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Regulation of Replication and Translation During KSHV Life Cycle
AdvisorVerma, Subhash C
Cell and Molecular Biology
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Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is a γ2-lymphotropic virus of the herpesvirus family that establishes asymptomatic infections in immunocompetent hosts but causes complications including tumors in an immunocompromised individual. KSHV is associated with various malignancies like Kaposi’s Sarcoma (KS), Primary Effusion lymphoma (PEL), and a plasmablastic form of Multicentric Castleman’s Disease (MCD). Like other members of the Herpesvirus family, KSHV’s life cycle consists of two phases; the latent and the lytic phase. During latency, the virus establishes a lifelong persistent infection in the host which cannot be eliminated as the virus interferes with the immune surveillance mechanism of the host. Because of the compromised immunity of the affected individuals and various external stimuli, the virus undergoes lytic reactivation leading to the production of infectious virions and tumorigenesis. Since latency is described as a dormant phase, virus persists in the infected host with restricted gene expression and viral replication to maintain itself in dividing tumor cells. A major latency associated protein, Latency Associated Nuclear Antigen (LANA) plays a significant role in maintaining the viral genome and regulating the growth of tumor cells. To achieve a better understanding of latency, it was essential to study how LANA interacts with cellular proteins and facilitates DNA replication. Our work determined the interaction between LANA and cellular helicases, the MCM complex in detail, where we determined the interactions of the MCM complex with LANA in different cell cycle phases. We also identified the domain of LANA that facilitated its interaction with MCM6 and was critical for latent DNA replication and maintenance of the viral genome. In addition, we demonstrated how MCMs are absolutely required for DNA replication and persistence of the viral genome along with their association with replicating DNA. Immune evasion is critical for maintaining viral persistence in the host for a lifetime, which also contributes in establishing a successful KSHV infection to a great extent. LANA, the master regulator of latency, has been shown to possess immunomodulatory functions. Studies described in Chapter 2, add to the existing knowledge about immune modulation during latency and highlight the role of secondary RNA structures called G-quadruplexes in regulating mRNA translation and antigen presentation of LANA. We successfully characterized the formation of RNA G-quadruplexes in LANA mRNA and their effect on the translation of LANA. The limited pool of LANA in cells leads to the inhibition of antigen presentation of LANA peptides, thereby thwarting the possibility of an immune response. We also demonstrated that a G-quadruplex binding protein; hnRNPA1, possibly destabilizes these structures and enhances the expression of LANA. Moreover, we share a brief insight into the autoregulation of protein expression by LANA through binding to the G-quadruplex forming RNA and reducing their export into the cytoplasm. Another critical aspect of KSHV pathogenesis involves lytic DNA replication, which leads to the synthesis of viral genome and production of viral proteins with roles in enhancing and promoting virion assembly. The initiation of lytic DNA replication occurs through the cis-acting oriLyt region along with the involvement of viral and cellular proteins. In Chapter 3, we discovered the presence of G-quadruplexes in the oriLyt, which were important for the initiation of lytic DNA replication as stabilizing those G-quadruplexes led to a reduction in replication initiation and viral genome copies. In addition, we also determined the potential of RecQ, a cellular helicase in facilitating lytic replication at these sites and how RecQ binding site in the oriLyt is crucial for lytic replication.