The primate lentiviruses have evolved to use at least three different proteins to counteract tetherin; Nef, Vpu and Env 
. Although a number of studies have addressed the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env 
, relatively little is known about the mechanism of tetherin antagonism by Nef–the viral gene product used by most SIVs to counteract the tetherin proteins of their respective hosts. In accordance with the species-dependent activity of Nef in opposing restriction by tetherin 
, we show for the first time that Nef selectively binds to rhesus macaque tetherin, but not to human tetherin. We identify residues in the N-terminus, globular core and flexible loop of Nef that are required for anti-tetherin activity, and demonstrate that, despite substantial overlap with sequences required for CD4 downregulation, tetherin antagonism is genetically separable from this activity, as well as from other Nef functions including MHC class I downregulation and infectivity enhancement. We also show that dominant-negative mutants of AP180 and Dyn2 impair tetherin antagonism by Nef, indicating that this activity is dependent, at least in part, on clathrin-mediated endocytosis.
Co-immunoprecipitation and surface plasmon resonance assays revealed a selective physical interaction between SIV Nef and rhesus tetherin. The specificity of this interaction is determined by binding of the core domain of Nef to the cytoplasmic domain of tetherin, since a truncated form of the SIVmac
239 Nef protein, containing the globular core of the protein, was sufficient for binding to a peptide corresponding to the cytoplasmic domain of rhesus tetherin. However, the affinity of this interaction was low (Kd
~400 µM), suggesting that additional Nef sequences, and perhaps one or more cellular co-factors, contribute to the stability of this interaction in virus-infected cells. In support of this, an analysis of alanine-scanning substitutions identified sequences in the N-terminal, globular core and flexible loop domains of Nef that participate in binding to rhesus tetherin. Although the N-terminal domain and flexible loop were dispensable for binding by SPR, these sequences were required to detect an interaction by co-immunoprecipitation. The contribution of the N-terminus of Nef to interactions with tetherin may reflect an indirect effect on membrane association, since the targeting of Nef to cellular membranes is dependent on the myristyolation of a glycine residue at position 2, and structural studies suggest that the N-terminus of Nef is disordered in the absence of phospholipids 
. Substitutions at positions 116–117, 174–175 in the globular core domain, and positions 181–182, 193–194, 195–196 and 199–200 in the flexible loop region also reduced binding to rhesus tetherin. Since the flexible loop contains a di-leucine and a di-acidic motif (E190
xxxLM and D204
D) required for binding to the AP-2 subunits (α2-σ2 and μ2, respectively) 
, and substitutions in these sites disrupt tetherin antagonism 
, it is conceivable that AP-2 stabilizes the binding of Nef to tetherin. Indeed, Nef was recently shown to form a trimolecular complex with the μ1 subunit of AP-1 to stabilize an otherwise low affinity bimolecular interaction with the cytoplasmic tail of MHC class I molecules 
. In support of a possible trimeric complex with AP-2, Nef and rhesus tetherin both co-immunoprecipitated with the μ2 and α2 subunits of AP-2 
. Taken together, these results suggest a model in which the specificity of SIV Nef for rhesus tetherin is driven by a direct physical interaction between the core domain of Nef and the N-terminus of tetherin, which is stabilized by residues in the N-terminal domain and flexible loop region, either through direct contacts or indirect effects on membrane association and/or the recruitment of additional cellular co-factor(s).
A systematic analysis of alanine-scanning substitutions throughout the SIVmac239 Nef protein identified a total of 43 mutations that impaired anti-tetherin activity. Substitutions in the C-terminal domain were not tested, since deletion of these sequences did not affect tetherin antagonism. Most of the mutations that disrupted the anti-tetherin activity of Nef also disrupted CD4-downregulation, MHC I-downregulation or infectivity enhancement. In some cases, both CD4− and MHC I-downregulation were lost due to effects on the association of Nef with cellular membranes, such as the G2A mutation and probably also the adjacent substitutions at positions 5–6. In other cases, these activities were lost due to a decrease in Nef expression or stability. While this was most evident for the changes at positions 178–179 and 180–181, some of the substitutions in the globular core domain also had partial effects on steady-state levels of Nef that may account for their reduced activity in CD4 and MHC class I downregulation assays. Although there was substantial overlap with sequences required for CD4-downregulation, five mutations were identified that disrupted the anti-tetherin activity of Nef, while retaining nearly wild-type levels of CD4-downregulation, as well as MHC class I-downregulation and infectivity enhancement. These mutations included alanine substitutions at positions 106–107 in the core domain and at positions 181–182, 193–194, 199–200 and 209–210 in the flexible loop region. Thus, tetherin antagonism by Nef is genetically separable from other functional activities of the protein.
