HIV-1 and 2 are members of lentivirus family of retroviruses and are grouped as complex retroviruses. The unique feature of this group in comparison to simple retroviruses is that the viral genome codes for several proteins in addition to the core structural proteins. In this regard, HIV-1 is known to code for six auxiliary proteins (Vif, Vpr, Tat, Rev, Vpu and Nef) besides the structural proteins. Previous studies have demonstrated that auxiliary proteins play an essential role in HIV-1 replication and pathogenesis . Our laboratory has been interested for several years in evaluating the contribution of auxiliary proteins including Vpr. In this study, we have analyzed the requirement of sequences in Vpr essential for oligomerization feature of Vpr and its relevance to the functions of Vpr. Specifically, Vpr shares this feature with other auxiliary proteins such as Vif, Rev, Vpu, and Nef.
HIV-1 Vpr is a small oligomeric protein that plays an important role in HIV pathogenesis [5, 17, 19, 23, 42]. The underlying reasons for the selection of Vpr for the present studies are the following: (i) Vpr is a virion associated protein; (ii) Vpr plays a critical role for the replication of virus in macrophages and positively regulates viral replication in T cells; (iii) Vpr is a transcriptional activator of HIV-1 and heterologous cellular genes; (iv) Vpr inhibits proliferation of cells at G2/M phase; (v) Vpr induces apoptosis in diverse cell types including T cells and neurons; (vi) Vpr exhibits immune suppressive effects. Further, studies from non human primates and analysis of viral genes in long term non progressors suggest a correlation between defective Vpr and delayed progression of the disease [5, 43, 44]. More importantly, several Vpr-mediated functions are known to be induced by both cell-associated and virion-associated Vpr [17, 18, 45]. Together these studies point out the biological significance of virion associated factors and its role in early events associated with virus infection. Therefore, understanding the role of oligomerization in Vpr functions and disease progression may provide useful information for the development of therapeutics against HIV-1 targeting Vpr.
The oligomerization feature of Vpr was evaluated by using a complementation system based on Venus protein as a reporter. A strategy involving the generation of chimeric Venus-Vpr protein has allowed us to monitor oligomerization in live cells. This system has the sensitivity to detect Vpr-Vpr and Vpr-Gag interactions. Studies on the oligomerization of Vpr, analyzed by site-specific mutagenesis, identified some residues located in specific domains of Vpr. However, there is no information available regarding the dimer interface structure of Vpr. To address this, we have utilized the available NMR structure and modeling approaches to identify the residues that form putative dimer and oligomer interfaces of Vpr. Deletion of residue at position 44, which is predominantly glutamine (Q), is known to be oligomerization defective and was used as a control in these experiments. We reasoned that moderate replacements, in particular those mostly affecting size, should show significant biological effects if these residues form part of the interfaces. Thus, for A30 and L68 we chose L and E replacements, respectively, conserving hydrophobicity but changing the size of the position. On the other hand, at position H71, we chose R, maintaining the positive charge. Mutant Vpr molecules were generated containing alterations in the selected residues. The results regarding the expression and steady state level of Vpr indicated that mutants lacking the ability to oligomerize exhibit a pattern similar to that of wild type Vpr. This observation suggests that monomeric Vpr molecules are stable in cells, which is in agreement with an earlier report .
