Browsing Theses and Dissertations by Author "Nale Lovett, Dakota J"
Contributions of the N and C – Termini of Varicella – Zoster Virus PortalNale Lovett, Dakota J; School of MedicineThe VZV portal protein is a multimeric protein found at a single vertex of the viral capsid that is essential for encapsidation (packaging) of viral DNA. All viruses within the herpesvirus family contain structurally homologous portal proteins. PORT compounds have been shown to target herpesvirus portal proteins and show potential as broad-spectrum herpesvirus antivirals. We are specifically interested in the interaction of PORT compounds with the VZV portal since the activity of our compounds against VZV was shown to be in the single nanomolar range. Unfortunately, VZV portal structure has yet to be resolved by transmission electron microscopy (TEM) due to complications with size and aggregation. We aimed to observe the effects of pORF54 (VZV portal protein) terminal truncation on virus viability to determine the minimal portal protein that can be used for structural analysis. Six recombinant viruses containing N and C- terminal mutations were created using bacterial artificial chromosome (BAC) technology and targeted recombineering to create the mutant VZV strains. PCR was used to engineer stop codons at 29, 49, and 75 AAs from the C-terminal end. In addition, a second set of recombinants was constructed where the first start codon of the 769 AA pORF54 open reading frame was deleted creating a 40 AA N-terminal truncation. Sequencing of all mutant strains confirmed the expected changes for the ORF54 gene and also that no gross off-target mutations occurred. All constructs showed a similar restriction digestion pattern, upon gel electrophoresis, compared to the parent BAC, pOKA, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious viral stocks after lipofectamine-based transfection into ARPE19 cells. Viruses with non-functional mutations in pORF54 were transfected into ARPE54 (complementing) cells, that constitutively express wild-type pORF54. The ∆29, ∆49, N40∆29, and N40∆49 viruses were replication competent in ARPE19 cells. Replication characteristics suggested that some of these viruses grew less efficiently in vitro. Overall, we confirmed that a protein of 679 AAs can form a functional viral portal. This portal multimer may represent a strong candidate for detailed structural studies.