Characterization of protein-protein interactions mediated by the second exon of the varicella-zoster virus ORF45/42 terminase subunit

Elizabeth A Syster-Clevenger, Purdue University

Abstract

Varicella-zoster virus (VZV), a member of the Herpesviridae, is ubiquitous human pathogen with significant morbidity and mortality in healthy and immunocompromised individuals. Primary infection causes a systemic viremia and skin rash known as varicella, and more commonly referred to as chickenpox. Latency is established in the nervous system during primary infection and can reactivate to cause zoster, more commonly known as shingles. Current treatments for VZV infection target viral DNA replication, primarily the DNA polymerase. However, novel small molecule inhibitors that target other viral replication processes, for example viral DNA encapsidation, offer additional therapeutic options. Seven conserved open reading frames (ORFs) encode proteins that were shown to be essential for herpesviral DNA encapsidation. This thesis explores the protein-protein interaction of one of the VZV encapsidation proteins, the small terminase protein, pORF45/42. Previous studies have explored interaction among the VZV terminase subunits, particularly exon II of pORF45/42. New studies were designed to identify potential small terminase protein self-interactions sites encoded by exon II. Full length pORF42 and four truncated ORF42Δ constructs were constructed based upon a proposed self-interaction site in the Human cytomegaloviurs (HCMV) homologue, pUL89. pORF42 constructs were expressed in E. coli, purified and then tested in GST pull down assays using aGST-pORF42 fusion protein (GST-ORF42-C269). GST pull-down assays demonstrated that pORF42, pORF42Δ1, and pORF42Δ2 interacted with immobilized GST-pORF42 fusion protein. Interestingly, pORF42Δ1 and pORF42Δ2 were developed with N-terminal deletions that removed the proposed interaction site of HCMV pUL89 yet both interacted with GST-ORF42-C269. pORF42Δ3 and pORF42Δ4 did not show any interaction with GST-ORF42-C269, suggesting that the self-interaction site was located within the C-terminus of pORF42. This unique VZV pORF45/42 self-interaction site may play a role in the formation of multimeric small terminase complexes and these may be required for proper terminase function. Assays targeting the VZV interaction site may reveal novel antivirals that target the VZV pORF45/42 terminase and the DNA encapsidation process.

Degree

M.S.

Advisors

Visalli, Purdue University.

Subject Area

Molecular biology|Microbiology|Virology

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