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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 mutations do not restore capsid stability in vitro. (A and B) Concentrated virions were subjected to ultracentrifugation through a detergent layer into a sucrose density gradient. Yields of cores were calculated as the percentage of the total CA that was detected in the peak fractions of cores. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation. (C) Disassembly of purified HIV-1 cores in vitro. Diluted HIV-1 cores were incubated at 37°C for the indicated times, followed by separation of free and core-associated CA by ultracentrifugation. Supernatants and pellets were analyzed by p24 ELISA. The extent of disassembly was determined as the percentage of the total CA protein in the reaction detected in the supernatant. Results shown are the average values of two independent experiments with duplicate determinations in each experiment. Error bars represent the spread of values obtained in the two experiments.
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Figure 4: Second-site suppressor mutations do not restore capsid stability in vitro. (A and B) Concentrated virions were subjected to ultracentrifugation through a detergent layer into a sucrose density gradient. Yields of cores were calculated as the percentage of the total CA that was detected in the peak fractions of cores. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation. (C) Disassembly of purified HIV-1 cores in vitro. Diluted HIV-1 cores were incubated at 37°C for the indicated times, followed by separation of free and core-associated CA by ultracentrifugation. Supernatants and pellets were analyzed by p24 ELISA. The extent of disassembly was determined as the percentage of the total CA protein in the reaction detected in the supernatant. Results shown are the average values of two independent experiments with duplicate determinations in each experiment. Error bars represent the spread of values obtained in the two experiments.

Mentions: To determine whether the compensatory mutations restore normal stability to the viral capsid, we isolated cores from each of the mutant viruses by centrifugation through a layer of nonionic detergent into a linear sucrose gradient. We quantified the recovery of core-associated CA in the fractions corresponding to the viral cores as a percentage of the total quantity of CA in the entire gradient. This value was approximately 10% for wild-type HIV-1 (Figure 4A and 4B). In contrast, the recovery of CA in P38A cores was about 2%, while that for E45A cores was approximately 20% (Figure 4B). Contrary to our expectation, cores recovered from the double mutants P38A/T216I and E45A/R132T exhibited CA levels that were similar to those of the corresponding single mutants (Figure 4A and 4B). To further evaluate the relative stability of E45A mutant HIV-1 cores, we assayed the rate of CA dissociation from purified cores in vitro. The poor recovery of core-associated CA from the P38A mutant particles precluded its analysis. Cores isolated from E45A and E45A/R132T both exhibited slower uncoating in vitro relative to wild-type cores (Figure 4C). Collectively, these results indicate that the suppressor mutations do not correct the aberrant intrinsic stability of the P38A and E45A mutant capsids observed in vitro.


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 mutations do not restore capsid stability in vitro. (A and B) Concentrated virions were subjected to ultracentrifugation through a detergent layer into a sucrose density gradient. Yields of cores were calculated as the percentage of the total CA that was detected in the peak fractions of cores. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation. (C) Disassembly of purified HIV-1 cores in vitro. Diluted HIV-1 cores were incubated at 37°C for the indicated times, followed by separation of free and core-associated CA by ultracentrifugation. Supernatants and pellets were analyzed by p24 ELISA. The extent of disassembly was determined as the percentage of the total CA protein in the reaction detected in the supernatant. Results shown are the average values of two independent experiments with duplicate determinations in each experiment. Error bars represent the spread of values obtained in the two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Second-site suppressor mutations do not restore capsid stability in vitro. (A and B) Concentrated virions were subjected to ultracentrifugation through a detergent layer into a sucrose density gradient. Yields of cores were calculated as the percentage of the total CA that was detected in the peak fractions of cores. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation. (C) Disassembly of purified HIV-1 cores in vitro. Diluted HIV-1 cores were incubated at 37°C for the indicated times, followed by separation of free and core-associated CA by ultracentrifugation. Supernatants and pellets were analyzed by p24 ELISA. The extent of disassembly was determined as the percentage of the total CA protein in the reaction detected in the supernatant. Results shown are the average values of two independent experiments with duplicate determinations in each experiment. Error bars represent the spread of values obtained in the two experiments.
Mentions: To determine whether the compensatory mutations restore normal stability to the viral capsid, we isolated cores from each of the mutant viruses by centrifugation through a layer of nonionic detergent into a linear sucrose gradient. We quantified the recovery of core-associated CA in the fractions corresponding to the viral cores as a percentage of the total quantity of CA in the entire gradient. This value was approximately 10% for wild-type HIV-1 (Figure 4A and 4B). In contrast, the recovery of CA in P38A cores was about 2%, while that for E45A cores was approximately 20% (Figure 4B). Contrary to our expectation, cores recovered from the double mutants P38A/T216I and E45A/R132T exhibited CA levels that were similar to those of the corresponding single mutants (Figure 4A and 4B). To further evaluate the relative stability of E45A mutant HIV-1 cores, we assayed the rate of CA dissociation from purified cores in vitro. The poor recovery of core-associated CA from the P38A mutant particles precluded its analysis. Cores isolated from E45A and E45A/R132T both exhibited slower uncoating in vitro relative to wild-type cores (Figure 4C). Collectively, these results indicate that the suppressor mutations do not correct the aberrant intrinsic stability of the P38A and E45A mutant capsids observed in vitro.

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