Self-association of the Lentivirus protein, Nef
© Kwak et al; licensee BioMed Central Ltd. 2010
Received: 20 April 2010
Accepted: 23 September 2010
Published: 23 September 2010
The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.
By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIVMAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.
We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.
The human immunodeficiency virus type I (HIV-1) accessory gene product, Nef, is a myristoylated protein with a decisive role in viral replication and pathogenesis [1–4]. HIV-1Nef has a canonical length of only 206 amino acids but is functionally complex. Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) Nefs are about 50 amino acids longer and are also functionally complex . In both cases functional complexity is reflected in overlapping effector domains that interact with multiple cellular proteins. These interactions bring about abnormal associations of host cell proteins that establish a favorable environment for viral replication [2, 5–8]. HIV-1Nef has a structured core (approximately amino acids 62-147 and 179-200), flexible N- and C- termini (2-61, 201-206) and an internal flexible loop (148-178). Homology between HIV-1 and SIV Nefs is largely restricted to the core region . It has been established that Nef can exist as a dimer, and to a much lesser extent a trimer, with the proposed oligomerization domain residing in the core [10–13].
Several groups have investigated the relationships between Nef's cellular localization, oligomerization, and its various activities. Specifically, a monomer of Nef has been proposed to be soluble, compact, and inactive with the myristate, the N-terminal flexible arm (2-62) and the internal flexible loop (148-178) all bound to the Nef core. In this conformation, soluble Nef may be refractory to oligomerization [14, 15]. Support for this model comes from the report that in vitro myristoylated full-length Nef is a compact monomer as determined by analytic gel filtration and ultracentrifugation . Insertion of the myristate alkane chain into the membrane would remove it from the Nef core, possibly favoring the dimeric state . Recent studies suggest that an interaction between positively charged residues within 22 amino acids of the N-terminus and the negatively charged surface of intracellular membranes act in concert with the myristoyl group to enhance and stabilize Nef binding to internal membranes [16, 17].
The large conformational changes thought to occur upon Nef binding to membrane are the basis of a tripartite regulatory model for Nef linking conformation, cellular localization, and function by Arold and Baur . In this model the initial cytosolic form of Nef is monomeric, non-functional, and described as "closed." Insertion of the myristate group into membrane, and association of the nearby cluster of arginines (R17, R19, R21, and R22) with phospholipids, detaches the flexible N-terminus from the Nef core giving a "semi-open" conformer. Subsequent extension of the flexible internal loop (amino acids 148-178) away from the Nef core gives a fully extended or "open" form of the protein. These changes could uncover Nef dimerization and effector domains in conjunction with the localization of the protein in proximity to its membrane-bound cellular targets [10, 17, 18]. The model further proposes that the sequence of Nef conformations, closed, semi-open, and open, directs subsets of Nef activities to appear in a temporal progression from synthesis on cytosolic ribosomes to membrane attachment and finally co-internalization with CD4 and other host cell plasma membrane proteins by the endocytotic machinery. In this way, Nef would be able to express functions important for early HIV-1 replication and then shift to functions appropriate for late HIV-1 replication . Arold and Baur rejected an alternate model of Nef function in which discrete conformers of Nef each with a different set of Nef activities occur randomly and simultaneously.
Reports from Arold and co-workers have also suggested an important role for Nef oligomerization in key Nef functions including CD4 downregulation, MHCI downregulation and enhancement of virion infectivity [10, 18]. These authors have described an oligomerization surface contained in the second α-helix (αB) of the Nef core (R105-I114) and 9 amino acids in the trailing loop. The basis for this proposed dimeric structure comes from analysis of Nef packing within hexagonal crystals. D123 is considered to be critical for Nef oligomerization since mutating this residue renders the mutant protein defective for all three of the above Nef activities . Poe and Smithgall have recently reported the presence of Nef dimers possibly linked by the 105-123 dimerization interface, but found Nef dimerization to be independent of membrane localization [13, 19]. Hence, the studies published to date raise a number of interesting questions regarding the mechanism and possible significance of Nef oligomerization. Crucial to all of these considerations is the robustness of the proposed inhibition of dimerization in the cytosol by the myristate group. If a significant proportion of oligomeric Nef is formed in the cytosol and transiently binds to membrane as a dimer; then a stochastic model is favored over a sequential model.
