Viral protein R regulates nuclear import of the HIV-1 pre-integration complex

Abstract

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vprmutant was rescued when recombinant Vpr was readded. In addition, import of Vpr -virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin α, a cellular receptor for NLSs. This association increased the affinity of karyopherin α for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 preintegration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.

Macrophages and HIV-1: dangerous liaisons

“…MA NLS mutants were found to be unable to replicate in macrophages in some studies but not in others (Fouchier et al, 1997;Reil et al, 1998;von Schwedler et al, 1994). The third viral protein involved in nuclear import of HIV-1 PIC is the viral protein R (Vpr) (Heinzinger et al, 1994;Popov et al, 1998) which is present only in lentiviruses. Vpr contains two non-canonical NLS that may contribute to nuclear transport of the PIC (Jenkins et al, 1998), and interacts with cellular import machinery through importin  and the nucleoporins (Popov et al, 1998;Vodicka et al, 1998).…”

“…The third viral protein involved in nuclear import of HIV-1 PIC is the viral protein R (Vpr) (Heinzinger et al, 1994;Popov et al, 1998) which is present only in lentiviruses. Vpr contains two non-canonical NLS that may contribute to nuclear transport of the PIC (Jenkins et al, 1998), and interacts with cellular import machinery through importin  and the nucleoporins (Popov et al, 1998;Vodicka et al, 1998). A study based on the overexpression of Vpr has suggested that PIC enters the nucleus by inducing herniations and partial breaking of the nuclear envelope ).…”

“…However, the role of Vpr in the nuclear import of PIC in macrophages is still controversial (Bouyac-Bertoia et al, 2001;Gallay et al, 1997). Vpr and MA may cooperate for the nuclear import of the PIC (Haffar et al, 2000;Popov et al, 1998). However, viruses lacking MA NLS and Vpr (Vpr.NLS) are able to replicate in macrophages, albeit less efficiently than the wild-type virus (Fouchier et al, 1997;Kootstra and Schuitemaker, 1999).…”

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

“…Vpr is a pleiotropic protein with multiple pathobiological mechanisms. These mechanisms include nuclear import of preintegration complex [14], transactivation of several viral promoters including long terminal repeat (LTR) [15,16], and induction of cell cycle arrest at G 2 /M phase (G 2 arrest) [17] and apoptosis [18,19]. Apoptosis induced by Vpr is believed to contribute to CD4+ T cell depletion [17,20].…”

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

“…Experiments in macrophages strongly suggest an important role for Vpr in mediating the nuclear import of HIV-1 PICs into the nucleus of nondividing cells [20]. The mechanism of Vpr-mediated nuclear import is not clear, it is likely that Vpr interacts directly or indirectly with cellular machinery regulating the nucleo-cytoplasmic shuttling [21][22][23][24][25].…”