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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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Infectivity of Env-pseudotyped viruses in cells expressing low CD4.Infectivity of Env-pseudotyped viruses was tested in (A) HeLa cells expressing low CD4 (RC49) [48]and (B) monocyte-derived macrophages (MDM). In both cases, effector cells were infected with Env-pseudotyped viruses with equal infectious units and the infectivity was assessed by immunostaining intracellular p24 and expressed as focus forming units (FFU) on Y-axis in a β-galactosidase assay as described earlier [46], [51]. (C). The β-galactosidase positive cells (in blue) representing MDM (from one donor) expressing intracellular p24 were scored as focus forming units (FFU) to assess the relative infectivity. For MDM assays, macrophage tropic pNLAD8 was used as positive. In HeLa (RC49) cells, YU2 and JR-CSF were used as controls.
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pone-0037157-g006: Infectivity of Env-pseudotyped viruses in cells expressing low CD4.Infectivity of Env-pseudotyped viruses was tested in (A) HeLa cells expressing low CD4 (RC49) [48]and (B) monocyte-derived macrophages (MDM). In both cases, effector cells were infected with Env-pseudotyped viruses with equal infectious units and the infectivity was assessed by immunostaining intracellular p24 and expressed as focus forming units (FFU) on Y-axis in a β-galactosidase assay as described earlier [46], [51]. (C). The β-galactosidase positive cells (in blue) representing MDM (from one donor) expressing intracellular p24 were scored as focus forming units (FFU) to assess the relative infectivity. For MDM assays, macrophage tropic pNLAD8 was used as positive. In HeLa (RC49) cells, YU2 and JR-CSF were used as controls.

Mentions: Since Envs expressing H681 showed enhanced sensitivity to sCD4 (Figure 4) and IgG1b12 (Table 1) and showed relatively increased binding with CD4-Ig and b12 over Envs expressing Y681, we hypothesized that H681 possibly modulated Env conformation at CD4 binding site in gp120. The enhanced interaction of Env trimers with CD4-Ig and IgG1b12 implied that Y681H substitution likely induced exposure of CD4bs enabling Envs towards low CD4 dependence for productive entry into the target cells. Thus, to test this, we examined the effect of N668S and H681Y on CD4 dependence of these Envs by infecting Env-pseudotyped viruses carrying both H681 and Y681 in HeLa cells expressing low CD4 but high CCR5 (Clone RC49) [48]. RC49 cells express high amount of CCR5 coreceptor but limiting amount of CD4 and virus infectivity in these cells is a measure of CD4 dependence of Env for entry. As expected, we found that 4.J2 (H681) showed greater infectivity in RC49 cells (Figure 6A) than 4.J2 (Y681) indicating that H681 modulated Env towards greater CD4bs exposure and possibly enhanced binding with CD4. In comparison to H681Y, N668S substitution in 4.J2 Env showed only modest reduction in infectivity of RC49 cells, suggesting H681 predominantly affected CD4bs in Env. Finally, we examined the effect of increased exposure of CD4bs due to presence of H681 in Env-pseudotyped viruses on their degree of infectivity in monocyte-derived macrophages (MDM) (obtained from three different donors) that naturally express low amounts of cell surface CD4 [49], [50]. Thus, MDMs were infected with equal TZM-bl infectious units of 4.J2 (H681) and 4.J2 (Y681) and infectivity measured by immunostaining of p24 positive cells as described before [51]. As shown in Figure 6B and C, 4.J2 expressing H681 showed significant enhanced infectivity in macrophages (P<0.05) compared to 4.J2 expressing Y681. In this assay, YU2 and JR-CSF Envs were used as positive and negative controls respectively [52]. Our data indicated that H681 modulated overall Env conformation that is more suitable for CD4 interaction and thus enhanced infectivity in low CD4 expressing cells.


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)

Infectivity of Env-pseudotyped viruses in cells expressing low CD4.Infectivity of Env-pseudotyped viruses was tested in (A) HeLa cells expressing low CD4 (RC49) [48]and (B) monocyte-derived macrophages (MDM). In both cases, effector cells were infected with Env-pseudotyped viruses with equal infectious units and the infectivity was assessed by immunostaining intracellular p24 and expressed as focus forming units (FFU) on Y-axis in a β-galactosidase assay as described earlier [46], [51]. (C). The β-galactosidase positive cells (in blue) representing MDM (from one donor) expressing intracellular p24 were scored as focus forming units (FFU) to assess the relative infectivity. For MDM assays, macrophage tropic pNLAD8 was used as positive. In HeLa (RC49) cells, YU2 and JR-CSF were used as controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351407&req=5

pone-0037157-g006: Infectivity of Env-pseudotyped viruses in cells expressing low CD4.Infectivity of Env-pseudotyped viruses was tested in (A) HeLa cells expressing low CD4 (RC49) [48]and (B) monocyte-derived macrophages (MDM). In both cases, effector cells were infected with Env-pseudotyped viruses with equal infectious units and the infectivity was assessed by immunostaining intracellular p24 and expressed as focus forming units (FFU) on Y-axis in a β-galactosidase assay as described earlier [46], [51]. (C). The β-galactosidase positive cells (in blue) representing MDM (from one donor) expressing intracellular p24 were scored as focus forming units (FFU) to assess the relative infectivity. For MDM assays, macrophage tropic pNLAD8 was used as positive. In HeLa (RC49) cells, YU2 and JR-CSF were used as controls.
Mentions: Since Envs expressing H681 showed enhanced sensitivity to sCD4 (Figure 4) and IgG1b12 (Table 1) and showed relatively increased binding with CD4-Ig and b12 over Envs expressing Y681, we hypothesized that H681 possibly modulated Env conformation at CD4 binding site in gp120. The enhanced interaction of Env trimers with CD4-Ig and IgG1b12 implied that Y681H substitution likely induced exposure of CD4bs enabling Envs towards low CD4 dependence for productive entry into the target cells. Thus, to test this, we examined the effect of N668S and H681Y on CD4 dependence of these Envs by infecting Env-pseudotyped viruses carrying both H681 and Y681 in HeLa cells expressing low CD4 but high CCR5 (Clone RC49) [48]. RC49 cells express high amount of CCR5 coreceptor but limiting amount of CD4 and virus infectivity in these cells is a measure of CD4 dependence of Env for entry. As expected, we found that 4.J2 (H681) showed greater infectivity in RC49 cells (Figure 6A) than 4.J2 (Y681) indicating that H681 modulated Env towards greater CD4bs exposure and possibly enhanced binding with CD4. In comparison to H681Y, N668S substitution in 4.J2 Env showed only modest reduction in infectivity of RC49 cells, suggesting H681 predominantly affected CD4bs in Env. Finally, we examined the effect of increased exposure of CD4bs due to presence of H681 in Env-pseudotyped viruses on their degree of infectivity in monocyte-derived macrophages (MDM) (obtained from three different donors) that naturally express low amounts of cell surface CD4 [49], [50]. Thus, MDMs were infected with equal TZM-bl infectious units of 4.J2 (H681) and 4.J2 (Y681) and infectivity measured by immunostaining of p24 positive cells as described before [51]. As shown in Figure 6B and C, 4.J2 expressing H681 showed significant enhanced infectivity in macrophages (P<0.05) compared to 4.J2 expressing Y681. In this assay, YU2 and JR-CSF Envs were used as positive and negative controls respectively [52]. Our data indicated that H681 modulated overall Env conformation that is more suitable for CD4 interaction and thus enhanced infectivity in low CD4 expressing cells.

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