Infectious Entry of Merkel Cell Polyomavirus

Abstract

27Merkel Cell Polyomavirus (MCPyV) is a small, non-enveloped tumor virus associated 28 with an aggressive form of skin cancer, the Merkel cell carcinoma (MCC). MCPyV 29 infections are highly prevalent in the human population with MCPyV virions being 30 continuously shed from human skin. However, the precise host cell tropism(s) of 31MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal 32fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. 33However, MCPyV appears different from other polyomaviruses as it requires sulfated 34 polysaccharides such as heparan sulfates and/or chondroitin sulfates for initial 35 attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co-36 )receptor. To explore the infectious entry process of MCPyV, we analyzed the cell 37 biological determinants of MCPyV entry into A549 cells, a highly transducible lung 38 carcinoma cell line, in comparison to well-studied simian virus 40 and a number of 39 other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-40 mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or 41 glycosphingolipid-enriched carriers. The viruses internalized in small endocytic pits 42 that led the virus to endosomes and from there to the endoplasmic reticulum (ER). 43Similar to other polyomaviruses, trafficking required microtubular transport, 44 acidification of endosomes, and a functional redox environment. To our surprise, the 45 virus was found to acquire a membrane envelope within endosomes, a phenomenon 46 not reported for other viruses. Only minor amounts of viruses reached the ER, while 47 the majority was retained in endosomal compartments suggesting that endosome-to-48 ER trafficking is a bottleneck during infectious entry. 49 50 51 52 3 Importance 53 MCPyV is the first polyomavirus directly implicated in the development of an 54 aggressive human cancer, the Merkel Cell Carcinoma (MCC). Although MCPyV is 55 constantly shed from healthy skin, MCC incidence increases among aging and 56 immunocompromised individuals. To date, the events connecting initial MCPyV 57 infection and subsequent transformation still remain elusive. MCPyV differs from 58 other known polyomaviruses concerning its cell tropism, entry receptor requirements, 59 and infection kinetics. In this study, we examined the cellular requirements for 60 endocytic entry as well as the subcellular localization of incoming virus particles. A 61 thorough understanding of the determinants of the infectious entry pathway and the 62 specific biological niche will benefit prevention of virus-derived cancers such as 63 MCC. 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 Polyomaviruses (PyV) are small, non-enveloped dsDNA viruses with a diameter of 80 45-50 nm. The icosahedral (T=7) capids consist of 72 homopentameric capsomers of 81 the major capid protein VP1 with minor capsid proteins VP2/VP3 located within a 82 cavity underneath the VP1 pentamers. The PyV capsid harbors a chromatinized, 83 circu...

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

“…MCPyV PsVs transduced target cells more slowly than SV40 infection, with detectable fluorescence observed between 48 to 72 hours post-transduction (hpt), consistent with previously reported timescales (Fig. 1E-F) (25). Using these systems, SV40 infections and MCPyV transductions could be performed in a 96- or 24-well plate format, respectively, providing a platform for high-throughput antiviral compound screening.…”

“…Following binding, JCPyV enters cells through clathrin-mediated endocytosis, whilst SV40, MCPyV and BKPyV enter via caveolar/lipid rafts (22–27). virions traffic through the endosomal system and in response to endosomal cues, including endosome acidification, initiate proteolytic rearrangements of the capsid prior to retrograde trafficking to the endoplasmic reticulum (ER) (25, 28–30). Within the ER, virions are further disassembled, exposing nuclear localisation signals (NLSs) that transport capsids to the nucleus via importins (31–37).…”

Isolation and Characterization of WUPyV in Polarized Human Airway Epithelial Cells

“…To date, there have been no cell lines available for the proliferation of HPyVs from clinical samples, with the exception of JCPyV and BKPyV [25]. Much of the research on polyomaviruses was performed using recombinant virions and pseudoviruses [26,27]. Previous studies showed that WUPyV-VP1 could be detected in cytokeratin + cells (epithelial cells), CD68 + cells (macrophage/monocyte lineage), and MUC5AC + cells (goblet cells) in the lung tissue by immunohistochemistry [11,28], indicating that WUPyV has obvious tropism for respiratory epithelia cells.…”

Taking the Scenic Route: Polyomaviruses Utilize Multiple Pathways to Reach the Same Destination

“…While there are significant commonalities between these viruses, they differ in their utilization of cellular receptors and entry routes for initiating infection. However, one distinct overlap in infection is clear: all polyomaviruses studied to date traffic to the endoplasmic reticulum following attachment and entry [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”