In addition to the sequences identified by Zhang et al. 
, we identified residues in the flexible loop region outside of the known AP-2 binding sites that separate tetherin antagonism from CD4-downregulation. Substitutions at positions 181–182 and 199–200 (residues N181
239 Nef) specifically impaired the anti-tetherin activity of Nef without affecting CD4-downregulation. These residues are well conserved among Nef alleles of SIVsmm/mac
and HIV-2 isolates, with identities of 61.1% for N181
, 75% for V182
, 94.4% for Q199
and 84.7% for T200
(Los Alamos database; http://www.hiv.lanl.gov/content/index
). Co-immunoprecipitation assays further demonstrated that these residues contribute to AP-2 binding. Our mutational analysis also identified residues in the N-terminal and globular core domains of Nef that are important for tetherin antagonism. Thus, our results reveal that the anti-tetherin activity of Nef is dependent on complex interactions involving multiple residues in the N-terminus, globular core and the flexible loop regions of the protein.
Nef is a multifunctional accessory protein that interacts with a number of different cellular factors to modulate cellular trafficking 
. Nef reroutes MHC I molecules from the trans
-Golgi network to lysosomes via AP-1 and promotes the internalization and lysosomal degradation of CD4 via AP-2 
. Nef also enhances virus infectivity by an undefined mechanism that depends on a physical interaction with Dyn2 
. We previously demonstrated that Nef downmodulates tetherin from the surface of SIV-infected and transfected cells 
, and this activity was later shown to occur by an AP-2-dependent pathway 
. Our experiments with dominant-negative mutants of AP180 and Dyn2 confirm that the internalization of tetherin by Nef, and the capacity of Nef to rescue virus release in the presence of tetherin, depends, at least in part, on clathrin-mediated endocytosis. A role for Dyn2 was further demonstrated by showing that Dynasore, a chemical inhibitor of dynamin, suppressed wild-type SIV replication to an extent comparable to nef
-deleted SIV under conditions of interferon-induced upregulation of tetherin. Since Dyn2 is also required for Nef-mediated infectivity enhancement, the inhibition of virus replication by Dynasore may reflect an additional effect of this compound on virus infectivity. Although tetherin antagonism and infectivity enhancement are genetically separable, 10 of the 12 Nef mutants that lost the ability to enhance virus infectivity, and to bind to Dyn2, also lost the ability to counteract tetherin. The concordance of these activities suggests that a physical interaction with Dyn2 may be necessary for both Nef functions. However, two of the mutations in the globular core disrupted infectivity enhancement and binding to Dyn2 without impairing tetherin antagonism. Moreover, three of the mutants with impaired infectivity enhancement and binding to Dyn2 (mutants 82–83, 146–147 and 168–169) did not lose binding to tetherin, suggesting that Nef uses distinct protein surfaces to bind to Dyn2 and to tetherin. Therefore, unlike infectivity enhancement, the anti-tetherin activity of Nef does not depend on a physical interaction with Dyn2.
Consistent with previous studies demonstrating the downmodulation of rhesus tetherin by Nef 
, SIV infection resulted in a striking redistribution of tetherin from the plasma membrane to compartments within the cell. An analysis of the distribution of tetherin in SIV-infected cells revealed co-localization with TGN46 and LAMP-1, but not with CD63, suggesting that in the presence of Nef, tetherin accumulates in the trans
-Golgi network and in lysosomes. Localization of tetherin to the trans
-Golgi network in uninfected cells has previously been reported 
. Thus, the contribution of Nef to directing tetherin to that compartment is unclear. The trafficking of tetherin to lysosomes raises the possibility that, similar to the effect of HIV-1 Nef on CD4 and MHC class I molecules 
, SIV Nef may direct rhesus tetherin for lysosomal degradation.
In summary, we show that the mechanism of tetherin antagonism by SIV Nef; (1) involves a direct physical interaction between the core domain of Nef and the cytoplasmic domain of rhesus tetherin, (2) requires sequences throughout the N-terminal, globular core and flexible loop domains, yet is genetically separable from other functional activities of Nef, and (3) depends, at least in part, on clathrin-mediated endocytosis. These results begin to reveal the molecular interactions and cellular pathways by which the majority of the primate lentiviruses counteract the tetherin proteins of their non-human primate hosts.