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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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Locations of mutations in the structure of the CA hexamer. (A) Top and side views of a CA hexamer showing E45 (green) and R132 (yellow) side chains from the NMR structure of CA151 (PDB ID: 3H47). (B) Same views, highlighting P38 and T216. P38 (red) in helix 2 is located at the CA NTD-NTD interface and is adjacent to helix 3 in the adjacent subunit. T216 (blue) in helix 11 is near the CA NTD-CTD interface and is close to helix 7 in the nearby subunit.
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Figure 10: Locations of mutations in the structure of the CA hexamer. (A) Top and side views of a CA hexamer showing E45 (green) and R132 (yellow) side chains from the NMR structure of CA151 (PDB ID: 3H47). (B) Same views, highlighting P38 and T216. P38 (red) in helix 2 is located at the CA NTD-NTD interface and is adjacent to helix 3 in the adjacent subunit. T216 (blue) in helix 11 is near the CA NTD-CTD interface and is close to helix 7 in the nearby subunit.

Mentions: Our NMR structural analysis of CA-NTD proteins suggests that the mutant phenotypes are not a consequence of major structural alterations in the capsid. NMR structural assessment of the effects of the mutations on the CA-NTD structure revealed no gross conformational changes in either P38A or E45A mutants, nor for the R132T mutant. The P38A substitution, however, resulted in more extensive changes that could be related to a general effect of the structural adjustment for the mutation. Examination of the locations of these amino acids in recently reported structures of the HIV-1 CA hexamer [9,10] revealed that R132 is positioned on the surface of the CA molecule facing the NTD-NTD interface (Figure 10A). E45 is in the vicinity of this interface as well; however, the distance between these two side chains prohibits a direct interact with one another, making it unlikely that R132T directly offsets the stability defect resulting from E45A. The R132T substitution could act by restoring the overall net charge of CA, which would be consistent with our previous findings that the stability of the HIV-1 capsid is sensitive to changes in ionic strength and pH [17]. Alternatively, the E45A mutation might affect the network of water-mediated hydrogen bonds observed at the NTD-NTD interface in the X-ray crystal structure [10], with R132T mutation partially restoring the H-bonding network. It should be noted that the hexamer crystal structure was determined with a mutant protein that contained two amino acid substitutions, one of which was a Glu to Cys change position 45.


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)

Locations of mutations in the structure of the CA hexamer. (A) Top and side views of a CA hexamer showing E45 (green) and R132 (yellow) side chains from the NMR structure of CA151 (PDB ID: 3H47). (B) Same views, highlighting P38 and T216. P38 (red) in helix 2 is located at the CA NTD-NTD interface and is adjacent to helix 3 in the adjacent subunit. T216 (blue) in helix 11 is near the CA NTD-CTD interface and is close to helix 7 in the nearby subunit.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Locations of mutations in the structure of the CA hexamer. (A) Top and side views of a CA hexamer showing E45 (green) and R132 (yellow) side chains from the NMR structure of CA151 (PDB ID: 3H47). (B) Same views, highlighting P38 and T216. P38 (red) in helix 2 is located at the CA NTD-NTD interface and is adjacent to helix 3 in the adjacent subunit. T216 (blue) in helix 11 is near the CA NTD-CTD interface and is close to helix 7 in the nearby subunit.
Mentions: Our NMR structural analysis of CA-NTD proteins suggests that the mutant phenotypes are not a consequence of major structural alterations in the capsid. NMR structural assessment of the effects of the mutations on the CA-NTD structure revealed no gross conformational changes in either P38A or E45A mutants, nor for the R132T mutant. The P38A substitution, however, resulted in more extensive changes that could be related to a general effect of the structural adjustment for the mutation. Examination of the locations of these amino acids in recently reported structures of the HIV-1 CA hexamer [9,10] revealed that R132 is positioned on the surface of the CA molecule facing the NTD-NTD interface (Figure 10A). E45 is in the vicinity of this interface as well; however, the distance between these two side chains prohibits a direct interact with one another, making it unlikely that R132T directly offsets the stability defect resulting from E45A. The R132T substitution could act by restoring the overall net charge of CA, which would be consistent with our previous findings that the stability of the HIV-1 capsid is sensitive to changes in ionic strength and pH [17]. Alternatively, the E45A mutation might affect the network of water-mediated hydrogen bonds observed at the NTD-NTD interface in the X-ray crystal structure [10], with R132T mutation partially restoring the H-bonding network. It should be noted that the hexamer crystal structure was determined with a mutant protein that contained two amino acid substitutions, one of which was a Glu to Cys change position 45.

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