Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

Khan, Raza; Giedroc, D.P.

doi:10.1016/s0021-9258(19)50481-1

Cited by 149 publications

(45 citation statements)

References 47 publications

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“…Thus, in contrast to polyeA data (Delahunty et al, 1992), there is no direct evidence for the involvement of the amino acid immediately following the His zinc-binding residue since the hydrophobic Ile 24 residue of their peptide is replaced by nonhydrophobic Thr 24 and Asn 45 residues in our peptides. Moreover, as the same nonelectrostatic contribution was found for the interaction of the whole NCp7 with poly A (Khan and Giedroc, 1992), we speculate that the nonelectrostatic interaction of NCp7 with nucleotides is due primarily to the stacking of one or both aromatic residues with the bases. The intercalation of Trp within the bases also modifies the tRNAPhe conformation, as can be observed from the strong negative band at 245 nm in the difference absorption spectra (Fig.…”

Section: Fm In the Absencesupporting

confidence: 74%

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study

Mély¹,

Piémont²,

Sorinas-Jimeno³

et al. 1993

Biophysical Journal

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The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.

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Section: Fm In the Absencesupporting

confidence: 74%

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study

Mély¹,

Piémont²,

Sorinas-Jimeno³

et al. 1993

Biophysical Journal

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“…Previous in vitro experiments have indicated that mature NC protein may contribute to a variety of functions, including initiation of reverse transcription, strand displacement during the final stage of DNA synthesis, improved efficiency of integrase, and protection of viral RNA from nuclease activity (13,26,28,31,47). The results described here suggest an in vivo function in addition to RNA packaging for the HIV-1 NC protein.…”

mentioning

confidence: 50%

“…As part of the precursor Pr55 Gag , it is involved in particle assembly and RNA packaging (reference 41 and references therein). The processed NC p7 may be involved in early steps of the infection process (13,26,28,31,39,47). We reasoned that the basic nature of this protein might be critical for its functions and have systematically substituted the basic amino acid residues of the NC domain in an HIV-1 viral clone by using alanine scanning mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

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Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity

Poon

Aldovini

1996

J Virol

136

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Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.

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“…The finding that N-terminal mutations can impair both proviral DNA synthesis and its integrity raises a number of questions. NCp7 was shown to promote melting of RNA secondary structures (22), and this property is probably linked to its ability to activate reverse transcription in vitro, by reducing the pausing FIG. 8.…”

Section: Discussionmentioning

confidence: 99%

Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis

Berthoux

Péchoux

Ottmann

et al. 1997

J Virol

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Nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein containing two zinc fingers of the form (CX2CX4HX4C) and is present at about 2,000 copies inside the viral core. NCp7 molecules are tightly associated with the genomic RNA dimer to form the nucleocapsid, which also includes reverse transcriptase and integrase proteins. In vitro, NCp7 has been shown to bind specifically to HIV-1 RNA, inducing NCp7-NCp7 interactions. In the viral context, mutagenesis of amino acid residues in the zinc finger domains showed that NCp7 is responsible for the specific incorporation of genomic RNA into virions and is necessary for correct virion assembly and maturation. In this work, we investigated the consequences of mutating conserved basic residues in the N-terminal region that precedes the first zinc finger. Two of the mutants were poorly infectious and showed only limited, though significant, defects in RNA encapsidation and viral protein maturation. Electron microscopy, together with sucrose gradient analysis, revealed defects in particle core structure and heterogeneity among mutant virions. These defects were associated with strong reduction of proviral DNA synthesis and stability in newly infected cells. Taken together, these data show multiple and probably interdependent implications for the NCp7 protein in both early and late phases of the HIV-1 replicative cycle and emphasize it as a target for antiviral drug development.

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Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

Cited by 149 publications

References 47 publications

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity

Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis

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