Because of the importance of retroviral long terminal repeats (LTRs) as determinants of tropism and pathogenic potential, in vitro mutagenesis techniques has been used to alter these elements in the context of an infectious molecular clones of the human immunodeficiency virus (HIV). Starting with a replication-incompetent molecular clone of HIV lacking the two NFkappaB and three Sp1 binding sites present in wild-type LTRS, proviruses containing reconstructed LTRs with individual or combinations of NFkappaB and Sp1 elements were generated and evaluated for their capacity to produce virus progeny following transfection-cocultivation. Virus stocks obtained exhibited a continuum of replicative capacities in different human T-cell types depending on which element(s) was present in the LTR. Of particular interest was the emergence of second-site revertants containing changes affecting the TATA box. An efficient system to monitor the cell-to-cell spread of HIV was developed employing chronically infected H9 donor cells. Under appropriate conditions of co-cultivation, the synthesis of unintegrated viral DNA, monitored by Southern blot hybridization, occurred between 2 and 4 hrs. following infection; viral proteins were detected 8 to 12 hrs following co-cultivation and progeny virions were released into the medium by 16 hrs. The use of metabolic inhibitors and/or specific antibodies revealed that the cell-to-cell spread of HIV required: 1) the interaction of gpl20 with the CD4 receptor and 2) reverse transcrption. Light and electron microscopy indicated that cell-cell fusion, occurring within 15 minutes of co-cultivation, mediated the rapid spread of the infection. Unexpectedly, the fusion observed did not appear to involve budding or free virus particles.
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