Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Puralpha in NIH3T3 cells

Levels of Puralpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Puralpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Puralpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Puralpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Puralpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Puralpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Puralpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Puralpha. Biochemical analysis of nuclear and cytoplasmic Puralpha proteins and confocal microscopic analysis of Puralpha location indicate that access of Puralpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Puralpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Puralpha colocalizes with CDK2 and cyclin A. Puralpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Puralpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Puralpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

Effect of HIV-1 Vpr on cell cycle regulators

Cell cycle is one of the most complex processes in the life of a dividing cell. It involves numerous regulatory proteins, which direct the cell through a specific sequence of events for the production of two daughter cells. Cyclin-dependent kinases (cdks), which complex with the cyclin proteins, are the main players in the cell cycle. They can regulate the progression of the cells through different stages regulated by several proteins including p53, p21(WAF1), p19, p16, and cdc25. Downstream targets of cyclin-cdk complexes include pRB and E2F. A cell cycle can be altered to the advantage of many viral agents, most notably polyomaviruses, papillomaviruses, adenoviruses, and retroviruses. In addition, viral protein R (Vpr) is a protein encoded by the human immunodeficiency virus type 1 (HIV-1). HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), is a member of the lentivirus class of retroviruses. This accessory protein plays an important role in the regulation of the cell cycle by causing G(2) arrest and affecting cell cycle regulators. Vpr prevents infected cells from proliferating, and collaborates with the matrix protein (MA) to enable HIV-1 to enter the nucleus of nondividing cells. Studies from different labs including ours showed that Vpr affects the functions of cell cycle proteins, including p53 and p21(WAF1). Thus, the replication of HIV-1, and ultimately its pathogenesis, are intrinsically tied to cell-cycle control.     

The TRIM family protein KAP1 inhibits HIV-1 integration

The integration of viral cDNA into the host genome is a critical step in the life cycle of HIV-1. This step is catalyzed by integrase (IN), a viral enzyme that is positively regulated by acetylation via the cellular histone acetyl transferase (HAT) p300. To investigate the relevance of IN acetylation, we searched for cellular proteins that selectively bind acetylated IN and identified KAP1, a protein belonging to the TRIM family of antiviral proteins. KAP1 binds acetylated IN and induces its deacetylation through the formation of a protein complex which includes the deacetylase HDAC1. Modulation of intracellular KAP1 levels in different cell types including T cells, the primary HIV-1 target, revealed that KAP1 curtails viral infectivity by selectively affecting HIV-1 integration. This study identifies KAP1 as a cellular factor restricting HIV-1 infection and underscores the relevance of IN acetylation as a crucial step in the viral infectious cycle.     

Role of the retinoblastoma tumor suppressor protein in cellular differentiation

The retinoblastoma protein (pRb105) is a true tumor suppressor as deregulation of the Rb pathway by either mutation of pRb105 itself or other proteins in the pathway, such as p16INK4a, occur in most cancers. This prototypical family member, along with the related p107 and p130, are involved in the control of cell cycle regulation, but pRb105 has also been shown to be involved in tissue development and differentiation. This prospective will discuss the increasing evidence for a role of pRb105 in cellular differentiation and the fact that various cancers, which contain mutant pRb105, or mutations in proteins in the pRb105 pathway, are perhaps a result of deregulation of differentiation.