The synthesis of DNA copies of the viral genome is an obligate step during the early phase of retroviral productive infections. Temperature sensitive (conditional) mutants have been invaluable for delineating structure-function relationships for proteins encoded by numerous bacteriophage and animal viruses. Virtually no mutants of this type exist for HIV-1. A temperature sensitive HIV-1 reverse transcriptase (RT) mutant was generated using the approach of clustered charged-to-alanine mutagenesis. Rather than affecting the catalytic function of the HIV-1 RT, the temperature sensitive step was shown to involve either the processing of the Gag-Pol precursor within nascent particles or the assembly of the p66/p51 heterodimeric RT molecule following the release of progeny virions. Like other cellular genes, integrated copies of HIV DNA are organized into chromatin in virus producing cells. The chromatin structure of the integrated HIV-1 LTRs was analyzed by digesting nuclei from the cloned cell lines with micrococcal nuclease or relevant restriction enzymes. In all of the cloned cell lines examined, the integrated LTR was packaged into two potential nucleosomes, designated Nuc-A and Nuc-C, separated by a 250 bp nuclease hypersensitive region that encompasses the enhancer and promoter elements. Under basal transcription conditions, Nuc-A remains resistant to nuclease digestion but Nuc-C, situated downstream of the promoter, becomes sensitive to enzymatic digestion. Mutagenesis (point and deletion mutations) of DNA sequences covered by Nuc-C prevents cellular proteins from interacting with cognate binding elements and inactivates the HIV-1. Taken together, these results indicate that the chromatin structure of integrated HIV DNA, downstream of the transcription initiation site, must undergo "remodeling" to allow basal and induced levels of RNA synthesis to occur.