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Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein.

ABSTRACT: 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.

SUBMITTER: Wu H

PROVIDER: S-EPMC4252577 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein.

Wu Hao H Wang Wei W Naiyer Nada N Fichtenbaum Eric E Qualley Dominic F DF McCauley Micah J MJ Gorelick Robert J RJ Rouzina Ioulia I Musier-Forsyth Karin K Williams Mark C MC

Virus research 20140607

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-t ...[more]

PMID: 24915282

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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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Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein.

Publications

Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein.

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