Here we describe a novel function for lentiviral Nef: it renders the HIV-1 virion refractory to the broadly-neutralizing antibodies 2F5 and 4E10. This effect was extraordinarily specific, and not the result of a global decrease in neutralization sensitivity, since Nef had no effect on sensitivity to nine other, well-characterized Env neutralizing agents. This Nef activity targets a property that is conserved among diverse HIV-1 Envs, irrespective of clade, chemokine receptor preference, or adaptation to tissue culture. Divergent HIV-1 Nef proteins, as well as an SIVAGM Nef that shares <40% amino acid identity with HIV-1 Nef, all have this activity, suggesting that it reflects a core Nef function. Analyses of Nef mutants revealed that the activity on neutralization susceptibility is a novel and yet unreported activity, mechanistically distinct from other Nef activities.
This same specific effect on HIV-1 sensitivity to neutralization by 2F5 and 4E10 was evident with MoMLV Glycogag, indicating that this activity is shared by an unrelated protein encoded by a gammaretrovirus. The TM glycoprotein of gammaretroviruses contains an MPER with clusters of aromatic residues like those in the HIV-1 MPER 
. Additionally, there are reports that the gammaretrovirus MPER is targeted by potent neutralizing antibodies 
. Taken together, these findings suggest that, like lentiviruses, gammaretroviruses encode a protein to protect from similar broadly-acting nAbs that target the MPER of TM.
Though HIV-1 particles pseudotyped with Env glycoproteins from a range of disparate HIV-1 isolates were all equally sensitive to the effect of Nef on neutralization sensitivity, the same virion pseudotypes responded very differently from one another with respect to the enhancing effect of Nef on virion infectivity. In particular, virions bearing EnvJRFL
were fully sensitive to the Nef effect on neutralization but totally unresponsive to the Nef effect on infectivity. Such large Env-dependent variation in the effect of Nef on infectivity has not previously been reported. Given the evidence that virions pseudotyped with virus glycoproteins driving entry to an endocytic compartment are not sensitive to the effect of Nef on infectivity 
, we hypothesize that different HIV-1 Envs could target cell entry of virus particles to different pathways, altering the requirement of Nef for optimal infectivity. Deciphering the primary sequence determinants for Env responsiveness may prove valuable for a better understanding of the mechanism by which Nef promotes virion infectivity. Though the neutralization experiments reported here were initiated to understand how Nef promotes virion infectivity, the lack of correlation indicates that the two phenotypes are independent. This conclusion is also supported by the evidence that several Nef mutants, which lack activity on virus infectivity, retain an unaltered ability to decrease sensitivity to neutralization.
A screen of Nef mutants revealed that Nef myristoylation is required for the activity on neutralization, while mutations impairing the interaction of Nef with some well characterized cellular partners, such as src kinases, adaptor proteins and dynamin 2, had no fundamental effect on this activity. Accordingly, overexpression of dominant-negative dynamin 2 had no effect on the ability of Nef to increase HIV resistance to 2F5 and 4E10 (Figure S7
). We therefore conclude that the effect on neutralization susceptibility is unrelated to other Nef activities.
Deletion of the TMgp41 cytoplasmic tail, and the intracellular trafficking signals that it possesses 
, did not alter the effect of Nef on neutralization sensitivity. This activity, then, is unlikely to involve intracellular redistribution of Env resulting from direct interaction with Nef, or from indirect effects of Nef on cellular signaling components. Additionally, the activity cannot be ascribed to an effect of Nef on Env encapsidation since the level of virion-associated Env was unchanged by Nef. The effect of Nef remained the same in the absence of CD4 expression in producer cells excluding more subtle interference of Env by its cognate receptor.
In response to sequential binding of gp120 to CD4 and chemokine receptors, gp41 undergoes a series of structural changes, first an extended conformation, followed by a fusogenic bundle of six α-helices called the trimer of hairpins. Previous reports proposed that 2F5 and 4E10 bind to the pre-hairpin intermediate with an extended conformation 
. However, Nef had no effect on sensitivity to T20 (), a fusion inhibitor that binds to the prehairpin intermediate 
, or to fusion of cell-free virions with cells 
. Perhaps the most compelling evidence that the extended conformation is not necessary for binding by 2F5 and 4E10 is the experiment presented here showing that these antibodies capture virus particles in the absence of receptor engagement (). These findings are in agreement with recent studies showing that the extent of 2F5 and 4E10 binding to cell-free virions correlates with neutralization 
, and causes gp120 shedding 
. Thus, the data here demonstrate that Nef attenuates the interaction of MPER-specific nAbs with cell-free virions, rather than modulating structural changes that occur subsequent to encounter with cells.
