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. Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a lymphotropic oncogenic virus, has been implicated in the pathogenesis of Kaposi's sarcoma; body cavity-based B-cell lymphoma (BCBL), or primary effusion lymphoma; and some forms of multicentric Castleman's disease. Similar to other herpesviruses, KSHV encodes many glycoproteins, which play important roles in virion morphogenesis and infectivity. The overall goal of this research is to investigate the structure and function of KSHV glycoproteins in the virus life-cycle with an initial focus on glycoproteins gM and gB. Two main approaches have been used to elucidate the structure and function of glycoproteins gM and gB: 1) Construction of KSHV mutants through the use of the KSHV genome cloned into an bacterial artificial chromosome (bac); 2) Conditional silencing of viral glycoprotein expression using siRNAs. gM-null KSHV mutant viruses were constructed by insertional inactivation of the gM gene into the KSHV-bac36 genome in E. coli. Transfection of the KSHV gM-null viral genome into 293 cells produced virions, whcih were able to egress out-of-the transfected cells and infect other cells indicating that gM was not important for virion egress and infectivity. KSHV gB is known to be important in virion egress. Our work on gB focused on the role of the gB cytoplasmic terminus in virus-induced cell fusion and virion egress. Mutant KSHV viruses were constructed that specified gB carboxyl terminal deletions. A gB deletion of 25 amino acids substantially increased virus-induced cell fusion. To delineate functional domains of gB involved in virion egress using the BCBL-1 cell line that produces large amounts of virus upon induction of the virus to lytic replication, siRNAs specific for gB were utilized. Transfection of siRNAs into BCBL-1 cells significantly reduced virion egress from BCBL-1 cells. Tranfection of BCBL-1 cells inhibited by anti-gB siRNAs with a codon optimized gB rescued virion egress. Similarly, transfections with plasmids expressing gB-truncations rescued virion egress indicating that the cytoplasmic terminus of gB is not important for virion egress.
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