Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro capsid assembly analysis. To initiate assembly, purified recombinant CA proteins were diluted into a buffer, resulting in a final NaCl concentration of 2.25 M. The turbidity of the samples was determined with a spectrophotometer at the indicated times. For reference, the results for the wild type CA protein are shown in both panels A and B. Values shown are the average of two parallel determinations. Shown are data from one representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351742&req=5

Figure 5: In vitro capsid assembly analysis. To initiate assembly, purified recombinant CA proteins were diluted into a buffer, resulting in a final NaCl concentration of 2.25 M. The turbidity of the samples was determined with a spectrophotometer at the indicated times. For reference, the results for the wild type CA protein are shown in both panels A and B. Values shown are the average of two parallel determinations. Shown are data from one representative of 3 independent experiments.

Mentions: Purified HIV-1 CA protein can assemble into tube-like structures in a high salt buffer system in vitro [27-30]. The E45A mutation was previously reported to accelerate HIV-1 CA assembly in vitro [31], suggesting that this mutation enhances protein-protein interactions between CA subunits. Reasoning that the hyperstability of the E45A viral capsid may be related to the accelerated CA assembly kinetics in vitro, we asked whether the suppressor mutations would affect the rate of CA assembly. Purified recombinant CA proteins encoding the E45A and E45A/R132T mutations were diluted into a high salt buffer, and the turbidity of the solution was monitored spectrophotometrically for 15 minutes following initiation of the reactions. The wild-type CA proteins assembled at a moderate rate, taking nearly 10 minutes for completion. In contrast, the E45A mutant assembled within 2 minutes (Figure 5A). However, the accelerated rate of assembly of E45A was not affected by the suppressor mutation R132T. The P38A and P38A/T216I mutant proteins also exhibited accelerated assembly, reaching completion within 3-4 minutes (Figure 5B). Thus, the effects of the E45A and P38A mutations on CA assembly in vitro were not correlated with the biological phenotypes of the corresponding CA mutant viruses.


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)

In vitro capsid assembly analysis. To initiate assembly, purified recombinant CA proteins were diluted into a buffer, resulting in a final NaCl concentration of 2.25 M. The turbidity of the samples was determined with a spectrophotometer at the indicated times. For reference, the results for the wild type CA protein are shown in both panels A and B. Values shown are the average of two parallel determinations. Shown are data from one representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In vitro capsid assembly analysis. To initiate assembly, purified recombinant CA proteins were diluted into a buffer, resulting in a final NaCl concentration of 2.25 M. The turbidity of the samples was determined with a spectrophotometer at the indicated times. For reference, the results for the wild type CA protein are shown in both panels A and B. Values shown are the average of two parallel determinations. Shown are data from one representative of 3 independent experiments.
Mentions: Purified HIV-1 CA protein can assemble into tube-like structures in a high salt buffer system in vitro [27-30]. The E45A mutation was previously reported to accelerate HIV-1 CA assembly in vitro [31], suggesting that this mutation enhances protein-protein interactions between CA subunits. Reasoning that the hyperstability of the E45A viral capsid may be related to the accelerated CA assembly kinetics in vitro, we asked whether the suppressor mutations would affect the rate of CA assembly. Purified recombinant CA proteins encoding the E45A and E45A/R132T mutations were diluted into a high salt buffer, and the turbidity of the solution was monitored spectrophotometrically for 15 minutes following initiation of the reactions. The wild-type CA proteins assembled at a moderate rate, taking nearly 10 minutes for completion. In contrast, the E45A mutant assembled within 2 minutes (Figure 5A). However, the accelerated rate of assembly of E45A was not affected by the suppressor mutation R132T. The P38A and P38A/T216I mutant proteins also exhibited accelerated assembly, reaching completion within 3-4 minutes (Figure 5B). Thus, the effects of the E45A and P38A mutations on CA assembly in vitro were not correlated with the biological phenotypes of the corresponding CA mutant viruses.

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