Here we report that Nef oligomers are present at levels consistent with functional significance. We also report evidence that supports an equilibrium model of different Nef conformations over the previously described sequential regulation model. For the first time, the evolutionally diverged SIVNef and HIVNef have been demonstrated to share at least one oligomerization interface. Finally, our data indicate the presence of one or more Nef oligomeric states in addition to the R105-D123 model previously proposed. Our modeling studies have suggested a new dimerization interface consistent with all of our data.
Membrane-bound Nef exists as monomers and dimers
Nef oligomers are also present in cytosol
The cytosolic fractions corresponding to the membrane-bound fractions presented in Figure 1A were also analyzed (Figure 1B). Unexpectedly, Nef oligomers were present in the cytosol with FD's for NA7-HFNef/NA7Nef and SF2-HF/NA7Nef of 0.35 and 0.23, respectively (Figure 2C). These values suggested a difference between the FDs for soluble Nef versus membrane-bound Nef (Figure 2C). Additional experiments confirmed a difference in FDs for soluble and membrane-bound Nefs (FDsoluble/FD membrane = 0.52 ± 0.05 with n = 4 and p = 0.015 by paired t-test). This 50% lower FD for soluble Nef is qualitatively consistent with the suggestion that the myristate group interacts with the Nef core in soluble Nef and diminishes dimerization [14, 15]. However, the fact that this is a partial effect means alternative mechanisms may account for the result.
The role of the N-terminal myristate in oligomerization
SIVMAC239Nef forms oligomers
Nef dimers in intact cells
The SF2Nef dimers that we observed by intracellular cross-linking with formaldehyde are present at 15% or less of the total Nef. The likely explanation is that much of the intracellular dimer is either not cross-linked or non-specifically cross-linked. Concentrations of formaldehyde higher than 0.5% resulted in weaker dimer bands due to non-specific cross-linking (not shown). Therefore, it is clear that the results of the formaldehyde cross-linking assay are not quantitative and may not be comparable to the immunoprecipitation assay. To test if the two assays yield similar results we took advantage of a tendency we noted in the immunoprecipitation assay for 2-fold higher FD's for oligomers composed of SF2Nef and SF2-HFNef relative to complexes containing NA7Nef or NA7-HFNef with each other or in hetero-dimeric combinations with SF2-HFNef or SF2Nef, respectively. To carefully compare the strength of self-interactions between SF2Nef and NA7Nef, we determined the FD's for the four possible combinations of SF2-HFNef with SF2Nef or NA7Nef, and NA7-HFNef with SF2Nef or NA7Nef by the immunoprecipitation assay of membrane-bound Nef (not shown). These FDs confirmed the initial observation. Combining all our relevant data, we found the FD for SF2Nef homodimers to be 0.71 ± 0.10 (n = 5) and for SF2Nef/NA7Nef heterodimers plus NA7Nef homodimers to be 0.30 ± 0.09 (n = 5). This difference was statistically significant (p = 0.032 by Mann Whitney test), and suggested that NA7Nef lacks one or more structural features present in the SF2Nef that stabilize dimerization. The observed difference in FDs allowed us to address whether the formaldehyde cross-linking assay was detecting the same oligomers as the immunoprecipitation assay. In that case there should also be a reduction in NA7Nef complexes relative to SF2Nef by formaldehyde cross-linking. The results in Figure 5B, lanes 3 and 5, demonstrate that this is in fact the case. Taking advantage of our finding we swapped the core domains of SF2Nef and NA7Nef to determine if the core sequences accounted for the observed differences in self-association. In Figure 5B, lanes 7 and 9 we observed that the NA7(SF2)Nef chimera with the SF2Nef core exhibited greater cross-linking than the SF2(NA7)Nef chimera with the NA7Nef core. These results strongly suggest that the formaldehyde cross-linking assay and the immunoprecipitation assay are detecting the same Nef oligomers and that oligomerization is dependent on core sequences.
The important finding that the immunoprecipitation assay and the intracellular cross-linking assay give similar results for SF2Nef and NA7Nef self-association led us to determine if the results of Figure 3 would also be confirmed. In Figure 5C we observed that SF2NefG2A did not exhibit enhanced dimerization by the formaldehyde cross-linking assay consistent with the results of the immunoprecipitation assay.
Can Nef dimers be cross-linked at cysteines?
