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

Identification of mutations responsible for the increased functional stability of Env.Individual Envs from (A) HC11, (B) GB2, and (C) HB2 stability-selected pools were sequenced. For each variant, the relevant numerical measurement of stability (T90 (i.e. the temperature at which viral infectivity is diminished by 90% in one hour) for heat and IC50 for GuHCl+/− SEM), the corresponding increase in stability relative to wild-type ADA, and the presence (+) or absence (−) of a given mutation is indicated. Mutations that arose de novo are shown in blue and mutations derived from the HIV-1 isolate LAI are indicated in red. Shown also in (B) and (C) are the numbers of residues in gp120 of each variant that originated from LAI rather than ADA and that differ from ADA. V1alt in (A) refers to the combinatorial deletion of amino acids N139 and I140 and the substitution N142S.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g003: Identification of mutations responsible for the increased functional stability of Env.Individual Envs from (A) HC11, (B) GB2, and (C) HB2 stability-selected pools were sequenced. For each variant, the relevant numerical measurement of stability (T90 (i.e. the temperature at which viral infectivity is diminished by 90% in one hour) for heat and IC50 for GuHCl+/− SEM), the corresponding increase in stability relative to wild-type ADA, and the presence (+) or absence (−) of a given mutation is indicated. Mutations that arose de novo are shown in blue and mutations derived from the HIV-1 isolate LAI are indicated in red. Shown also in (B) and (C) are the numbers of residues in gp120 of each variant that originated from LAI rather than ADA and that differ from ADA. V1alt in (A) refers to the combinatorial deletion of amino acids N139 and I140 and the substitution N142S.

Mentions: To understand the basis of the hyperstable phenotypes of the rescued mutant clones, we determined the primary Env sequences. Mutations were identified, remarkably, only in regions of ADA Env not targeted by our mutagenesis procedure, most likely because the targeted mutations negatively impacted infectivity and any mutants were out-competed by the small number of virions incorporating mostly wild-type Env sequence. From the heat-stable library pool HC11, all of the clones contained the substitution S649A in the CHR region of gp41, whereas some but not all clones contained two substitutions in gp120 C5 (S462G and D474N) and an alteration to gp120 V1 (i.e. deletion of N139/I140 plus an N142S substitution that will hereby be referred to as “V1alt”). These mutations in the HC11 clones appeared to arise de novo. (Figure 3A).


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

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

Identification of mutations responsible for the increased functional stability of Env.Individual Envs from (A) HC11, (B) GB2, and (C) HB2 stability-selected pools were sequenced. For each variant, the relevant numerical measurement of stability (T90 (i.e. the temperature at which viral infectivity is diminished by 90% in one hour) for heat and IC50 for GuHCl+/− SEM), the corresponding increase in stability relative to wild-type ADA, and the presence (+) or absence (−) of a given mutation is indicated. Mutations that arose de novo are shown in blue and mutations derived from the HIV-1 isolate LAI are indicated in red. Shown also in (B) and (C) are the numbers of residues in gp120 of each variant that originated from LAI rather than ADA and that differ from ADA. V1alt in (A) refers to the combinatorial deletion of amino acids N139 and I140 and the substitution N142S.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g003: Identification of mutations responsible for the increased functional stability of Env.Individual Envs from (A) HC11, (B) GB2, and (C) HB2 stability-selected pools were sequenced. For each variant, the relevant numerical measurement of stability (T90 (i.e. the temperature at which viral infectivity is diminished by 90% in one hour) for heat and IC50 for GuHCl+/− SEM), the corresponding increase in stability relative to wild-type ADA, and the presence (+) or absence (−) of a given mutation is indicated. Mutations that arose de novo are shown in blue and mutations derived from the HIV-1 isolate LAI are indicated in red. Shown also in (B) and (C) are the numbers of residues in gp120 of each variant that originated from LAI rather than ADA and that differ from ADA. V1alt in (A) refers to the combinatorial deletion of amino acids N139 and I140 and the substitution N142S.
Mentions: To understand the basis of the hyperstable phenotypes of the rescued mutant clones, we determined the primary Env sequences. Mutations were identified, remarkably, only in regions of ADA Env not targeted by our mutagenesis procedure, most likely because the targeted mutations negatively impacted infectivity and any mutants were out-competed by the small number of virions incorporating mostly wild-type Env sequence. From the heat-stable library pool HC11, all of the clones contained the substitution S649A in the CHR region of gp41, whereas some but not all clones contained two substitutions in gp120 C5 (S462G and D474N) and an alteration to gp120 V1 (i.e. deletion of N139/I140 plus an N142S substitution that will hereby be referred to as “V1alt”). These mutations in the HC11 clones appeared to arise de novo. (Figure 3A).

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