Although not essential for viral replication, HIV-1 Nef is important for disease progression and, therefore, is considered a pathogenic factor in primate lentiviridae . For its function, Nef depends on specific surfaces to interact with host cell proteins. Therefore, a strong positive selection pressure exists to keep the property of these surfaces conserved . It is key to identify conserved sites in Nef with functional importance in protein-protein interactions, since they might serve as potential target sites for pharmacological intervention.
In the present study, we identified, by functional analysis of a panel of clinical HIV-1 and HIV-2 nef alleles, a previously unrecognized conserved region. A HIV-1 group O nef allele, was mutated in an amphipathic stretch of amino acids located in the core domain encompassing the acidic cluster, a VGF region and the PxxP motif, resulting in the loss of downregulation of MHC-I and CXCR4, but not of CD4. By selective mutation of this O8 nef allele back to the consensus sequence, we show that both an intact PxxP motif in conjunction with an intact VGF region are needed to restore the defect in receptor trafficking. We could extend this observation by showing that the VGF region is essential for the association of Nef with active PAK2 and consequently hyper-phosphorylation of cofilin resulting in the inhibition of actin remodeling following TCR triggering. In addition, Nef-induced Lck accumulation in the TGN also requires integrity of the VGF region. The importance of the proline-rich region for this Nef function has been studied extensively [10, 47]. However, despite its high conservation in HIV-1 and SIVcpz nef alleles, the function of the VGF region remained poorly investigated.
Modulation of cell surface molecules such as MHC-I and chemokine receptors or the TNF receptor-associated factor TRAF2 were shown to depend on the conservation of the PxxP motif as well as the acidic cluster (EEEE) [19, 48, 49]. How the acidic cluster contributes to downregulation of MHC-I is controversial. Several studies have observed that the four contiguous glutamate residues interact with PACS-1 and PACS-2 to initiate assembly of a multiprotein complex which targets MHC-I to the trans-Golgi network for subsequent degradation [50, 51]. However, in an alternative model for MHC-I downregulation, in which Nef interacts with both MHC-I and the μ subunit of the AP-1 endosomal coat complex, the EEEE motif plays only a stabilizing role [52, 53]. The PxxP motif on the other hand is essential for the interaction of Nef with Src kinases (like Hck); misrouting of Lck as well as association of Nef with the cellular kinase PAK2, results in elevated cellular levels of inactivated phosphorylated cofilin, a deregulation that is instrumental for the inhibition of TCR- or chemokine-induced F-actin remodeling [26, 28–31, 34].
Triple mutants of the VGF motif (VGF→AAA) lost both ‘trafficking’ (MHC-I, CXCR4) as well as ‘signaling' (PAK2 association, cofilin hyper-phosphorylation and inhibition of actin ring formation) functions, suggesting that the VGF region is functionally linked with the PxxP motif. Moreover, Baugh et al. showed that similar to the PxxP motif, MHC-I downregulation, interaction with PAK2, and to a lesser degree enhancement of virion infectivity are dependent on the acidic cluster . Our observation that mutation of VGF into triple alanines similarly abrogates these multiple effects of Nef is a hint that the acidic cluster-VGF-PxxP triplet forms a functional unit important for protein-protein interactions with host cell factors.
In the dysfunctional Nef O8 allele, the VGF motif is in fact deleted, but not replaced to varying amino acids by gene tropism. The effect of the deletion mutation could therefore be indirect, meaning that only the correct spacing between the acidic cluster EEEE motif and the PxxP motif is required for Nef functionality, i.e. the correct positioning of the EEEE motif and potentially the preceding CAWL protease recognition motif with respect to the Nef core domain. Alternatively, the effect of Nef dysfunction could be direct through binding of these amino acids to the primary receptors itself or to any stimulatory co-factor required for receptor downregulation. As mutation of the VGF motif to AAA in O8 Nef did not restore the Nef trafficking functions, the effect of mutation is most likely direct. Our finding that individual mutation of the three amino acids (AGF, VAF, VGA) is sufficient to interfere with the effect of Nef on receptor trafficking and TCR signaling further supports this hypothesis and shows that the function of V-G-F is sequence specific. The integrity of the phenylalanine appears to be specifically essential for functions related to the proline-rich motif. In contrast to single mutants of the valine and the glycine (VGF→AGF and VGF→VAF), single mutants of this amino-acid (VGF→VGA) did not accumulate Lck. Of note, several SH3 binding regions of mammalian proteins contain a N-terminal phenylalanine, which is required for efficient ligand binding . These observations suggest that the effect of Nef on TCR induced actin remodeling and on Lck targeting to the TGN are mechanistically different, as was shown before .
