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."

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The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents infected cells from passing through mitosis by arresting them in the G 2 phase of the cell cycle. Vpr is conserved among all primate lentiviruses, suggesting an important role in the virus life cycle. Moreover, in this study we show that the ability to cause cell cycle arrest is also conserved in Vpr proteins from a wide variety of both tissue culture-passaged and uncultured human (HIV-1 and HIV-2), sooty mangabey (simian immunodeficiency virus SIV SM), African green monkey (SIV AGM), and Sykes' monkey (SIV SYK) isolates. However, this property is cell type specific and appears to depend on the particular primate species from which the cells are derived. SIV AGM and SIV SYK Vpr proteins are capable of arresting African green monkey cells but are completely inactive in human cells. By contrast, HIV-1, HIV-2, and SIV SM Vpr proteins function in both simian and human cell types, although SIV SM Vpr functions more efficiently in simian cells than it does in human cells. Neither differential protein stability nor subcellular localization explains the species-specific activities of these proteins. These results thus suggest that Vpr exerts its G 2 arrest function by interacting with cellular factors that have evolved differently among the various primate species.

Lentiviruses share the common characteristic of infecting non-dividing target cells, distinguishing them from the oncogenic retroviruses which only productively infect dividing cells. The search for determinants for infection of non-dividing cells has produced a number of candidates. From HIV-1, the viral proteins matrix, integrase and Vpr have all been implicated. A structural determinant, the central DNA flap, has also been implicated. The supporting evidence for each of these proposed determinants will be examined and compared to how other viruses, non-retroviruses, transport their genomes to the nucleus. With currently available data, integrase and the central DNA flap appear to be the key players, and yet the mechanism for infection of non-dividing cells remains undefined.

Spemann's Organizer, located in the dorsal marginal zone of the amphibian gastrula, induces and differentiates dorsal axial structures characteristic of this and other vertebrates. To trace the cellular origins of the Xenopus Organizer, we labelled dorsal blastomeres of three of the four tiers (A, B and C) of the 32-cell embryo with green, red and blue fluorescent lineage tracers. A strong vegetalward displacement of labelled clones occurs between the late blastula and early gastrula stages but clones mix only slightly at their borders. The typical early gastrula Organizer is composed of approximately 10% A1 progeny in its animalmost region, 70% B1 progeny in the central region, and 20% C1 progeny in vegetal and deep regions. Variability in the composition of the early gastrula Organizer results from variability in the position of early cleavage planes and in pregastrulation movements. As the Organizer involutes during gastrulation, forming dorsal axial mesoderm, clonal boundaries are greatly dispersed by cell intermixing. Within a clone, deep cells are displaced and intermixed more than superficial cells. Variability in the distribution of progeny in the dorsal axial mesoderm of the late gastrula results mostly from variable intermixing of cells during gastrulation. Experiments to perturb later developmental events by molecular or embryonic manipulations at an early stage must take this variability into account along with the majority distributions of the fate map. Within the early gastrula Organizer, the genes Xbra, goosecoid, noggin and xNR3 are expressed differently in the animal-vegetal and superficial-deep dimensions. In situ hybridization and lineage labelling define distinct regions of the dorsal marginal zone. By the end of gastrulation, dorsal axial mesoderm cells derived from the Organizer have altered their expression of the genes Xbra, goosecoid, noggin and xNR3. At a given stage, a cell's position in the embryo rather than its lineage may be more important in determining which genes it will express.