To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G 2 cell-cycle arrest. Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells. Like the importin-␤ subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-␣ subunit and nucleoporins. Moreover, overexpression of either Vpr or importin-␤ in yeast blocks nuclear transport of mRNAs. A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-␣ and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently. Vpr F34I, however, still causes a G 2 arrest, demonstrating that the dual functions of Vpr are genetically separable. Our data suggest Vpr functionally resembles importin-␤ in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G 2 arrest.

CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."