Journal of General Virology - Volume 97, Issue 2, 2016
Small DNA Viruses
Disc large 1 expression is altered by human papillomavirus E6/E7 proteins in organotypic cultures of human keratinocytes
M. Bugnon Valdano, A. L. Cavatorta, M. G. Morale, F. Marziali, V. de Souza Lino, R. D. M. Steenbergen, E. Boccardo and D. Gardiol
https://doi.org/10.1099/jgv.0.000364
Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions.
The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8
Sandra Heuser, Martin Hufbauer, Benjamin Marx, Ali Tok, Slawomir Majewski, Herbert Pfister and Baki Akgül
Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.
Large DNA Viruses
Pseudorabies virus isolates from domestic pigs and wild boars show no apparent in vitro differences in replication kinetics and sensitivity to interferon-induced antiviral status
Sara Verpoest, Ann Brigitte Cay, Herman Favoreel and Nick De Regge
Pseudorabies virus is the causative agent of Aujeszky's disease. Domestic pigs and wild boars are its natural hosts, and strains circulating within both populations differ in their capacity to induce clinical disease. Cell biological and molecular explanations for the observed differences in virulence are, however, lacking. Different virulence determinants that can be assessed in vitro were determined for five domestic swine strains, four wild boar strains and the NIA3 reference strain. Replication kinetics and plaque formation capacity in continuous swine testicular cells and different primary porcine cell lines were highly similar for isolates from both populations. Treatment of these cell lines with IFNα, IFNγ or a combination of both provoked similar plaque-reducing effects for all strains. In conclusion, our results indicate that isolates from domestic swine and wild boar differ neither in intrinsic replication and dissemination capacity nor in sensitivity to antiviral effects of IFNs.
Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus
Caroline Denesvre, Sylvie Rémy, Laetitia Trapp-Fragnet, Lorraine P. Smith, Sonia Georgeault, Jean-François Vautherot and Venugopal Nair
T-lymphocytes are central targets of Marek's disease, a major chicken disease induced by the oncogenic alphaherpesvirus Marek's disease virus (MDV). T-lymphocyte infection is also associated with immunosuppression and virus latency. To decipher viral morphogenesis in T-lymphocytes, we used the recombinant vRB-1B 47EGFP marker virus to generate a new lymphoblastoid cell line, 3867K, that exhibited typical properties of other MDV-transformed chicken cell lines in term of cell markers, reactivation rate and infectivity. Examination of reactivating EGFP-positive 3867K cells by transmission electron microscopy revealed the presence of most types of herpesvirus particles inside the cells but no extracellular ones. Quantification of virion types indicated only 5 % cytoplasmic particles, with 0.5 % being mature. This study demonstrated that MDV morphogenesis is complete upon reactivation in T-lymphocytes, albeit with poor efficiency, with a defect in the exit of virions from the nucleus and secondary envelopment, as occurs in infected fibroblasts.
Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates
Sara Verpoest, Ann Brigitte Cay, Willem Van Campe, Laurent Mostin, Sarah Welby, Herman Favoreel and Nick De Regge
Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.
ΔPK oncolytic activity includes modulation of the tumour cell milieu
Dominique Bollino, Aric Colunga, Baiquan Li and Laure Aurelian
Oncolytic virotherapy is a unique cancer therapeutic that encompasses tumour cell lysis through both virus replication and programmed cell death (PCD) pathways. Nonetheless, clinical efficacy is relatively modest, likely related to the immunosuppressive tumour milieu. Our studies use the herpes simplex virus type 2 (HSV-2)-based oncolytic virus ΔPK that has documented anti-tumour activity associated with virus replication, PCD and cancer stem cell lysis. They are designed to examine whether ΔPK-mediated oncolysis includes the ability to reverse the immunosuppressive tumour microenvironment by altering the balance of cytokines directly secreted by the melanoma cells and to define its mechanism. Here, we show that melanoma cells secreted the immunosuppressive cytokine IL-10, and that secretion was inhibited by ΔPK through virus replication and c-Jun N-terminal kinase/c-Jun activation. ΔPK-induced IL-10 inhibition upregulated surface expression of MHC class I chain-related protein A, the ligand for the activating NKG2D receptor expressed on NK- and cytotoxic T-cells. Concomitantly, ΔPK also upregulated the secretion of inflammatory cytokines TNF-α, granulocyte macrophage colony-stimulating factor and IL-1β through autophagy-mediated activation of Toll-like receptor 2 pathways and pyroptosis, and it inhibited the expression of the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen 4. Pharmacologic inhibition of these processes significantly reduces the oncolytic activity of ΔPK.
