Wild-type and mutant HIV type 1 nucleocapsid proteins increase the proportion of long cDNA transcripts by viral reverse transcriptase
ABSTRACT HIV-1 nucleocapsid, p7, contains two retroviral zinc fingers, which are both necessary for efficient packaging of genomic RNA and infectivity. The nucleocapsid protein is bound tightly to genomic RNA in the mature virion. In this study, the effect of p7 on polymerization of nascent cDNA by viral reverse transcriptase (RT) was examined. An 874-base RNA of HIV-1 was synthesized and used as a template in RT assays with varying concentrations of intact p7, mutants of p7 that have transposed or repeated zinc fingers, and several different peptides that represent various structural regions of p7. Results indicate that at greater than or equal to 50% saturation of p7-binding sites, with p7, there is up to a 90% reduction in total cDNA synthesis, as measured by nucleotide incorporation. However, the cDNA products that are made are almost exclusively full length. Three zinc finger mutants exhibited effects similar to those of wild-type p7. N-terminal and C-terminal halves of p7 inhibited total nucleotide incorporation, but also inhibited synthesis of long cDNA products by RT. In the absence of p7 an array of short transcripts (< 200 bases) was produced by RT. These studies show that full-length p7 is necessary to increase the proportion of long cDNA transcripts produced by RT. The relative position of the two zinc fingers is not critical for this effect.
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- "Based on these in vitro experiments, it is clear that a major function of NC is as a nucleic acid chaperone; that is, NC assists nucleic acids in obtaining the most thermodynamically stable annealed structures (Levin et al., 2005; Rein et al., 1998). During HIV-1 infections, NC melts nucleic acid secondary structures that would otherwise result in pausing or premature termination of reverse transcription, which has been observed in numerous in vitro studies (Drummond et al., 1997; Ji et al., 1996; Klasens et al., 1999; Wu et al., 1996). The two zinc-fingers have been shown to be important for this chaperone activity, as mutations eliminating either finger abrogate this function (Levin et al., Virology 353 (2006) 41 – 51 www.elsevier.com/locate/yviro "
ABSTRACT: The nucleocapsid (NC) protein from HIV-1 contains two zinc-fingers, both of which are necessary for virus replication. This is the first in-depth study that presents the effects of nucleocapsid zinc-finger substitutions on the kinetics of reverse transcription and integration. Over a 72-h time-course of infection, the quantities of viral DNA (vDNA) observed with viruses containing either the nucleocapsid His23Cys or His44Cys mutations were significantly lower than those observed in infections with virus containing wild-type NC. In addition, the kinetics of vDNA formation and loss were significantly different from wild-type. The kinetic profiles observed indicated reduced vDNA stability, as well as defects in reverse transcription and integration. Overall, the defect in integration was much more pronounced than the reverse transcription defects. This suggests that the principal reason for the replication defectiveness of these mutant viruses is impairment of integration, and thus demonstrates the critical importance of NC in HIV-1 infection.Virology 10/2006; 353(1):41-51. DOI:10.1016/j.virol.2006.05.014 · 3.28 Impact Factor
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ABSTRACT: The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H- mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H- RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.Journal of Biological Chemistry 04/1998; 273(16):9976-86. DOI:10.1074/jbc.273.16.9976 · 4.57 Impact Factor
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ABSTRACT: To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lys primer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(-)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (-)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.Journal of Virology 12/1998; 72(11):9353-8. · 4.65 Impact Factor