In Figure 6B, top, we checked the impact of C55S and C206X on the ability of SF2Nef to downregulate CD4 downregulation and MHCI downregulation; both mutants were found to be functional. Neither mutation reduced the level of Nef expression relative to SF2Nef (Figure 6B, bottom). In contrast, SF2NefC142S was significantly less stable than the other two cysteine mutants (not shown), confirming previous reports [33, 34]. This mutant was partially defective for MHCI downregulation but fully functional for CD4 downregulation. Since MHCI downregulation is more sensitive to reduced levels of Nef expression than CD4 downregulation, this result is expected . Residue 142 is completely buried in the hydrophobic core of Nef and unlikely to be involved in intermolecular S-S bonds between native Nefs .
On the basis of these cross-linking experiments, it appears that SF2Nef exhibits a dimeric structure that brings C-terminal regions into proximity. Inspection of space filling models of Nef dimers associated at the previously proposed R105-D123 interface shows the two C-terminal cysteines to be distant from each other (not shown). A second Nef/Nef interaction surface found in cubic crystals of Nef extends from R188 to E197 plus F139 but does not include C206 . This surface is viewed by Arold et al. as a biologically irrelevant result of crystal packing . However, it is clear from inspection of Nef crystal structures that if Nef self-association was mediated by this interface in the C-terminal domain, then cross-linking of C206 by the BM[PEG]3 reagent would be possible (not shown). These considerations suggest that the cysteine cross-linking result in Figure 6A may reflect proximity of C-terminal cysteines in the SF2Nef BM[PEG]3 cross-linked dimer, but not a structural role in dimerization per se. To test this conclusion we cross-linked SF2Nef, SF2NefC55S, and SF2NefC206X with formaldehyde and found no impact of cysteine mutations on dimerization (Figure 6C). Therefore, we interpret our cysteine cross-linking results as reflecting the location of an interaction interface but not defining C206 as a critical structural element for dimerization. It is interesting to note in this regard that 15% of subtype B Nefs have no C-terminal cysteine but a termination codon instead (TGY to TGA).
Salt bridges flanking the αB helix are not necessary for oligomerization
It has been proposed that R105 forms salt bridges with D123 on the flanks of the amphipathic αB helix, and that mutation of D123 disrupts oligomerization [10, 18]. Several groups have reported multiple functional consequences of mutating D123, including CD4 downregulation, MHCI downregulation, and enhancement of virion infectivity. The multiple defective phenotype of D123 mutants has been attributed to disruption of Nef dimerization . The conservative mutation D123E gives the same defective phenotype as non-conservative mutations, and has also been found to enhance p21-activated protein kinase 2 (PAK2) activation relative to unmodified SF2Nef . The significant impacts on four separate Nef activities define a distinctly complex D123 mutant phenotype [8, 14, 18, 28, 35, 36]. However, the fact that aspartate replaced by the isoelectric glutamate generates the equivalent phenotype as non-conservative mutations suggests that the isoelectric mutation involves disruption of precise structural considerations beyond mere charge interactions. A phenotypic impact as a result of mutating D123 to glutamate is consistent with its extreme conservation (> 99%). In contrast, HIV-1 subtype B Nefs are weakly conserved at position 105 with 71% lysine, 22% arginine, and 6% glutamine .
The replacement mutations for R105 and D123 were designed to test the proposal that R105 and D123 were components of a critical salt bridge within a functionally significant dimerization domain. Since our results failed to verify the salt bridge model, we considered alternative explanations for the roles of R105 and D123. One explanation is that mutation of D123 locks Nef into a conformation that is competent for dimerization, but nonetheless different from the conformation of the bulk of wild type SF2Nef. This hypothesis is based on the fact that adding the R105D mutation to give the double mutant has little effect on dimerization per se, and no effect on the functional properties of Nef mutated at D123.
