Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates

Department of Biochemistry, University of Rochester, NY.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 08/1992; 1131(3):270-80. DOI: 10.1016/0167-4781(92)90025-U
Source: PubMed


We have examined the parameters that determine the length and distribution of products synthesized processively by the human immunodeficiency virus reverse transcriptase (HIV-RT). On native or homopolymer templates, the overall length distribution of processively synthesized products is increased by increased temperature or deoxynucleoside triphosphate concentration, or decreased ionic strength. Specific terminations of processive synthesis on either native DNA or RNA templates occur most frequently at positions where the reverse transcriptase (RT) pauses during synthesis. These sites correlate with the template sequence 3'-(A/U)(A/U)(G/C)-5', particularly when this sequence is predicted to be base paired with another region of the template in a secondary structure. Many positions of termination are in similar positions on DNA or RNA templates. Notable exceptions are runs of A residues, which promote termination on DNA but not RNA templates. Termination intensities vary when different RTs are used demonstrating an influence of RT structure.

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    • "In the case of set #2 (Figure 1C), premature terminations are apparent (with product lengths of mostly 47, 40 and 36-nt), whereas with set #1 these longer premature terminations are less evident (see also below, Figures 2A and 4A, regarding set #1). These results support the concept that the extent of RT-dependent processivity and, consequently pausing, can depend, for a given RT, on the sequences of the copied templates, for example, (12,13). "
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    ABSTRACT: We present evidence that the reverse transcriptase (RT) of human immunodeficiency virus type-1 stabilizes in vitro very short (2-nt) duplexes of 3'-overhangs of the primer strand that are annealed to complementary dinucleotides tails of DNA or RNA template strands, provided that these sequences contain at least one C or G. This RT-induced strand 'clamping' activity promotes RT-directed DNA synthesis. This function is achieved only when the functional template strand is adjacent to a second DNA or RNA segment, annealed upstream to most of the primer (without gaps). The combined clamp/polymerase activity is typical to RTs, as it was found in different RTs from diverse retroviral groups, whereas cellular DNA-polymerases (devoid of 3'→5' exonucleolytic activity) showed no clamp activity. The clamp-associated DNA-binding activity is markedly stabilized by dGTP, even when dGTP is not incorporated into the nascent DNA strand. The hereby-described function can help RTs in bridging over nicks in the copied RNA or DNA templates, encountered during reverse transcription. Moreover, the template-independent blunt-end synthesis of RTs can allow strand transfers onto compatible acceptor strands while synthesizing DNA. These RT properties can shed light on potentially-new roles of RTs in the reverse-transcription process and define new targets for anti-retroviral drugs.
    Nucleic Acids Research 09/2010; 39(3):1042-53. DOI:10.1093/nar/gkq786 · 9.11 Impact Factor
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    • "Expression of lentivirus accessory genes requires splicing or internal ribosome entry sites, both of which are defined by specific secondary structure in the RNA. These stable RNA structures may facilitate recombination by decreasing processivity of the viral reverse transcriptase (DeStefano et al., 1992). The outcome of most strand switches is not detected in natural infection because many recombinant progeny will be replication incompetent. "
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    Virology 09/2007; 364(2):362-70. DOI:10.1016/j.virol.2007.03.023 · 3.32 Impact Factor
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    ABSTRACT: System requirements: World Wide Web browser and PDF reader. Mode of access: Available through the Internet. Title from document title page. Document formatted into pages; contains vi, 119 p. : ill. Thesis (Ph. D.)--West Virginia University, 1999. Vita. Includes abstract. Includes bibliographical references.
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