Limits...
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.

Show MeSH

Related in: MedlinePlus

Comb-mut Env spikes exhibit increased oligomeric stability.(A) Wild-type ADA and comb-mut viruses were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The Western blot shown was stained with an anti-gp41 cocktail of mAbs, and similar results were seen using anti-gp120 mAbs. (B) The intensity of the Env trimer bands from each lane in panel A was quantified using ImageJ software and plotted as the percent of the trimer band remaining relative to the corresponding band in the 37°C treated lane. The samples exposed to higher temperatures (58–67°C; the blot on the right for each virus) were run on a separate gel and the band intensities were calculated relative to 37°C treated controls on those gels that are not shown. (C) Wild-type ADA and comb-mut virions were incubated at 37°C with a protease cocktail as in Figure 12D and the oligomeric state of Env was analyzed by BN-PAGE. The Western blot was stained as in panel A. (D) Env trimer band intensities, as quantified for panel B. (E) Wild-type ADA and comb-mut virions were incubated at 37°C for up to 24 hours in 1% DDM detergent. The oligomeric state of DDM-treated Env was subsequently visualized using BN-PAGE. The Western blot was stained as in panel A. (F) Env trimer band intensities, as quantified for panel B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g013: Comb-mut Env spikes exhibit increased oligomeric stability.(A) Wild-type ADA and comb-mut viruses were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The Western blot shown was stained with an anti-gp41 cocktail of mAbs, and similar results were seen using anti-gp120 mAbs. (B) The intensity of the Env trimer bands from each lane in panel A was quantified using ImageJ software and plotted as the percent of the trimer band remaining relative to the corresponding band in the 37°C treated lane. The samples exposed to higher temperatures (58–67°C; the blot on the right for each virus) were run on a separate gel and the band intensities were calculated relative to 37°C treated controls on those gels that are not shown. (C) Wild-type ADA and comb-mut virions were incubated at 37°C with a protease cocktail as in Figure 12D and the oligomeric state of Env was analyzed by BN-PAGE. The Western blot was stained as in panel A. (D) Env trimer band intensities, as quantified for panel B. (E) Wild-type ADA and comb-mut virions were incubated at 37°C for up to 24 hours in 1% DDM detergent. The oligomeric state of DDM-treated Env was subsequently visualized using BN-PAGE. The Western blot was stained as in panel A. (F) Env trimer band intensities, as quantified for panel B.

Mentions: We further examined the direct effect of heat on Env trimer dissociation by comparing wild-type ADA and comb-mut using BN-PAGE. As expected, the Env trimer of wild-type ADA dissociated on heat treatment at a temperature slightly above the T90 of ADA (Figure 13A and B) [7]. In contrast, the Env trimer of comb-mut was much more resistant to this treatment and did not significantly dissociate until an incubation temperature of 63.6°C. The observed increase in oligomeric stability of mutant Env trimers might be due at least in part to an increase in the level of uncleaved gp160 incorporated into the virus. We therefore analyzed the relative levels of cleaved gp120 and uncleaved gp160 associated with each virus by reducing SDS-PAGE. We observed no effect on cleavage as a result of the stabilizing mutations, as all Env variants appear to be ∼95% cleaved (Figure S2).


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

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

Comb-mut Env spikes exhibit increased oligomeric stability.(A) Wild-type ADA and comb-mut viruses were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The Western blot shown was stained with an anti-gp41 cocktail of mAbs, and similar results were seen using anti-gp120 mAbs. (B) The intensity of the Env trimer bands from each lane in panel A was quantified using ImageJ software and plotted as the percent of the trimer band remaining relative to the corresponding band in the 37°C treated lane. The samples exposed to higher temperatures (58–67°C; the blot on the right for each virus) were run on a separate gel and the band intensities were calculated relative to 37°C treated controls on those gels that are not shown. (C) Wild-type ADA and comb-mut virions were incubated at 37°C with a protease cocktail as in Figure 12D and the oligomeric state of Env was analyzed by BN-PAGE. The Western blot was stained as in panel A. (D) Env trimer band intensities, as quantified for panel B. (E) Wild-type ADA and comb-mut virions were incubated at 37°C for up to 24 hours in 1% DDM detergent. The oligomeric state of DDM-treated Env was subsequently visualized using BN-PAGE. The Western blot was stained as in panel A. (F) Env trimer band intensities, as quantified for panel B.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g013: Comb-mut Env spikes exhibit increased oligomeric stability.(A) Wild-type ADA and comb-mut viruses were subjected to a heat gradient for 1 hour and then visualized using BN-PAGE. The Western blot shown was stained with an anti-gp41 cocktail of mAbs, and similar results were seen using anti-gp120 mAbs. (B) The intensity of the Env trimer bands from each lane in panel A was quantified using ImageJ software and plotted as the percent of the trimer band remaining relative to the corresponding band in the 37°C treated lane. The samples exposed to higher temperatures (58–67°C; the blot on the right for each virus) were run on a separate gel and the band intensities were calculated relative to 37°C treated controls on those gels that are not shown. (C) Wild-type ADA and comb-mut virions were incubated at 37°C with a protease cocktail as in Figure 12D and the oligomeric state of Env was analyzed by BN-PAGE. The Western blot was stained as in panel A. (D) Env trimer band intensities, as quantified for panel B. (E) Wild-type ADA and comb-mut virions were incubated at 37°C for up to 24 hours in 1% DDM detergent. The oligomeric state of DDM-treated Env was subsequently visualized using BN-PAGE. The Western blot was stained as in panel A. (F) Env trimer band intensities, as quantified for panel B.
Mentions: We further examined the direct effect of heat on Env trimer dissociation by comparing wild-type ADA and comb-mut using BN-PAGE. As expected, the Env trimer of wild-type ADA dissociated on heat treatment at a temperature slightly above the T90 of ADA (Figure 13A and B) [7]. In contrast, the Env trimer of comb-mut was much more resistant to this treatment and did not significantly dissociate until an incubation temperature of 63.6°C. The observed increase in oligomeric stability of mutant Env trimers might be due at least in part to an increase in the level of uncleaved gp160 incorporated into the virus. We therefore analyzed the relative levels of cleaved gp120 and uncleaved gp160 associated with each virus by reducing SDS-PAGE. We observed no effect on cleavage as a result of the stabilizing mutations, as all Env variants appear to be ∼95% cleaved (Figure S2).

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