The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control.

We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (N pro ) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from N pro -expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as "community effect".

International audienceAvailable empirical data on the natural occurrence of ruminant pestiviruses has shown that bovine viral diarrhoea virus (BVDV) is nearly exclusively found in cattle, whereas both border disease virus (BDV) and BVDV can be isolated from sheep. During routine genetic typing of pestivirus RNA from UK cattle diagnosed as BVDV positive between 2006 and 2008, five samples that were classified as BDV positive yielded positive virus isolates in cell cultures. The samples originated from animals that had shown signs typical for BVD. Phylogenetic analysis of the bovine BDVs showed that two belonged to the BDV-1a group and three to the BDV-1b group, thereby matching the genetic diversity seen for previously described UK ovine BDVs. Antigenic typing with a set of monoclonal antibodies (MABs) showed that all bovine BDVs lacked one or more epitopes conserved among ovine BDV-1 isolates, and that they had gained reactivity with at least one BVDV-1 specific MAB. Serial passaging of two of the virus isolates in ovine cell cultures did not change the epitope expression pattern. These findings suggest that the presumed natural resistance of cattle against infection with BDV no longer holds. A consequence of this is that BVD diagnostic assays should be checked for their ability to also detect BDV, and also highlights the need for monitoring of the BDV status in sheep that may be in contact with cattle in areas with organised BVD control programmes