This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A KSHV gM-null virus was constructed using transposon-insertion mutagenesis. The KSV gMnull genome was transfected into Vero cells and a permanently transformed cell line that harbors the viral genome was isolated and characterized. Real-time PCR TaqMan assays were developed against the lytic KSHV gene ORF59 and employed to assess the levels of infectious virus production intracellularly, as well as in extracellular spaces after induction of lytic replication. In addition, supernatant viruses were collected and tested in specialized infectivity assays. These experiments revealed that the gM-null virus egressed from cells and retained infectivity showing that KSHV gM is not essential for virion egress and infectivity. A strategy was devised to silence the gB gene through the use of two siRNAs. Transient transfection of gene cassettes expressing anti-gB siRNAs efficiently silenced transcription of the gB gene in cotransfection experiments in 293 cells, as well as in BCBL-1 cells. Real time PCR (TaqMan) and infectivity assays showed that gB is essential for virion egress and infectivity. A novel aspect of this work is the construction of a partially codon-optimized gB gene in which the first 500 nucleotides of the gB gene have been altered to not be recognized by the anti-gB siRNAs. Transfection of a gene construct expressing the codon optimized gB gene efficiently rescued virion egress and infectivity in BCBL-1 cells. These results constitute proof-of-principle that the structure and function of gB and other viral proteins can be effectively studied using conditional inhibition of KSHV mRNA transcription by siRNAs.
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