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). A chimeric RT having the murine leukemia virus (MuLV) polymerase domain fused to E. coli RNase H has been expressed and purified and its RNase H and polymerase activities compared to those of wild-type RT. In the absence of polymerization, the chimeric protein behaves like E. coli RNase H. When there is limited extension of the primer (i.e., addition of 3 dNTPs and 1 ddNTP), we find that with wild-type RT, the RNA is shortened by the same number of bases as the DNA is extended. This result indicates that cleavage by RNase H is normally linked to DNA synthesis. With the chimeric RT, there is little or no effect of dNTPs on RNA cleavage, suggesting that this enzyme is deficient in polymerization. Indeed, direct measurement of polymerase activity shows that unlike wild-type, the chimeric RT makes small products with either an RNA or DNA template. These results indicate that despite the fact that the chimeric RT has a wild-type polymerase region, it is unable to synthesize DNA in a processive manner. Future efforts will include analysis of the RNase H and polymerase activities of other mutant RTs. In other work, translational control of viral gene expression is being investigated by studying readthrough suppression of the UAG codon at the MuLV gag-pol junction. We have now identified the readthrough signal and find that it is entirely contained within the first 57 nucleotides on the 3' side of the UAG codon. The signal is complex and consists of two distinct elements: a highly conserved, eight-nucleotide, purine-rich sequence immediately downstream of the UAG codon, followed by a pseudoknot structure spanning the next 49 nucleotides. Studies to determine how the signal affects translation of a transcript containing CAG instead of UAG at the gag-pol junction will be performed.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
1992
Total Cost
Indirect Cost
City
State
Country
United States
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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
Iwatani, Yasumasa; Chan, Denise S B; Wang, F et al. (2007) Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Res 35:7096-108
Iwatani, Yasumasa; Takeuchi, Hiroaki; Strebel, Klaus et al. (2006) Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J Virol 80:5992-6002
Opi, Sandrine; Takeuchi, Hiroaki; Kao, Sandra et al. (2006) Monomeric APOBEC3G is catalytically active and has antiviral activity. J Virol 80:4673-82
Levin, Judith G; Guo, Jianhui; Rouzina, Ioulia et al. (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80:217-86
Miles, Lesa R; Agresta, Beth E; Khan, Mahfuz B et al. (2005) Effect of polypurine tract (PPT) mutations on human immunodeficiency virus type 1 replication: a virus with a completely randomized PPT retains low infectivity. J Virol 79:6859-67
Heilman-Miller, Susan L; Wu, Tiyun; Levin, Judith G (2004) Alteration of nucleic acid structure and stability modulates the efficiency of minus-strand transfer mediated by the HIV-1 nucleocapsid protein. J Biol Chem 279:44154-65
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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