Cyclosporine A-resistant human immunodeficiency virus type 1 mutants demonstrate that Gag encodes the functional target of cyclophilin A

Abstract

The cellular peptidyl-prolyl isomerase cyclophilin A is incorporated into human immunodeficiency virus type 1 virions via contacts with the proline-rich domain of the Gag polyprotein. Cyclosporine A and nonimmunosuppressive analogs bind with high affinity to cyclophilin A, compete with Gag for binding to cyclophilin A, and prevent incorporation of cyclophilin A into virions; in parallel with the disruption of cyclophilin A incorporation into virions, there is a linear reduction in the initiation of reverse transcription after infection of a T cell. Passage of human immunodeficiency virus type 1 in the presence of the drug selects one of two mutations, either of which alters the proline-rich domain of Gag and is sufficient to confer drug resistance on the cloned wild-type provirus. Neither mutation alters Gag's cyclophilin A-binding properties in vitro, and cyclophilin A incorporation into drug-resistant virions is effectively disrupted by cyclosporine A, indicating that the drugresistant mutants do not require virion-associated cyclophilin A to initiate infection. That Gag's functional dependence on cyclophilin A can be differentiated genetically from its ability to bind cyclophilin A is further demonstrated by the rescue of a mutation precluding cyclophilin A packaging by a mutation conferring cyclosporine A resistance.These experiments demonstrate that, in addition to its ability to package cyclophilin A into virions, gag encodes the functional target of cyclophilin A.

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Human Cells by Debio-025, a Novel Cyclophilin Binding Agent

“…We did not detect the previously described mutation A92E or G94D (1,12) in the sequences of the CypA binding loop from naturally resistant virus isolates. Mutants carrying these mutations had been selected from strain NL4-3 when it infected CD4 ϩ HeLa cells and was passaged in the presence of NIM811.…”

“…So far, all CypA variants with reduced catalytic activities have a concomitant reduction in substrate affinity (26); thus, the isomerase activity cannot be separated from the binding function. The binding of CypA to the HIV-1 CA protein as well as PPIase activity are known to be inhibited by Cs (12,48) and the nonimmunosuppressive cyclosporine NIM811 (10,87).…”

Productive Replication and Evolution of HIV-1 in Ferret Cells

“…A92T has not been previously reported, but a charge-adding mutation at this residue, A92E, is known to arise when HIV-1 is passaged in HeLa cells in the presence of CsA, thus conferring CsA resistance and in some cell lines actually conferring CsA dependence on the virus (1,12); this phenomenon was later shown to reflect high levels of CypA in HeLa cells (70). Our experiments make it clear that CypA is present at substantial levels in the Mus- telidae cell lines that we used ( Fig.…”

Host factors involved in resistance to retroviral infection

“…3). Disruption of CypA incorporation c 2008 The Societies and Blackwell Publishing Asia Pty Ltd into the virion by CsA administration or by Gag mutations resulted in reduction in infectivity of the produced viruses (85,89,91,(95)(96)(97)(98). Several reports have shown that both CA dimerization and CypA multimerization are required for the efficient CA-CypA binding which is critical for HIV-1 infectivity (99,100).…”