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. Studies are being carried out on the functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT), using purified murine leukemia virus (MuLV) RT proteins. These include wild-type RT and three mutants: (i) deltaH, which is missing the entire RNase H domain; (ii) a chimeric RT in which the MuLV RNase H domain is replaced with E. coli RNase H; and (iii) deltaSX, having a large deletion in a polymerase region corresponding to the """"""""connection"""""""" subdomain in the p66 subunit of HIV RT. Our data show that each of these mutations, while occurring in only one domain, has profound effects on both polymerase and RNase H cleavage activities. We find, for example, that wild-type RT undergoes two types of RNase H cleavages. The major cleavage event is coordinated with DNA synthesis and occurs at a site in the RNA 14-18 nucleotides from the 3'-terminus of the primer; we refer to this cleavage as """"""""3'-OH-dependent"""""""" since in this case, the 3'-OH of the primer is bound to the polymerase active site. Additional cleavages occurring closer to the 5'-end of the RNA are termed """"""""3-OH-independent"""""""" i.e., the 3'-OH of the primer is no longer bound to the polymerase site and addition of dNTPs has little or no effect on the cleavage pattern. Interestingly, cleavages catalyzed by the deltaSX deletion mutant and the chimeric RT are exclusively 3'-OH-independent. Recent results evaluating the polymerase activity of the mutants indicate that the RTs with RNase H mutations cannot synthesize DNA in a processive manner. Experiments to determine whether these results reflect abnormal binding of primer/template will be performed. Future efforts will also include construction and analysis of other mutant RTs. In other work on translational control of viral gene expression, we have previously identified the cis-acting signal responsible for readthrough suppression of the UAG codon at the MuLV gag- pol junction. We are currently investigating the effect of this signal on normal translation of a transcript containing the glutamine codon CAG instead of UAG. Experiments are being carried out with infected cells and with an in vitro translation system.

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Tang, Shixing; Ablan, Sherimay; Dueck, Megan et al. (2007) A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein. Virology 359:105-15
Wu, Tiyun; Heilman-Miller, Susan L; Levin, Judith G (2007) Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein. Nucleic Acids Res 35:3974-87
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Opi, Sandrine; Takeuchi, Hiroaki; Kao, Sandra et al. (2006) Monomeric APOBEC3G is catalytically active and has antiviral activity. J Virol 80:4673-82
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Imamichi, Tomozumi; Murphy, Michael A; Adelsberger, Joseph W et al. (2003) Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression. J Virol 77:1011-20
Iwatani, Yasumasa; Rosen, Abbey E; Guo, Jianhui et al. (2003) Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions. J Biol Chem 278:14185-95

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