TY - JOUR
T1 - A novel aminoacid determinant of HIV-1 restriction in the TRIM5α variable 1 region isolated in a random mutagenic screen
AU - Pham, Quang Toan
AU - Veillette, Maxime
AU - Brandariz-Nuñez, Alberto
AU - Pawlica, Paulina
AU - Thibert-Lefebvre, Caroline
AU - Chandonnet, Nadia
AU - Diaz-Griffero, Felipe
AU - Berthoux, Lionel
N1 - Funding Information:
We thank Michel J. Tremblay and Réjean Cantin for sharing their BSL3 facility and the p24 ELISA assay. This work was supported by the Canadian Institutes for Health Research and the Canada Research Chairs program (L.B.). M.V. was supported by a FRQS Master's Training Award . Work in F.D.-G.’s laboratory was supported by NIH R01 AI087390 to F.D.-G, and a K99/R00 Pathway to Independence Award from the National Institutes of Health .
PY - 2013/5
Y1 - 2013/5
N2 - Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G-R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G-R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G-R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties.
AB - Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G-R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G-R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G-R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties.
KW - Genetic screen
KW - HIV-1
KW - Random mutagenesis
KW - Restriction
KW - TRIM5α
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U2 - 10.1016/j.virusres.2013.01.013
DO - 10.1016/j.virusres.2013.01.013
M3 - Article
C2 - 23357295
AN - SCOPUS:84876858334
SN - 0168-1702
VL - 173
SP - 306
EP - 314
JO - Virus Research
JF - Virus Research
IS - 2
ER -