Goals of this proposal include elucidating the role that CD4 affinity of HIV-1 gp120 plays in determining lab adapted or primary phenotype, generating cell lines which will support the growth of T-cell tropic isolates, and establishing quantitative infectivity assays with which to analyze macrophage-tropic isolates of HIV-1. It is proposed to dissect out distinct mutational changes that occur in gp120 as HIV-1 undergoes lab adaptation, and to compare infectious properties, and gp120 biochemical attributes such as CD4 affinity, to those of the parental primary isolate, thus gaining insight into ways to grow primary isolates. T-cell lines capable of supporting primary isolate growth will be generated by using a retroviral vector to introduce high amounts of CD4 into leukemic T-cell lines, thus making it easier for primary isolates to attach to and establish an infection. A quantitative infectivity assay for macrophage-tropic isolates of HIV-1 will be established by expressing CD44S in adherent cell lines such as HeLa CD4 or CEM-SS. The most susceptible line will be used for infectivity assays. Soluble CD4 and gp120 subunit vaccines (both based and initially tested on lab adapted strains) have failed to inactivate primary strains of HIV- 1. Understanding how infectious properties of primary isolates differ from those of lab adapted isolates will aid in the development of cell culture systems allowing propagation of primary strains of HIV-1 for use in the development of vaccines and other antiviral strategies.
