Master of Science
Robert J. Visalli
Date of Award
Varicella-zoster virus (VZV) is a double-stranded DNA virus that causes a self-limiting primary infection known as chickenpox and can manifest into a painful, debilitating disease known as shingles in its latent form. Current therapies target the DNA replication process, but novel studies propose targeting the encapsidation process of VZV. The encapsidation process of VZV is based on the well-studied Herpes Simplex 1 (HSV-1) homologs. Seven genes or open reading frames (ORFs) are experimentally shown to be essential for HSV-1 DNA encapsidation. This thesis explores two specific strategies to characterize functional regions of the VZV encapsidation proteins: 1) stable cell lines expressing putative VZV encapsidation proteins to grow VZV mutant viruses and 2) VZV ORF54 encapsidation protein transposon library development for use in functional studies. It is hypothesized that specific regions of the encapsidation proteins will be essential for DNA packaging since it is known that any one deletion of HSV-1 homologs results in accumulation of empty capsids and un-cleaved DNA in the nucleus of infected cells.
Currently, VZV mutants lacking encapsidation genes do not exist. It is expected that the deletion of each putative DNA encapsidation gene will yield a phenotype consistent with a defect in DNA cleavage and/or packaging. Essential to developing VZV mutant viruses is the isolation of stable cell lines expressing the encapsidation protein. The cell lines would complement the VZV mutant virus and can be used for further encapsidation studies. Invitrogen’s Flp-In two-plasmid system was used to isolate isogenic cells lines from the encapsidation genes. With the Flp-In system, stable cell lines expressing a protein of interest can be rapidly generated.
The encapsidation protein known as the portal homolog, pORF54, could prove to be a novel target for antiviral therapy. Little is known about the role of specific functional regions within portal polypeptides including HSV-1. Preparing an ORF54 transposon library for use in functional studies could be used for characterization of functional regions within the VZV portal protein. It is expected that specific regions of pORF54 will be essential for DNA encapsidation. Briefly, ORF54 with flanking regions was isolated from the VZV genome of Ellen strain by PCR. This fragment was inserted into Invitrogen’s Zero Blunt TOPO PCR cloning vector, and transformed into E. coli. The Epicentre EZ-Tn5 In-Frame Linker Insertion Kit was used to prepare in frame insertions of the 19 amino acid transposon throughout the ORF54 plasmid for mutagenesis. Characterization of wild type ORF54 and mutated ORF54 can be accomplished by complementation with a null ORF54 bacterial artificial chromosome (BAC) via transfection (Roche’s FuGene6) into human retina cells (ARPE-19). The importance of ORF54 regions will be determined by complementing the ORF54 null virus with the mutated ORF54 gene. It is hypothesized that specific regions of pORF54 will be essential since it is known that deletion of the HSV-1 portal protein homolog is lethal. This will be the first comprehensive study to characterize potential important function regions within any large DNA virus portal protein.
Pearl C. Pfiester (2012).
Strategies to Characterize Functional Regions of the Varicella-Zoster Virus Encapsidation Proteins.