VIRAL PROTEIN-R REGULATES NUCLEAR IMPORT OF THE HIV-1 PREINTEGRATION COMPLEX

Replication of human immunodeficiency virus type 1 (HIV-1) in non-divi ding cells critically depends on import of the viral pre-integration c omplex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import pr ocess. An ill vitro assay that reconstitutes nuclear import of HIV-1 p re-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 t o be imported in vitro, whereas mutants that Lack a functional MA nucl ear localization sequence (NLS) were only partially defective, Strikin gly, the import defect of the Vpr(-) mutant was rescued when recombina nt Vpr was readded, In addition, import of Vpr virus was rescued by ad ding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent, In a solution binding assay, Vpr associated wi th karyopherin alpha, a cellular receptor for NLSs. This association i ncreased the affinity of karyopherin alpha for basic-type NLSs, includ ing that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 preintegration complex and BSA-NLS conjugates, The se results identify the biochemical mechanism of Vpr function in trans port of the viral pre-integration complex to, and across, the nuclear membrane.