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Association of enhanced HIV-1 neutralization by a single Y681H substitution in gp41 with increased gp120-CD4 interaction and macrophage infectivity.

Ringe R, Bhattacharya J - PLoS ONE (2012)

Bottom Line: In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies.Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes.In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, National AIDS Research Institute, Indian Council of Medical Research, Bhosari, Pune, India.

ABSTRACT
HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

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gp120 shedding assay.(A) The CD4-induced gp120 shedding assay was done by Western blot analysis of Envs obtained from supernatants of 293T cells transfected with the plasmid DNA encoding 4.J2 and YU2 Env variants as well as JRFL Env. HIV-1 gp120 shedding upon binding of sCD4 and VRC01 from the Env trimers on the surface of 293T cells was assessed by probing with the anti-gp120 human monoclonal antibodies (VRC01, 447-52D, 3074). (B). gp120 shedding of viral variants with respect to time. Env-pseudotyped viruses harvested from transfected 293T cells were incubated for indicated times at 37°C. The particle associated gp120 shedding was assessed by calculating the ratio of infectivity in TZM-bl cells and p24 (left panel) and ratio of band intensities of gp120 and p24 by densitometric analysis (right panel).
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pone-0037157-g007: gp120 shedding assay.(A) The CD4-induced gp120 shedding assay was done by Western blot analysis of Envs obtained from supernatants of 293T cells transfected with the plasmid DNA encoding 4.J2 and YU2 Env variants as well as JRFL Env. HIV-1 gp120 shedding upon binding of sCD4 and VRC01 from the Env trimers on the surface of 293T cells was assessed by probing with the anti-gp120 human monoclonal antibodies (VRC01, 447-52D, 3074). (B). gp120 shedding of viral variants with respect to time. Env-pseudotyped viruses harvested from transfected 293T cells were incubated for indicated times at 37°C. The particle associated gp120 shedding was assessed by calculating the ratio of infectivity in TZM-bl cells and p24 (left panel) and ratio of band intensities of gp120 and p24 by densitometric analysis (right panel).

Mentions: The possibility of Y681H substitution to reduce the subunit (gp120 and gp41) association on the viral membrane and spontaneous shedding of gp120 from the Env trimer was also considered and examined to explain its contribution in neutralization. To test this, we selected 4.J2, 4.J2 (H681Y), YU2 and YU2 (Y681H) and JRFL as control. We examined both CD4-induced shedding as well as spontaneous shedding with respect of time. Cell free viral variants were first quantitated by p24 antigen ELISA and normalized for quantitative assessment of dissociated gp120 in Western blot analysis. To test CD4-incuded gp120 shedding, 293T cells expressing Envs were treated with different concentrations of sCD4 and VRC01 MAb at 72 hours; cell supernatants were collected subsequently and run in 8% SDS-PAGE as described by Li et al[55]. As shown in Figure 7A, the shedding induced by sCD4 at various concentrations of sCD4 was found to be comparable between Envs expressing Y681 and H681. VRC01 MAb was taken as a negative control inducer of gp120 shedding as recently shown by Li et al.[55]. Furthermore, to evaluate time-dependent spontaneous gp120 shedding, we examined loss of infectivity and virus particle-associated gp120 and p24. Thus, cell supernatants containing Env-pseudotyped viruses were incubated at 37°C for different indicated times and while infectivity was assessed in TZM-bl cells, p24 was measured by ELISA. Virus containing supernatants collected at indicated times were pelleted in an ultracentrifuge and gp120 and p24 resolved in 10% SDS-PAGE under denaturing conditions. As shown in Figure 7B, the shedding of gp120 with respect to time was found to be comparable between Env-pseudotyped viruses expressing H681 and Y681. Thus, our data indicated that the Y681H substitution had no impact on the association of gp120 and gp41 subunits in the Env trimers and did not contribute to the increased neutralization sensitivity.


Association of enhanced HIV-1 neutralization by a single Y681H substitution in gp41 with increased gp120-CD4 interaction and macrophage infectivity.

