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.