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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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Related in: MedlinePlus

BN-PAGE and infectivity decay analysis reveals that functional stability of Env correlates with oligomeric stability.Two stable clones that were selected to be resistant to GuHCl (A and B) or heat (C and D) were exposed to the corresponding treatment. Afterwards, virus was assayed for infectivity in TZM-bl cells (A and C) and Env trimer dissociation was visualized by BN-PAGE Western blot using an anti-gp41 mAb cocktail (B and D).
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ppat-1003184-g006: BN-PAGE and infectivity decay analysis reveals that functional stability of Env correlates with oligomeric stability.Two stable clones that were selected to be resistant to GuHCl (A and B) or heat (C and D) were exposed to the corresponding treatment. Afterwards, virus was assayed for infectivity in TZM-bl cells (A and C) and Env trimer dissociation was visualized by BN-PAGE Western blot using an anti-gp41 mAb cocktail (B and D).

Mentions: To determine the relationship between functional stability and oligomeric stability of the unliganded mutant Env trimers, we turned to BN-PAGE. Two GuHCl resistant and two heat resistant clones were chosen for the analysis: GB21-6, GB22-1, HB22-5 and HC11-4, the latter of which shares similar stability and most of the same mutations as HC11-1 (Figure 3). The clones were subjected to increasing temperature or GuHCl concentrations, and samples were analyzed for infectivity and by BN-PAGE. In each case, dissociation of the Env trimer on BN-PAGE closely correlated with the loss of infectivity, with stability selected Envs clearly maintaining trimeric association under conditions that caused dissociation of wild-type ADA trimers (Figure 6). HC11-1, used above and in subsequent analyses, was also found to be more stable (data not shown). Importantly, the stable Env variants also appear more homogeneous than that of wild-type ADA as non-trimeric species in the former are much less apparent than in the latter.


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

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

BN-PAGE and infectivity decay analysis reveals that functional stability of Env correlates with oligomeric stability.Two stable clones that were selected to be resistant to GuHCl (A and B) or heat (C and D) were exposed to the corresponding treatment. Afterwards, virus was assayed for infectivity in TZM-bl cells (A and C) and Env trimer dissociation was visualized by BN-PAGE Western blot using an anti-gp41 mAb cocktail (B and D).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g006: BN-PAGE and infectivity decay analysis reveals that functional stability of Env correlates with oligomeric stability.Two stable clones that were selected to be resistant to GuHCl (A and B) or heat (C and D) were exposed to the corresponding treatment. Afterwards, virus was assayed for infectivity in TZM-bl cells (A and C) and Env trimer dissociation was visualized by BN-PAGE Western blot using an anti-gp41 mAb cocktail (B and D).
Mentions: To determine the relationship between functional stability and oligomeric stability of the unliganded mutant Env trimers, we turned to BN-PAGE. Two GuHCl resistant and two heat resistant clones were chosen for the analysis: GB21-6, GB22-1, HB22-5 and HC11-4, the latter of which shares similar stability and most of the same mutations as HC11-1 (Figure 3). The clones were subjected to increasing temperature or GuHCl concentrations, and samples were analyzed for infectivity and by BN-PAGE. In each case, dissociation of the Env trimer on BN-PAGE closely correlated with the loss of infectivity, with stability selected Envs clearly maintaining trimeric association under conditions that caused dissociation of wild-type ADA trimers (Figure 6). HC11-1, used above and in subsequent analyses, was also found to be more stable (data not shown). Importantly, the stable Env variants also appear more homogeneous than that of wild-type ADA as non-trimeric species in the former are much less apparent than in the latter.

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