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Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications.

ABSTRACT: The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing activities required for strand transfer. Surprisingly, unlike NC, with increasing concentrations, Gag proteins drastically inhibited the DNA elongation step. This result is consistent with "nucleic acid-driven multimerization" of Gag and the reported slow dissociation of Gag from bound nucleic acid, which prevent reverse transcriptase from traversing the template ("roadblock" mechanism). Our findings illustrate one reason why NC (and not Gag) has evolved as a critical cofactor in reverse transcription, a paradigm that might also extend to other retrovirus systems.

SUBMITTER: Wu T

PROVIDER: S-EPMC2963451 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications.

Wu Tiyun T Datta Siddhartha A K SA Mitra Mithun M Gorelick Robert J RJ Rein Alan A Levin Judith G JG

Virology 20100723 2

The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing act ...[more]

PMID: 20655566

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Project description:BackgroundThe nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein.ResultsWe have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity.ConclusionOur data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may influence the efficiency of reverse transcription and other key steps of the HIV-2 replication cycle.

Project description:The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is an essential protein for retroviral replication. Among its numerous functions, NC is a nucleic acid (NA) chaperone protein that catalyzes NA rearrangements leading to the formation of thermodynamically more stable conformations. In vitro, NC chaperone activity is typically assayed under conditions of low or no Mg(2+), even though reverse transcription requires the presence of divalent cations. Here, the chaperone activity of HIV-1 NC was studied as a function of varying Na(+) and Mg(2+) concentrations by investigating the annealing of complementary DNA and RNA hairpins derived from the trans-activation response domain of the HIV genome. This reaction mimics the annealing step of the minus-strand transfer process in reverse transcription. Gel-shift annealing and sedimentation assays were used to monitor the annealing kinetics and aggregation activity of NC, respectively. In the absence of protein, a limited ability of Na(+) and Mg(2+) cations to facilitate hairpin annealing was observed, whereas NC stimulated the annealing 10(3)- to 10(5)-fold. The major effect of either NC or the cations is on the rate of bimolecular association of the hairpins. This effect is especially strong under conditions wherein NC induces NA aggregation. Titration with NC and NC/Mg(2+) competition studies showed that the annealing kinetics depends only on the level of NA saturation with NC. NC competes with Mg(2+) or Na(+) for sequence-nonspecific NA binding similar to a simple trivalent cation. Upon saturation, NC induces attraction between NA molecules corresponding to approximately 0.3 kcal/mol/nucleotide, in agreement with an electrostatic mechanism of NC-induced NA aggregation. These data provide insights into the variable effects of NC's chaperone activity observed during in vitro studies of divalent metal-dependent reverse transcription reactions and suggest the feasibility of NC-facilitated proviral DNA synthesis within the mature capsid core.

Project description:The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein plays an essential role in several stages of HIV-1 replication. One important function of HIV-1 NC is to act as a nucleic acid chaperone, in which the protein facilitates nucleic acid rearrangements important for reverse transcription and recombination. NC contains only 55 amino acids, with 15 basic residues and two zinc fingers, each having a single aromatic residue (Phe16 and Trp37). Despite its simple structure, HIV-1 NC appears to have optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid nucleic acid annealing. Here we combine single molecule DNA stretching experiments with ensemble solution studies of protein-nucleic acid binding affinity, oligonucleotide annealing, and nucleic acid aggregation to measure the characteristics of wild-type (WT) and aromatic residue mutants of HIV-1 NC that are important for nucleic acid chaperone activity. These in vitro results are compared to in vivo HIV-1 replication for viruses containing the same mutations. This work allows us to directly relate HIV-1 NC structure with its function as a nucleic acid chaperone in vitro and in vivo. We show that replacement of either aromatic residue with another aromatic residue results in a protein that strongly resembles WT NC. In contrast, single amino acid substitutions of either Phe16Ala or Trp37Ala significantly slow down NC's DNA interaction kinetics, while retaining some helix-destabilization capability. A double Phe16Ala/Trp37Ala substitution further reduces the latter activity. Surprisingly, the ensemble nucleic acid binding, annealing, and aggregation properties are not significantly altered for any mutant except the double aromatic substitution with Ala. Thus, elimination of a single aromatic residue from either zinc finger strongly reduces NC's chaperone activity as determined by single molecule DNA stretching experiments without significantly altering its ensemble-averaged biochemical properties. Importantly, the substitution of aromatic residues with Ala progressively decreases NC's nucleic acid chaperone activity while also progressively inhibiting viral replication. Taken together, these data support the critical role of HIV-1 NC's aromatic residues, and establish a direct and statistically significant correlation between nucleic acid chaperone activity and viral replication.

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Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications.

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Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications.

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