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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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Stable HIV-1 Env mutants are resistant to ligand destabilization in infectivity assays.Wild type HIV-1 ADA and corresponding mutants were treated with the indicated neutralizing mAbs or inhibitors for either one hour or 20 hours, overlaid on TZM-bl target cells, and luciferase activity was measured 48 hours later. PF-348089 is an analogue of the inhibitor BMS-378806. IC50s were calculated from experiments performed in triplicate. (A) Plotted on the Y-axis for each ligand is the ratio of IC50 following a 20 hour pre-incubation period relative to a 1 hour pre-incubation period. (B) Plotted on the Y-axis is the ratio of IC50 of the stable Env mutant relative to wild-type ADA (mutant IC50/wild-type IC50) for each ligand using a 1 hour pre-incubation period.
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ppat-1003184-g008: Stable HIV-1 Env mutants are resistant to ligand destabilization in infectivity assays.Wild type HIV-1 ADA and corresponding mutants were treated with the indicated neutralizing mAbs or inhibitors for either one hour or 20 hours, overlaid on TZM-bl target cells, and luciferase activity was measured 48 hours later. PF-348089 is an analogue of the inhibitor BMS-378806. IC50s were calculated from experiments performed in triplicate. (A) Plotted on the Y-axis for each ligand is the ratio of IC50 following a 20 hour pre-incubation period relative to a 1 hour pre-incubation period. (B) Plotted on the Y-axis is the ratio of IC50 of the stable Env mutant relative to wild-type ADA (mutant IC50/wild-type IC50) for each ligand using a 1 hour pre-incubation period.

Mentions: If an inhibitor binds and inactivates Env, the IC50 of that inhibitor is expected to decrease over time [37]. In a second approach to assay ligand-induced Env destabilization, GB21-6 and HC11-1 viruses were pre-incubated with mAbs or inhibitors for two different periods of time prior to measuring viral infectivity on target cells. Of the inhibitors we tested, mAbs b12 (anti-CD4 binding site; CD4bs), 4E10 and 2F5 (anti-membrane proximal external region; MPER), as well as sCD4 have all been shown to destabilize Env trimers [36], [37]; mAb VRC01 (anti-CD4bs) has a much weaker destabilizing effect [40]; mAb PG9 (anti-V2/V3) has not been well studied in this context [41]; C34 (anti-NHR) should have no activity towards unliganded Env as it only binds Env post-CD4 engagement [42]; and PF-348089 is an analogue of BMS-378806 that binds to gp120 and prevents CD4-induced conformational changes [43], [44]. Consistent with the VCA results above, both GB21-6 and HC11-1 resisted inactivation by sCD4; similar resistance was also observed using b12, 4E10 and 2F5 (Figure 8A; Table S1). Thus, whereas the IC50s of these inhibitors against wild-type ADA decreased 9–15-fold from the first (1 h) to the second (20 h) pre-incubation time point, the IC50 decreases with GB21-6 and HC11-1 were only 3–6-fold and 4–8-fold, respectively. Notably, VRC01 affected all three viruses equivalently. PG9 inactivated both wild-type ADA and clone GB21-6 with IC50 decreases of ∼10-fold between pre-incubation times. In contrast, HC11-1 resisted PG9 inactivation, but this clone contains an alteration in V1 that may directly affect the PG9 epitope. Finally and as expected, C34 and PF-348089 did not inactivate any of the viruses over time.


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

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

Stable HIV-1 Env mutants are resistant to ligand destabilization in infectivity assays.Wild type HIV-1 ADA and corresponding mutants were treated with the indicated neutralizing mAbs or inhibitors for either one hour or 20 hours, overlaid on TZM-bl target cells, and luciferase activity was measured 48 hours later. PF-348089 is an analogue of the inhibitor BMS-378806. IC50s were calculated from experiments performed in triplicate. (A) Plotted on the Y-axis for each ligand is the ratio of IC50 following a 20 hour pre-incubation period relative to a 1 hour pre-incubation period. (B) Plotted on the Y-axis is the ratio of IC50 of the stable Env mutant relative to wild-type ADA (mutant IC50/wild-type IC50) for each ligand using a 1 hour pre-incubation period.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g008: Stable HIV-1 Env mutants are resistant to ligand destabilization in infectivity assays.Wild type HIV-1 ADA and corresponding mutants were treated with the indicated neutralizing mAbs or inhibitors for either one hour or 20 hours, overlaid on TZM-bl target cells, and luciferase activity was measured 48 hours later. PF-348089 is an analogue of the inhibitor BMS-378806. IC50s were calculated from experiments performed in triplicate. (A) Plotted on the Y-axis for each ligand is the ratio of IC50 following a 20 hour pre-incubation period relative to a 1 hour pre-incubation period. (B) Plotted on the Y-axis is the ratio of IC50 of the stable Env mutant relative to wild-type ADA (mutant IC50/wild-type IC50) for each ligand using a 1 hour pre-incubation period.
Mentions: If an inhibitor binds and inactivates Env, the IC50 of that inhibitor is expected to decrease over time [37]. In a second approach to assay ligand-induced Env destabilization, GB21-6 and HC11-1 viruses were pre-incubated with mAbs or inhibitors for two different periods of time prior to measuring viral infectivity on target cells. Of the inhibitors we tested, mAbs b12 (anti-CD4 binding site; CD4bs), 4E10 and 2F5 (anti-membrane proximal external region; MPER), as well as sCD4 have all been shown to destabilize Env trimers [36], [37]; mAb VRC01 (anti-CD4bs) has a much weaker destabilizing effect [40]; mAb PG9 (anti-V2/V3) has not been well studied in this context [41]; C34 (anti-NHR) should have no activity towards unliganded Env as it only binds Env post-CD4 engagement [42]; and PF-348089 is an analogue of BMS-378806 that binds to gp120 and prevents CD4-induced conformational changes [43], [44]. Consistent with the VCA results above, both GB21-6 and HC11-1 resisted inactivation by sCD4; similar resistance was also observed using b12, 4E10 and 2F5 (Figure 8A; Table S1). Thus, whereas the IC50s of these inhibitors against wild-type ADA decreased 9–15-fold from the first (1 h) to the second (20 h) pre-incubation time point, the IC50 decreases with GB21-6 and HC11-1 were only 3–6-fold and 4–8-fold, respectively. Notably, VRC01 affected all three viruses equivalently. PG9 inactivated both wild-type ADA and clone GB21-6 with IC50 decreases of ∼10-fold between pre-incubation times. In contrast, HC11-1 resisted PG9 inactivation, but this clone contains an alteration in V1 that may directly affect the PG9 epitope. Finally and as expected, C34 and PF-348089 did not inactivate any of the viruses over time.

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