Document Detail

Aromatic residue mutations reveal direct correlation between HIV-1 nucleocapsid protein's nucleic acid chaperone activity and retroviral replication.
MedLine Citation:
PMID:  22814429     Owner:  NLM     Status:  MEDLINE    
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.
Hao Wu; Mithun Mitra; Micah J McCauley; James A Thomas; Ioulia Rouzina; Karin Musier-Forsyth; Mark C Williams; Robert J Gorelick
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-07-16
Journal Detail:
Title:  Virus research     Volume:  171     ISSN:  1872-7492     ISO Abbreviation:  Virus Res.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-18     Completed Date:  2013-07-29     Revised Date:  2014-02-04    
Medline Journal Info:
Nlm Unique ID:  8410979     Medline TA:  Virus Res     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  263-77     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier B.V. All rights reserved.
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MeSH Terms
Amino Acid Motifs
Base Sequence
DNA, Viral / chemistry,  genetics
HIV Infections / virology*
HIV-1 / chemistry,  genetics*,  physiology
Molecular Chaperones / chemistry*,  genetics,  metabolism*
Molecular Sequence Data
Nucleic Acid Conformation
Recombination, Genetic
Reverse Transcription
Virus Replication*
gag Gene Products, Human Immunodeficiency Virus / chemistry*,  genetics,  metabolism*
Grant Support
Reg. No./Substance:
0/DNA, Viral; 0/Molecular Chaperones; 0/NCP7 protein, Human immunodeficiency virus 1; 0/gag Gene Products, Human Immunodeficiency Virus

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