Structural studies have been invaluable to understanding HIV-1 viral interaction with host cells, including non-dividing macrophages. Relatively recent structural studies have identified three alpha helical domains, α-H1 (13-33), α-H2 (38-50), and α-H3 (55-77) as well as other structural features capable of mediating diverse biological functions . Indeed, Vpr's structure allows for direct binding to many cellular proteins, which likely enables Vpr to mediate functions such as nuclear import and G2 arrest. All three alpha helices have been implicated in Vpr mediated nuclear localization [12, 13, 59–62], while the G2 arrest property has been attributed mainly to the C-terminal region of Vpr . However, as the nuclear import, promoter transactivation, and G2 arrest properties of Vpr seem to not only be related, at least on a structural level, they also may be jointly attributed to specific physiological properties of Vpr in productive HIV-1 infection of macrophages .
Vpr mediates nuclear localization by binding to importin-α via residues located within the alpha helices. While some studies initially reported a low affinity of Vpr for importin-α , others have found that Vpr binds to importin-α using other techniques [50, 51, 64]. Vpr/importin-α binding was shown to be non-competitive with that of the classical the NLS found on MA . Kamata and others demonstrated that regions 17-34 (αH1) and 46-74 (αH2+αH3) can both independently localize to the nucleus, albeit to a lower extent than an identified bona fide Vpr NLS consisting of residues 17-74 . Mutations in αH1, αLA (L20,22,23,26A), as well as in αH2+αH3, I60P and L69P, completely ablated the ability of the individual peptides to localize to the nucleus. Later, Kamata and others found that Vpr αH1 and αH3 both bind importin-α, that the IBB domain of importin-α primarily determines this interaction, and that the C-terminal domain of importin-α, 393-462, is necessary for nuclear localization of Vpr . Although, an importin-α lacking an IBB still facilitated import of Vpr, a mutation in Vpr's first alpha helix, αLA, impaired importin-α binding and nuclear localization but still showed perinuclear accumulation. In contrast, a mutation in the third alpha helix, L67P, failed to localize to both the nuclear and perinuclear areas, but still permitted binding to importin-α. The authors concluded that binding to importin-α requires only the first alpha helix and that the third alpha helix serves to localize Vpr to the perinuclear area independently of importin binding. Previous findings from other investigators also showed that the use of IBB peptides failed to inhibit Vpr mediated nuclear localization. This suggests that importin-α may be essential for Vpr's karyophilic properties but that the direct interaction between importin-α and Vpr may not be essential . Hitahara-Kasahara and others showed that importin-α1, α3, and α5 isoforms are all able to induce Vpr mediated nuclear import . Importin-α was shown to be essential for HIV-1 replication in macrophages, suggesting that importin-α nuclear import is a vital process in the infection of these cells. Furthermore, a recent study found that Vpr does not bind to importin-α2 or importin-α2/β1 heterodimers, suggesting that cell-line specific expression of importins may regulate Vpr's karyophilic properties . In summary, these studies suggest that importin-α is important for Vpr-mediated nuclear translocation, but the exact nature of this mechanism is still under investigation.
In addition to the reported binding interaction with importin-α, Vpr has been demonstrated to bind directly to nuclear pore proteins [47, 49–51, 67]. Vpr mutants F34I and H71R have been found to lose the ability to localize to perinuclear areas, suggesting that these residues are involved in nuclear pore interaction . These mutants were still found in the nucleus, which is not surprising considering that Vpr is less than 40kDa. The F34I mutant showed lower binding to importin-α and Nsp1p, a member of the nuclear pore complex. WT Vpr colocalizes with importin-β and nuclear pores in perinuclear regions and binds both Pom 121 and very weakly to Nsp1p . An A30P mutant lacked these abilities.
