Structure/Function Mapping of Amino Acids in the N- Terminal Zinc Finger of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein: Residues Responsible for Nucleic Acid Helix Destabilizing Activity †
The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild-type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W, which had substantially greater helix destabilizing activity than that of the wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a dominant negative effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.
"The nucleic acid aggregating activity of NC resides primarily in the basic N-terminal domain (29,56–58), and the duplex destabilizing activity of NC has been mapped to its zinc finger structures (22,59–62). NC exhibits sequence-specific binding to single-stranded regions through interactions that involve the zinc fingers (51,53,63). "
[Show abstract][Hide abstract] ABSTRACT: Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA-DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop-loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop-loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open 'Y' conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the 'Y' conformation exhibiting at least 12 unpaired nucleotides in its lower part.
Nucleic Acids Research 06/2011; 39(18):8148-62. DOI:10.1093/nar/gkr526 · 9.11 Impact Factor
"More recently, it has been reported that the nucleocapsid protein of HIV-1 can destabilize DNA  via its DNA-bending activity . More specifically, the DNAdestabilization function involves the protein's first zinc finger , bearing residues Ile24 and Asn27 . Similarly, a DNA destabilization process was attributed to prion protein. "
[Show abstract][Hide abstract] ABSTRACT: DNA targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia, both in terms of drug brands and prescription volumes. Small DNA-interacting molecules share the ability of certain proteins to change the DNA helix's overall organization and geometrical orientation via tilt, roll, twist, slip, and flip effects. In this ocean of DNA-interacting compounds, most stabilize both DNA strands and very few display helix-destabilizing properties. These types of DNA-destabilizing effect are observed with certain mono- or bis-intercalators and DNA alkylating agents (some of which have been or are being developed as cancer drugs). The formation of locally destabilized DNA portions could interfere with protein/DNA recognition and potentially affect several crucial cellular processes, such as DNA repair, replication, and transcription. The present paper describes the molecular basis of DNA destabilization, the cellular impact on protein recognition, and DNA repair processes and the latter's relationships with antitumour efficacy.
Journal of nucleic acids 07/2010; 2010. DOI:10.4061/2010/290935
"The lack of potent inhibition by NC with a DNA primer could then be explained by RT's higher affinity for the DNA 3′ end in contrast to low affinity for RNA 3′ recessed termini (see Results). We also tested other NC point mutants known to bind with different affinities to nucleic acid including I24Q, N27D, N17K, and F16W (Narayanan et al., 2006). In general, the level of RNA primer inhibition with these mutants was also consistent with binding affinity for the substrate (data not shown). "
[Show abstract][Hide abstract] ABSTRACT: The current study indicates a new role for HIV nucleocapsid protein (NC) in modulating the specificity of plus strand priming. RNase H cleavage by reverse transcriptase (RT) during minus strand synthesis gives rise to RNA fragments that could potentially be used as primers for synthesis of the plus strand, leading to the initiation of priming from multiple points as has been observed for other retroviruses. For HIV, the central and 3' polypurine tracts (PPTs) are the major sites of plus strand initiation. Using reconstituted in vitro assays, results showed that NC greatly reduced the efficiency of extension of non-PPT RNA primers, but not PPT. Experiments mimicking HIV replication showed that RT generated and used both PPT and non-PPT RNAs to initiate "plus strand" synthesis, but non-PPT usage was strongly inhibited by NC. The results support a role for NC in specifying primer usage during plus strand synthesis.
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