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

Yang R, Shi J, Byeon IJ, Ahn J, Sheehan JH, Meiler J, Gronenborn AM, Aiken C - Retrovirology (2012)

Bottom Line: Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT

Background: 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.

Results: 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 TRIM5α.

Conclusions: 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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The second-site mutation T216I restores the ability of P38A particles to saturate TRIM5 restrictions in simian cells. Cultures of OMK (A) and FRhK-4 (B) cells were inoculated with the indicated quantities of VSV-G-pseudotyped CA mutant HIV-1 particles and a fixed subsaturating quantity of GFP-encoding pseudotyped HIV-1 particles. Results shown are representative of two independent experiments.
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Figure 9: The second-site mutation T216I restores the ability of P38A particles to saturate TRIM5 restrictions in simian cells. Cultures of OMK (A) and FRhK-4 (B) cells were inoculated with the indicated quantities of VSV-G-pseudotyped CA mutant HIV-1 particles and a fixed subsaturating quantity of GFP-encoding pseudotyped HIV-1 particles. Results shown are representative of two independent experiments.

Mentions: In previous studies, we observed that the P38A mutation impairs the ability of HIV-1 particles to abrogate host restriction in simian cells owing to capsid instability. To test whether the second-site suppressor restores capsid stability in target cells, we asked whether T216I restores the ability of P38A particles to abrogate restriction. Owl monkey (OMK) cells, which express TRIMCyp, were inoculated with a fixed quantity of HIV-GFP reporter virions with various quantities of wild type or mutant HIV-1 particles (non-GFP-encoding). Titration of the wild-type particles led to a marked enhancement of infection by the reporter virus (Figure 9A). By contrast, P38A mutant particles were approximately 80% less effective at abrogating restriction, as we previously reported [40]. For P38A/T216I mutant particles, the activity was intermediate between wild type and P38A levels. In rhesus macaque cells, in which HIV-1 infection is restricted by TRIM5α, the impaired abrogation activity of P38A mutant particles was also rescued by the T216I suppressor mutation (Figure 9B). In both cell lines, the T216I single mutant exhibited a similar level of activity as the wild type, demonstrating that the rescue of the P38A mutant activity was not due to a general enhancement of activity by the suppressor mutation. Collectively, these results suggest that the T216I mutation prevents premature disassembly of the P38A mutant core in target cells, thereby relieving the impaired ability of the mutant core to interact with restriction factors.


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

Yang R, Shi J, Byeon IJ, Ahn J, Sheehan JH, Meiler J, Gronenborn AM, Aiken C - Retrovirology (2012)

The second-site mutation T216I restores the ability of P38A particles to saturate TRIM5 restrictions in simian cells. Cultures of OMK (A) and FRhK-4 (B) cells were inoculated with the indicated quantities of VSV-G-pseudotyped CA mutant HIV-1 particles and a fixed subsaturating quantity of GFP-encoding pseudotyped HIV-1 particles. Results shown are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351742&req=5

Figure 9: The second-site mutation T216I restores the ability of P38A particles to saturate TRIM5 restrictions in simian cells. Cultures of OMK (A) and FRhK-4 (B) cells were inoculated with the indicated quantities of VSV-G-pseudotyped CA mutant HIV-1 particles and a fixed subsaturating quantity of GFP-encoding pseudotyped HIV-1 particles. Results shown are representative of two independent experiments.
Mentions: In previous studies, we observed that the P38A mutation impairs the ability of HIV-1 particles to abrogate host restriction in simian cells owing to capsid instability. To test whether the second-site suppressor restores capsid stability in target cells, we asked whether T216I restores the ability of P38A particles to abrogate restriction. Owl monkey (OMK) cells, which express TRIMCyp, were inoculated with a fixed quantity of HIV-GFP reporter virions with various quantities of wild type or mutant HIV-1 particles (non-GFP-encoding). Titration of the wild-type particles led to a marked enhancement of infection by the reporter virus (Figure 9A). By contrast, P38A mutant particles were approximately 80% less effective at abrogating restriction, as we previously reported [40]. For P38A/T216I mutant particles, the activity was intermediate between wild type and P38A levels. In rhesus macaque cells, in which HIV-1 infection is restricted by TRIM5α, the impaired abrogation activity of P38A mutant particles was also rescued by the T216I suppressor mutation (Figure 9B). In both cell lines, the T216I single mutant exhibited a similar level of activity as the wild type, demonstrating that the rescue of the P38A mutant activity was not due to a general enhancement of activity by the suppressor mutation. Collectively, these results suggest that the T216I mutation prevents premature disassembly of the P38A mutant core in target cells, thereby relieving the impaired ability of the mutant core to interact with restriction factors.

Bottom Line: Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT

Background: 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.

Results: 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 TRIM5α.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus