Second-site suppressors of HIV-1 capsid mutations: restoration of intracellular activities without correction of intrinsic capsid stability defects

Ruifeng Yang,Jinwoo Ahn,Jens Meiler,Jiong Shi,Jonathan H Sheehan,In-Ja L Byeon,Angela M Gronenborn,Christopher Aiken

doi:10.1186/1742-4690-9-30

Ruifeng Yang, Jinwoo Ahn + Show 6 more

Open Access

https://doi.org/10.1186/1742-4690-9-30

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Abstract

BackgroundDisassembly 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.ResultsTo 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 TRIM5α.ConclusionsThe 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.

Highlights

Of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection
Second-site suppressor mutations restore replication to the P38A and E45A mutants To probe the role of domain interactions in maintaining optimal Human immunodeficiency virus type 1 (HIV-1) capsid stability, second-site suppressor mutations were isolated by serial passage of P38A and E45A mutant viruses in human CEM T cells
To determine whether phenotypic reversion was associated with acquisition of compensatory HIV-1 mutations, DNA was purified from infected CEM cells harvested near the peak of growth

Summary

Introduction

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. The viral capsid protein (CA) assembles into a shell, referred to as the capsid, surrounding the viral genomic ribonucleoprotein (RNP) complex. HIV-1 CA consists of 231 residues that fold into two distinct domains connected by a flexible linker. Various surfaces of CA are involved in HIV-1 capsid formation. The existence of an NTD-CTD interface in the retroviral capsid was originally inferred from studies of the Rous sarcoma virus (RSV), in which two lethal MHR mutations in the CTD were rescued by compensatory mutations in the NTD [6]. In HIV-1, NTD-CTD contacts were detected by hydrogen-deuterium exchange [7] and chemical cross-linking [8], and in structures of CA hexamers and pentamers [9,10]

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