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Updated: Jun 14, 2026
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Published on: August 31, 2014
Generation of human TRIM5alpha mutants with high HIV-1 restriction activity
Q T Pham1, A Bouchard, M G Grütter
1Laboratory of Retrovirology, University of Québec, Trois-Rivières, Canada.
Abstract:
Rhesus macaque tripartite motif (TRIM)5alpha potently inhibits early stages of human immunodeficiency virus (HIV)-1 replication, while the human orthologue has little effect on this virus. We used PCR-based random mutagenesis to construct a large library of human TRIM5alpha variants containing mutations in the PRYSPRY domain. We then applied a functional screen to isolate human cells made resistant to HIV-1 infection by the expression of a mutated TRIM5alpha. This protocol led to the characterization of a human TRIM5alpha variant containing a mutation at arginine 335 as conferring resistance to HIV-1 infection. The level of protection stemming from expression of this mutant was comparable to that of previously described mutations at position 332. R332/R335 double mutants decreased permissiveness to HIV-1 and to other lentiviruses by 20- to 50-fold in TE671 fibroblasts and in the T-cell line SUP-T1, and prevented HIV-1 spreading infection as efficiently as the rhesus macaque TRIM5alpha orthologue did. The finding that only two substitutions in human TRIM5alpha can confer resistance to HIV-1 at levels as high as one of the most potent natural orthologues of TRIM5alpha removes a roadblock toward the use of this restriction factor in human gene therapy applications.
Insights
Researchers engineered human TRIM5alpha to resist HIV-1 infection. Specific mutations provide potent antiviral activity, paving the way for gene therapy applications against human immunodeficiency virus.
Area of Science:
- Virology
- Immunology
- Genetics
Background:
- Rhesus TRIM5alpha potently inhibits human immunodeficiency virus (HIV)-1, but human TRIM5alpha has limited effect.
- Understanding TRIM5alpha's antiviral mechanisms is crucial for developing HIV therapies.
Purpose of the Study:
- To engineer human TRIM5alpha to confer resistance to HIV-1 infection.
- To identify specific mutations in human TRIM5alpha that enhance its antiviral activity.
Main Methods:
- Constructed a library of human TRIM5alpha variants using PCR-based random mutagenesis targeting the PRYSPRY domain.
- Applied a functional screen to isolate human cells expressing resistant TRIM5alpha variants.
- Characterized the antiviral activity of mutated TRIM5alpha against HIV-1 and other lentiviruses.
Main Results:
- Identified a human TRIM5alpha variant with an R335 mutation conferring resistance to HIV-1.
- Double mutants (R332/R335) demonstrated 20- to 50-fold decreased permissiveness to HIV-1 and other lentiviruses.
- Engineered human TRIM5alpha variants achieved resistance levels comparable to potent rhesus TRIM5alpha.
Conclusions:
- Two substitutions in human TRIM5alpha can confer potent resistance to HIV-1, similar to natural orthologues.
- This engineered TRIM5alpha shows promise for gene therapy applications to combat HIV infection.
