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The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here, we investigate the effect of lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals., Competing Interests: KF, JW, NR, CM, VS, AT, MC, MP, ST, GT, LJ, DJ, TB No competing interests declared, (© 2024, Faysal, Walsh et al.)

The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection., Competing Interests: The authors have declared that no competing interests exist.

HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP 6 ) for viral production and primary cell replication. HIV-1 recruits IP 6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP 6 packaging and reduces viral production. Loss of IP 6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP 6 incorporation and infection remain impaired, consistent with an independent role for IP 6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP 6 availability limits the production of diverse lentiviruses, but in the absence of IP 6 , HIV-1 packages IP 5 without loss of infectivity. Together, these data suggest that IP 6 is a critical cofactor for HIV-1 replication., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: 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. 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)., Results: We 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., Conclusions: The 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.