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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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Amide chemical shift differences between the wild type and E45A, E45A/R132T and P38A mutant CA-NTD proteins. Combined 1H, 15 N chemical shift differences between wild-type and mutants E45A (A), E45A/R132T (B) and P38A (C) are plotted versus residue number. Structural mapping of the chemical shift differences onto the X-ray structure of CA-NTD [1AK4] in ribbon representation is provided in the inset. The locations of the mutation sites are marked with large green spheres. Positions of residues whose amide resonances exhibit significant chemical shift differences are color-coded according to the magnitude of the change: red, Δδ > (Δδaverage + 2 × SD); orange, (Δδaverage + 2 × SD) > Δδ > (Δδaverage + 1xSD).
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Figure 6: Amide chemical shift differences between the wild type and E45A, E45A/R132T and P38A mutant CA-NTD proteins. Combined 1H, 15 N chemical shift differences between wild-type and mutants E45A (A), E45A/R132T (B) and P38A (C) are plotted versus residue number. Structural mapping of the chemical shift differences onto the X-ray structure of CA-NTD [1AK4] in ribbon representation is provided in the inset. The locations of the mutation sites are marked with large green spheres. Positions of residues whose amide resonances exhibit significant chemical shift differences are color-coded according to the magnitude of the change: red, Δδ > (Δδaverage + 2 × SD); orange, (Δδaverage + 2 × SD) > Δδ > (Δδaverage + 1xSD).

Mentions: 1H-15 N HSQC NMR experiments were performed to assess the effects of the E45A, P38A, E45A/R132T and R132T mutations on the structure of the N-terminal domain of CA (CA-NTD). Superpositions of the 1H-15 N HSQC spectra of wild-type and E45A (A), P38A (B), E45A/R132T (C) and R132T (D) mutants are shown in Additional file 1. The spectra of all the mutants are very similar to that of wild type, demonstrating conservation of the overall global fold. Chemical shift changes due to the mutations are plotted versus residue number in Figure 6. Resonance changes caused by the E45A and R132T mutations when compared with the wild type spectrum were essentially confined to the mutation sites. For example, in the E45A mutant only Gly46 (except the mutated residue Glu45) exhibited a large (~ 0.8 ppm) chemical shift change. Similarly, only neighboring residues (Ile129, Trp133, and Leu136) were associated with noticeable changes, ranging from 0.3 to 1 ppm in the R132T mutant. In contrast, the spectrum of the P38A mutant exhibited overall smaller changes (< 0.42 ppm), but the mutation affected more residues that were dispersed over a wider region. For example, eighteen residues exhibit chemical shift changes larger than the average change plus one standard deviation (SD) (0.14 ppm). These residues are located around the mutation site (such as Va36, Ile37 and Met39 on helix α2), but also in the preceding loop (Lys30-Ser33), helices α1 (Trp23-Glu28), α3 (Met55) and α7 (L136 and V142). This suggests that a more general effect such as an overall loosening of the structure may have occurred. We conclude that the E45A and R132T mutations induce highly localized structural changes, indicating that the effects of the mutations simply result from the change in chemical nature of the substituted amino acid. The small, but more widespread effects of the P38A mutation on the protein structure suggest that modulation of structural properties occurs in the mutant. Thus, the reduced capsid stability of the mutant may result from subtle changes in the overall structure and from Pro38's involvement in the NTD-NTD interaction in the assembled capsid. Note that Pro38 is in close proximity (< 5A) to Thr54, Asn57, and Thr58 of the neighboring CA molecule in the CA hexamer structure (3H47)[10].


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)

Amide chemical shift differences between the wild type and E45A, E45A/R132T and P38A mutant CA-NTD proteins. Combined 1H, 15 N chemical shift differences between wild-type and mutants E45A (A), E45A/R132T (B) and P38A (C) are plotted versus residue number. Structural mapping of the chemical shift differences onto the X-ray structure of CA-NTD [1AK4] in ribbon representation is provided in the inset. The locations of the mutation sites are marked with large green spheres. Positions of residues whose amide resonances exhibit significant chemical shift differences are color-coded according to the magnitude of the change: red, Δδ > (Δδaverage + 2 × SD); orange, (Δδaverage + 2 × SD) > Δδ > (Δδaverage + 1xSD).
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Figure 6: Amide chemical shift differences between the wild type and E45A, E45A/R132T and P38A mutant CA-NTD proteins. Combined 1H, 15 N chemical shift differences between wild-type and mutants E45A (A), E45A/R132T (B) and P38A (C) are plotted versus residue number. Structural mapping of the chemical shift differences onto the X-ray structure of CA-NTD [1AK4] in ribbon representation is provided in the inset. The locations of the mutation sites are marked with large green spheres. Positions of residues whose amide resonances exhibit significant chemical shift differences are color-coded according to the magnitude of the change: red, Δδ > (Δδaverage + 2 × SD); orange, (Δδaverage + 2 × SD) > Δδ > (Δδaverage + 1xSD).
Mentions: 1H-15 N HSQC NMR experiments were performed to assess the effects of the E45A, P38A, E45A/R132T and R132T mutations on the structure of the N-terminal domain of CA (CA-NTD). Superpositions of the 1H-15 N HSQC spectra of wild-type and E45A (A), P38A (B), E45A/R132T (C) and R132T (D) mutants are shown in Additional file 1. The spectra of all the mutants are very similar to that of wild type, demonstrating conservation of the overall global fold. Chemical shift changes due to the mutations are plotted versus residue number in Figure 6. Resonance changes caused by the E45A and R132T mutations when compared with the wild type spectrum were essentially confined to the mutation sites. For example, in the E45A mutant only Gly46 (except the mutated residue Glu45) exhibited a large (~ 0.8 ppm) chemical shift change. Similarly, only neighboring residues (Ile129, Trp133, and Leu136) were associated with noticeable changes, ranging from 0.3 to 1 ppm in the R132T mutant. In contrast, the spectrum of the P38A mutant exhibited overall smaller changes (< 0.42 ppm), but the mutation affected more residues that were dispersed over a wider region. For example, eighteen residues exhibit chemical shift changes larger than the average change plus one standard deviation (SD) (0.14 ppm). These residues are located around the mutation site (such as Va36, Ile37 and Met39 on helix α2), but also in the preceding loop (Lys30-Ser33), helices α1 (Trp23-Glu28), α3 (Met55) and α7 (L136 and V142). This suggests that a more general effect such as an overall loosening of the structure may have occurred. We conclude that the E45A and R132T mutations induce highly localized structural changes, indicating that the effects of the mutations simply result from the change in chemical nature of the substituted amino acid. The small, but more widespread effects of the P38A mutation on the protein structure suggest that modulation of structural properties occurs in the mutant. Thus, the reduced capsid stability of the mutant may result from subtle changes in the overall structure and from Pro38's involvement in the NTD-NTD interaction in the assembled capsid. Note that Pro38 is in close proximity (< 5A) to Thr54, Asn57, and Thr58 of the neighboring CA molecule in the CA hexamer structure (3H47)[10].

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