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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

Env stability is inversely correlated with LAI-derived gp120 amino acid residues in HIV-1 variants.The T90 of each clone is plotted against the number of gp120 residues in each clone derived from LAI rather than ADA. Data for GB21-6, which contains LAI gp41 and ADA gp120, and that of LAI are also plotted and are the most and least stable viruses, respectively. The inverse correlation is statistically significant (p = 0.0009, F test).
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ppat-1003184-g004: Env stability is inversely correlated with LAI-derived gp120 amino acid residues in HIV-1 variants.The T90 of each clone is plotted against the number of gp120 residues in each clone derived from LAI rather than ADA. Data for GB21-6, which contains LAI gp41 and ADA gp120, and that of LAI are also plotted and are the most and least stable viruses, respectively. The inverse correlation is statistically significant (p = 0.0009, F test).

Mentions: Sequencing of Env from the GuHCl-stable and heat-stable library pools, G and HB2, surprisingly revealed mutations to residues in common with the LAI strain that was used in engineering the display vector, pLAI (Figure 3B and 3C). In these clones, recombination events involving a very small amount of LAI DNA appear to have occurred during the PCR used in library production. Importantly, all of the selected clones were more stable than either the parental strain, ADA, or the LAI strain (Figure 3C). Multiple recombination events appear to have taken place during the selection process resulting in virions containing different amounts of LAI-derived gp120 sequence, and among the most stable clones, LAI-derived sequence from gp41, but not from gp120, was associated with improved Env stability. Thus, the presence of LAI gp120 was significantly inversely correlated with Env stability (Figure 4) and the most stable clone (GB21-6) contained full LAI gp41 and ADA gp120. In total, 9 amino acid residues in the gp41 ectodomain differ between ADA and LAI (Figure 3). In addition, a tenth mutation, K574R, arose de novo and was conserved amongst the stable clones.


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

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

Env stability is inversely correlated with LAI-derived gp120 amino acid residues in HIV-1 variants.The T90 of each clone is plotted against the number of gp120 residues in each clone derived from LAI rather than ADA. Data for GB21-6, which contains LAI gp41 and ADA gp120, and that of LAI are also plotted and are the most and least stable viruses, respectively. The inverse correlation is statistically significant (p = 0.0009, F test).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003184-g004: Env stability is inversely correlated with LAI-derived gp120 amino acid residues in HIV-1 variants.The T90 of each clone is plotted against the number of gp120 residues in each clone derived from LAI rather than ADA. Data for GB21-6, which contains LAI gp41 and ADA gp120, and that of LAI are also plotted and are the most and least stable viruses, respectively. The inverse correlation is statistically significant (p = 0.0009, F test).
Mentions: Sequencing of Env from the GuHCl-stable and heat-stable library pools, G and HB2, surprisingly revealed mutations to residues in common with the LAI strain that was used in engineering the display vector, pLAI (Figure 3B and 3C). In these clones, recombination events involving a very small amount of LAI DNA appear to have occurred during the PCR used in library production. Importantly, all of the selected clones were more stable than either the parental strain, ADA, or the LAI strain (Figure 3C). Multiple recombination events appear to have taken place during the selection process resulting in virions containing different amounts of LAI-derived gp120 sequence, and among the most stable clones, LAI-derived sequence from gp41, but not from gp120, was associated with improved Env stability. Thus, the presence of LAI gp120 was significantly inversely correlated with Env stability (Figure 4) and the most stable clone (GB21-6) contained full LAI gp41 and ADA gp120. In total, 9 amino acid residues in the gp41 ectodomain differ between ADA and LAI (Figure 3). In addition, a tenth mutation, K574R, arose de novo and was conserved amongst the stable clones.

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