We next considered a role for R105 in dimerization of wild type Nef distinct from D123. Also, of potential interest was the adjacent R106. Unlike R105 R106 is highly conserved (99%). The isoelectric mutation of R106 to lysine, in analogy to D123E, gives a complex phenotype that is quite different from D123E. As previously reported, R106K is highly defective for MHCI downregulation, partially defective for PAK2 activation but functional for CD4 downregulation and enhancement of infectivity. R106A has a more drastic phenotype- defective for MHCI downregulation, fully defective for PAK2 activation, partially defective for CD4 downregulation, and fully defective for enhancement of infectivity. R106L like R106A is fully defective for MHCI downregulation and PAK2 activation but also fully defective for CD4 downregulation . To try and discern if dimerization possibly plays a role in these complex phenotypes we compared the effects of mutating R105 and R106 (R106A, R105A, and R105D) to mutating D123 (D123R and R105D/D123R). In Figure 7C the R105A and the R106A mutant proteins had reduced levels of Nef dimerization, though not to the extent of the reduction observed for R105D. This suggests that the region of Nef containing R105 and R106 is significant for dimerization and that having the negatively charged D at position 105 may disrupt the normal interactions of the positively charged R106. It is important to note that 93% of Nefs are lysine or arginine at residue 105 and 99% arginine at 106 suggesting that a +2 charge for these two adjacent residues is necessary for optimal function.
Modeling Nef dimerization
To gain further insight into potential Nef dimerization interfaces, we employed ClusPro 2.0 http://cluspro.bu.edu for a rigid body docking analysis of Nef. The structure of the monomer used in the ClusPro search, was extracted from the hexagonal crystal structure of Nef (PDB ID 1AVZ, Chain A). For our initial ClusPro analysis, we used the same structure of the Nef monomer as both receptor and ligand. The Nef-Nef interface in the crystal structure providing our search model is the basis for the proposal that the R105-D123 interaction forms a critical component of the dimerization interface. Thus, the Nef structure employed in the ClusPro analysis has R105 and D123 side chains in positions appropriate and optimal for formation of the proposed dimerization interface involving the R105-D123 salt bridge. We chose the 10 most strongly interacting models of dimeric Nef structures for analysis. Seven of the structures involved at least part of the αB helix hydrophobic domain. Interestingly, none of the 10 models included the R105/D123 salt bridge that was evident in the crystal structure.
For the Nef homodimer (Figure 8B, light orange background) the residues that interact between the Nef monomers are presented. For example, ligand R106 interacts with L100, R106, I109, and W183 in β5 (8B; light orange background). In total, the ligand HIV-1Nef has 17 residues that interact with the receptor Nef, 14 of which react with the same side chains in receptor Nef as residues in SIVMAC239Nef (identical interactions shown in bold, Figure 8B). In the homodimer receptor, 11 residues interact with the ligand with ten of these residues matching ligand residues in SIVMAC239Nef residues.
In the HIV-1Nef/SIVMAC239Nef heterodimer (Figure 8B, light blue background) the ligand SIVMAC239Nef has 21 residues that interact with the receptor Nef compared to 17 for the HIV-1Nef ligand. Three of the four extra residues (S201, Q202, and W203) are from the C-terminal portion of the internal loop that is present in SIVMAC239Nef core construct that is deleted in the HIV-1 Nef core. In the heterodimer receptor, 18 residues interact with the ligand SIVMAC239Nef compared to 11 for the HIV-1Nef homodimer receptor. The seven extra amino acids mostly interact with residues in the SIVMAC239Nef ligand that are not present in the Nef core structure, accounting for 12 of 16 interactions. The extra SIVMAC239Nef residues are in internal loop or the N-terminal side of PQVPLR near the beginning of the Nef core region. Potentially, the agreement between heterodimer and homodimer would be even greater if the Nef structure contained the extra residues.
Prior to the present report, the extent of Nef oligomerization was not known nor had the regulatory model of Arold and Baur been directly tested . Poe and Smithgall reported the presence of Nef dimers in intact cells by bimolecular fluoresecence complementation but the fractional dimerization was not assessed . To investigate Nef self-association, we have employed two assays. First, the immunoprecipitation assay which utilizes a tagged Nef and an untagged Nef. The tag allowed us to differentiate between the two otherwise identical Nefs immunologically and by different mobilities on SDS/PAGE electrophoresis. This approach allowed us to demonstrate, for the first time, that Nef oligomers are distributed between the membrane and cytosol compartments of the cell. We placed the tag at the C-terminus to preserve Nef myristoylation. The results from this assay were compared to intracellular cross-linking by formaldehyde. We have also demonstrated for the first time that SF2Nef can be cross-linked by BM[PEG]3. This cysteine reactive agent cross-linked only at C206 and provided new information of a structural nature regarding the location of a Nef dimerization interface. With these new approaches we have investigated previously unaddressed questions regarding Nef oligomerization, and identified R105 and R106 as important constituents of a dimeric interface.
What is the proportion of Nef that is oligomeric in cells?
