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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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Identification of stable clones from virion pools.(A–D) Stable Env cDNAs were rescued using RT-PCR on pooled viral RNA. Variant Env clones derived from the heat-selected B2 (A; HB2) and C11 (B; HC11) virion pools, the GuHCl-selected B2 pools (C; GB2), and the 37°C-selected M1 pool (D; PM1) were tested for resistance to the destabilizing treatment with which they were selected alongside the originating virion pool. (E and F) Stable HIV-1 variants identified via limiting dilution. Polyclonal pools of virus derived from the 37°C-selected M1 (E; PM1) and C12 (F; PC12) virion pools were subjected to limiting dilution and individual clonal/oligoclonal viruses were tested for infectivity decay at 37°C.
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ppat-1003184-g002: Identification of stable clones from virion pools.(A–D) Stable Env cDNAs were rescued using RT-PCR on pooled viral RNA. Variant Env clones derived from the heat-selected B2 (A; HB2) and C11 (B; HC11) virion pools, the GuHCl-selected B2 pools (C; GB2), and the 37°C-selected M1 pool (D; PM1) were tested for resistance to the destabilizing treatment with which they were selected alongside the originating virion pool. (E and F) Stable HIV-1 variants identified via limiting dilution. Polyclonal pools of virus derived from the 37°C-selected M1 (E; PM1) and C12 (F; PC12) virion pools were subjected to limiting dilution and individual clonal/oligoclonal viruses were tested for infectivity decay at 37°C.

Mentions: To identify individual Env mutants of increased stability, viral RNA was purified from the stability-enriched pools and env was amplified using RT-PCR. Only the ectodomain portion of Env was subcloned back into the pLAI display vector in order to rule out mutations in the gp41 TM and cytoplasmic tail (CT) domains that might affect interactions below the viral membrane, such as between the gp41 CT and Gag [35], and would add further complexity to the analysis. Individual Env clones were picked from each pool and the corresponding virions were assayed for resistance to each of the selection conditions (Figure 2). A variety of stability phenotypes were observed. Notably, some clones from the GuHCl and heat-treated pools fully recapitulated the stability phenotype of the originating viral populations (Figure 2A, B, and C).


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

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

Identification of stable clones from virion pools.(A–D) Stable Env cDNAs were rescued using RT-PCR on pooled viral RNA. Variant Env clones derived from the heat-selected B2 (A; HB2) and C11 (B; HC11) virion pools, the GuHCl-selected B2 pools (C; GB2), and the 37°C-selected M1 pool (D; PM1) were tested for resistance to the destabilizing treatment with which they were selected alongside the originating virion pool. (E and F) Stable HIV-1 variants identified via limiting dilution. Polyclonal pools of virus derived from the 37°C-selected M1 (E; PM1) and C12 (F; PC12) virion pools were subjected to limiting dilution and individual clonal/oligoclonal viruses were tested for infectivity decay at 37°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585149&req=5

ppat-1003184-g002: Identification of stable clones from virion pools.(A–D) Stable Env cDNAs were rescued using RT-PCR on pooled viral RNA. Variant Env clones derived from the heat-selected B2 (A; HB2) and C11 (B; HC11) virion pools, the GuHCl-selected B2 pools (C; GB2), and the 37°C-selected M1 pool (D; PM1) were tested for resistance to the destabilizing treatment with which they were selected alongside the originating virion pool. (E and F) Stable HIV-1 variants identified via limiting dilution. Polyclonal pools of virus derived from the 37°C-selected M1 (E; PM1) and C12 (F; PC12) virion pools were subjected to limiting dilution and individual clonal/oligoclonal viruses were tested for infectivity decay at 37°C.
Mentions: To identify individual Env mutants of increased stability, viral RNA was purified from the stability-enriched pools and env was amplified using RT-PCR. Only the ectodomain portion of Env was subcloned back into the pLAI display vector in order to rule out mutations in the gp41 TM and cytoplasmic tail (CT) domains that might affect interactions below the viral membrane, such as between the gp41 CT and Gag [35], and would add further complexity to the analysis. Individual Env clones were picked from each pool and the corresponding virions were assayed for resistance to each of the selection conditions (Figure 2). A variety of stability phenotypes were observed. Notably, some clones from the GuHCl and heat-treated pools fully recapitulated the stability phenotype of the originating viral populations (Figure 2A, B, and C).

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