Based on recent structural studies, the MPER epitopes targeted by 2F5 and 4E10 are believed to be partially embedded within the lipid bilayer of the virion 
–. The neutralizing activity of these antibodies is proposed to rely on their ability to interact with membranes 
, and is dependent on the long hydrophobic CDR3 loop 
. This might facilitate their interaction with the lipid bilayer and be instrumental for the ability to dock with the epitope, to extract hydrophobic residues from the lipid environment 
and ultimately contribute to the neutralizing activity 
. In contrast, neutralization with Z13e1 might not require an interaction with lipids 
, in line with evidence that the crucial residues of the epitope are located on the solvent exposed face of MPER 
. Interestingly, several studies have reported a link between Nef and lipid biosynthesis and trafficking. Nef was reported to induce expression of genes involved in cholesterol biosynthesis 
, to reduce cholesterol efflux 
, to enhance the raft-like character of virions via
an increase in their cholesterol content 
and/or a preferential incorporation of sphingomyelin 
, a phospholipid with a neutral head group. The efficiency of the interaction of 2F5 is favored by phospholipids with negatively charged head groups 
, and the association of the MPER with membranes is favored by the presence of sphingomyelin and cholesterol 
. We therefore propose that Nef, by altering the lipid composition of the virion, alters the susceptibility to neutralization by 2F5 and 4E10, either by reducing the preliminary contact of the antibodies with the virus particle, and/or by increasing the strength of the association of the MPER with the viral membrane (), which would make the epitopes less accessible to the antibodies. Supporting this hypothesis, our experiments revealed that the effect on neutralization is totally dependent on Nef myristoylation, which is essential for the localization of Nef into lipid rafts 
and was found to be required for enhanced synthesis and efflux of cholesterol. We found that mutating the cholesterol binding motif, which had been initially linked to an increased cholesterol content of virus particles, does not abrogate the activity on neutralization. However, the role of such a binding motif, however remains unclear, in light of a more recent study 
which failed to confirm its cholesterol binding function.
Model depicting the possible mechanism by which Nef specifically alters the accessibility of 2F5 and 4E10 MPER epitopes to nAbs.
We have observed variability of the magnitude of the Nef activity on neutralization when screening virus produced by different cell lines, observing that virus generated by HEK293T cells is only minimally responsive. Interestingly, it has been recently reported that the lipid composition of cellular membranes and viruses derived from different cell lines can vary significantly. The composition of the cell membrane isolated from HEK293T and from the T-cell line MT4, as well as the lipid pool of progeny viruses derived from these cell lines were found to vary significantly in their sphingomyelin content 
. It is therefore plausible that cell-type specific variabilities of the membrane lipid composition modulate the responsiveness of the progeny virus to the effect of Nef on neutralization.
The most potent monoclonal antibodies targeting Env have been cloned from bone marrow and B-cells of HIV-1 infected patients, and were instrumental in identifying crucial antibody specificities associated with protection. Within gp120, these antibodies were found to target the CD4 binding site (e.g. b12 and VRC01 
), CD4 inducible epitopes (e.g. 17B 
and E51 
), a carbohydrate dependent epitope (2G12 
) and a quaternary structure-dependent epitope comprising the V2 and V3 loops (e.g. PG16) 
. Within gp41, antibodies targeting the membrane proximal external region (MPER) were found to be the most potent and broadly neutralizing, including the monoclonal antibodies 2F5 and 4E10 
and Z13e1 
. The use of MPER as an antigen to induce a protective immune response in vivo
has therefore been widely tested 
. However, MPER-specific neutralizing antibodies are only rarely found in HIV-1 infected subjects 
and different immunization strategies using the MPER have failed to induce significant neutralizing immunity 
. This could be the result of limited accessibility of nAbs to such native epitopes which are located at the interface with the retroviral membrane. By favoring the interaction of MPER with the membrane Nef might contribute to hiding these crucial viral epitopes from the humoral immune response. Long-term nonprogression to AIDS has been reported in people infected with Nef-defective HIV-1 
. It would be of interest to determine if the strong, broadly neutralizing antibody responses observed in some of these individuals 
are caused by MPER targeting antibodies.