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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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Second-site suppressor mutation R132T relieves the cell-cycle dependence of the E45A mutant in a single-cycle infectivity assay. Control and aphidicolin-arrested HeLa-P4 cells were inoculated with the indicated viruses. Infectivity was determined as described in the legend to Figure 2. Results shown are from one representative of three independent experiments.
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Figure 7: Second-site suppressor mutation R132T relieves the cell-cycle dependence of the E45A mutant in a single-cycle infectivity assay. Control and aphidicolin-arrested HeLa-P4 cells were inoculated with the indicated viruses. Infectivity was determined as described in the legend to Figure 2. Results shown are from one representative of three independent experiments.

Mentions: HIV-1 efficiently infects both dividing and nondividing cells, and the capacity to infect nondividing cells has been linked genetically to CA [32]. Several mutations in CA, including E45A, result in selective impairment of infection of mitotically-arrested cells [25]. We therefore asked whether introduction of the R132T mutation in the E45A mutant virus CA would relieve its cell-cycle-dependence in arrested HeLa cells. Infection by the E45A mutant was reduced by eight-fold in arrested vs. control cells, while the wild type and E45A/R132T double mutant were only slightly (less than 50%) reduced in arrested cells. Thus, the R132T second-site substitution rescued the impaired ability of E45A infection in nondividing cells. In contrast, the P38A and P38A/T216I mutants behaved similar to wild type HIV-1 and were only slightly less infectious for arrested vs. dividing cells (Figure 7). These results indicate that the R132T mutation corrects the selective impairment of infection of nondividing cells associated with the E45A mutant virus.


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)

Second-site suppressor mutation R132T relieves the cell-cycle dependence of the E45A mutant in a single-cycle infectivity assay. Control and aphidicolin-arrested HeLa-P4 cells were inoculated with the indicated viruses. Infectivity was determined as described in the legend to Figure 2. Results shown are from one representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Second-site suppressor mutation R132T relieves the cell-cycle dependence of the E45A mutant in a single-cycle infectivity assay. Control and aphidicolin-arrested HeLa-P4 cells were inoculated with the indicated viruses. Infectivity was determined as described in the legend to Figure 2. Results shown are from one representative of three independent experiments.
Mentions: HIV-1 efficiently infects both dividing and nondividing cells, and the capacity to infect nondividing cells has been linked genetically to CA [32]. Several mutations in CA, including E45A, result in selective impairment of infection of mitotically-arrested cells [25]. We therefore asked whether introduction of the R132T mutation in the E45A mutant virus CA would relieve its cell-cycle-dependence in arrested HeLa cells. Infection by the E45A mutant was reduced by eight-fold in arrested vs. control cells, while the wild type and E45A/R132T double mutant were only slightly (less than 50%) reduced in arrested cells. Thus, the R132T second-site substitution rescued the impaired ability of E45A infection in nondividing cells. In contrast, the P38A and P38A/T216I mutants behaved similar to wild type HIV-1 and were only slightly less infectious for arrested vs. dividing cells (Figure 7). These results indicate that the R132T mutation corrects the selective impairment of infection of nondividing cells associated with the E45A mutant virus.

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