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)ORULGD6WDWH8QLYHUVLW\/LEUDULHV 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 By 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 3 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. 4 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). 5 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). 6 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