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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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Effect of individual point mutations on stability of functional Env.Each of the sequence changes identified in the stable variants were introduced as point mutants into wild-type ADA. The mutations identified in the HB2 and GB2 variants were tested against heat (A) and GuHCl (B). The mutations derived from the HC11 variants were assayed only against heat (C). The change in T90 or in the IC50 for mutant relative to wild-type ADA is plotted for each point mutant.
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ppat-1003184-g010: Effect of individual point mutations on stability of functional Env.Each of the sequence changes identified in the stable variants were introduced as point mutants into wild-type ADA. The mutations identified in the HB2 and GB2 variants were tested against heat (A) and GuHCl (B). The mutations derived from the HC11 variants were assayed only against heat (C). The change in T90 or in the IC50 for mutant relative to wild-type ADA is plotted for each point mutant.

Mentions: Based on the stable mutant Env sequences we selected single amino acid residue changes to introduce into wild-type ADA and examined their effect on Env stability. H625N and T626M were introduced as a double substitution, as these residues were adjacent to one another and seemed to co-vary. Although none of the point mutants completely recapitulated the phenotype of the stable clones, stabilizing effects were clearly observed and could be narrowed down to a few residues in each case (Figure 10). Thus, from the B2 pools, I535M, L543Q, and K574R in the NHR and H625N/T626M in the CHR each partially stabilized ADA Env to both heat and GuHCl treatment. In the case of the HC11 clones, the CHR mutation S649A played the largest role in stabilization and the V1alt substitution provided a more limited increase in Env stability.


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

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

Effect of individual point mutations on stability of functional Env.Each of the sequence changes identified in the stable variants were introduced as point mutants into wild-type ADA. The mutations identified in the HB2 and GB2 variants were tested against heat (A) and GuHCl (B). The mutations derived from the HC11 variants were assayed only against heat (C). The change in T90 or in the IC50 for mutant relative to wild-type ADA is plotted for each point mutant.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g010: Effect of individual point mutations on stability of functional Env.Each of the sequence changes identified in the stable variants were introduced as point mutants into wild-type ADA. The mutations identified in the HB2 and GB2 variants were tested against heat (A) and GuHCl (B). The mutations derived from the HC11 variants were assayed only against heat (C). The change in T90 or in the IC50 for mutant relative to wild-type ADA is plotted for each point mutant.
Mentions: Based on the stable mutant Env sequences we selected single amino acid residue changes to introduce into wild-type ADA and examined their effect on Env stability. H625N and T626M were introduced as a double substitution, as these residues were adjacent to one another and seemed to co-vary. Although none of the point mutants completely recapitulated the phenotype of the stable clones, stabilizing effects were clearly observed and could be narrowed down to a few residues in each case (Figure 10). Thus, from the B2 pools, I535M, L543Q, and K574R in the NHR and H625N/T626M in the CHR each partially stabilized ADA Env to both heat and GuHCl treatment. In the case of the HC11 clones, the CHR mutation S649A played the largest role in stabilization and the V1alt substitution provided a more limited increase in Env stability.

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