Characterization of the Varicella-zoster virus ORF25 gene product, pORF25

Jacquelyn R Knepper, Purdue University

Abstract

The Herpesviridae encode a group of highly conserved proteins designated the Herpes UL33 Superfamily (pfam03581). The UL33 Superfamily is believed to be part of the trimeric terminase complex, which is responsible for cleaving viral genomes from concatamers and inserting a single genome into each viral capsid. The goal of this study was to characterize protein-protein interactions for the Varicella-zoster virus (VZV) homolog encoded by the ORF25 gene. ORF25 was used to generate a GST-ORF25 fusion protein in E. coli. In pull-down assays, several encapsidation proteins including pORF30, pORF42, pORF43 and pORF25 were shown to specifically interact with the GST-ORF25 fusion protein but not GST alone. The self-interaction was confirmed via a yeast-two hybrid assay as follows. The ORF25 gene was cloned into a plasmid containing either the activation domain (AD) or DNA binding domain (BD) of the GAL4 transcriptional activator. Fusion protein expression was confirmed via immublot analysis of total protein extracts from yeast cells transformed with the ORF25-containing plasmids. The results confirmed that yeast transformed with both of these plasmids produced two fusion proteins: pGADT7-ORF25 and pGBKT7-ORF25. An &agr;- galactosidase assay performed on yeast supernatants revealed significant &agr;-gal activity from yeast cotransformed with pGADT7-ORF25 and pGBKT7-ORF25. Co-transformed yeast were only expected to express &agr;-gal activity if the pGADT7-ORF25 and pGBKT7-ORF25 fusion proteins interacted via their pORF25 portions. In total, the results support my hypothesis that pORF25 can form complexes with multiple viral proteins including itself.

Degree

M.S.

Advisors

Visalli, Purdue University.

Subject Area

Molecular biology|Virology

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