGAGGA GGAGAGGTTC GTGGGCTACG ACGATACCGA TTCCTCTGAC GAGACATTTT CTGATGTGGA CTGCGAGAAT GACGGCAAGA GCGTGGATGA GGACACCAGC GACGATTCCT ATAGCTCCAA CGAGATGGAG AGCATGGTGA CCAAGTCCCT GAATATCGTG GAGACAGATT CTAGCTCCTC TGATGAGGAG GACGAGTTCG TGTACCTGAG GCCACCAATC GGACACACCG CAGACATGGG CTCTAAGAGG AGCCGCACCC CTACAATCGG CACCGTGCCA GAGACACAGA AGAAGCTGAA GCCCCACGTG GATTACTCCG AGCACGAGAT GCTGGACACA CCTCCAATCA AGGACGATGA CTATAACTGG CCTTGGCTGA ATTGACTCGA G Retroperitoneal RFHV GGATCCAAAT GGCCATGTTC CTGGACGATT CCCCCCCTCC AATGGACGAT GACAGGCTGT fibromatosis-associated TTCCATACGA GGGAGCACCA AGGAGAGTGC

herpesvirus CACCTAGGCG CTTCATCTTT CCACCACCTA GGCCAGTGCA GCCTTATGGA GGACCACCCG ORF45 TGATCGATCT GAGCGCCCCT GATGACGTGT TCGCCGAGGA TGACACCAGC CCTCCAGCCA CACCACTGGA CCTGCTGCCT TCCCCAGGAC CAGCAGATGA CAGAGCCCTG GGCATCAATG CACACCACCC TGGAGCAGGC GATTGGATGC CAGCCGCCCT GCCACCTCCA CTGGCAGGCG ACAGAGGCAG GCCCCTGGCC AGCACCGTGC GGAGAACAGT GCCTGTGGCA GTGGTGACCG GACACGTGCG CTCTCCACTG CGGAGCGAGA CAGTGAGCTC CGATTCCTTC CCAGACTGGG AGTCTGAGGG AGAGGGCTTC AGCCCAGGAG AGAGCTTTTC CGATGGCGAG ACCACAGACA CCTTTCTGTC CGAGTCTAGC GTGACCAGGG ATTCTACAAG CGAGGGAGCA CACCTGGAGA TGGACCCCGA CGAGGGACCA AGCTGGAGAC CTCTGAGGGC CGAGGTGGTG GCCACCATCG ATCTGCTGTC CGATTCTGAC AGCGAGTCCC CAACCGAGGG AACACACGGA CACTCCTCTG AGGAGACCCT GTCCGCCGAC GAGGGACCTT CTACCGCAGT GGCACAGGTG AGAGAGACAG TGATCAAGCG CAAGCGGCAG TCCACCGCCA GCTCCGCCAG CGAGGCATCC GGCAGGCCAT CTAGGCGCCG GCTGTGGTCT AGCGGCCACA CAAGCATCAT CATCATCTCC TCTGATTCTG ACAGCGAGCC TGAGCCAACC CAGCGCAGCT CCTTTGGCCC ACCTACAGGA CAGGCAGATG CAGCACAGCG GCTGCCAGAG AACCTGGATG ACGAGTCCAC CTCTGCCACA AGCCTGTCTA GCAGATCCTC TGACAGCTCC TCTGGCGATG GCGACGCCTC CAGGGCCCTG ACCTCTACAC CACCCCTGAG CGGCAACGGC AATTACAACT GGCCTTGGCT GGACTGACTC GAG Table 3 Full optimized DNA sequences of ORF45 homologues synthesized by Genscript.

References Alba, M. M. (2001). Genomewide Function Conservation and Phylogeny in the Herpesviridae. Genome Research,11(1), 43-54. doi:10.1101/gr.149801 Avey, D., Tepper, S., Pifer, B., Bahga, A., Williams, H., Gillen, J., . . . Zhu, F. (2016). Discovery of a Coregulatory Interaction between Kaposis Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36. Journal of Virology,90(13), 5953-5964. Bortz, E., Whitelegge, J. P., Jia, Q., Zhou, Z. H., Stewart, J. P., Wu, T., & Sun, R. (2003). Identification of Proteins Associated with Murine Gammaherpesvirus 68 Virions. Journal of Virology,77(24), 13425-13432. doi:10.1128/jvi.77.24.13425-13432.2003 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. http://doi.org/10.1128/MMBR.00031-10 Chang, Y., & Moore, P. (2014). Twenty Years of KSHV. Viruses,6(12), 4258-4264. doi:10.3390/v6114258 Davison AJ. Overview of classification. In: Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007. Chapter 1. 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. doi:10.3410/f.722183224.793516429 Jha , H. C., Banerjee, S., & Robertson, E. S. (2016). The role of Gammaherpesviruses in Cancer Pathogenesis [Review]. Pathogens ,5(18), 1-43. Jia, X., Shen, S., Lv, Y., Zhang, Z., Guo, H., & Deng, H. (2016). Tegument Protein ORF45 Plays an Essential Role in Virion Morphogenesis of Murine Gammaherpesvirus 68. Journal of Virology,90(16), 7587-7592. doi:10.1128/jvi.03231-15 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. Kuang, E., Wu, F., & Zhu, F. (2009). Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45. Journal of Biological Chemistry,284(20), 13958-13968. doi:10.1074/jbc.m900025200 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. doi:10.1128/jvi.02209-08 Mahmood, T., & Yang, P. (2012, September). Western Blot: Technique, Theory, and Trouble Shooting. Retrieved February 14, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/ Morrison, D. K. (2012). MAP Kinase Pathways. Cold Spring Harbor Perspectives in Biology, 4(11), a011254. http://doi.org/10.1101/cshperspect.a011254 Pearson, W. R. (2013). An Introduction to Sequence Similarity (“Homology”) Searching. Current Protocols in Bioinformatics,1-7. doi:10.1002/0471250953.bi0301s42 Russo , J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M., Maddalena, D., . . . Moore, P. S. (1996). Nucleotide Sequence of the Kaposi sarcoma associated herpesvirus (HHV8) . Microbiology,93, 14862-14867.

Skike, N. D., Minkah, N. K., Hogan, C. H., Wu, G., Benziger, P. T., Oldenburg, D. G., . . . Krug, L. T. (2018). Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice. PLOS Pathogens,14(2). doi:10.1371/journal.ppat.1006843 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. doi:10.1128/jvi.77.7.4221-4230.2003