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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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Inhibition of wild type, E45A, and E45A/R132T mutant HIV-1 by PF74. Viruses were added to HeLa-P4 target cells at predetermined dilutions in the presence of the indicated concentrations of PF74. Infected cells were identified two days later by X-gal staining and quantified. Results were expressed as the extent of infection normalized by the values obtained with the no-drug controls.
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Figure 8: Inhibition of wild type, E45A, and E45A/R132T mutant HIV-1 by PF74. Viruses were added to HeLa-P4 target cells at predetermined dilutions in the presence of the indicated concentrations of PF74. Infected cells were identified two days later by X-gal staining and quantified. Results were expressed as the extent of infection normalized by the values obtained with the no-drug controls.

Mentions: In a recent study, we showed that the small molecule HIV-1 inhibitor PF74 binds and destabilizes the viral capsid, leading to premature HIV-1 uncoating in target cells [33]. The E45A substitution rendered HIV-1 resistant to PF74, presumably by stabilizing the capsid, as the mutation did not reduce PF74 binding to mature HIV-1 particles. To further probe the effects of the suppressor mutation R132T on the phenotype of the E45A mutant in target cells, we compared the sensitivity of wild type, E45A, and E45A/R132T mutant viruses to inhibition by PF74 in single-cycle infection assays. The compound was added at various doses to the cells at the time of virus inoculation, and the number of infected cells quantified after two days of culture. As previously shown, the E45A mutant virus exhibited strong resistance to PF74 (Figure 8). By contrast, the E45A/R132T mutant exhibited nearly wild type sensitivity (IC50 = 0.38 μM vs. 0.26 μM for the wild type virus). The restored sensitivity of the double mutant virus to PF74 suggests that the R132T mutation partially reverses the E45A-induced uncoating defect in target cells.


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)

Inhibition of wild type, E45A, and E45A/R132T mutant HIV-1 by PF74. Viruses were added to HeLa-P4 target cells at predetermined dilutions in the presence of the indicated concentrations of PF74. Infected cells were identified two days later by X-gal staining and quantified. Results were expressed as the extent of infection normalized by the values obtained with the no-drug controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Inhibition of wild type, E45A, and E45A/R132T mutant HIV-1 by PF74. Viruses were added to HeLa-P4 target cells at predetermined dilutions in the presence of the indicated concentrations of PF74. Infected cells were identified two days later by X-gal staining and quantified. Results were expressed as the extent of infection normalized by the values obtained with the no-drug controls.
Mentions: In a recent study, we showed that the small molecule HIV-1 inhibitor PF74 binds and destabilizes the viral capsid, leading to premature HIV-1 uncoating in target cells [33]. The E45A substitution rendered HIV-1 resistant to PF74, presumably by stabilizing the capsid, as the mutation did not reduce PF74 binding to mature HIV-1 particles. To further probe the effects of the suppressor mutation R132T on the phenotype of the E45A mutant in target cells, we compared the sensitivity of wild type, E45A, and E45A/R132T mutant viruses to inhibition by PF74 in single-cycle infection assays. The compound was added at various doses to the cells at the time of virus inoculation, and the number of infected cells quantified after two days of culture. As previously shown, the E45A mutant virus exhibited strong resistance to PF74 (Figure 8). By contrast, the E45A/R132T mutant exhibited nearly wild type sensitivity (IC50 = 0.38 μM vs. 0.26 μM for the wild type virus). The restored sensitivity of the double mutant virus to PF74 suggests that the R132T mutation partially reverses the E45A-induced uncoating defect in target cells.

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