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
ABSTRACT 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.
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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
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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.Virology 09/2008; 378(2):385-96. DOI:10.1016/j.virol.2008.06.002 · 3.28 Impact Factor
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ABSTRACT: The nucleocapsid protein (NC) plays an important role in retroviral replication, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) NC has been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of the activity of this highly basic protein (pI ~10). The chaperone activity of other NC proteins is not well understood. We used single molecule DNA stretching to characterize the activity of HIV-1, RSV, and MMLV NC. We found distinct differences in the chaperone activities of each protein, which reflect the requirements for nucleic acid chaperone activity in each retroviral replication system. HTLV-1 NC exhibited overall poor nucleic acid chaperone acitivity. This result is explained by its poor aggregating activity and slow dissociation from single-stranded DNA. This NC protein is overall neutral at pH=7.5 and possesses a unique, acidic C-terminal domain. By studying different HTLV-1 NC mutants, the role of C-terminal domains to the chaperone activity was elucidated. The results suggest that the electrostatic interaction between HTLV-1 NC and nucleic acids is the major factor determining the kinetics. We also examine the nucleic acid interaction properties of the Apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like 3G (APOBEC3G/A3G) that is known to inhibit HIV-1 reverse transcription in absence of viral infectivity factor (Vif). Our stretching experiments suggested a novel mechanism for deaminase-independent inhibition of reverse transcription due to vital differences of nucleic acid binding kinetics between NC, A3G and reverse transcriptase (RT). Finally, Long interspersed nucleic elements (LINE) are highly repeated nucleic acids sequences in mammal genomes. Our single DNA molecule stretching experiments characterized the nucleic acid chaperone function of ORF1p in the mouse LINE-1 retrotransposon. We found that a single amino acid substitution altered retrotransposition efficiency by a factor of 15 due to a reduction in nucleic acid chaperone activity exhibited by ORF1p. For all of the studies presented here, we used single molecule methods to characterize the nucleic acid interactions of proteins involved in reverse transcription in retroviruses or retrotransposons. In each case, complementary bulk experiments were done by collaborators. The results are presented together in each chapter of the thesis.