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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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Quantitative analysis of reverse transcription of CA mutants in target cells. HeLa-P4 cells were inoculated with the indicated HIV-1 mutants, and the second-strand transfer viral DNA products accumulated at 8 hours were quantified by PCR. In half of the samples, the reverse transcriptase inhibitor Efavirenz (EFV) was included as a control for plasmid DNA contamination carryover from the transfections used to produce the virus stocks. Error bars represent the standard deviations of triplicate infections, and the results are representative of three independent experiments.
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Figure 3: Quantitative analysis of reverse transcription of CA mutants in target cells. HeLa-P4 cells were inoculated with the indicated HIV-1 mutants, and the second-strand transfer viral DNA products accumulated at 8 hours were quantified by PCR. In half of the samples, the reverse transcriptase inhibitor Efavirenz (EFV) was included as a control for plasmid DNA contamination carryover from the transfections used to produce the virus stocks. Error bars represent the standard deviations of triplicate infections, and the results are representative of three independent experiments.

Mentions: Our group previously showed that the P38A and E45A mutant capsids exhibited altered stability without major structural defects, and were impaired for reverse transcription, suggesting that the mutations result in aberrant uncoating in target cells [17]. Accordingly, the P38A substitution destabilized the viral capsid, while E45A resulted in a hyperstable capsid. In follow-up studies, we have repeatedly observed that the E45A mutation does not impair reverse transcription, but results in impairment at later stages of infection, after nuclear entry (Figure 3). In this respect, E45A resembles the Q63A/Q67A CA mutant, which exhibits delayed uncoating in target cells and is impaired for nuclear import and integration [16,17]. Importantly, the T216I mutation partially rescued the impaired reverse transcription exhibited by P38A (Figure 3), consistent with the interpretation that the impaired infectivity exhibited by P38A is owing to its reduced reverse transcription capacity. Collectively, these results suggest that a stable capsid is necessary for efficient reverse transcription in target cells, while mutants with hyperstable capsids are impaired at later steps of infection.


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)

Quantitative analysis of reverse transcription of CA mutants in target cells. HeLa-P4 cells were inoculated with the indicated HIV-1 mutants, and the second-strand transfer viral DNA products accumulated at 8 hours were quantified by PCR. In half of the samples, the reverse transcriptase inhibitor Efavirenz (EFV) was included as a control for plasmid DNA contamination carryover from the transfections used to produce the virus stocks. Error bars represent the standard deviations of triplicate infections, and the results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Quantitative analysis of reverse transcription of CA mutants in target cells. HeLa-P4 cells were inoculated with the indicated HIV-1 mutants, and the second-strand transfer viral DNA products accumulated at 8 hours were quantified by PCR. In half of the samples, the reverse transcriptase inhibitor Efavirenz (EFV) was included as a control for plasmid DNA contamination carryover from the transfections used to produce the virus stocks. Error bars represent the standard deviations of triplicate infections, and the results are representative of three independent experiments.
Mentions: Our group previously showed that the P38A and E45A mutant capsids exhibited altered stability without major structural defects, and were impaired for reverse transcription, suggesting that the mutations result in aberrant uncoating in target cells [17]. Accordingly, the P38A substitution destabilized the viral capsid, while E45A resulted in a hyperstable capsid. In follow-up studies, we have repeatedly observed that the E45A mutation does not impair reverse transcription, but results in impairment at later stages of infection, after nuclear entry (Figure 3). In this respect, E45A resembles the Q63A/Q67A CA mutant, which exhibits delayed uncoating in target cells and is impaired for nuclear import and integration [16,17]. Importantly, the T216I mutation partially rescued the impaired reverse transcription exhibited by P38A (Figure 3), consistent with the interpretation that the impaired infectivity exhibited by P38A is owing to its reduced reverse transcription capacity. Collectively, these results suggest that a stable capsid is necessary for efficient reverse transcription in target cells, while mutants with hyperstable capsids are impaired at later steps of infection.

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