Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1 V165A , originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including 186 KRK 188 / 211 KELQKQITK 219 and Cys-130. Previous work established HIV-1 K186Q , HIV-1 Q214L/Q216L , and HIV-1 C130G as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1 V165A synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1 C130G was also defective for reverse transcription, but Vpr-integrase C130G did not efficiently complement class I mutant HIV-1. Since HIV-1 C130A grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1 C130G phenotype.

Cyclin-dependent kinase inhibitors (CKIs) and Notch receptor activation have been shown to influence adult stem cells and progenitors by altering stem cell self-renewal and proliferation. Yet, no interaction between these molecular pathways has been defined. Here we show that ligand-independent and ligand-dependent activation of Notch1 induces transcription of the S phase kinase–associated protein 2 (SKP2), the F-box subunit of the ubiquitin-ligase complex SCFSKP2 that targets proteins for degradation. Up-regulation of SKP2 by Notch signaling enhances proteasome-mediated degradation of the CKIs, p27Kip1 and p21Cip1, and causes premature entry into S phase. Silencing of SKP2 by RNA interference in G1 stabilizes p27Kip1 and p21Cip1 and abolishes Notch effect on G1-S progression. Thus, SKP2 serves to link Notch1 activation with the cell cycle machinery. This novel pathway involving Notch/SKP2/CKIs connects a cell surface receptor with proximate mediators of cell cycle activity, and suggests a mechanism by which a known physiologic mediator of cell fate determination interfaces with cell cycle control.

Retroviral replication requires the accomplishment of certain key steps in the early phase of the viral life cycle. Soon after entering a cell, the viral enzyme reverse transcriptase (RT) copies genomic RNA into linear double-stranded cDNA. The viral enzyme integrase (IN) then inserts this DNA into a host cell chromosome. In vivo, reverse transcription and integration take place in the context of large nucleoprotein complexes that are called reverse transcription complexes (RTCs) and preintegration complexes (PICs), respectively (reviewed in reference 29).Two different IN activities are required for integration. During an initial 3Ј processing reaction, IN cleaves each cDNA end adjacent to the phylogenetically conserved sequence CA. For both Moloney murine leukemia virus (MoMLV) (7,28,45) and human immunodeficiency virus type 1 (HIV-1) (12, 13, 39), 3Ј processing can occur in the cell cytoplasm. Following nuclear entry, IN transfers the processed 3Ј ends to the 5Ј phosphates of a double-stranded staggered cut in chromosomal DNA (7, 28). The final step of the integration process, which involves repairing the gaps between the unjoined viral 5Ј ends and the chromosome, can be completed by host cell enzymes (4, 54).The double lipid bilayer that envelops animal cell nuclei presents a formidable barrier for retroviral PICs. The nuclear envelope contains numerous nuclear pore complexes (NPCs) that permit the passive diffusion of relatively small macromolecules with diameters up to about 9 nm, which corresponds roughly to a 40-to 60-kDa globular protein (reviewed in reference 37). The relatively large size of retroviral PICs, estimated to be roughly the size of a ribosome for HIV-1 (25), precludes their passive transport through intact NPCs (reviewed in reference 27). Different retroviruses have apparently evolved different strategies to access the cell nucleus. HIV-1, for example, can be transported by an energy-dependent process in nondividing cells (9), suggesting that HIV-1 PICs contain specific nuclear localization sequences (NLSs) that govern their transport through intact NPCs (reviewed in references 27 and 52). Productive infection by MoMLV, in contrast, requires cells to pass through the M phase of the cell cycle (36, 44). Since animal cell nuclei disassemble during mitosis, MoMLV apparently reaches cell chromosomes in the absence of active nuclear transport by waiting for the dissolution of nuclear membranes.Recently, replication-defective MoMLV (55) and HIV-1 (3, 57) mutants were described as blocked at the nuclear import step in rapidly dividing cells. This suggested that retroviruses

In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.