Whitehouse, A. | bioRxiv Science

bioRxiv2020

Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion

Merkel cell polyomavirus (MCV) small T antigen (sT) is the main oncoprotein for the development of Merkel cell carcinoma (MCC). MCC is a rare, clinically aggressive neuroendocrine tumor of the skin with a high propensity for local, regional, and distant spread. The dysregulation of matrix metalloproteinase-9 (MMP-9) has been implicated in multiple essential roles in the development of various malignant tumor cell invasion and metastasis. Previously, MCV sT was shown to induce the migratory and invasive phenotype of MCC cells through the transcriptional activation of the Sheddase molecule, ADAM 10 (A disintegrin and metalloprotease domain-containing protein 10). In this study, we show that MCV sT protein stimulates differential expression of epithelial-mesenchymal transition (EMT) associated genes, including MMP-9 and Snail. This effect is dependent on the presence of the large T stabilization domain (LSD), which is known to be responsible for cell transformation through targeting of promiscuous E3 ligases, including FBW7, a known MMP-9 and Snail regulator. Chemical treatments of MMP-9 markedly inhibited sT-induced cell migration and invasion. These results suggest that MCV sT contributes to the activation of MMP-9 as a result of FBW7 targeting, and increases the invasive potential of cells, which can be used for targeted therapeutic intervention. IMPORTANCEMerkel cell carcinoma (MCC) is the most aggressive cutaneous tumor without clearly defined treatment. Although MCC has a high propensity for metastasis, little is known about the underlying mechanisms that drive MCC invasion and metastatic progression. MMP-9 has shown to play a detrimental role in many metastatic human cancers, including melanoma and other non-melanoma skin cancers. Our study shows that MCV sT-mediated MMP-9 activation is driven through the LSD, a known E3 ligase targeting domain, in MCC. MMP-9 may serve as the biochemical culprit to target and develop a novel approach for the treatment of metastatic MCC.

microbiology↗

bioRxiv2019

A requirement for Potassium and Calcium Channels during the Endosomal Trafficking of Polyomavirus Virions

Following internalisation viruses have to escape the endocytic pathway and deliver their genomes to initiate replication. Members of the Polyomaviridae transit through the endolysosomal network and through the endoplasmic reticulum (ER), from which heavily degraded capsids escape into the cytoplasm prior to nuclear entry. Acidification of endosomes and ER entry are essential in the lifecycle of polyomaviruses, however many mechanistic requirements are yet to be elucidated. Alteration of endocytic pH relies upon the activity of ion channels. Using two polyomaviruses with differing capsid architecture, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we firstly describe methods to rapidly quantify infection using an IncuCyte ZOOM instrument, prior to investigating the role of K+ and Ca2+ channels during early stages of infection. Broad spectrum inhibitors identified that MCPyV, but not SV40, is sensitive to K+ channel modulation. In contrast, both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil, however specific targeting of transient or long lasting Ca2+ channel subfamilies had no detrimental effect. Further investigation revealed that tetrandrine blockage of two-pore channels (TPCs), the activity of which is essential for endolysosomal-ER fusion, ablates infectivity of both MCPyV and SV40 by preventing disassembly of the capsid, which is required for the exposure of minor capsid protein nuclear signals necessary for nuclear transport. This study therefore identifies a novel target to restrict the entry of polyomaviruses.\n\nIMPORTANCEPolyomaviruses establish ubiquitous, asymptomatic infection in their host. However, in the immunocompromised these viruses can cause a range of potentially fatal diseases. Through the use of SV40 and MCPyV, two polyomaviruses with different capsid organisation, we have investigated the role of ion channels during infection. Here, we show that Ca2+ channel activity is essential for both polyomaviruses and that MCPyV is also sensitive to K+ channel blockage, highlighting new mechanistic requirements of ion channels during polyomavirus infection. In particular, tetrandrine blockage of endolysosomal-ER fusion is highlighted as an essential modulator of both SV40 and MCPyV. Given that the role of ion channels in disease have been well characterised, there is a large panel of clinically available therapeutics that could be repurposed to restrict persistent polyomavirus infection and may ultimately prevent polyomavirus-associated disease.

microbiology↗