Analysis of Human Cell Heterokaryons Demonstrates that Target Cell Restriction of Cyclosporine-Resistant Human Immunodeficiency Virus Type 1 Mutants Is Genetically Dominant

Abstract

The host cell protein cyclophilin A (CypA) binds to CA of human immunodeficiency virus type 1 (HIV-1) and promotes HIV-1 infection of target cells. Disruption of the CypA-CA interaction, either by mutation of the CA residue at G89 or P90 or with the immunosuppressive drug cyclosporine (CsA), reduces HIV-1 infection. Two CA mutants, A92E and G94D, previously were identified by selection for growth of wild-type HIV-1 in cultures of CD4؉ HeLa cell cultures containing CsA. Interestingly, infection of some cell lines by these mutants is enhanced in the presence of CsA, while in other cell lines these mutants are minimally affected by the drug. Little is known about this cell-dependent phenotype of the A92E and G94D mutants, except that it is not dependent on expression of the host factor TRIM5␣. Here, we show that infection by the A92E and G94D mutants is restricted at an early postentry stage of the HIV-1 life cycle. Analysis of heterokaryons between CsA-dependent HeLa-P4 cells and CsA-independent 293T cells indicated that the CsA-dependent infection by A92E and G94D mutants is due to a dominant cellular restriction. We also show that addition of CsA to target cells inhibits infection by wild-type HIV-1 prior to reverse transcription. Collectively, these results support the existence of a cell-specific human cellular factor capable of restricting HIV-1 at an early postentry step by a CypA-dependent mechanism.

Dynamic allostery governs cyclophilin A–HIV capsid interplay

“…In humans, the HIV-1 infectivity is enhanced by the interaction of CypA with the capsid (29,30). On the contrary, in other primates, CypA interferes with the viral infection by enhancing the activity of the restriction factors TRIM5α and TRIM-Cyp (31,32).…”

Cyclophilin A-Dependent Restriction of Human Immunodeficiency Virus Type 1 Capsid Mutants for Infection of Nondividing Cells

“…Early reverse transcription products (minus-strand strong-stop DNA) were amplified with the primers ERT-SS-F (5Ј-GCTAACTAGGGAACCCACTG CTT-3Ј) and ERT-SS-R (5Ј-ACAACAGACGGGCACACACTAC-3Ј) and were detected with the probe ERT-SS (5Ј-6-carboxyfluorescein-AGCCTCAATA AAGCTTGCCTTGAGTGCTTC-6-carboxytetramethylrhodamine-3Ј). Late reverse transcription products (U5-Gag) were amplified with primers MH531 (5Ј-TG TGTGCCCGTCTGTTGTGT-3Ј) and MH532 (5Ј-GAGTCCTGCGTCGAGAGA GC-3Ј) and were detected with the probe LTR-P (5Ј-6-carboxyfluorescein-CAGTGGCGCCCGAACAGGGA-6-carboxytetramethylrhodamine-3Ј) as previously described (12,43).…”

“…Infections were performed in triplicate, and only values within the linear range of the infection assay (up to 1,000 blue cells per well) were used to calculate infectivity. Infections with HIV-GFP reporter viruses were analyzed by flow cytometric analysis of GFP expression as previously described (43).…”

“…Additional CA mutations conferring a CsA-resistant/dependent phenotype have been identified outside the CypA binding loop, including T54A and R132K (40,54). The mechanism by which these substitutions render HIV-1 dependent on CsA is unclear, but heterokaryons generated between permissive and nonpermissive cells exhibit the nonpermissive cell phenotype, suggestive of a dominant restriction that depends on binding of CypA to the viral capsid (43).…”

Tissue-specific restriction of cyclophilin A-independent HIV-1- and SIV-derived lentiviral vectors

“…Emerging reports also postulate the existence of another restriction factor(s) in human cells that is different from TRIM5a Hu , but that is modulated by CypA. 7,8,15,22,27,28 In certain human epithelial and hematopoietic cell lines, transduction with HIV-1 capsid mutants (such as A 92 E) is inhibited. Careful titration studies have shown that this restriction is not saturable and that infectivity can be rescued by CypA.…”