Analysis of NCp7-dependent activation of HIV-1 cDNA integration and its conservation among retroviral nucleocapsid proteins.
ABSTRACT HIV-1 nucleocapsid protein NCp7 is a small basic protein with two zinc fingers, found in the virion core where several hundred molecules coat the genomic RNA. NCp7 has nucleic acid chaperone properties that guide reverse transcriptase (RT) to synthesize the proviral DNA flanked by the long terminal repeats (LTR). In vitro, NCp7 can strongly activate magnesium-dependent LTR-DNA strand transfer by integrase (IN). Here we show that IN activation relies on both the basic residues and the zinc fingers of NCp7. NCp7 lacking the zinc fingers binds DNA but moderately stimulates strand transfer by IN. The NCp7 zinc-finger domain binds DNA poorly and does not efficiently stimulate IN activity. However, the NC zinc-finger domain can complement DNA binding to restore full activation of strand transfer by IN. We propose that the basic residues and the zinc fingers function together to stabilize IN at the LTR ends and promote the formation of a nucleoprotein complex competent for integration. We also show that these properties of HIV-1 NCp7 are remarkably conserved among nucleocapsid proteins of retrotransposon and retrovirus origins.
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ABSTRACT: The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.Journal of Molecular Biology 02/1994; 235(1):287-301. · 3.91 Impact Factor
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ABSTRACT: Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively.In vitro binding of HIV-1 NCp7 to viral 5′ RNA and primer tRNA3 Lys catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNAi Met to a 5′-3′ bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5′ PBS (1). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5′-3′ PBS, primer tRNAi Met, NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.Journal of Biological Chemistry 12/1999; 274(51):36643-36648. · 4.65 Impact Factor
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ABSTRACT: Retroviral nucleocapsid (NC) protein, in addition to its structural roles in the virion core, is involved in the early and late phases of the viral replication cycle. To further characterise the role of NC protein of MoMuLV (NCp10) in the replication of the viral genome, the influence of NCp10 on self-primed versus primer-specific reverse transcription has been analysed in vitro. The results show that NCp10 can enhance the specificity of proviral DNA synthesis by inhibiting self-primed cDNA synthesis while promoting primer-specific DNA synthesis within active NCp10-RNA nucleoprotein complexes. Retroviruses are known to show a high degree of variability and this prompted us to examine the possible implication of NCp10 in the genetic variability of MoMuLV. The ability of reverse transcriptase (RT) to extend different mutated primers using an RNA or a DNA template has been investigated in the presence or in the absence of NCp10. NCp10 was found to have different effects on RT depending on the nature of the template: an enhancement at the elongation level of mutated primers using RNA as template versus a slight inhibition using DNA as template. These observations suggest that NCp10 could be implicated in the genetic variability of MoMuLV by allowing nucleotide misincorporation principally during minus strand DNA synthesis.Journal of Molecular Biology 08/1998; 280(2):215-25. · 3.91 Impact Factor