Using a novel PCR-based method (Quantitative Competitive-PCR, QC-PCR), for quantifying HIV-1RNA we recently discovered unexpectedly high levels of cell-free virus in human plasma. Virus titers determined by this technique were significantly related to disease stage, CD4+ lymphocyte counts, p24 antigen levels, and therapy with AZT, and correlated with, but exceeded infectious virus titers in plasma by an average of 60,000-fold (range 8 to 1,200,000; n+66). The biophysical, biological, and genetic basis of this high but variable ratio of total HIV-1 particles to infectious units is completely unknown, as are the contribution of these virus populations to pathogenesis. We will pursue four approaches to rigorously examine the content, composition, biophysical and biological properties of HIV-1 virions in plasma, and the clinical correlates of these properties: (1) Using HIV-1 isolated from human plasma, we will determine total virion counts; culturable infectious units; and virion content of (viral) RNA and DNA, structural proteins including gag, pol, env gp 120/gp41, an host cell derived proteins, including MHC Class I, MHC Class II, and beta2- microglobulin, as well as immunoglobulin and complement. We also determine the buoyant density and ultrastructure of virus and/or viral immune complexes in plasma. (2) We will define the biologic and genetic basis for the observed high ratios of total virus to infectious units in plasma by determining the stage(s) in the HIV-1 replication cycle where replication is aborted, and by directly measuring the genetic and functional integrity of tat and RT genes from plasma virus. (3) In patients undergoing rapid changes in viral replication (e.g., in acute infection), and in patients identified in prospective natural history studies as exhibiting rapid or slow disease progression, we will determine if quantitative or qualitative measures of plasma virus content or composition are significantly associated with clinical course and outcome. (4) Using plasma samples from patients treated with novel single agent and combination antiretroviral therapies, we will determine the kinetic profile and magnitude of change in plasma virus as determined by quantitative measurements of viral RNA, infectious units, an p24 antigen. We will also determine the effect of these novel therapies on the biochemical and genetic composition of virus that evolves during and after therapy. The studies proposed will be performed by a collaborative group of investigators each of whom has specialized technical and/or clinical expertise directly related to the aims of this project. The anticipated results are likely to expand, in a new direction, our understanding of basic virologic and host mechanisms underlying HIV-1 pathogenesis, and in addition, should contribute importantly to the development of improved markers for assessing the activity of antiretroviral therapies in vivo.
