Generation of human TRIM5α mutants with high HIV-1 restriction activity

• Published in: Gene therapy Vol. 17; no. 7; pp. 859 - 871
• Main Authors: Pham, Q T, Bouchard, A, Grütter, M G, Berthoux, L
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-07-2010; Nature Publishing Group
• Subjects: 631/1647/666/2261; 631/208/737; 631/250/255/1901; 631/61/201; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Biotechnology; Care and treatment; Cell Biology; Fibroblasts; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene Therapy; Genetic aspects; Health aspects; Health. Pharmaceutical industry; HIV; HIV infection; Human Genetics; Human immunodeficiency virus; Industrial applications and implications. Economical aspects; Infections; Lymphocytes T; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Mutants; Mutation; Nanotechnology; original-article; Random mutagenesis; Replication; Viral proteins; Canada; Switzerland; HIV-1; restriction factor; TRIM5α; retroviral replication; random mutagenesis; lentivirus; Human; Retroviridae; Lentivirus; Virus; Mutagenesis; TRlM5α; Replication; Mutation; Human immunodeficiency virus; Gene therapy
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/gt.2010.40

Rhesus macaque tripartite motif (TRIM)5α 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 TRIM5α 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 TRIM5α. This protocol led to the characterization of a human TRIM5α 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 TRIM5α orthologue did. The finding that only two substitutions in human TRIM5α can confer resistance to HIV-1 at levels as high as one of the most potent natural orthologues of TRIM5α removes a roadblock toward the use of this restriction factor in human gene therapy applications.