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

Combining stabilizing point mutations from different Env variants produces a hyperstable virus, comb-mut.Point mutations derived from GB21-6 or HC11-1 that were also found to increase stability of ADA (Figure 10) were combined into viruses, Gmut and Hmut, for comparison to the original clones, while all seven stabilizing mutations were combined to produce comb-mut. (A) These viruses with combined Env mutations were tested for resistance to GuHCl (top), heat (center), or 37°C decay (bottom). Results shown are from a representative experiment performed in duplicate. (B) The relative resistance of comb-mut to ligand-induced infectivity decay is shown as a function of the fold decrease in IC50 after 20 hour pre-incubation relative to 1 hour pre-incubation as described in Figure 8. (C) The relative sensitivity of comb-mut to neutralization by a panel of ligands is shown as the ratio of mutant IC50 to wild-type IC50 as described in Figure 8. (D) ADA and comb-mut virions were incubated with a cocktail of proteases (trypsin, chymotrypsin, and proteinase K) for various time points and the remaining infectivity of the samples was determined by infection of TZM-bl cells. The percent infectivity remaining was calculated relative to virus incubated without protease present for the same time periods.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g012: Combining stabilizing point mutations from different Env variants produces a hyperstable virus, comb-mut.Point mutations derived from GB21-6 or HC11-1 that were also found to increase stability of ADA (Figure 10) were combined into viruses, Gmut and Hmut, for comparison to the original clones, while all seven stabilizing mutations were combined to produce comb-mut. (A) These viruses with combined Env mutations were tested for resistance to GuHCl (top), heat (center), or 37°C decay (bottom). Results shown are from a representative experiment performed in duplicate. (B) The relative resistance of comb-mut to ligand-induced infectivity decay is shown as a function of the fold decrease in IC50 after 20 hour pre-incubation relative to 1 hour pre-incubation as described in Figure 8. (C) The relative sensitivity of comb-mut to neutralization by a panel of ligands is shown as the ratio of mutant IC50 to wild-type IC50 as described in Figure 8. (D) ADA and comb-mut virions were incubated with a cocktail of proteases (trypsin, chymotrypsin, and proteinase K) for various time points and the remaining infectivity of the samples was determined by infection of TZM-bl cells. The percent infectivity remaining was calculated relative to virus incubated without protease present for the same time periods.

Mentions: In an attempt to reconstitute the phenotypes of the most stable variants, we combined the stabilizing mutations identified from the GB2 and HC11 viral pools to produce Gmut (I535M, L543Q, K574R and H625N/T626M) and Hmut (S649A and V1alt). We also generated comb-mut, which combines both sets of consensus mutations from the two unrelated stable clones. When challenged with GuHCl, both Gmut and Hmut clearly recapitulated the phenotype of the clones from which they were derived. Notably, comb-mut was even more stable than either variant, being resistant to GuHCl at 2 M (Figure 12A). Similar results were seen with heat treatment, although in this case comb-mut was only slightly more stable than the others. Similarly, following prolonged incubation at 37°C, all of the stabilized mutants showed much improved half-lives compared to wild-type ADA, but no significant differences could be seen between the mutants. Loss of infectivity due to heat or 37°C incubation involves multiple viral components and we have seen that functional Env stability beyond a T90 of ∼50°C or a half-life of ∼20 h at 37°C cannot be quantified under the conditions of the assay [7].


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

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

Combining stabilizing point mutations from different Env variants produces a hyperstable virus, comb-mut.Point mutations derived from GB21-6 or HC11-1 that were also found to increase stability of ADA (Figure 10) were combined into viruses, Gmut and Hmut, for comparison to the original clones, while all seven stabilizing mutations were combined to produce comb-mut. (A) These viruses with combined Env mutations were tested for resistance to GuHCl (top), heat (center), or 37°C decay (bottom). Results shown are from a representative experiment performed in duplicate. (B) The relative resistance of comb-mut to ligand-induced infectivity decay is shown as a function of the fold decrease in IC50 after 20 hour pre-incubation relative to 1 hour pre-incubation as described in Figure 8. (C) The relative sensitivity of comb-mut to neutralization by a panel of ligands is shown as the ratio of mutant IC50 to wild-type IC50 as described in Figure 8. (D) ADA and comb-mut virions were incubated with a cocktail of proteases (trypsin, chymotrypsin, and proteinase K) for various time points and the remaining infectivity of the samples was determined by infection of TZM-bl cells. The percent infectivity remaining was calculated relative to virus incubated without protease present for the same time periods.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g012: Combining stabilizing point mutations from different Env variants produces a hyperstable virus, comb-mut.Point mutations derived from GB21-6 or HC11-1 that were also found to increase stability of ADA (Figure 10) were combined into viruses, Gmut and Hmut, for comparison to the original clones, while all seven stabilizing mutations were combined to produce comb-mut. (A) These viruses with combined Env mutations were tested for resistance to GuHCl (top), heat (center), or 37°C decay (bottom). Results shown are from a representative experiment performed in duplicate. (B) The relative resistance of comb-mut to ligand-induced infectivity decay is shown as a function of the fold decrease in IC50 after 20 hour pre-incubation relative to 1 hour pre-incubation as described in Figure 8. (C) The relative sensitivity of comb-mut to neutralization by a panel of ligands is shown as the ratio of mutant IC50 to wild-type IC50 as described in Figure 8. (D) ADA and comb-mut virions were incubated with a cocktail of proteases (trypsin, chymotrypsin, and proteinase K) for various time points and the remaining infectivity of the samples was determined by infection of TZM-bl cells. The percent infectivity remaining was calculated relative to virus incubated without protease present for the same time periods.
Mentions: In an attempt to reconstitute the phenotypes of the most stable variants, we combined the stabilizing mutations identified from the GB2 and HC11 viral pools to produce Gmut (I535M, L543Q, K574R and H625N/T626M) and Hmut (S649A and V1alt). We also generated comb-mut, which combines both sets of consensus mutations from the two unrelated stable clones. When challenged with GuHCl, both Gmut and Hmut clearly recapitulated the phenotype of the clones from which they were derived. Notably, comb-mut was even more stable than either variant, being resistant to GuHCl at 2 M (Figure 12A). Similar results were seen with heat treatment, although in this case comb-mut was only slightly more stable than the others. Similarly, following prolonged incubation at 37°C, all of the stabilized mutants showed much improved half-lives compared to wild-type ADA, but no significant differences could be seen between the mutants. Loss of infectivity due to heat or 37°C incubation involves multiple viral components and we have seen that functional Env stability beyond a T90 of ∼50°C or a half-life of ∼20 h at 37°C cannot be quantified under the conditions of the assay [7].

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