FXFG regions on nucleoporins, a form of phenylalanine-glycine (FG) repeat, have been reported to interact with cytoplasmic proteins involved in nuclear import [22, 68, 69]. Vpr was reported to bind to FXFG containing proteins p54 and p58 as well as to the FXFG region of Nup1 . Further, addition of Vpr was shown to stabilize the binding of importin-α/β to Nup1 FXFG. Another report failed to show interaction between Vpr and FXFG of Pom121, but instead demonstrated that the alpha helices of Vpr interact with hCG1 by binding to a non-FG repeat region located in the N-terminal region on residues 49-170 . This area has no known homology to binding motifs and has no known binding partners. In a later study, it was found that four Vpr mutants L23F, K27M, A30L, and F34I, which all occur on one face of the first alpha helix, have impaired hCG1 binding and fail to show nuclear localization . Thus, it seems that Vpr is able to bind to importin-α as well as nucleoporin using the same residues on the first helix. In both cases, there is evidence that Vpr binding to nucleoporin components occurs in a way that is distinct from the classical NLS pathway.
The role of importin-β in the nuclear transport of Vpr is an aspect of the mechanism of Vpr's karyophilic properties that remains to be fully understood. Early studies showed that Vpr fails to bind importin-β  or that it binds at a low affinity . Oddly, the latter study found greater affinity of Vpr to importin-β than to -α. Subsequent studies argued that Vpr's localization is importin-α, but not -β, dependent. Addition of importin-β to digitonin permeabilized cells, which was required for the classical SV40-NLS localization, was unnecessary for Vpr N17C74, a construct containing the minimal region for nuclear localization [38, 66]. These studies also found that ΔIBB importin-α, which is unable to bind to importin-β, still caused nuclear translocation of N17C74. Previous studies demonstrating that the use of IBB peptides failed to inhibit Vpr localization also lend some support to these findings . Further, importin-β siRNA failed to prevent N17C74 localization to the nucleus . Vpr has also been shown to physiologically behave in ways similar to importin-β, leading some authors to suggest that Vpr replaces the role of importin-β, which, like Vpr, also binds to both importin-α and nuclear pores, in the nuclear translocation process . Other studies, however, suggest that importin-β is necessary for Vpr's karyophilic properties. Papov and others found that Vpr prevents FXFG Nup 1 peptide mediated dissociation of MA with importin-α/β complexes and increases the affinity of importin-α to NLS [51, 65]. Based on these findings Papov and others proposed that Vpr stabilizes the MA and IN NLS complex with importin-α/β to promote nuclear entry. A dominant negative form of importin-β, residues 71-876  has also been shown to inhibit Vpr localization, further suggesting that importin-β plays a role in Vpr mediated nuclear targeting . Recent studies have clearly shown binding of Vpr to importin-β3, but not to importin-β1 or to importin-α2/β1 complexes . This may explain discrepancies in early findings that failed to find effects of isolated importin-β which were not necessarily applicable to other importin-β isoforms.
The respective roles of the alpha helices and the C-terminal region in nuclear localization and G2 arrest remain controversial. Through extensive mutational analysis, Mahalingam and others put forth a hypothesis that the nuclear localization function resides primarily in the alpha helices while the G2 arrest property is determined by the carboxyl-terminus . Previous studies lend support to this assertion as the alpha helices, but not N-terminal or C-terminal regions were involved in nucleoporin binding by Vpr . Other reports found that N17C74 Vpr, which lacks the C and N terminal regions and other Vpr constructs lacking the C-terminus are unimpaired in nuclear localization [11, 66]. Although the C-terminal region closely resembles a classical NLS, this region does not have NLS function and Vpr functions independently of NLS binding [14, 71]. Conversely, many other studies found that the C-terminal is necessary or sufficient for nuclear entry of Vpr [12, 34, 47, 62, 72]. The discrepancy between these studies remains unexplained. Interestingly, recent studies have shown that all three alpha helices are involved in Vpr oligomerization . The authors reported that mutations that affected oligomerization did not prevent apoptosis induction by Vpr (a G2 arrest dependent property ). Nuclear localization, however, was perturbed for these mutants. These studies may suggest that karyophilic and cell cycle arrest properties rely on multiple domains that may be separable to some degree.