A transient expression assay in human Jurkat T cells was used to study the effect of SIV Nef and HIV Nef on CXCR4 cell surface expression. Cells were transfected with plasmids expressing Nef and a GFP reporter protein from the same bicistronic transcription unit and CXCR4 expression and GFP expression were detected by flow cytometry. As shown in Fig. , expression of SIVmac239 Nef (SIVmac239 Nef) resulted in a dose-dependent decrease in CXCR4 expression on the cell surface. In contrast, expression of HIV-1 Nef protein encoded by the NA7 allele had a diminished effect on CXCR4 cell surface levels, in agreement with previous observations (29
FIG. 1. SIVmac239 Nef downregulates CXCR4 cell surface expression. (A) Flow cytometry analysis of CXCR4 and GFP expression in Jurkat T cells transiently coexpressing SIVmac239 or HIV-1 NA7 Nef and GFP reporter from bicistronic transcription units (panels 2 and (more ...)
To confirm that CXCR4 is downregulated in virally infected cells, we characterized its expression on the surface of cells expressing the SIVmac239 provirus. As controls, we also measured cell surface expression of CD4, CD3, CD28 and class I MHC, which are all downmodulated by SIVmac239 Nef (3
). To directly visualize the productively infected cells we used viral constructs expressing GFP from an IRES element placed immediately downstream of the nef
gene. The viruses also carried a frameshift mutation in the env
gene to permit only a single infection cycle. Viral stocks pseudotyped with Env protein of the vesicular stomatitis virus (vesicular stomatitis virus G) were produced from HEK 293T cells cotransfected with proviral constructs carrying either the nef
open or nef
-defective reading frame and a plasmid expressing vesicular stomatitis virus G and were then used to infect CD4-positive Jurkat T cells. As shown in Fig. , CXCR4 was strongly downregulated from the surface of cells infected with viruses containing only the intact nef
gene. We also found that SIVmac239 Nef also downregulated rhesus CXCR4 in transient expression assays in monkey CV1 cells (data not shown). We concluded that SIVmac239 Nef strongly downregulates cell surface expression of CXCR4 receptor for SDF-1 chemokine in infected cells.
The observation that SIVmac239 Nef strongly downregulates CXCR4 but HIV-1 NA7 Nef does not suggested that the NA7 allele may be an exceptional loss-of-function allele. Alternatively, it was possible that the ability to downregulate CXCR4 is conserved only in Nef proteins from a certain subset of primate lentiviruses. To distinguish between these possibilities we tested additional SIV and HIV-1 Nef proteins for their ability to downregulate CXCR4 from the cell surface. We also characterized Nef proteins from several HIV-2 strains. HIV-2 is closely related to SIVmac yet it frequently uses CXCR4 as an entry coreceptor, while SIVmac does not (13
). We also characterized the abilities of all Nef proteins tested to downmodulate CD4, since this function is known to be widespread among lentiviral Nef and therefore this assay could identify generally inactive proteins.
As shown in Fig. , all SIV nef
alleles tested downmodulated CXCR4. Nef protein encoded by SIV naturally infecting sooty mangabeys (SIVsm, FWR1), which is closely related to SIVmac239, caused a strong decrease in CXCR4 surface expression. This decrease was comparable to that seen with the SIVmac239 protein. Nef proteins of two SIV strains isolated from African green monkeys (Cercopithecus aethiops
, SIVagm, Sab1, and Tan1) had similar effects even though SIVagm is only distantly related to SIVmac and SIVsm. Nef proteins of SIV isolated from De Brazza's monkey (Cercopithecus neglectus
, SIVcne5) (7
) and from blue monkeys (Cercopithecus mitis
, SIVblu31) also downmodulated CXCR4, but their effects were not as strong as those seen with SIVmac-, SIVsm-, and SIVagm-derived alleles. Additional SIVcne and SIVblu Nef proteins need to be characterized to determine whether the alleles tested are representative or rather unusual weak alleles.
FIG. 2. Effects of SIV, HIV-1 and HIV-2 Nef proteins on CXCR4 and CD4 cell surface expression. (A) Flow cytometry analysis of CXCR4 and CD4 expression on the surface of Jurkat T cells transiently transfected to coexpress the indicated Nef proteins and GFP marker (more ...)
The effects of HIV-2 Nef proteins on CXCR4 cell surface expression were somewhat less pronounced. Of five alleles tested only the Ben allele decreased CXCR4 expression to levels seen with SIVmac- and SIVsm-derived nef genes. The remaining four HIV-2 nef alleles also diminished cell surface CXCR4 expression but to lesser extents. Finally, all six HIV-1 nef alleles tested affected CXCR4 cell surface expression but only marginally. We concluded that Nef proteins from diverse SIV strains strongly downregulate CXCR4, those from HIV-1 do so weakly, and those from HIV-2 have intermediate effects. In contrast, all Nef proteins tested strongly downregulated cell surface expression of CD4. Clearly, there was no correlation between the ability of various Nef proteins to downregulate CXCR4 and CD4.
