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DNA damage removal by nucleotide excision repair (NER) and replicative bypass via translesion synthesis (TLS) and template switch (TSw) are important in ensuring genome stability. In this study, we tested the applicability of an SV40 large T antigen-based replication system for the simultaneous examination of these damage tolerance processes. Using both Sanger and next-generation sequencing combined with lesion-specific qPCR and replication efficiency studies, we demonstrate that this system works well for studying NER and TLS, especially its one-polymerase branch, while it is less suited to investigations of homology-related repair processes, such as TSw. Cis-syn cyclobutane pyrimidine dimer photoproducts were replicated with equal efficiency to lesion-free plasmids in vitro, and the majority of TLS on this lesion could be inhibited by a peptide (PIR) specific for the polη-PCNA interaction interface. TLS on 6-4 pyrimidine-pyrimidone photoproduct proved to be inefficient and was slightly facilitated by PIR as well as by a recombinant ubiquitin-binding zinc finger domain of polη in HeLa extract, possibly by promoting polymerase exchange. Supplementation of the extract with recombinant PCNA variants indicated the dependence of TLS on PCNA ubiquitylation. In contrast to active TLS and NER, we found no evidence of successful TSw in cellular extracts. The established methods can promote in vitro investigations of replicative DNA damage bypass., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mucin 5AC (MUC5AC) is a highly O-glycosylated mucin secreted by human bronchial epithelial cells during pulmonary inflammatory diseases. T antigen, a component of the MUC5AC glycans, is the product of the O-glycosylation transferase T-synthase and its chaperone Cosmc. Since the expression of Cosmc is mediated by signaling pathways and inflammatory factors affecting mucin O-glycosylation, we analyzed the impact of neutrophil elastase (NE)-mediated Cosmc and T antigen expression in BEAS-2B cells derived from human bronchial epithelial cells. The expression of Cosmc and T antigen in human lung tissue was analyzed by immunohistochemistry. Cellular immunohistochemistry and western blot analysis demonstrated elevated expression of T antigen in BEAS-2B cells after NE stimulation. Altered Cosmc expression in BEAS-2B cells after NE stimulation was analyzed by confocal microscopy, western blot analysis and quantitative RT-PCR. To assess the biological implications of Cosmc function for T-synthase activity and T antigen synthesis after NE stimulation, BEAS-2B cells were transfected with shRNA to silence the expression of Cosmc. The changes in signaling pathways were analyzed by western blotting. The expression of Cosmc and T antigen increased in lung tissue exposed to chronic inflammation. The expression of Cosmc and T antigen increased in NE-stimulated BEAS-2B cells. NE induced increases in T antigen expression and T-synthase transferase activity in BEAS-2B cells expressing Cosmc, highlighting the importance of Cosmc in the relationship between NE and T antigen. Cosmc and phosphatidylinositol-3-kinase (PI3K) played important roles in the signaling pathway that stimulated hyperexpression of T antigen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell-cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflects their distinctive roles in cell biology., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r , 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets., (Copyright © 2018 American Society for Microbiology.)

A new immunological investigation was carried out to study the association between non-Hodgkin lymphoma and Simian virus 40 (SV40). To this end, a new indirect ELISA was employed with two mimotopes from SV40 large T antigen (Tag), the viral oncoprotein, to analyse for specific reactions to antibodies in sera from non-Hodgkin lymphoma patients and controls, represented by healthy subjects (HS) and breast carcinoma (BC) patients. This study allowed us to assay a new sera collection from non-Hodgkin lymphoma patients (NHL, n = 254). To verify the association between NHL and SV40 Tag, two totally independent cohorts were analysed: NHL1 n = 150 and NHL2 n = 104. The epidemiological survey included sera from HS1, n = 150; HS2, n = 104 and BC, n = 78. This new indirect ELISA revealed that antibodies against SV40 Tag mimotopes are detectable in NHL1 and NHL2 sera with a prevalence of 37 and 36%, respectively. The prevalence of SV40-antibodies detected in both NHL1 and NHL2 cohorts differs statistically from controls, at 19% for HS1 (p < 0.01), HS2 (p < 0.05) and BC patients (p < 0.05). This study, carried out with an immunological assay with specific Tag oncoprotein mimotopes of Simian virus 40, reports the presence of IgG antibodies against the large Tumour antigen in non-Hodgkin lymphomas for the first time. Our immunological data with two independent NHL cohorts show a statistically significant association between Simian virus 40 Tag and non-Hodgkin lymphoma. These results suggest that SV40-positive non-Hodgkin lymphomas could be treated differently from those tested SV40-negative.

Glycosylation is one of the major posttranslational modifications of proteins. N-glycosylation (Asn-linked) and O-glycosylation (Ser/Thr-linked) are the two main forms. Abnormal O-glycosylation is frequently observed on the surface of tumor cells, and is associated with an adverse outcome and poor prognosis in patients with cancer. O-glycans (Tn, sTn, and T antigen) can be synthesized in the Golgi apparatus with the aid of several glycosyltransferases (such as T-synthase and ST6GalNAc-I) in a suitable environment. The unique molecular chaperone of T-synthase is Cosmc, which helps T-synthase to fold correctly in the endoplasmic reticulum. Dysregulation of these glycosyltransferases, molecular chaperones, or the environment is involved in the dysregulation of O-glycans. Tn, sTn, and T antigen neo- or over-expression occurs in many types of cancer including gastric, colon, breast, lung, esophageal, prostate, and endometrial cancer. This review discusses the major synthetic pathway of O-glycans and the mechanism by which Tn, sTn, and T antigens promote tumor metastasis., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

JC virus (JCV), a ubiquitous polyoma virus that commonly infects the human, is identified as the etiologic agent for progressive multifocal leukoencephalopathy and some malignancies. To clarify the oncogenic role of JCV T antigen, we established two transgenic mice of T antigen using either α-crystallin A (αAT) or cytokeratin 19(KT) promoter. Lens tumors were found in high-copy αAT mice with the immunopositivity of T antigen, p53, β-catenin and N-cadherin. Enlarged eyeballs were observed and tumor invaded into the brain by magnetic resonance imaging and hematoxylin-and-eosin staining. The overall survival time of homozygous mice was shorter than that of hemizygous mice (p<0.01), the latter than wild-type mice (p<0.01). The spontaneous salivary tumor and hepatocellular carcinoma were seen in αAT5 transgenic mice with no positivity of T antigen. KT7 mice suffered from lung tumor although JCV T antigen was strongly expressed in gastric epithelial cells. The alternative splicing of T antigen intron was detectable in the lens tumor of αAT mice, gastric mucosa of KT mice, and various cells transfected with pEGFP-N1-T antigen. It was suggested that JCV T antigen might induce carcinogenesis at a manner of cell specificity, which is not linked to alternative splicing of its intron. Both spontaneous lens and lung tumor models provide good tools to investigate the oncogenic role of JCV T antigen.