Validating VIL-6 as a target for KSHV-associated disease

DESCRIPTION (provided by applicant): Preliminary studies in rhesus macaques reveal that macaques experimentally infected with the simian immunodeficiency virus (SIV) and a rhesus human herpesvirus 8 homologue (RhHHV8) develop B cell hyperplasia compared to macaques infected with SIV alone. The RhHHV8, like HHV8/Kaposi's sarcoma-associated herpesvirus (KSHV), encodes an intedeukin-6 (IL-6) homologue. The viral IL-6 homologue in HHVS/KSHV is thought to be a necessary growth factor for Kaposis sarcoma and the B cell-derived malignancy referred to as body cavity based lymphomas or primary effusion lymphoma in AIDS patients also infected with HHV8/KSHV. The long-term objectives of this study aim to evaluate the role of the RhHHV8 IL-6 homologue in viral-mediated B cell hyperplasia in the context of an SIV infection. To determine this the following Specific Aims are proposed: Specific Aim #1 is to construct mutant RhHHV8 isolates that lack an intact IL-6 gene. Viral IL-6(-) mutants will be generated via recombination through the use of infectious viral DNA and a plasmid encoding the enhanced green fluorescent protein (EGFP) driven by a constitutive promoter and flanked by homologous viral DNA sequences to facilitate recombination. EGFP wilt be utilized to facilitate the identification of recombinant viruses containing the desired mutation. To ensure that the phenotype associated with the vlL-6(-) mutant is in fact due to the insertion of EGFP, a rescuant will be constructed that re-introduces the vlL-6 back into the viral genome. Confirmation that the vlL-6 gene is deleted and then re-introduced into the correct location will be determined by restriction endonuclease digestion and Southern blot analysis using the vlL-6 gene as probe, as well as sequence analysis. Specific Aim #2 is to evaluate the phenotype of the vlL-6 (-) virus versus the wild type and rescuant in the context of an SIV infection. Parameters that will be measured include: viral load as defined by PCR; differential leukocyte counts in peripheral blood; serum protein electrophoresis for hypergammagtobulinemia; host and viral IL-6 by ELISA and bioassay; lymph node and bone marrow biopsies, and physical examination. The results should help elucidate the role of the vlL-6 in virus infection and provide new insights into the future development of diagnostics and therapies for gamma-2 herpesvirus-induced malignancies.