Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

AbstractPolyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis

Glycosaminoglycans and Sialylated Glycans Sequentially Facilitate Merkel Cell Polyomavirus Infectious Entry

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus

The Merkel Cell Polyomavirus Minor Capsid Protein

<div><p>The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.</p></div

Differing effects of the MCV VP2 protein on pseudovirus transduction.

<p>(A) Purified MCV pseudovirions with no VP2 (None) a low level of VP2 (Low) or a high level of VP2 (High) were analyzed by SDS-PAGE and SYPRO Ruby protein stain. (B) The infectivity of MCV pseudoviruses with differing levels of VP2 was analyzed in 293TT cells by flow cytometry. The average percentage of GFP-positive cells from three experiments is shown and error bars represent the standard error of the mean. (C–F) Analysis of transduction as above, in other cell lines previously shown to be highly transducible with MCV VP1/VP2 pseudoviruses.</p

Mutation of VP2 internal methionine residues has little effect on MCV infectivity.

<p>(A) Purified native MCV carrying mutations in the first internal methionine (Met₄₆) of VP2 (dVP3a), the second internal methionine (Met₁₂₉) of VP2 (dVP3b) or a double Met mutant (dVP3d) were analyzed alongside WT MCV by SDS-PAGE and western blot with a mixture of anti-VP1 and anti-VP2 rabbit sera. These native virus stocks were not produced simultaneously and differed slightly in concentration. Four µl of WT and dVP3d and 2 µl of dVP3a and dVP3b were run on the gel. (B) Equal amounts of WT and dVP3 mutant viruses, normalized by MCV genomic copies, were inoculated onto 293-4T cells. Samples taken one day or four days post-infection were analyzed for viral DNA concentration by qPCR to determine replication of virus-delivered genomic DNA as a measure of infectivity. The average of three experiments performed in duplicate is shown and error bars represent the standard error of the mean.</p

Entry tropism of BK and Merkel cell polyomaviruses in cell culture.

Merkel Cell Polyomavirus (MCV or MCPyV) was recently discovered in an aggressive form of skin cancer known as Merkel cell carcinoma (MCC). Integration of MCV DNA into the host genome likely contributes to the development of MCC in humans. MCV infection is common and many healthy people shed MCV virions from the surface of their skin. MCV DNA has also been detected in samples from a variety of other tissues. Although MCC tumors serve as a record that MCV can infect the Merkel cell lineage, the true tissue tropism and natural reservoirs of MCV infection in the host are not known. In an effort to gain insight into the tissue tropism of MCV, and to possibly identify cellular factors responsible for mediating infectious entry of the virus, the infection potential of human cells derived from a variety of tissues was evaluated. MCV gene transfer vectors (pseudoviruses) carrying reporter plasmid DNA encoding GFP or luciferase genes were used to transduce keratinocytes and melanocytes, as well as lines derived from MCC tumors and the NCI-60 panel of human tumor cell lines. MCV transduction was compared to transduction with pseudoviruses based on the better-studied human BK polyomavirus (BKV). The efficiency of MCV and BKV transduction of various cell types occasionally overlapped, but often differed greatly, and no clear tissue type preference emerged. Application of native MCV virions to a subset of highly transducible cell types suggested that the lines do not support robust replication of MCV, consistent with recent proposals that the MCV late phase may be governed by cellular differentiation in vivo. The availability of carefully curated gene expression data for the NCI-60 panel should make the MCV and BKV transduction data for these lines a useful reference for future studies aimed at elucidation of the infectious entry pathways of these viruses

Nuclear localization of MCV VP2 expressed +/− VP1.

<p>Confocal immunofluorescent microscopy of 293TT cells transfected with VP1 (red), VP2 (green) or VP1 and VP2 from a bicistronic plasmid. A mouse monoclonal antibody was used to detect VP1, and a rabbit polyclonal antibody was used to detect VP2. Immunostained coverslips were mounted with DAPI (blue).</p

No discernible effect of VP2 on MCV trafficking.

<p>Purified MCV VP1-only, MCV VP1+VP2, or BKV pseudovirions produced with EdU-labeled encapsidated DNA were examined by confocal immunofluorescent microscopy in NCI/ADR-RES cells, 48 hours after pseudovirion inoculation. EdU was reacted with Alexa Fluor 488 (green), and then (A) LAMP-1-positive (late endosome/lysosome) or (B) ERp72-positive (ER) subcellular compartments were immunostained (red) and mounted with DAPI (blue).</p

The MCV VP2 protein is myristoylated.

<p>MCV pseudovirions produced in the presence (+Myr) or absence (−Myr) of myristic acid-azide were reacted with TAMRA-alkyne. Proteins were precipitated and separated by SDS-PAGE for in-gel examination of TAMRA fluorescence. The same gel was then stained with SYPRO Ruby to detect all proteins.</p