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C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

ABSTRACT: Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from single-stranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability.

SUBMITTER: Qualley DF

PROVIDER: S-EPMC2804176 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

Qualley Dominic F DF Stewart-Maynard Kristen M KM Wang Fei F Mitra Mithun M Gorelick Robert J RJ Rouzina Ioulia I Williams Mark C MC Musier-Forsyth Karin K

The Journal of biological chemistry 20091103 1

Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral ...[more]

PMID: 19887455

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Project description:Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic residue switches the binding mode of FIV NC from primarily electrostatic binding to more non-electrostatic binding, conferring upon it NA interaction properties comparable to that of HIV-1 NC.

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.

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.

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C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

Publications

C-terminal domain modulates the nucleic acid chaperone activity of human T-cell leukemia virus type 1 nucleocapsid protein via an electrostatic mechanism.

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