Merkel cell polyomavirus (MCPyV)-positive Merkel cell carcinoma (MCC) tumor cell growth is dependent on the expression of a viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. This RB-binding domain in MCPyV-LT is-in contrast to other polyomavirus LTs (e.g., SV40)-embedded between two large MCPyV unique regions (MUR1 and MUR2). To identify elements of the MCPyV-LT necessary for tumor cell growth, we analyzed the rescue activity of LT variants following knockdown of the endogenous LT in MCC cells. These experiments demonstrate that nuclear localization is essential for LT function, but that a motif previously described to be a nuclear localization sequence is neither required for nuclear accumulation of truncated MCPyV-LT nor for promotion of MCC cell proliferation. Furthermore, large parts of the MURs distal to the RB binding domain as well as ALTO-a second protein encoded by an alternative reading frame in the MCPyV-LT mRNA-are completely dispensable for MCPyV-driven tumor cell proliferation. Notably, even MCPyV-LTs in which the entire MURs have been removed are still able to promote MCC cellular growth although rescue activity is reduced which may be due to MUR1 being required for stable LT expression in MCC cells. Finally, we provide evidence implying that-while binding to Vam6p is not essential-HSC-70 interaction is significantly involved in mediating MCPyV-LT function in MCC cells including growth promotion and induction of E2F target genes.Polyomaviruses are small DNA viruses with a circular double-stranded genome coding for 5-9 proteins. 1 The genome is divided into an early and a late region. From the early region the T antigens (TA) are derived by differential splicing of a common transcript while the late region encodes for capsid proteins into which the viral genome is packaged. 2 It has long been known that polyomaviruses-as the name indicates-can induce tumor formation. 3 Until recently, however, this had only been observed in animal models whiledespite extensive research and controversial discussion-no convincing evidence for a contribution of the Simian Virus 40 (SV40), a monkey polyomavirus that is also present in the human population, or the two well-studied human polyomaviruses JC and BK to human malignancies could be obtained. 4,5 In contrast, one of the seven newly identified human polyomaviruses discovered during the last 6 years, the Merkel cell polyomavirus (MCPyV), is now well established as a causal factor of Merkel cell carcinoma (MCC). 2,4,6 MCC is a rare but very aggressive skin cancer with high mortality rates. Immunosuppression, UV exposure and old age are risk factors for developing MCC. 7 The molecular pathogenesis of MCC, however, remained enigmatic until MCPyV was discovered as monoclonally integrated into the genome of most MCCs indicating that integration occurred prior to clonal expansion of the tumor cells. 8 In many different countries the presence of MCPyV in the vast majority of MCC tumors has been confirmed, 9-12 and a recent publication s...

Merkel cell polyomavirus (MCPyV) is regarded as a major causal factor for Merkel cell carcinoma (MCC). Indeed, tumor cell growth of MCPyV-positive MCC cells is dependent on the expression of a truncated viral Large T antigen (LT) with an intact retinoblastoma protein (RB)-binding site. Here we determined the phosphorylation pattern of a truncated MCPyV-LT characteristically for MCC by mass spectrometry revealing MCPyV-LT as multi-phospho-protein phosphorylated at several serine and threonine residues. Remarkably, disruption of most of these phosphorylation sites did not affect its ability to rescue knockdown of endogenous T antigens in MCC cells indicating that phosphorylation of the respective amino acids is not essential for the growth promoting function of MCPyV-LT. However, alteration of serine 220 to alanine completely abolished the ability of MCPyV-LT to support proliferation of MCC cells. Conversely, mimicking the phosphorylated state by mutation of serine 220 to glutamic acid resulted in a fully functional LT. Moreover, MCPyV-LT S220A demonstrated reduced binding to RB in co-immunoprecipitation experiments as well as weaker induction of RB target genes in MCC cells. In conclusion, we provide evidence that phosphorylation of serine 220 is required for efficient RB inactivation in MCC and may therefore be a potential target for future therapeutic approaches.Merkel cell carcinoma (MCC) is a very aggressive skin cancer translating into high mortality rates. Immunosuppression is one of the risk factors for developing MCC. 1 This may be explained by the fact that a viral infection contributes to MCC. In this regard, DNA of the Merkel cell polyomavirus (MCPyV) is detected in the vast majority of MCC cases, 2 and monoclonal integration of MCPyV into the tumor genome indicates that viral integration occurs prior to clonal expansion of the tumor cells. 3 Polyomaviruses are small DNA viruses with a circular double-stranded genome encoding five -nine proteins. 4 From the early region different oncoproteins termed T antigens (TA) are derived by translation of differentially spliced mRNAs. In MCPyV-positive MCC cells the viral small and Large T antigen (sT and LT) are expressed. 5,6 LT and sT share the same Nterminal region comprising 78 amino acids but differ in their C-terminus. Importantly, MCC-associated MCPyV-LTs are characterized by large C-terminal deletions. 5,7 These truncated LTs result from point mutations leading to premature stop codons, or are due to integration break points. Generally, however, the retinoblastoma protein (RB) binding motif is preserved, and a mutation affecting RB binding abolishes the ability of MCPyV-LT to promote growth of MCC cells. 8 The molecular function of a protein is not only dependent on its amino acid sequence, but also determined by posttranslational modifications. A prominent type of posttranslational modification is phosphorylation. 9 It has been estimated that 30% of all cellular proteins contain at least one phosphorylated residue. 10 Hence, it is not surprising that po...

Supplemental Digital Content is Available in the Text.Deep learning-based analysis of large-scale bioimages of the dorsal root ganglion after nerve injury reveals satellite glial cell plasticity but no loss of sensory neurons.

Phosphodiesterase 3 (PDE3) exists in two isoforms (PDE3A and PDE3B) and is known to act as cGMP-inhibited cAMP-degrading PDE. Therefore, PDE3 may likely be involved in the interaction between the two second messenger pathways. NO-sensitive guanylyl cyclase (NO-GC) is the most important cytosolic generator of cGMP. Here, we investigated the effect of NO-GC deletion on PDE3A-mediated signaling in animals lacking NO-GC either globally (GCKO) or specifically in smooth muscle cells (SMC-GCKO). PDE3A expression is detected in murine aortic smooth muscle, platelets, and heart tissue. Expression and activity of PDE3A in aortae from GCKO and SMC-GCKO mice was reduced by approx. 50% compared to that in control animals. PDE3A downregulation can be linked to the reduction in NO-GC and is not an effect of the increased blood pressure levels resulting from NO-GC deletion. Despite the different PDE3A expression levels, smooth muscle relaxation induced by forskolin to stimulate cAMP signaling was similar in all genotypes. Basal and forskolin-stimulated cAMP levels in aortic tissue were not different between KO and control strains. However, the potency of milrinone, a selective inhibitor of PDE3A, to induce relaxation was higher in aortae from GCKO and SMC-GCKO than that in aorta from control animals. These data were corroborated by the effect of milrinone in vivo, which led to an increase in systolic blood pressure in both KO strains but not in control mice. We conclude that NO-GC modulates PDE3A expression and activity in SMC in vivo conceivably to preserve functional cAMP signaling.

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