With the immunoprecipitation assay we found approximately two thirds of membrane-bound SF2Nef is oligomerized. Lower levels of oligomerization by half were observed for membrane-bound NA7Nef and SIVMAC239Nef. All three of these Nefs are highly functional, well-studied alleles [2, 26–28, 43] though NA7Nef exhibits weak PAK2 activation relative to SF2Nef . In the case of NA7Nef and SF2Nef, the structured cores accounted for the allelic variation.
Is Nef oligomerization inhibited by the N-terminal myristate group?
To assess whether or not Nef oligomerization is regulated by membrane association, we determined the fractional dimerization of Nef from the membrane-bound and cytosolic fractions with the immunoprecipitation assay. Nef oligomers were demonstrated in both of these two compartments with the fraction of oligomeric Nef approximately two-fold higher in the membrane compartment. The immunoprecipitation assay should reflect the in vivo state of Nef except for very weakly associated Nef that would disassociate during cell lysis and immunoprecipitation. We have shown that the short HF tag does not generally interfere with Nef function though larger tags do in some instances . Importantly, immunoprecipitation from cellular extracts would be expected to suppress non-specific interactions including aggregation of Nef protomers . Thus, our observations are distinct from the previous observations of very weak self-association by purified Nef from bacteria (Kd > 0.5 mM) [10, 45, 46]. It is not clear that the weak association observed in vitro is reflective of functionally significant interactions inside cells.
The presence of Nef oligomers in the cytosol argues that Nef oligomerization is not strongly inhibited by the myristoylated N-terminal arm of Nef. If the binding of the N-terminal arm of Nef to the core blocked oligomerization, then the intramolecular nature of this interaction would overwhelm any weak tendency to self-association unless the N-terminal arm is dissociated from the core by stable binding to membrane [47–49]. These considerations argue strongly that a negative regulation of Nef oligomerzation by its N-terminal segment would result in cytosolic Nef being entirely monomeric. Our observation that relatively high levels of dimers are present in the cytosol demonstrates that the N-terminal domain does not significantly regulate dimerization and suggests that self-association of Nef is a constitutive property of the protein [10, 14, 18]. In the absence of data demonstrating sequestration of Nef oligomers to membranes, we conclude that Nef is unlikely to be sequentially regulated. Instead Nef appears to be in equilibrium between monomeric and oligomeric forms.
Is oligomerization evolutionarily conserved?
We addressed the question of evolutionary conservation of Nef oligomerization by investigating whether or not SIVMAC239Nef oligomerized and whether SIVMAC239Nef and SF2Nef associate. Our positive findings for both cases demonstrate the existence of a conserved mechanism of dimerization. Although the location of the dimerization interface for this heterologous association is not known, it is certainly expected that it will be within a highly conserved region. This is in contrast to several other shared functions of HIV-1Nefs and SIVNefs with variant structural bases [2, 28]. From this evolutionary perspective, conserved but mechanistically distinct activities like CD4 downregulation and MHCI downregulation would then represent evolutionarily malleable functions relative to oligomerization.
What regions of Nef interact to form multimers?
The interface or interfaces responsible for Nef oligomerization have not yet been defined. Previously proposed dimerization interfaces within the Nef core are not sufficiently conserved between SIVNefs and HIV-1Nefs to be operative in these assays [10, 11, 18]. Nonetheless, our results do suggest the Nef core as the site of Nef dimerization interfaces. The Nef core exhibits the highest level of identity between SF2Nef and SIVMAC239Nef, especially the region largely encoded by nucleotides of the polypurine tract (amino acids, 88-99, in HIV-1Nef with 11 out of 12 identical). Also, our finding that dimer formation by the SF2Nef core is strong relative to the NA7Nef core further supports this conclusion (Figure 5B).
It does not seem likely that either the N-terminal arm of Nef or the internal flexible loop of Nef are involved in dimerization as they are weakly conserved between HIV-1Nef and SIVNef. The myristoylated N-terminal Nef synthetic peptide (amino acids 2-57) does not dimerize , and we have observed dimerization of SF2NefΔ160-175 despite removal of much of the internal loop (not shown). Therefore, Nef dimerization domains appear to be located in the highly conserved core of Nef . These considerations validate the use of Nef structures from crystals, even though the Nefs are truncated for the N-terminal arm and flexible internal loop.
