MOLECULAR CLONING AND CHARACTERIZATION OF HUMAN RETROVIRUSES

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Heterogeneity among retroviral genomes is a distinct feature of lentiviruses. Molecular cloning, restriction enzyme analysis, southern blotting, nucleotide sequencing, and polymerase chain reaction techniques were used to characterize isolates of human immunodeficiency virus type 1 (HIV-1). The objective was to obtain information pertaining to the structure and diversity of HIV with respect to its biochemical, pathogenic, and immunological variability. Most HIV isolates cloned are T-cell tropic. Recently, we succeeded in obtaining a complete and infectious molecular clone from a macrophage-tropic viral isolate (AD8). T-cells are known to be the major target for the replication of HIV in peripheral blood. However, macrophages represent the predominant HIV-infected cell type in most tissues. Macrophages are the primary reservoir of HIV and sustain a persistent infection in individuals for many years. Because of its capacity to generate high titers of progeny virus in infected macrophages, and the vital role of macrophages in the establishment of an HIV-1 infection, the AD8 molecular clone should be a useful reagent for the biochemical analyses of productively infected macrophages. Southern blot analysis of lung tissue obtained from a [SIV-HIV hybrid virus (SHIV)] infected, CD4 depleted cynomolgus macaque showed two distinct bands by autoradiography; one approximately 10Kb (full length SHIV) and one very pronounced signal approximately 6Kb (deleted SHIV). Molecularly cloning directly from the infected lung tissue resulted in the isolation of 6 individual clones. Two were full length in size while the other 4 appeared to have been deleted (6Kb). These clones are presently under study.