To interpret the mutagenesis data in structural terms, we created structural models of the Vpr dimer and oligomers through molecular docking based on the available full length monomer structure . Our models are shown in Figure 3, highlighting the positions of the residues that resulted in oligomerization defective properties of Vpr in vivo. The docked models revealed propensities for both parallel and antiparallel orientations of the monomers within a dimer indicating that both of the conformations are plausible. Regardless of orientation, helices II and III constitute the dimer interface in the majority of the models. In the parallel orientation, residues 30, 44, 68, 71, all of which fail to oligomerize when mutated, even with the conservative substitutions chosen, were part of the interface; while in the antiparallel orientation residues 44, 68 and 71 contributed to the binding energy in the interface. Further supporting our predicted interfaces, recent studies of Vpr mutants I60A and I67A indicated that these residues play a major role in trafficking the Vpr to the nuclear rim . Both of these residues are part of the interface in the predicted parallel conformation, whereas I60 is part of the antiparallel model. In agreement with the experimental results presented here suggest that both of these two dimer conformations are likely to occur in vivo. The parallel orientation fits the experimental data better, but further studies will be needed to fully differentiate between the two models. Furthermore, assays are needed that can clearly distinguish dimers from higher order oligomers. Based on the combined modeling and experimental results, we propose that dimerization primarily involves helices II and III, while oligomerization includes helix I also. The fact that the Vpr mutation A30L reported here and the recently studied nuclear rim localization defective mutant L23F  are located in helix I supports the proposed role of helix I in oligomerization. We predict that because helix I in the dimer models faces away from the dimer interface, it may play a pivotal role in mediating higher order Vpr-Vpr interactions. We therefore built models for higher order multimeric forms, and a hexamer model (Fig. 3C and Fig. 3D) exhibited an interface where all the three helices participated in interaction interfaces. In this hexamer model, both parallel and antiparallel dimeric units were present and the hydrophobic residues faced to interior of the helices (Fig. 3D).
HIV-1 Vpr is one of the non-structural proteins that is packaged in significant quantities in virus particles. Virion-associated Vpr is present in the infected cells prior to de novo synthesis and is known to cause the host cellular dysfunctions during early infection [17, 19, 42]. Studies have indicated that the p6 domain of Gag is critical for the incorporation of Vpr into virus particles [15, 39, 40, 46]. However, it is not clear whether Vpr oligomerization is a prerequisite for virion incorporation. As expected, chimeric Venus containing wild type Vpr and chimeric Venus containing Gag resulted in the reconstitution of Venus with fluorescence suggesting an interaction between these two proteins. On the other hand, chimeric Venus containing mutant Vpr failed to interact with Gag. Vpr mutants that showed oligomerization negative phenotype also failed to incorporate into virus particles. Several studies have reported that virus particle contains between 14-275 molecules of Vpr in comparison to approximately 2500-2750 molecules of Gag protein depending on the system and methods used [38, 47]. This suggests that low amount of Vpr to Gag may be due to the interaction restricted to the specific configuration of Gag. Several studies evaluated Vpr-Gag interaction and reported that helical domain I (residues E25 and A30), P35 and helical domain III (isoleucine-leucine residues) in Vpr are required for interaction with Gag, thus virion incorporation [26, 39, 48]. Our results on Vpr-Gag interaction using BiFC (Table 2) are in agreement with these studies and further supports the utility of BiFC assay for evaluating the interactions of Vpr with interacting partner proteins. Oligomerization defective mutants, A30L, Δ44, L68E and H71R lack the ability to incorporate into the virus particles, suggesting that Vpr oligomerization might be directly linked to virion associated Vpr functions, pathogenesis and disease progression. A very recent publication further confirms our findings that Vpr oligomerization is required for interaction with Gag and oligomerization deficient mutants of Gag interacted with Vpr .
An understanding of HIV-1 Vpr functions and its properties, in our view, is likely to shed light on the mechanisms involved in Vpr incorporation into the virus particle and how oligomerization feature influences infection of non dividing target cells. Although Vpr is not essential for virus replication in in vitro studies using established cell lines, it is well established that virion- associated Vpr play a major role in macrophage infection by aiding the transport of PIC into the nucleus [50, 51]. More importantly, virion-associated Vpr is known to mediate several host cellular events and immune evasive functions that are very similar to de novo synthesized Vpr [18, 52, 53]. This further bolsters the significance of virion-associated proteins present both in infectious and noninfectious particles and their role in HIV-1 pathogenesis. These studies further support the idea of developing potential therapeutic agents including small molecules against Vpr-Vpr interaction, Vpr-Gag interaction, virion incorporation and virion associated Vpr induced host cell dysregulation to combat HIV-1 infection.