ClusPro analysis of HIV-1Nef homodimers and the HIV-1Nef and SIVNef heterodimers suggests a candidate interface for Nef dimerization. The critical residues of this proposed interface are L100, I101, R105, R106, I109, L112, W113, and H116. Four considerations support this interface. First, the residues are conserved, especially R106, I109, L112, and W113. Second, D123 is excluded from the model. Third, R105 and R106 interact within a region that is highly conserved between HIV-1Nef and SIVNef (HIV-1Nef residues 88-99). Fourth, ClusPro models for HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers exhibit nearly identical binding (Figure 8). Extensive mutational and phenotypic analysis will be required to establish this model.
How many oligomeric forms of Nef exist?
The existence of one or more dimerization domains distinct from the αB plus flanking residues does not exclude the presence of Nef dimers linked at this site. In fact, Poe and Smithgall have provided evidence that Nefs linked by this interface exists in intact cells . However, our inability to demonstrate a role for the R105-D123 interface by formaldehyde cross-linking assays suggests that the dimer detected by Poe and Smithgall is a quantitatively minor form of total Nef dimers. In this regard, the biomolecular fluorescence complementation assays employed by Poe and Smithgard freezes in place the conformations of Nef that allow the molecular complementation of the N-terminal 85 amino acids of GFP to the C-terminal 153 amino acids of GFP. Once formed, the reconstituted GFP may block the dynamic equilibrium between Nef conformers and result in the accumulation of a minor form . Another HIV-1Nef dimer that could be a minor form is the BM[PEG]3 cross-linked dimer. A second crystal interface could account for this interaction in the C-terminal region (Figure 9B, Magenta and Lilac).
Interaction between SIVNefs and HIV-1Nefs with host cell proteins
There are minor Nef complexes that run above the SF2Nef dimer band in SDS/PAGE following formaldehyde cross-linking (See Figure 5). Whether these bands represent different forms of lysine cross-linked Nef with an altered SDS/PAGE mobility or Nef cross-linked to a different protein remains to be determined. However, the situation for SIVMAC239Nef appears to be different. Two prominent species of cross-linked SIVMAC239Nef are evident in Figure 5A, right. The lower of the two bands at approximately 82 kD could plausibly be a heterodimer of SIVMAC239-HFNef and a host cell protein. Alternatively, the different mobilities could result from variant lysine cross-links of SIVMAC239Nef dimers. We favor the latter interpretation for the following reasons. Of the various proteins shown to bind tightly to Nef only Rac1 (211 amino acids) is approximately the correct size [29, 30]. Skowronski and co-workers report that Rac1 immunoprecipitates with NA7-HFNef and SIVMAC239Nef. As a result, either the tagged SF2-HFNef or the untagged SF2Nef in a cross-linked heterodimer with Rac-1 should migrate separately from SF2Nef dimer in SDS/PAGE. This is not the case (Figure 5A and 5C). Therefore, we conclude that the major Nef binding protein in intact cells is Nef.
The density of potential protein/protein interaction domains displayed in Figure 9 suggests that Nef is capable of initiating complex host cell associations in which two host cell proteins are brought together to achieve abnormal situations within the infected cell that are advantageous to the virus. Two ternary complexes have been recently reported between Nef, the cytoplasmic tail of MHCI, and AP-1, and Nef, the cytoplasmic tail of CD4, and AP-2 [40–42]. These interactions are proposed to downregulate cell surface expression or MHCI and CD4. Another interesting mechanistic explanation for explaning Nef functional complexity is the possibility of allosteric interactions . Finally, Nef self-association may create new interfaces for protein association with each Nef contributing part of the interface. All of these mechanisms should be considered in efforts to resolve the complexities of Nef function made possible by the fastest genome evolution ever described .
We have demonstrated significant levels of oligomerization of Nef in cells. This property of Nef is conserved between HIV-1Nef and SIVNef which strongly suggests functional significance. We found that the R105D mutation greatly reduced the fraction of dimeric Nef, but exhibited only partial defects in MHCI downregulation and PAK2 activation, and only a small effect on CD4 downregulation (Figure 7). For this reason, we favor the explanation that the functional role of Nef dimerization is a function not presently known.
