Figure 1.
Nef increases HIV-1NL4-3 resistance to 2F5 and 4E10 but not to other neutralizing agents targeting Env.
Neutralization of wild-type and Nef-defective HIV-1NL4-3 by the indicated neutralizing agents targeting gp120 (A) or gp41 (B). The residual infectivity is relative to that of untreated viruses considered as 100%. C, Fold-change of IC50 values, derived from the fitted sigmoidal curves, caused by Nef. Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 2.
Nef does not alter the efficiency of Env incorporation into virus particles.
A. Western blotting of pelleted wild-type and Nef-defective HIV-1NL4-3 virus particles and producer cells extracts (Jurkat), showing abundance of gp120, gp41 and Gag. A sample expressing an env-defective HIV-1NL4-3 (no Env) and a sample expressing a gag-defective provirus construct (no Gag) were used to control the assay specificity. B. Quantitative gp120 Elisa of the same samples shown in A. Results show average and standard deviation of triplicate determinations.
Figure 3.
Virus particles pseudotyped with Env Glycoproteins derived from diverse HIV-1 isolates are responsive to the Nef effect on the susceptibility to 2F5 and 4E10.
Neutralization of Nef-positive and Nef-defective HIV-1NL4-3 virus particles pseudotyped with Env glycoproteins derived from HIV-1JRFL (A), from HIV-1SF162 (B) and from two HIV-1 subgroup C isolates (C). Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 4.
The effect of Nef on neutralization does not depend on the effect of Nef on infectivity.
A, fold-increase of infectivity caused by Nef on HIV-1NL4-3 pesudotyped with Env glycoproteins derived from the indicated HIV-1 isolates, measured on TZM-bl reporter cells. B, fold-increase of 4E10 IC50 for the same viral pseudotypes, derived from the experiments shown in Figure 3, obtained by dividing the IC50 of the Nef-positive virus with that of the Nef-defective virus.
Figure 5.
The effect of Nef on neutralization does not depend on the presence of CD4 in producer cells and is observed with HIV-1 derived from various cell lines.
A, neutralization of wild-type and Nef-defective HIV-1NL4-3 produced in two CD4-negative T lymphoid cell lines. B, Neutralization of wild type and Nef-defective NL4-3 pseudotyped with EnvJRFL produced in the indicated cell lines. C, Fold-change of IC50 values for 4E10 and 2F5, derived from the fitted sigmoidal curves shown in B, caused by Nef. Neutralization was performed three times independently. Shown are the mean values and SD. The significance of the differences of IC50 values were assessed by 2-tail Mann-Whitney test, which retrieved for all samples p<0.05.
Figure 6.
Nef alters the neutralization sensitivity of virus derived from primary cells.
Neutralization of wild-type and Nef-defective HIV-1NL4-3 pseudotyped with JRFL Env produced in PBMC from three different donors, with the indicated nAbs. Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 7.
The effect of Nef on neutralization is conserved among different Nef alleles.
Neutralizion of HIV-1 HXB2 by 2F5 (A) and neutralization of HIV-1NL4-3 pseudotyped with EnvJRFL neutralized by 4E10 (B). IC50 derived from the sigmoidal curves are shown on the right. As indicated, nef alleles were expressed in trans in producer cells. Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 8.
MoMLV Glycogag, like Nef, increases HIV-1 resistance to 2F5 and 4E10.
Comparison of the activity of Nef and MLV Glycogag on HIV-1 neutralization by MPER targeting nAbs (A) and IC50 values (B). Nef-positive and Nef-defective HIV-1NL4-3 pseudotyped with EnvJRFL were neutralized by 2F5 and 4E10. As indicated, Glycosylated Gag or an empty vector control were expressed in trans in producer cells. Neutralization was performed three times independently. Shown are the mean values and SD.
Table 1.
Properties of the Nef mutants analyzed in this study.
Figure 9.
Nef myristoylation is required for its activity on neutralization.
IC50 of 2F5 and 4E10 for HIV-1NL4-3 pseudotyped with EnvJRFL and carrying the indicated Nef mutations. Values were derived from the sigmoidal curves shown in Figure S6. Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 10.
The effect of Nef on neutralization does not depend on the cytoplasmic tail of HIV-1 Env.
Neutralization of wild type and Nef-defective HIV-1NL4-3 virus particles pseudotyped with EnvJRFL mutants lacking the cytoplasmic tail, by 2G12 and 4E10 (A). Fold-increase of IC50 caused by Nef (B). Neutralization was performed three times independently. Shown are the mean values and SD.
Figure 11.
Nef and MoMLV Glycogag specifically alter the capture efficiency of HIV-1 by 2F5 and 4E10.
EnvJRFL virus pseudotypes (Env+) captured by magnetic beads conjugated with the indicated antibodies, targeting gp120 (A) or gp41 (B), in the presence or absence of Nef, quantified by RT activity and expressed as µU of HIV-1 RT. C, direct comparison of the abilities of Nef and Glycogag to alter virus capture by 2F5, 4E10 and 2G12. The specificity of the virus capture was assessed by measuring capture of Env-defective virus particles (Env-) by magnetic beads conjugated with each antibody, or capture of EnvJRFL pseudotypes by non-conjugated magnetic beads (noAb). Data show average values and standard deviation from triplicate independent captures.
Figure 12.
Model depicting the possible mechanism by which Nef specifically alters the accessibility of 2F5 and 4E10 MPER epitopes to nAbs.
Schematic conformation of the MPER in relation to the viral membrane in the absence (A) or presence (B) of Nef. Nef, by altering the virion lipid composition, increases the strength of the association of the MPER with the viral membrane, decreasing specifically the accessibility of the epitopes for 2F5 and 4E10, but not Z13e1, which is located on the exposed side of the MPER.