Shelton et al. showed recently that the VGFPV region, in their study identified as the secretion modification region, forms a specific binding surface for mortalin which subsequently promotes cellular secretion of extracellular Nef . The phenotypes which we analysed in this study are all cell intrinsic and are unlikely to be explained by differences in levels of extracellular Nef. The results of Shelton et al. and ours together show that the VGF region is not just a mere spacer between the acidic cluster and the proline rich region. Instead, it acts as an interaction surface, which together with its neighboring motifs forms a functional unit.
Replication of HIV-1 in primary T lymphocytes is tightly coupled to their activation state. While HIV-1 undergoes early replication events in quiescent CD4+ T cells, subsequent steps in the viral life cycle require T cell activation. One of the prime roles of Nef in vivo is to fine tune activation states in infected T cells . Nef interferes with TCR proximal signaling to prevent T cell activation and activation–induced cell death upon antigenic stimulation [28, 30, 31, 55], while at the same time enhancing distal TCR signaling effects to promote proviral expression [33, 57, 58]. Both functions of Nef depend on the interaction of several specific SH3-domain containing host cell proteins with the conserved PxxP motif. For example, the PxxP motif interacts with the SH3-domain of Lck, misrouting the TCR proximal kinase Lck from the plasma membrane to the trans Golgi-network (TGN), altering TCR proximal signaling events [28, 30, 33, 59]. In our HIV replication assay, WT HIV viruses replicated much more efficiently then HIV Nefstop viruses. The requirement of PxxP residues for efficient HIV-1 replication in T-cells is controversial [35, 39, 44, 59]. Lundquist and coworkers did not observe significant differences between the replication-efficiency of WT, AxxA mutant and polyproline deleted viruses , while Saksela and co-workers reported that AxxA mutant replicate as delta-Nef viruses . In our hands, the replication of the HIV Nef AxxA viruses was significantly attenuated compared to WT viruses. The fact that we use a different proviral backbone and other culture methods for CD4+ lymphocyte stimulation may account for the discrepancy between our results and observations from others. Similar to Nef AxxA viruses, VGF→AAA mutants replicated much less efficiently than WT HIV viruses. We recently showed that the accumulation of Lck is required for TGN-associated Ras-Erk signaling, which in turn was shown to promote IL-2 production and enhance virus spread . As HIV viruses harboring AxxA or VGF→AAA mutations in Nef fail to efficiently accumulate Lck , this could explain the altered replication kinetics of these mutants. Interestingly, our previous observation that the HIV O8 isolate showed reduced fitness compared to other isolates of the same group might be due to the deletion of the VGF domain .
Despite the opposing findings concerning the role of the PxxP motif for replication of HIV, there is general consensus that this domain is required for Nef to enhance the infectivity of HIV virions [61–63]. The mechanism by which Nef enhances viral infectivity remains unclear, but is thought to involve alterations of specific signaling and trafficking events in the producer cell. . We observed that, in line with our other results, VGF→AAA mutants were similarly less infective than wild type viruses.
In our study, we only analyzed the importance of the VGF motif in Nef for HIV-1 function. Despite VGF conservation in HIV-1 and SIVcpz; HIV-2 and SIVsmm instead harbor VGV. There are no indications that this change would result in major functional differences, since PxxP dependent functions such as downregulation of MHC-I, accumulation of Lck, deregulation of actin remodeling and association with PAK2 are well conserved between both HIV-1 Nef and HIV-2 Nef [26, 28]. Moreover, both phenylalanine and valine are hydrophobic amino acids, conserving the lipophilic character of the entire VGF/V region, and they are not expected to impose a different ultrastructure that would imply different host protein interactions. Rather, the strong conservation of VGF/VGV in HIV/SIV Nef suggests that such a structural motif is required for function of acidic cluster-VGF/V-PxxP motifs in general.