KINETIC-ANALYSIS OF HIV-1 EARLY REPLICATIVE STEPS IN A COCULTURE SYSTEM

During an acute human immunodeficiency virus (HIV) infection in vitro, different forms of unintegrated viral DNA accumulate in target cells. They include linear full-length HIV DNA molecules, which are the prec ursors of the integrated provirus, and two types of circular molecules (with one or two LTRs), whose role and mode of formation are not full y understood. To evaluate the intracellular fate of HIV unintegrated D NA, and to follow the formation of the two types of circular DNA molec ules, the nuclear transport of viral DNA, and its integration in host cell DNA, we have designed a ''DNA chase'' assay. This assay is based on cocultivation of persistently HIV-1-infected H9 cells with uninfect ed MT4, allowing rapid accumulatian of viral DNA, which is then blocke d by addition of AZT. In this highly efficient, synchronous, one-step cycle infection system, HIV linear DNA can be detected on Southern blo ts as early as 4 hr after the start of the coculture. Subsequently, vi ral DNA that had been synthesized before the addition of AZT could be ''chased,'' establishing that almost all linear DNA molecules are rapi dly transported to the nucleus, where they are either processed into t he two types of circles or integrated. We could estimate that from the number of viral DNA molecules synthesized in 6 hr in this system, at least a third will become integrated and another third will circulariz e within 24 hr.