Recent studies clearly indicate that the course of human immunodeficiency virus type-1 disease is intimately related to persistent viral replication and massive viral burden in the host. Previous therapeutic interventions have demonstrated only transient and modest efficacy because they have failed to provide lasting control of viral replication and thus have had only transitory impact on viral burden. Effective therapeutic strategies need to address the massive viral reservoirs in the infected patient and their capacity either to harbor or generate resistant variants. These notions have emerged from the application of new PCR based technologies to measure, localize and characterize HIV- 1 nucleic acids in the blood and tissues of HIV-1 infected individuals at various stages of disease and during therapy with antiretrovirals. Therefore, the Projects comprising this SPIRAT application describe therapeutic strategies designed to reduce viral burden below levels previously achieved and sustain control over viral replication in the patient. The function of the molecular virology core is to perform a common set of state of the art virological assessments in the patient samples from each Project in this SPIRAT Program. Viral burden and expression will be measured in peripheral blood and lymphoid tissue samples generated in the clinical trials described in Projects 1-3 to determine the kinetics of viral turnover in these compartments. Quantitative automated sequence analysis of virus will be performed to determine the relationships between clinically relevant viral reservoirs and examine interactions of drug resistance mutations. In situ hybridization combined with flow cytometry or automated image analysis will be conducted to identify the nature and location of cellular viral reservoirs and their response to therapeutic intervention. To accomplish these aims we have assembled leading experts in the relevant fields of HIV- 1 virology to participate in this Core. The specific virological assays to be conducted by members of this core include: 1) plasma HIV-1 RNA levels; 2) PBMC HIV-1 DNA and RNA levels; 3) Lymph Node HIV-I DNA and RNA levels; 4) plasma immune complex dissociated (ICD) viral p24 antigen levels; 5) Quantitative plasma and PBMC viral cultures; 6) Quantitative plasma HIV-l RNA, PBMC proviral DNA, and Lymph Node mononuclear cell (MC) proviral DNA reverse transcriptase (RT) and protease genotypic analysis; 7) Phenotypic drug sensitivity analysis of plasma virus and PBMC viral isolates; 8) in situ/PCR HIV-1 DNA and direct in situ hybridization RNA analysis in PBMC and Lymph Node MC; 9) Lymph Node architectural analysis combined with in situ hybridization HIV-1 RNA analysis. These studies will explore the potential clinical benefits of novel combinations of antiretroviral, immunoablative, and immune reconstitutive therapies and substantially increase our understanding of the virologic and immunologic mechanisms of HIV-1 pathogenesis.
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