Nef proteins downmodulate cell surface levels of target receptors by recruiting them to sites of endocytosis (9
). Therefore we asked whether SIVmac239 Nef enhances CXCR4 endocytosis rate. Jurkat T cells were transfected to transiently express SIVmac239 Nef or HIV-1 NA7 Nef and GFP or GFP alone as controls. Cells were reacted with anti-CXCR4 monoclonal antibody labeled with phycoerythrin. Subsequently, the rates of CXCR4 internalization in Nef-expressing and in control cells were determined for populations showing identical levels of GFP fluorescence using a flow cytometry-based endocytosis assay (67
). As a control, we determined the rates of CD4 endocytosis in the same transfected cell populations.
As shown in Fig. , expression of SIVmac239 Nef resulted in an approximately 10-fold increase in the rate of CXCR4 internalization over that seen in cells expressing GFP protein alone. The magnitude of this increase was almost as high as that seen for CD4 in cells expressing SIVmac239 and HIV-1 NA7 Nef proteins. Thus, the accelerated endocytosis of CXCR4 was probably responsible for its downregulation from the surface of SIVmac239 Nef-expressing cells. Notably, HIV-1 Nef also increased the CXCR4 endocytosis rate compared to that in control cells, but to a much lesser extent than SIVmac239 Nef. This is consistent with the observations that the NA7 Nef modestly decreased CXCR4 cell surface expression and likely explains this phenomenon (Fig. and ) (29).
FIG. 3. SIVmac239 Nef accelerates CXCR4 endocytosis rate. The rates of CXCR4 endocytosis (left panel) and CD4 endocytosis in Jurkat T cells expressing SIVmac239 Nef (○), HIV-1 NA7 Nef (), and control cells () were measured by a flow (more ...)
To elucidate how SIVmac239 Nef induces CXCR4 endocytosis, we studied which surfaces and elements of the protein are required for this effect. Previous studies revealed that SIV Nef induces endocytosis of CD4, CD28, and CD3 via the AP-2 clathrin adaptor pathway and class I MHC via an AP-2-independent pathway (40
). Mutations in SIVmac239 Nef that selectively disrupt molecular interactions required for these different functions were previously identified (40
). Therefore we tested the effects of these mutations on the ability of SIVmac239 Nef to downregulate CXCR4 cell surface expression. Figure shows the results of these experiments.
FIG. 4. Effects of mutations in SIVmac239 Nef on downregulation of CXCR4 and other cell surface receptors. Flow cytometry analysis of CXCR4, CD4, CD3, and class I MHC expression on the surface of Jurkat T cells transiently expressing the indicated Nef proteins (more ...)
We first tested a mutation that disrupts the interaction of SIVmac239 Nef with the AP-2 clathrin adaptor by deleting two constitutively strong AP-2 binding elements located in the N-terminal region of this Nef molecule (Δ23-74) (40
). As shown in Fig. , this mutation severely disrupted CXCR4 downregulation (Fig. , panel 9). Next we tested two smaller deletions in the same region that each removes a different constitutively strong AP-2 binding element in Nef (40
). Deletion of amino acid residues including lysine L31 through aspartic acid D37 (Δ31-37) removes the N-proximal AP-2 binding element that is dispensable for known functions of Nef (40
). This mutation had a weak disruptive effect on the ability of Nef to downregulate CXCR4 (Fig. , panel 13). A more limited disruption of this region by alanine substitutions for tyrosines Y28 and Y39, which were implicated as important for the interaction with the μ2 subunit of the AP-2 complex (51
), had no detectable effect on downregulation of CXCR4 or any of the other receptors tested (Fig. , panels 45 to 48). Thus, another aspect of the N-proximal element likely contributes to CXCR4 downregulation.
Deletion of amino acids Q64 to N67 (Δ64-67) removes the N-distal AP-2 binding element in SIVmac239 Nef, which is required for downregulation of CD4 and CD28 through their recruitment to AP-2. Notably, the Δ64-67 mutation severely disrupted the ability of Nef to downregulate cell surface CXCR4 (Fig. , panel 17). This observation further suggested that Nef uses the N-distal element to downregulate CXCR4 through the AP-2 mechanism, similar to how it downregulates CD4 and CD28 (40, 67) (data not shown).
