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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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Stability selected ADA mutant HIV-1 are resistant to multiple destabilizing treatments.Two representative stable clones (GB21-6 and HC11-1) were tested along with ADA and LAI for resistance to GuHCl (A), heat (B), and 37°C decay (C). Shown are the results of a representative experiment performed in duplicate.
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ppat-1003184-g005: Stability selected ADA mutant HIV-1 are resistant to multiple destabilizing treatments.Two representative stable clones (GB21-6 and HC11-1) were tested along with ADA and LAI for resistance to GuHCl (A), heat (B), and 37°C decay (C). Shown are the results of a representative experiment performed in duplicate.

Mentions: The stability of the selected Env clones can either be specific to the selection conditions, or can impart a broader resistance to multiple destabilizing treatments. To investigate, the most stable clones from the GuHCl and heat-selected pools, GB21-6 and HC11-1, were subjected in parallel to GuHCl, heat and prolonged incubation at physiological temperature. Both GB21-6 and HC11-1 maintained infectivity with increased resistance to each of these conditions relative to wild-type ADA (Figure 5). Clone GB21-6 was consistently the most stable in each case. In keeping with our prior observation of a negative correlation between the number of LAI gp120 residues and Env stability (Figure 4), LAI was less stable than GB21-6 when treated with heat or GuHCl, but notably the two viruses displayed similar rates of decay at physiological temperature (Figure 5).


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

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

Stability selected ADA mutant HIV-1 are resistant to multiple destabilizing treatments.Two representative stable clones (GB21-6 and HC11-1) were tested along with ADA and LAI for resistance to GuHCl (A), heat (B), and 37°C decay (C). Shown are the results of a representative experiment performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g005: Stability selected ADA mutant HIV-1 are resistant to multiple destabilizing treatments.Two representative stable clones (GB21-6 and HC11-1) were tested along with ADA and LAI for resistance to GuHCl (A), heat (B), and 37°C decay (C). Shown are the results of a representative experiment performed in duplicate.
Mentions: The stability of the selected Env clones can either be specific to the selection conditions, or can impart a broader resistance to multiple destabilizing treatments. To investigate, the most stable clones from the GuHCl and heat-selected pools, GB21-6 and HC11-1, were subjected in parallel to GuHCl, heat and prolonged incubation at physiological temperature. Both GB21-6 and HC11-1 maintained infectivity with increased resistance to each of these conditions relative to wild-type ADA (Figure 5). Clone GB21-6 was consistently the most stable in each case. In keeping with our prior observation of a negative correlation between the number of LAI gp120 residues and Env stability (Figure 4), LAI was less stable than GB21-6 when treated with heat or GuHCl, but notably the two viruses displayed similar rates of decay at physiological temperature (Figure 5).

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