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Increased functional stability and homogeneity of viral envelope spikes through directed evolution.

Leaman DP, Zwick MB - PLoS Pathog. (2013)

Bottom Line: Combining the seven mutations generated a variant Env with superior homogeneity and stability.Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9.The latter result may reflect a change in glycans on the stabilized Envs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure.

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Substitution K574A in gp41 decreases the thermostability of HIV-1 functional Env.HIV-1 mutant K574A was tested for resistance to heat in either the LAI (A) or JR-FL (B) backgrounds relative to wild-type. Viruses were exposed to varying temperatures for one hour and assayed for infectivity using TZM-bl target cells. Results shown are the average of two experiments performed in duplicate.
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ppat-1003184-g011: Substitution K574A in gp41 decreases the thermostability of HIV-1 functional Env.HIV-1 mutant K574A was tested for resistance to heat in either the LAI (A) or JR-FL (B) backgrounds relative to wild-type. Viruses were exposed to varying temperatures for one hour and assayed for infectivity using TZM-bl target cells. Results shown are the average of two experiments performed in duplicate.

Mentions: Among the gp41 amino acid changes identified in the GB2 and HB2 virion pools that increased functional trimer stability, K574R was the only mutation that was not of LAI origin. The conserved Lys at position 574 residue has previously been shown to be crucial for stability of the six-helix bindle (6HB) protein that is a mimetic of gp41 in a post-fusion form [45]. Other studies have shown that mutations to the NHR of gp41 can affect neutralization sensitivity of HIV-1 [46], [47], and as shown above Env trimer stability can also alter sensitivity to certain inhibitors. To further characterize the relationship between Env stability and neutralization sensitivity due to mutation in the NHR, we examined how non-conservative mutation at position 574 affects trimer stability. We first performed this analysis using the mutant K574A in the LAI strain, which was generated in a previous study [48]. The K574A mutation profoundly decreased the T90 (the temperature at which viral infectivity is diminished by 90% in one hour) of HIV-1 LAI by ∼4°C and also globally increased sensitivity to a number of Env-destabilizing ligands (e.g. b12, sCD4, 2F5, 4E10, and b6; Figure 11A and Table 1). The most profound effect was observed with the weakly neutralizing CD4bs mAb b6 which was 250-fold more potent against K574A than wild-type LAI. In contrast, the substitution K574A had a less pronounced effect on neutralization by the non-destabilizing mAb 2G12 [37], and was only 2-fold more sensitive to mAbs and inhibitors that target Env in a pre-fusion intermediate state.


Increased functional stability and homogeneity of viral envelope spikes through directed evolution.

Leaman DP, Zwick MB - PLoS Pathog. (2013)

Substitution K574A in gp41 decreases the thermostability of HIV-1 functional Env.HIV-1 mutant K574A was tested for resistance to heat in either the LAI (A) or JR-FL (B) backgrounds relative to wild-type. Viruses were exposed to varying temperatures for one hour and assayed for infectivity using TZM-bl target cells. Results shown are the average of two experiments performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g011: Substitution K574A in gp41 decreases the thermostability of HIV-1 functional Env.HIV-1 mutant K574A was tested for resistance to heat in either the LAI (A) or JR-FL (B) backgrounds relative to wild-type. Viruses were exposed to varying temperatures for one hour and assayed for infectivity using TZM-bl target cells. Results shown are the average of two experiments performed in duplicate.
Mentions: Among the gp41 amino acid changes identified in the GB2 and HB2 virion pools that increased functional trimer stability, K574R was the only mutation that was not of LAI origin. The conserved Lys at position 574 residue has previously been shown to be crucial for stability of the six-helix bindle (6HB) protein that is a mimetic of gp41 in a post-fusion form [45]. Other studies have shown that mutations to the NHR of gp41 can affect neutralization sensitivity of HIV-1 [46], [47], and as shown above Env trimer stability can also alter sensitivity to certain inhibitors. To further characterize the relationship between Env stability and neutralization sensitivity due to mutation in the NHR, we examined how non-conservative mutation at position 574 affects trimer stability. We first performed this analysis using the mutant K574A in the LAI strain, which was generated in a previous study [48]. The K574A mutation profoundly decreased the T90 (the temperature at which viral infectivity is diminished by 90% in one hour) of HIV-1 LAI by ∼4°C and also globally increased sensitivity to a number of Env-destabilizing ligands (e.g. b12, sCD4, 2F5, 4E10, and b6; Figure 11A and Table 1). The most profound effect was observed with the weakly neutralizing CD4bs mAb b6 which was 250-fold more potent against K574A than wild-type LAI. In contrast, the substitution K574A had a less pronounced effect on neutralization by the non-destabilizing mAb 2G12 [37], and was only 2-fold more sensitive to mAbs and inhibitors that target Env in a pre-fusion intermediate state.

Bottom Line: Combining the seven mutations generated a variant Env with superior homogeneity and stability.Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9.The latter result may reflect a change in glycans on the stabilized Envs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT
The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure.

Show MeSH
Related in: MedlinePlus