Then we analyzed additional mutations in Nef that compromise CD4 downregulation and/or other Nef functions. Initially we focused on mutations that do not affect constitutively strong Nef binding to AP-2 but rather disrupt other molecular interactions required for CD4 downregulation (40
). Alanine substitutions for leucine L194 and methionine M195 (LM194AA) selectively diminished CXCR4 and CD4 downregulation without affecting T-cell receptor-CD3 and class I MHC cell surface expression (Fig. , panels 25, 27, and 28). Mutation in acidic element (Aci1, residues D88 to D95) (12) disrupted in part the effect of Nef on CXCR4 (Fig. , panel 29). This mutation was shown previously (28
) to have a pleiotropic effect on several Nef functions including CD4 and class I MHC downregulation (Fig. , panels 30 and 32). The mutation of the arginine residues R137 and R138 (RR137GG) also had a pleiotropic effect on receptor expression but had little detectable effect on CXCR4 downregulation (Fig. , panel 37). Similarly, two mutations that selectively disrupted downregulation of class I MHC (LL20AA and Y223A) (67) had little detectable effect on CXCR4 downregulation (Fig. , panels 21 and 41).
Finally, alanine substitutions for prolines P104 and P107, which in HIV-1 Nef are required for association with PAK2 kinase (58
), did not have a noticeable effect on the downregulation of any of the four receptors under study (Fig. , panel 33 to 36). Together, these data are consistent with the possibility that the ability of SIVmac239 Nef to bind AP-2 via its N-distal AP-2 binding element is crucial for CXCR4 downregulation and support the possibility that Nef induces CXCR4 endocytosis via the AP-2 clathrin adaptor.
CXCR4 mediates migration of cells to SDF-1 chemokine (10
). Since CXCR4 levels on the surface of SIVmac239 Nef-expressing cells were at least 10-fold lower than those on control cells, we characterized the migration of these cells to SDF-1. Jurkat T cells were transiently transfected with a control plasmid expressing GFP marker protein alone or with a plasmid that coexpresses Nef and a GFP marker protein from the same bicistronic transcription unit. Migration of the cell populations to SDF-1 was measured using a Transwell migration assay. The relative frequency of Nef-expressing and control cells in the migrated populations was then determined by flow cytometric measurement of GFP fluorescence (29
Consistent with previous observations, approximately 30% of cells in the control populations migrated regardless of the level of GFP expression (29, data not shown). In contrast, in cell populations expressing SIVmac239 Nef migration was inhibited with increasing Nef/GFP expression in a dose dependent manner up to 100-fold (Fig. ). Notably, the SIV Nef was more potent in inhibiting SDF-1-induced migration than HIV-1 Nef. We showed previously that inhibition of lymphocyte migration by HIV-1 NA7 Nef is independent of a slight decrease in CXCR4 expression levels seen on the surface of cells expressing this Nef protein (29
). These observations together suggested that strong downmodulation of CXCR4 cell surface expression is an important component to the SIVmac239 induced inhibition of migration to SDF-1.
FIG. 5. SIVmac239 Nef inhibits lymphocyte migration to SDF-1. (A) SIV Nef strongly inhibits migration to SDF-1. Jurkat T cells transiently expressing SIVmac239 Nef () or HIV-1 NA7 Nef (•) and GFP or GFP alone () were used in transwell (more ...)
To demonstrate this directly we determined effects of mutations in SIVmac239 Nef that disrupted CXCR4 downmodulation on the inhibition of migration to SDF-1. The results are shown in Fig. in a scatter plot as a function of CXCR4 expression levels on the surface of the transfected cell populations. Mutations such as Δ64-67, Δ23-74, Aci1, and LM194AA severely diminished the ability of SIVmac239 Nef to downmodulate CXCR4. All these mutations also severely disrupted the ability of SIVmac239 Nef to inhibit cell migration to SDF-1. Mutations such as LL20AA, Δ31-37, RR137GG, and Y223A disrupted CXCR4 downmodulation by SIVmac239 Nef to a lesser extent. However, these mutations had only a marginally negative effect on the ability of SIVmac239 Nef to inhibit lymphocyte migration to SDF-1. Thus, generally there was a strong positive correlation between the ability of mutant Nef proteins to downmodulate CXCR4 and to inhibit SDF-1-induced lymphocyte migration (Pearson correlation coefficient r = 0.93, P < 0.0001).
Interestingly, a closer examination of the data revealed that the Nef variant carrying the Y223A mutation inhibited migration more strongly than the wild-type SIVmac239 Nef even though the effect of the two proteins on CXCR4 expression was comparable. Therefore the robust disruption of lymphocyte migration to SDF-1 cannot be explained only by the ability of SIV Nef to downmodulate CXCR4, and additional components that contribute to this effect likely exist.