Generation of human TRIM5alpha mutants with high HIV-1 restriction activity

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.