Retroviruses
Reciprocal relationship of T regulatory cells and monocytic myeloid-derived suppressor cells in LP-BM5 murine retrovirus-induced immunodeficiency
Megan A. O'Connor, Jennifer L. Vella and William R. Green
Immunomodulatory cellular subsets, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumour microenvironment and are targets of immunotherapy, but their role in retroviral-associated immunosuppression is less well understood. Due to known crosstalk between Tregs and MDSCs in the tumour microenvironment, and also their hypothesized involvement during human immunodeficiency virus/simian immunodeficiency virus infection, studying the interplay between these immune cells during LP-BM5 retrovirus-induced murine AIDS is of interest. IL-10-producing FoxP3+ Tregs expanded after LP-BM5 infection. Following in vivo adoptive transfer of natural Treg (nTreg)-depleted CD4+T-cells, and subsequent LP-BM5 retroviral infection, enriched monocytic MDSCs (M-MDSCs) from these nTreg-depleted mice displayed altered phenotypic subsets. In addition, M-MDSCs from LP-BM5-infected nTreg-depleted mice exhibited increased suppression of T-cell, but not B-cell, responses, compared with M-MDSCs derived from non-depleted LP-BM5-infected controls. Additionally, LP-BM5-induced M-MDSCs modulated the production of IL-10 by FoxP3+ Tregs in vitro. These collective data highlight in vitro and for the first time, to the best of our knowledge, in vivo reciprocal modulation between retroviral-induced M-MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of such regulatory cells during retroviral infection.
RNA Viruses
Additional heritable virus in the parasitic wasp Leptopilina boulardi: prevalence, transmission and phenotypic effects
Julien Martinez, David Lepetit, Marc Ravallec, Frédéric Fleury and Julien Varaldi
Parasitoid wasps can be found in association with heritable viruses. Although some viruses have been shown to profoundly affect the biology and evolution of parasitoid wasps, the genetic and phenotypic diversity of parasitoid-associated viruses remains largely unexplored. We previously discovered a behaviour-manipulating DNA virus in the parasitoid wasp Leptopilina boulardi. In this species, which lays its eggs inside Drosophila larvae, Leptopilina boulardi filamentous virus (LbFV) forces the females to lay their eggs in already parasitized Drosophila larvae. This behavioural manipulation increases the chances for the horizontal transmission of the virus. Here, we describe in the same parasitoid species another virus, which we propose to call Leptopilina boulardi toti-like virus (LbTV). This double-stranded RNA virus is highly prevalent in insect laboratory lines as well as in parasitoids caught in the field. In some cases, LbTV was found in coinfection with LbFV, but did not affect the behaviour of the wasp. Instead we found that the presence of LbTV correlates with an increase in the number of offspring, mostly due to increased survival of parasitoid larvae. LbTV is vertically transmitted mostly through the maternal lineage even if frequent paternal transmission also occurs. Unlike LbFV, LbTV is not horizontally transmitted. Its genome encodes a putative RNA-dependent RNA polymerase (RdRp) showing similarities with RdRps of Totiviridae. These results underline the high incidence and diversity of inherited viruses in parasitoids as well as their potential impact on the phenotype of their hosts.
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