In this work, we investigate the potential of highly sulfated synthetic glycomimetics to act as inhibitors of viral binding/infection. Our results indicate that both long-chain glycopolymers and short-chain glycooligomers are capable of preventing viral infection. Notably, glycopolymers efficiently inhibit Human Papillomavirus (HPV16) infection in vitro and maintain their antiviral activity in vivo, while the glycooligomers exert their inhibitory function post attachment of viruses to cells. Moreover, when we tested the potential for broader activity against several other human pathogenic viruses, we observed broad-spectrum antiviral activity of these compounds beyond our initial assumptions. While the compounds tested displayed a range of antiviral efficacies, viruses with rather diverse glycan specificities such as Herpes Simplex Virus (HSV), Influenza A Virus (IAV), and Merkel Cell Polyomavirus (MCPyV) could be targeted. This opens new opportunities to develop broadly active glycomimetic inhibitors of viral entry and infection.

Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry. IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

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...

Background/Aims: Cellular senescence, or permanent growth arrest, is known as an effective tumor suppressor mechanism that can be induced by different stressors, such as oncogenes, chemotherapeutics or cytokine cocktails. Previous studies demonstrated that the growth-repressing state of oncogene-induced senescent cells depends on argonaute protein 2 (Ago2)-mediated transcriptional gene silencing and Ago2/Rb corepression of E2F-dependent cell cycle genes. Cytokine-induced senescence (CIS) likewise depends on activation of the p16^Ink4a/Rb pathway, and consecutive inactivation of the E2F family of transcription factors. In the present study, we therefore analyzed the role of Ago2 in CIS. Methods: Human cancer cell lines were treated with interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) to induce senescence. Senescence was determined by growth assays and measurement of senescence-associated β-galactosidase (SA-β-gal) activity, Ago2 translocation by Ago2/ Ki67 immunofluorescence staining and western blot analysis, and gene transcription by quantitative polymerase chain reaction (qPCR). Results: IFN-γ and TNF permanently stopped cell proliferation and time-dependently increased SA-β-gal activity. After 24 – 48 h of cytokine treatment, Ago2 translocated from the cytoplasm into the nucleus of Ki67-negative cells, an effect which was shown to be reversible. Importantly, the proinflammatory cytokine cocktail suppressed Ago2-regulated cell cycle control genes, and siRNA-mediated depletion of Ago2 interfered with cytokine-induced growth inhibition. Conclusion: IFN-γ and TNF induce a stable cell cycle arrest of cancer cells that is accompanied by a fast nuclear Ago2 translocation and repression of Ago2-regulated cell cycle control genes. As Ago2 downregulation impairs cytokine-induced growth regulation, Ago2 may contribute to tissue homeostasis in human cancers.

Streptococcus pyogenes tonsillar infection is well known to trigger and exacerbate psoriasis lesions in both guttate and plaque forms of the disease. Although mucosal and cutaneous tissues are closely involved in psoriasis pathology, the interaction between their specific immune responses has not been deeply explored. This work aims to address and characterize the presence of humoral responses against S. pyogenes in patients with psoriasis and its putative association with cytokine responses detected in vitro in our psoriasis ex vivo model, based on the coculture of cutaneous lymphocyte-associated antigen þ/À T cells with autologous epidermal cells. Patients with psoriasis presented increased IgA response to S. pyogenes when compared with control subjects. In patients with plaque psoriasis, despite being negative for anti-streptolysin O antibody titer, IgA plasma levels against S. pyogenes correlated with cutaneous lymphocyte-associated antigen þ T-celledependent IL-17F response in vitro. No association is observed for IgG levels in plaque psoriasis. Similar association is observed for IgA anti-S. pyogenes extract and IL-17A in patients with guttate psoriasis. We propose S. pyogenesespecific IgA as a potential new perspective for better understanding the role of S. pyogenes in psoriasis development.

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA− cutaneous lymphocyte antigen (CLA)+/− T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA+ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA+ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.