Identifier

etd-06282008-141328

Degree

Master of Science (MS)

Department

Veterinary Medical Sciences - Pathobiological Sciences

Document Type

Thesis

Abstract

A conditional silencing system using anti-gB siRNAs was devised to investigate the structure and function of the KSHV gB. Transient co-transfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the basal cavity-based lymphoma cells (BCBL-1) caused substantial reduction of gB transcription and protein synthesis. TaqMan real-time PCR and infectivity assays showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB not recognized by the anti-gB siRNAs, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. Virion egress experiments with truncated gBs revealed that removal of the entire predicted cytoplasmic domain of gB increased virion egress suggesting the presence of a egress-regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity. To investigate the potential role of gB in tumorigenesis, BCBL-1 cells transiently transfected with anti-gB siRNAs and codon optimized gB were mixed with Matrigel and injected subcutaneously in nude mice. Direct measurement of tumors revealed that BCBL-1 cells transfected with anti-gB siRNAs produced tumors significantly smaller than mock-transfected BCBL-1 cells. Co-transfection of codon optimized gB appeared to abrogate the inhibition of tumor formation by siRNAs. These results show that gB is important for infectivity, virion egress and pleural effusion lymphoma (PEL) formation in mice. Current work focuses on the use of a lentiviral expression system to generate BCBL-1 cells that constitutively express anti-gB siRNAs to improve inhibition of endogenous gB expression.

Date

2008

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Konstantin Gus Kousoulas

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