Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A

Wang Peng,K M Rifat Faysal,Jiong Shi,Christopher Aiken,Chang H Byeon,James Walsh,Till Böcking,Chantal L Márquez,Derrick Lau,In-Ja L Byeon

doi:10.1186/s12977-019-0471-4

Abstract

BackgroundEfficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016).ResultsWe performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1.ConclusionsThe amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

Highlights

Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA)
The peptidylprolyl isomerase cyclophilin A (CypA) is an abundant cytosolic protein that binds via its active site to the CypA binding loop located on the N-terminal domain of CA (CA-NTD)
CypA enhances HIV-1 infection in Jurkat cells [30, 36] and we found that infection of Jurkat cells with an HIV-1 reporter virus expressing GFP was about twice that in the absence of cyclosporin A (CsA) vs. in the presence of CsA (Fig. 6a)

Summary

Introduction

Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. The interaction of CypA molecules with multiple of these loops exposed on the outside of the capsid in the cytoplasm of the target cell modulates HIV infection in a cell type-specific manner [16, 17], possibly by regulating capsid stability and uncoating [18, 19]. Inhibition of this interaction in myeloid cells, either by using the competitive inhibitor cyclosporin A (CsA) or by mutation of residues in the CypA binding loop on CA, results in HIV-1 induction of host cell immune responses, which has been attributed to premature uncoating and release of viral DNA in the cytoplasm [20, 21]. The molecular mechanisms underlying modulation of infection are not fully understood but may involve dynamic allostery [22]

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