Lee, Hyunah; (2020) Structural and biochemical studies of proteins implicated in Kaposi’s Sarcoma-associated Herpesvirus pathobiology. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Kaposi’s sarcoma-associated Herpes virus (KSHV) infects hundreds of millions of people world-wide contributing to the development of Kaposi’s sarcoma (the most common HIV-related cancer), Multicentric Castleman’s disease and Primary effusion lymphoma. The project focused on two key proteins: KSHV SOX and KSHV-vFLIP that operate during the lytic and latent phases of the KSHV life-cycle respectively. KSHV SOX is a virally encoded highly conserved alkaline exonuclease which plays a key role in the global and rapid degradation of host mRNA in a process termed host shutoff as well as the packaging of viral genomes into capsids following replication. Although published (Bagneries et al., 2011) and unpublished (Lee, 2015, Master’s thesis, UCL) crystal structures of SOX bound to DNA has given key insights into the mechanism of DNA recognition and cleavage, it was unclear how RNA could be recognised owing to the lack of canonical RNA binding motifs and therefore uncertain whether the mechanism of cleavage proposed for the exonucleolytic cleavage of DNA substrates would apply. A combination of structural and biochemical approaches were therefore used to address these key outstanding questions which are presented in this thesis. They include the first crystal structure of SOX-E244S mutant bound to the KSHV pre-miRNA K2-31 which was determined at 3.3 Å by molecular replacement. Analysis of this structure has revealed a distinct binding mode for RNA binding relative to DNA in which the “bridge” motif, spanning the C- and N-terminal lobes of the SOX molecule, has an essential role in substrate recognition. Despite these differences, endonucleolytic processing of RNA transcripts could still be achieved by the SN2 (biomolecular nucleophilic substitution) mechanism originally proposed for the exonucleolytic cleavage of DNA. The results of biochemical and biophysical assays performed on wild-type and mutant proteins defective in host shut off further revealed that the degradation of RNA is largely sequence non-specific, but requires structured elements such as stem loop or bulge motifs. They also revealed that impaired host shut off activity could be explained by impaired RNase activity against structured substrates for a subset of the mutants. The ability of compare SOX-DNA and SOX-RNA structures afforded by these studies lead to the identification of phytic acid as a potential inhibitor of both its DNase and RNase activities confirmed by the crystal structure of SOX-phytic acid complex refined to 2.3 Å. Here it is shown that phytic acid not only has the potential to physically block the association of DNA/RNA substrates but also inhibit nucleolytic cleavage by directly co-ordinating to a catalytically important magnesium ion. During latency, KSHV produces a limited number of proteins essential for its survival. One of these is the viral FLICE inhibitory proteins (vFLIPs) that directly activates the canonical and alternative NF-κB pathways resulting in increased cell proliferation, transformation, cytokine secretion, and protection against growth factor withdrawal-induced apoptosis. Previous studies have shown that KSHV-vFLIP forms a ternary complex with the transcription factor p100 and the kinase IKKα for persistent activation of the alternative pathway shown to have an important role in cellular transformation. Although the mechanism underlying this process is unclear, a physical interaction between KSHV-vFLIP and p100 has been implicated. Having successfully produced all three proteins in E. Coli and baculovirus expression systems, the nature of this complex was investigated using pull down assays and site-directed mutagenesis. From these results, it has been possible to deduce that KSHV-vFLIP interacts with residues 860-900 located at the C-terminal end of p100 and does not interact with the death domain of p100.

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