Our results are contrary to a regulatory model in which Nef is induced by membrane binding to produce oligomers [14, 15]. These models require cytosolic Nef to be monomeric with Nef dimers residing strictly on membranes, but we did not observe this to be the case. We further observed that the myristoylation-defective G2A mutation does not enhance oligomerization. For this reason it does not appear that Nef's association with membrane and Nef dimerization are mechanistically related. Since Nef exhibits different functions in multiple cellular compartments including the Golgi, endosomes, plasma membrane and cytosol, it logically follows that multiple conformers and oligomers of Nef would all have extensive distributions with monomers as well as oligomers being functionally active . The existence of multiple oligomeric forms could greatly expand the activities exhibited by this small protein. Here we found that a major contributor to one or more Nef dimeric interfaces is the dibasic motif R105-R106. In the future it will be important to not only consider Nef as acting at specific cellular locations to elicit specific derangements in host cell physiology but also as acting in an expanded role throughout the cell.
Cell lines and culture conditions
293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS; Cellgro), 100 IU/ml of penicillin, 100 μg/ml streptomycin, 2 mM glutamine (Cellgro) in 10% CO2 at 37°C. CEM cells were cultured in RPMI 1640 medium supplemented with 5% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate (Cellgro) and were maintained at 37 °C in a humidified incubator with 5% CO2.
pCG and pCGCG expression vectors encoding two wild type HIV-1Nefs, NA7Nef and SF2Nef, and HF-tagged Nefs, NA7-HFNef and SF2-HFNef, were described in Raney et al. . pCG, pCGCG, and pCG239ahf (SIVMAC239Nef with the HF C-terminal tag) were provided by Jacek Skowronski [29, 30]. The HF tag contains the HA epitope, YPYDVPDYA. pCG and pCGCG give equivalent expression of Nef. SF2Nef oligomerization mutants (R105D, D123R, R105D/D123R, R105A and R106A) and cysteine mutants (C55S, C142S, C206X), myristoylation-defective mutant G2A, and the Nef deletion mutant Δ160-175 were constructed using a site-directed mutagenesis kit (Stratagene) with oligonucleotides designed for each mutation. All mutated Nefs were confirmed by DNA sequencing. NA7/SF2 Nef chimeras were made by cloning the BlpI-BspEI fragment from SF2Nef (coding for amino acids 24-200) into pCGCGNA7Nef and vice versa. SF2Nef is four amino acids longer than the canonical 206 amino acids. To avoid confusion the position of amino acid residues are referenced to a canonical length of 206 amino acids .
Co-immunoprecipitation and Western blot analysis
Expression vectors encoding NA7Nef, NA7-HFNef, SF2Nef, SF2-HFNef, SIVMAC239Nef, and SIVMAC239-HFNef were transfected into 293T cells in 6-well plates with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were washed in cold phosphate buffered saline (PBS) then removed from the plate in 1 ml of 25 mM Tris-HCl, pH = 7.5, 150 mM NaCl, and 5 mM EDTA (Buffer A). Cell suspensions were disrupted by sonication (ten, 10 second bursts at power level 3.5) at 4°C. Nuclei were removed by centrifugation at 800 Xg for 10 minutes. The low speed supernatant fraction was re-spun at 27,000 rpm for 25 minutes in a MLA 130 fixed angle rotor (Beckman). The high speed supernatant fraction was separated from the pellet and adjusted to 1 ml to contain 50 mM Tris, pH = 8.0, 100 mM NaCl, 25 mM NaF, 25 mM benzamidine, 20 mM β-glycerophosphate, 2 mM Na3VO2, 3 mM EDTA, 10% glycerol, plus Roche protease inhibitors (Buffer B). The buffer-adjusted high speed supernatant sample was used to determine total soluble Nef expression (100 μl) and for immunprecipitation studies (900 μl). The pellet from the high speed centrifugation was rinsed with 0.5 ml of Buffer A. The membrane pellet was then re-suspended in 1 ml of Buffer A by Dounce homogenization and spun at 27,000 rpm. The pellet was saved and solubilized in 1 ml of Buffer B plus 0.5% IGEPAL CA-630 with a Dounce homogenizer and respun at 27,000 rpm for 10 minutes. The supernatant fraction of the detergent solubilized membrane pellet was used for determining total membrane-bound Nef expression (100 μl) and for immunoprecipitation studies (900 μl). With this procedure there was no detectable contamination of soluble protein in the membrane fraction or membrane protein in the soluble fraction (Additional file 2; Figure S2, Left and Middle). The distribution of membrane-bound and soluble SF2Nef and membrane-bound and soluble SF2NefG2A were nearly the same as previously reported values (Additional file 2; Figure S2, Right). Nef proteins were detected by Western blot analysis. Three monoclonal antibodies were used for detection: EH1 from James Hoxie , anti-SIV Nef 17.2 from the AIDS Repository, anti-HA (Covance). Two polyclonal antibodies were used for immunoprecipitations: sheep anti-SF2Nef  and sheep anti-SIVMAC239Nef antibody (Rogers, RP, Foster, JL and Garcia JV, unpublished.) Bands were quantitated by scanning the autoradiograms and determining density using ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2009). Density values are reported as the ImageJ value divided by 1000 to simplify the presentation.
