The goal of this project is to define the molecular mechanisms involved in the replication of mammalian retroviruses and in particular, to understand the factors which influence the regulated expression of viral genetic information. We have been using an in vitro approach to study events in reverse transcription. We have shown that during (-) strand DNA elongation on a murine leukemia virus (MuLV) RNA template, there is a major pause site preceding a stable stem-loop structure, formed by bases from the polypurine tract (PPT) and downstream sequences. HIV-1 nucleocapsid (NC) protein, which destabilizes secondary structure, reduces pausing and increases the rate of synthesis of full-length DNA. This shows that NC stimulates processivity of reverse transcriptase (RT). Experiments with chemically modified NC reveal that the cysteine residues in the Zn fingers are required for NC activity in the pausing assay and facilitate NC binding to the template. In studies with HIV-1 RT, we have found that initiation of (+) strand viral DNA synthesis is dictated by the PPT alone and not by the surrounding context. Based on the results of assays measuring the ability of wild-type and mutant PPT RNA oligonucleotides to act as primers and CD spectra of short PPT-containing RNA-DNA hybrids, we also conclude that a combination of an ususual helical structure and a requirement for certain bases at the 3~ end of the PPT appear to be important determinants for primer recognition and extension. Analysis of RNase H* activity (degradation of double-stranded (ds)RNA) shows that an active RNase H domain is required; activity is enhanced by fusion of the RNase H domain to polymerase, which stimulates binding of the dsRNA substrate to RT. In studies on in vitro formation of retroviral RNA dimers, we have demonstrated that NC protein can convert an unstable dimer to a more thermostable form. Thus, we now have an in vitro model for NC-mediated maturation of the genomic RNA dimer in virus particles. Experiments to test the activity of mutant NC proteins in the pausing and dimerization assays are in progress.