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Second site reversion of a mutation near the amino terminus of the HIV-1 capsid protein.

ABSTRACT: During HIV-1 morphogenesis, the precursor Gag protein is processed to release capsid (CA) proteins that form the mature virus core. In this process, the CA proteins assemble a lattice in which N-terminal domain (NTD) helices 1-3 are critical for multimer formation. Mature core assembly requires refolding of the N-terminus of CA into a ?-hairpin, but the precise contribution of the hairpin core morphogenesis is unclear. We found that mutations at isoleucine 15 (I15), between the ?-hairpin and NTD helix 1 are incompatible with proper mature core assembly. However, a compensatory mutation of histidine 12 in the ?-hairpin to a tyrosine was selected by long term passage of an I15 mutant virus in T cells. The tyrosine does not interact directly with residue 15, but with NTD helix 3, supporting a model in which ?-hairpin folding serves to align helix 3 for mature NTD multimerization.

SUBMITTER: Lopez CS

PROVIDER: S-EPMC3868222 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Second site reversion of a mutation near the amino terminus of the HIV-1 capsid protein.

López Claudia S CS Tsagli Seyram M SM Sloan Rachel R Eccles Jacob J Barklis Eric E

Virology 20130921 1-2

During HIV-1 morphogenesis, the precursor Gag protein is processed to release capsid (CA) proteins that form the mature virus core. In this process, the CA proteins assemble a lattice in which N-terminal domain (NTD) helices 1-3 are critical for multimer formation. Mature core assembly requires refolding of the N-terminus of CA into a β-hairpin, but the precise contribution of the hairpin core morphogenesis is unclear. We found that mutations at isoleucine 15 (I15), between the β-hairpin and NTD ...[more]

PMID: 24210103

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Project description:The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

Project description:Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection.To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5?.The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

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Dataset Information

Second site reversion of a mutation near the amino terminus of the HIV-1 capsid protein.

Publications

Second site reversion of a mutation near the amino terminus of the HIV-1 capsid protein.

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