Nef cross-linking by formaldehyde or 1,11-bis(maleimido)triethylene glycol (BM[PEG]3)
For formaldehyde-mediated Nef cross-linking, expression vectors encoding wild type or Nef mutants were transfected into 293T cells. Prior to harvest intact cells were incubated with or without formaldehyde for 10 min at room temperature and washed with PBS. Then, whole cell lysates were prepared in Buffer B plus 0.5% IGEPAL CA-630, centrifuged at 13,000 Xg for 30 minutes and the supernatant fraction collected. Cross-linking of Nef by BM[PEG]3 (Pierce) was performed as follows: Transfected 293T cells were lysed in Buffer B plus 0.5% IGEPAL-630 and the lysate centrifuged. Clarified lysates were incubated with or without BM[PEG]3 dissolved in DMSO for 1 h at room temperature. The analysis of Nef cross-linking by formaldehyde (at lysine residues) or BM[PEG]3 (at cysteine residues) was performed by SDS-PAGE Western blotting.
Retrovirus vector preparation, transduction and flow cytometry analysis
293T cells were transfected with the amphotropic packaging vector pEQPAM and pLXSN (vector control) or appropriate pLNefSN vectors with Lipofectamine 2000 (Invtrogen). Medium was harvested approximately 48 hours post-transfection and was filtered through a 0.45 μm filter.
A 24-well plate was coated with 40 μg/well of retronectin (Takara Biomedicals, Kyoto, Japan). After 2 h at room temperature, the retronectin was removed, 0.5 ml 2% bovine serum albumin in PBS was added for 30 min at room temperature, and the wells were washed once with PBS (0.5 ml). Filtrate (0.5 ml) containing amphotrophic vector was then added and left on the plate for 45 min at 37°C, and this procedure was repeated. CEM cells (300,000) were transduced by the retronectin-bound retroviral vector in 0.5 ml of complete RPMI overnight at 37°C. An additional 0.5 ml of vector was added the next day. On the following day, the cells were re-suspended in a 12-well plate containing 1.5 mg/ml G418 (Gibco) in a final volume of 2 ml. After 48 hours, the cells were re-plated in a 6-well plate and 1 ml of medium plus G418. Transduced cells then were expanded into a T75 flask without G418 for subsequent analysis.
The transduced CEM cells were labeled for analysis of cell surface CD4 and MHC-1 levels. Cells (500,000) were first incubated with a mouse monoclonal antibody recognizing haplotypes A1, A11, and A26 of MHC class 1 (One Lambda, Canoga Park, CA) for 20 min on ice in the dark, and then the cells were washed twice in 2 ml of ice-cold PBS containing 5% FBS. Cells were then incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG for 20 min on ice. Cells were washed as indicated above and incubated for 20 min on ice with 2 μg of mouse IgG, and washed again. Cells were then incubated with phycoerythrin (PE)-conjugated IgG monoclonal antibody to human CD4 (Exalpha, Maynard, MA) for 20 min on ice. Stained cells were washed and analyzed on a Becton Dickinson FACSCalibur instrument equipped with Cellquest-Pro software. All fluorescence data were collected in log mode. CEM cells transduced with LXSN served as the positive control for CD4 and MHCI cell surface expression. For negative controls, mouse isotype antibody replaced anti-MHC class I, and PE-conjugated mouse IgG (Exalpha) replaced PE-conjugated anti-CD4.
The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: SIVmac251 Nef Monoclonal Antibody (17.2). We thank James Hoxie for EHI monoclonal antibody and Carlos Gonzalez for critically reading the manuscript. This work was supported by grant AI33331 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, USA, and UNC CFAR P30 AI50410.
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