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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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Rescue of P38A (A) and E45A (B) mutants by second-site mutations T216I and R132T, respectively. Replication of mutant and wild-type viruses was analyzed in CEM cells. Samples were collected on the days indicated and analyzed for reverse transcriptase (RT) activity. Shown are the average values obtained from duplicate parallel cultures.
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Figure 1: Rescue of P38A (A) and E45A (B) mutants by second-site mutations T216I and R132T, respectively. Replication of mutant and wild-type viruses was analyzed in CEM cells. Samples were collected on the days indicated and analyzed for reverse transcriptase (RT) activity. Shown are the average values obtained from duplicate parallel cultures.

Mentions: To probe the role of domain interactions in maintaining optimal HIV-1 capsid stability, second-site suppressor mutations were isolated by serial passage of P38A and E45A mutant viruses in human CEM T cells. Virus supernatants were harvested immediately following the peak of growth of the mutant virions and were inoculated into fresh cells and the cultures were maintained until the reemergence of HIV-1 in the cultures. 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. Sequence analysis of PCR products from the P38A culture revealed the presence of a mutation at codon 216 resulting in a substitution of Thr to Ile (T216I) in addition to the original P38A mutation. Sequence analysis of PCR products from the E45A culture revealed the presence of a mutation at codon 132 resulting in a substitution of Arg to Thr (R132T) in addition to the original E45A mutation. No other changes were observed in the PCR-amplified viral sequences. To determine whether these second-site substitutions were responsible for the accelerated growth kinetics of the adapted viruses, the mutations were transferred into the original P38A and E45A mutant viral clones. A replication assay was performed with the resulting double mutant viruses in CEM cells. In contrast to the original P38A and E45A mutants, which failed to replicate in CEM cells within 26 days, the P38A/T216I and E45A/R132T double mutants both replicated, though each was delayed relative to the wild type (Figure 1A, B). These results demonstrate that the respective second-site mutations, T216I and R132T, partially restore the ability of the corresponding P38A and E45A mutant viruses to replicate in CEM 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)

Rescue of P38A (A) and E45A (B) mutants by second-site mutations T216I and R132T, respectively. Replication of mutant and wild-type viruses was analyzed in CEM cells. Samples were collected on the days indicated and analyzed for reverse transcriptase (RT) activity. Shown are the average values obtained from duplicate parallel cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Rescue of P38A (A) and E45A (B) mutants by second-site mutations T216I and R132T, respectively. Replication of mutant and wild-type viruses was analyzed in CEM cells. Samples were collected on the days indicated and analyzed for reverse transcriptase (RT) activity. Shown are the average values obtained from duplicate parallel cultures.
Mentions: To probe the role of domain interactions in maintaining optimal HIV-1 capsid stability, second-site suppressor mutations were isolated by serial passage of P38A and E45A mutant viruses in human CEM T cells. Virus supernatants were harvested immediately following the peak of growth of the mutant virions and were inoculated into fresh cells and the cultures were maintained until the reemergence of HIV-1 in the cultures. 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. Sequence analysis of PCR products from the P38A culture revealed the presence of a mutation at codon 216 resulting in a substitution of Thr to Ile (T216I) in addition to the original P38A mutation. Sequence analysis of PCR products from the E45A culture revealed the presence of a mutation at codon 132 resulting in a substitution of Arg to Thr (R132T) in addition to the original E45A mutation. No other changes were observed in the PCR-amplified viral sequences. To determine whether these second-site substitutions were responsible for the accelerated growth kinetics of the adapted viruses, the mutations were transferred into the original P38A and E45A mutant viral clones. A replication assay was performed with the resulting double mutant viruses in CEM cells. In contrast to the original P38A and E45A mutants, which failed to replicate in CEM cells within 26 days, the P38A/T216I and E45A/R132T double mutants both replicated, though each was delayed relative to the wild type (Figure 1A, B). These results demonstrate that the respective second-site mutations, T216I and R132T, partially restore the ability of the corresponding P38A and E45A mutant viruses to replicate in CEM 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