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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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Virion pools are resistant to destabilizing conditions after three rounds of selection.Virion pools that had already been selected for two rounds in duplicate were treated with either GuHCl (A), urea (B), or heat (C) for one hour, or incubated at 37°C for up to 144 hours (D). After treatment, an aliquot of virus was removed and titered on TZM-bl cells. Percent infectivity remaining was calculated relative to untreated virus.
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ppat-1003184-g001: Virion pools are resistant to destabilizing conditions after three rounds of selection.Virion pools that had already been selected for two rounds in duplicate were treated with either GuHCl (A), urea (B), or heat (C) for one hour, or incubated at 37°C for up to 144 hours (D). After treatment, an aliquot of virus was removed and titered on TZM-bl cells. Percent infectivity remaining was calculated relative to untreated virus.

Mentions: To select for stable ADA variants, the pools of virions were treated separately with incremental concentrations of GuHCl, urea, hyper-physiological temperatures or prolonged incubations at physiological temperature that inactivates wild-type ADA [7]. After destabilizing treatment, the surviving infectious viruses were rescued on MT2-CCR5ΔCT cells. Following three rounds of selection, some virion pools were more stable than ADA wild-type to GuHCl and heat treatment (e.g. B21 and B22), one was resistant to heat (e.g. C11), and two pools were resistant to 37°C decay (e.g. C12 and M2; Figure 1). None of the selected library pools were found to have increased stability to urea.


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

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

Virion pools are resistant to destabilizing conditions after three rounds of selection.Virion pools that had already been selected for two rounds in duplicate were treated with either GuHCl (A), urea (B), or heat (C) for one hour, or incubated at 37°C for up to 144 hours (D). After treatment, an aliquot of virus was removed and titered on TZM-bl cells. Percent infectivity remaining was calculated relative to untreated virus.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g001: Virion pools are resistant to destabilizing conditions after three rounds of selection.Virion pools that had already been selected for two rounds in duplicate were treated with either GuHCl (A), urea (B), or heat (C) for one hour, or incubated at 37°C for up to 144 hours (D). After treatment, an aliquot of virus was removed and titered on TZM-bl cells. Percent infectivity remaining was calculated relative to untreated virus.
Mentions: To select for stable ADA variants, the pools of virions were treated separately with incremental concentrations of GuHCl, urea, hyper-physiological temperatures or prolonged incubations at physiological temperature that inactivates wild-type ADA [7]. After destabilizing treatment, the surviving infectious viruses were rescued on MT2-CCR5ΔCT cells. Following three rounds of selection, some virion pools were more stable than ADA wild-type to GuHCl and heat treatment (e.g. B21 and B22), one was resistant to heat (e.g. C11), and two pools were resistant to 37°C decay (e.g. C12 and M2; Figure 1). None of the selected library pools were found to have increased stability to urea.

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