Ringe R, Bhattacharya J - PLoS ONE (2012)

gp120 shedding assay.(A) The CD4-induced gp120 shedding assay was done by Western blot analysis of Envs obtained from supernatants of 293T cells transfected with the plasmid DNA encoding 4.J2 and YU2 Env variants as well as JRFL Env. HIV-1 gp120 shedding upon binding of sCD4 and VRC01 from the Env trimers on the surface of 293T cells was assessed by probing with the anti-gp120 human monoclonal antibodies (VRC01, 447-52D, 3074). (B). gp120 shedding of viral variants with respect to time. Env-pseudotyped viruses harvested from transfected 293T cells were incubated for indicated times at 37°C. The particle associated gp120 shedding was assessed by calculating the ratio of infectivity in TZM-bl cells and p24 (left panel) and ratio of band intensities of gp120 and p24 by densitometric analysis (right panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351407&req=5

pone-0037157-g007: gp120 shedding assay.(A) The CD4-induced gp120 shedding assay was done by Western blot analysis of Envs obtained from supernatants of 293T cells transfected with the plasmid DNA encoding 4.J2 and YU2 Env variants as well as JRFL Env. HIV-1 gp120 shedding upon binding of sCD4 and VRC01 from the Env trimers on the surface of 293T cells was assessed by probing with the anti-gp120 human monoclonal antibodies (VRC01, 447-52D, 3074). (B). gp120 shedding of viral variants with respect to time. Env-pseudotyped viruses harvested from transfected 293T cells were incubated for indicated times at 37°C. The particle associated gp120 shedding was assessed by calculating the ratio of infectivity in TZM-bl cells and p24 (left panel) and ratio of band intensities of gp120 and p24 by densitometric analysis (right panel).
Mentions: The possibility of Y681H substitution to reduce the subunit (gp120 and gp41) association on the viral membrane and spontaneous shedding of gp120 from the Env trimer was also considered and examined to explain its contribution in neutralization. To test this, we selected 4.J2, 4.J2 (H681Y), YU2 and YU2 (Y681H) and JRFL as control. We examined both CD4-induced shedding as well as spontaneous shedding with respect of time. Cell free viral variants were first quantitated by p24 antigen ELISA and normalized for quantitative assessment of dissociated gp120 in Western blot analysis. To test CD4-incuded gp120 shedding, 293T cells expressing Envs were treated with different concentrations of sCD4 and VRC01 MAb at 72 hours; cell supernatants were collected subsequently and run in 8% SDS-PAGE as described by Li et al[55]. As shown in Figure 7A, the shedding induced by sCD4 at various concentrations of sCD4 was found to be comparable between Envs expressing Y681 and H681. VRC01 MAb was taken as a negative control inducer of gp120 shedding as recently shown by Li et al.[55]. Furthermore, to evaluate time-dependent spontaneous gp120 shedding, we examined loss of infectivity and virus particle-associated gp120 and p24. Thus, cell supernatants containing Env-pseudotyped viruses were incubated at 37°C for different indicated times and while infectivity was assessed in TZM-bl cells, p24 was measured by ELISA. Virus containing supernatants collected at indicated times were pelleted in an ultracentrifuge and gp120 and p24 resolved in 10% SDS-PAGE under denaturing conditions. As shown in Figure 7B, the shedding of gp120 with respect to time was found to be comparable between Env-pseudotyped viruses expressing H681 and Y681. Thus, our data indicated that the Y681H substitution had no impact on the association of gp120 and gp41 subunits in the Env trimers and did not contribute to the increased neutralization sensitivity.

Bottom Line: In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies.Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes.In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, National AIDS Research Institute, Indian Council of Medical Research, Bhosari, Pune, India.

ABSTRACT
HIV-1 variants that show unusual sensitivity to autologous antibodies due to presence of critical neutralization signatures would likely contribute towards rational envelope based HIV-1 vaccine design. In the present study, we found that presence of a naturally occurring H681 in gp41 membrane proximal external region (MPER) of a clade C envelope (Env) obtained from a recently infected Indian patient conferred increased sensitivity to autologous and heterologous plasma antibodies. Furthermore, Env-pseudotyped viruses expressing H681 showed increased sensitivity to soluble CD4, b12 and 4E10 monoclonal antibodies both in related and unrelated Envs and was corroborated with increased Env susceptibility and binding to cellular CD4 as well as with prolonged exposure of MPER epitopes. The increased gp120-CD4 interaction was further associated with relative exposure of CD4-induced epitopes and macrophage infectivity. In summary, our data indicate that Y681H substitution exposes neutralizing epitopes in CD4bs and MPER towards comprehensive interference in HIV-1 entry.

Show MeSH
Related in: MedlinePlus