Search

Poly(ADP-ribosyl)ation, catalysed by a family of poly(ADP-ribose) polymerases (PARPs), plays an important role in a large variety of physiological processes, including cell proliferation, but its role in cell cycle progression is not yet completely defined. As reported here, the examination of early times following serum stimulation of quiescent fibroblasts suggests that poly(ADP-ribosyl)ation is necessary for the transition from the G0 phase to the G1 phase. We show that PARP activity is involved in this step through the regulation of immediate-early response genes, such as c-Fos and c-Myc. This is supported by the finding that exogenous Myc expression substantially restores cell cycle reactivation in the absence of polymer synthesis. Furthermore, using RNA interference, we show that PARP-1 is the PARP family member playing the most prominent role in the upregulation of c-Fos and c-Myc during G0-G1 transition. We report that even in lectin-stimulated peripheral blood mononucleated cells, the inhibition of PARP activity interferes with the upregulation of immediate-early genes and delays the induction of proliferation, suggesting a general role for PARP-1 in linking growth factor signaling with cell cycle entry.

The first contact of a virus with the host cell surface and further entry are important steps for a successful outcome of the infection process and for the virus-associated pathogenicity. We have previously shown that the entry of the murine Polyomavirus (Py) into fibroblasts is a multi-step process involving, at least, the attachment to primary sialic acids (SA)-containing cell receptors followed by post-binding interaction with secondary receptors, such as the alpha4beta1 integrin, likely through the VP1-LDV motif. Here we report on the functional role of the VP1-LDV motif in Py infectivity and in vivo virus tissue tropism. For this purpose, we have characterized a recombinant virus mutant, PyLNV, harboring a single aa substitution in this motif (D138N). Although not critical for virus viability, the D138N substitution abrogates the post-attachment Py-alpha4beta1 interaction, rendering the PyLNV mutant virus twofold less infectious than the Py wild-type (Wt) in alpha4beta1-positive fibroblasts. To study the putative role of the VP1-LDV motif in vivo, newborn C57BL/6 mice were inoculated with PyWt or PyLNV and, after six days, organs were analyzed for the presence of viral DNA. Intriguingly, PyLNV showed an altered spectrum of in vivo replication compared with PyWt, particularly in the skin and in the kidney. The implication of Py-alpha4beta1 integrin interaction in conditioning tissue-specificity of virus replication is discussed.

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.

Murine polyomavirus (MPyV) infection occurs through recognition of sialic acid (SA) residues present on the host cell membrane, but the nature of the molecules involved and the exact role of this interaction in virus cell entry still need to be clarified. In this work, mutations at residues R(77) or H(298) of the MPyV VP1 protein were shown to lead to a complete loss of virus infectivity, which, however, could be restored by lipofection of virus particles into the cytoplasm of the host cells. Using virus-like particles (VLPs), it was demonstrated that the non-infectivity of these mutants was due to impaired cell entry caused by total abrogation of SA-dependent cell binding. This indicates that SA residues are essential primary cell receptors for MPyV. As the alpha4beta1 integrin has been identified recently as a cell receptor for MPyV, the relationship, if any, was investigated between SA-containing and alpha4beta1 integrin receptors. The ability of mutants R(77)Q and H(298)Q and wt VLPs to bind to cells overexpressing the alpha4beta1 integrin was studied in SA-positive (BALB/c 3T3 cells and Pro-5 cells) and SA-deficient (Pro5-derived Lec-2 cells) backgrounds. Overexpression of alpha4beta1 integrin did not restore binding of mutant VLPs in any of these cell lines or, indeed, that of wt VLPs in a SA-deficient background. Moreover, evidence is provided that overexpression of the sialylated alpha4beta1 integrin enhances wt VLP cell binding, suggesting that, in addition to its function at a post-attachment level, alpha4beta1 integrin acts also as one of the SA-containing receptors for initial cell binding.

Dystroglycan is a transmembrane heterodimeric complex of alpha and beta subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin-soluble extract from bovine brain synaptosomes by laminin-affinity chromatography and characterized the protein components. The 120-kDa alpha-dystroglycan was the major 125I-laminin-labeled protein detected by overlay assay. This complex, in addition to beta-dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti-FAK antibodies co-immunoprecipitated Grb2 with FAK. However, no direct interaction between beta-dystroglycan and FAK was detected by co-precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind beta-dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant beta-dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK-beta-dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.

In normal mouse superior cervical ganglion, dystrophin immunoreactivity is present in ganglionic neurons, satellite cells and Schwann cells. It is associated with several cytoplasmic organelles and specialized plasma membrane domains, including two types of structurally and functionally different intercellular junctions: synapses, where it is located at postsynaptic densities, and adherens junctions. Dystrophin immunostaining can be ascribed to the 427,000 mol. wt full-length dystrophin, as well as to the several dystrophin isoforms present in superior cervical ganglion, as revealed by western immunoblots. In mdx mouse superior cervical ganglion, which lacks the 427,000 mol. wt dystrophin, the unchanged pattern of dystrophin immunolabelling observed at several subcellular structures indicates the presence of dystrophin isoforms at these sites. Moreover, the absence of labelled adherens junctions indicates the presence of full-length dystrophin at this type of junction in the normal mouse superior cervical ganglion. The lower number of immunopositive postsynaptic densities in mdx mouse superior cervical ganglion than in normal mouse ganglion suggests the presence, in the latter, of postsynaptic densities with differently organized dystrophin cytoskeleton: some containing dystrophin isoforms alone or together with 427,000 mol. wt dystrophin, and others containing 427,000 mol. wt dystrophin alone.

Alpha- and beta-dystroglycan form a heteromeric transmembrane complex linking the extracellular matrix to the cytoskeleton. In muscle beta-dystroglycan interacts with dystrophin on the inside of the cell and with alpha-dystroglycan, which binds the extracellular matrix protein laminin, on the outside. Dystroglycan is expressed not only in muscle but also in other tissues. We cloned beta-dystroglycan from rabbit brain by RT-PCR and expressed deletion mutants of the beta-dystroglycan cytoplasmic domain as GST-fusion proteins. We identified the dystrophin binding region on beta-dystroglycan by protein overlay and co-precipitation assays with skeletal muscle dystrophin and recombinant apo-dystrophin I. We demonstrate that the beta-dystroglycan carboxyl terminus interacts with dystrophin and that the binding site is restricted to the last 20 amino acids. Our data also suggest that the region adjacent to the beta-dystroglycan transmembrane domain might modulate beta-dystroglycan-dystrophin interaction.