DNA damage stress control is a tLT- and EHMT2-dependent central feature of Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. MC polyomavirus (MCPyV) causes 80% of MCCs, encoding the viral oncogenes small T (sT) and truncated large T antigens (tLT). These proteins impair the Rb1-dependent G1/S checkpoint blockade and subvert the host cell epigenome to promote cancer. Whole proteome analysis and proximal interactomics identified a tLT-dependent deregulation of DNA damage response (DDR). Our investigation revealed a previously unreported interaction between tLT and the histone methyltransferase EHMT2, to our knowledge. T Antigens knockdown reduced DDR protein levels and increased levels of the DNA damage marker γH2Ax. EHMT2 normally promotes H3K9 methylation and DDR signaling. Given that inhibition of EHMT2 did not significantly change the MCC cells proteome, tLT-EHMT2 interaction could affect the DDR. With tLT, we report that EHMT2 gained DNA damage repair proximal interactors. EHMT2 inhibition rescued proliferation in MCC cells depleted for their T antigens, suggesting impaired DDR and/or lack of checkpoint efficiency. Combined tLT and EHMT2 inhibition led to altered DDR, evidenced by multiple signaling alterations. Here we show that tLT hijacks multiple components of the DNA damage machinery to enhance tolerance to DNA damage in MCC cells, which could explain the genetic stability of these cancers.

Viral and cellular telomerase RNAs possess host-specific anti-apoptotic functions.

Human telomerase RNA (hTR) is overexpressed in many cancers and protects T cells from apoptosis in a telomerase-independent manner. The most prevalent cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a viral telomerase RNA (vTR) that plays a crucial role in MDV-induced tumorigenesis and shares all four conserved functional domains with hTR. In this study, we assessed whether hTR drives tumor formation in this natural model of herpesvirus-induced tumorigenesis. Therefore, we replaced vTR with hTR in the genome of a highly oncogenic MDV. Furthermore, we investigated the anti-apoptotic activity of vTR, hTR, and their counterpart in the chicken [chicken telomerase RNA (cTR)]. hTR was efficiently expressed and did not alter replication of the recombinant virus. Despite its conserved structure, hTR did not complement the loss of vTR in virus-induced tumorigenesis. Strikingly, hTR did not inhibit apoptosis in chicken cells, but efficiently inhibited apoptosis in human cells. Inverse host restriction has been observed for vTR and cTR in human cells. Our data revealed that vTR, cTR, and hTR possess conserved but host-specific anti-apoptotic functions that likely contribute to MDV-induced tumorigenesis. IMPORTANCE hTR is overexpressed in many cancers and used as a cancer biomarker. However, the contribution of hTR to tumorigenesis remains elusive. In this study, we assessed the tumor-promoting properties of hTR using a natural virus/host model of herpesvirus-induced tumorigenesis. This avian herpesvirus encodes a telomerase RNA subunit (vTR) that plays a crucial role in viral tumorigenesis and shares all conserved functional domains with hTR. Our data revealed that vTR and cellular TRs of humans and chickens possess host-specific anti-apoptotic functions. This provides important translational insights into therapeutic strategies, as inhibition of apoptosis is crucial for tumorigenesis.

The sulphated polysaccharides extract ulvans from Ulva armoricana limits Marek's disease virus dissemination in vitro and promotes viral reactivation in lymphoid cells.

BACKGROUND: Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by an alphaherpesvirus, Marek's disease virus (MDV). MD is presently controlled by systematic vaccination of animals, which protects efficiently against the development of clinical disease. However, MDV vaccines do not prevent the multiplication and spread of MDV field strains and may favor the emergence of strains with increased virulence. Therefore, MDV persists to be a major problem for the poultry industry and the development of new alternative strategies to control MDV is needed. Seaweed extracts have previously been shown to exert immunomodulatory and antiviral activities, especially against herpesviruses. The objective of the present study was to explore the effect of Ulva armoricana extracts on MDV infection in vitro. RESULTS: We could demonstrate that the ulvan extract as well as its vitamin-enriched formulation reduce the viral load by about 80% at 24 h post-infection in infected chicken fibroblasts at concentrations that are innocuous for the cells. We also observed a substantial decrease in MDV plaque size suggesting that ulvans impede MDV cell-to-cell spread in vitro. Moreover, we showed that ulvan extract could promote MDV reactivation in lymphoid cells. CONCLUSIONS: Our data provide the first evidence that the use of the ulvan extract could be a good alternative to limit MDV infection in poultry.

Hypoxia and HIF-1 Trigger Marek's Disease Virus Reactivation in Lymphoma-Derived Latently Infected T Lymphocytes.

Latency is a hallmark of herpesviruses, allowing them to persist in their host without virion production. Acute exposure to hypoxia (below 3% O2) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gammaherpesviruses (Kaposi's sarcoma-associated virus [KSHV] and Epstein-Barr virus [EBV]). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek's disease virus (MDV), which shares biological properties with EBV and KSHV (notably oncogenic properties), in lymphocytes. Acute exposure to hypoxia (1% O2) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia response element consensus sequences on 119 open reading frames. Reverse transcriptase quantitative PCR (RT-qPCR) analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl2) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor 1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. Under such conditions, the lytic cycle was detected in cells with a sustainable HIF-1α expression but also in HIF-1α-negative cells, indicating that MDV reactivation is mediated by HIF-1 in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown to be upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors. IMPORTANCE Latent-to-lytic switch of herpesviruses (also known as reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alphaherpesvirus establishing latency in T lymphocytes and causing lethal T lymphomas in chickens. In vivo, a second lytic phase is observed during the tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently infected T lymphocytes, like previously shown for EBV and KSHV in B lymphocytes. In this study, we demonstrated that acute hypoxia (1% O2) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor 1 (HIF-1) overexpression induces MDV reactivation to an extent similar to that of hypoxia after 24 h. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genera.

Marek's disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype.

Marek's disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.

Identification of Marek's Disease Virus VP22 Tegument Protein Domains Essential for Virus Cell-to-Cell Spread, Nuclear Localization, Histone Association and Cell-Cycle Arrest.

VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek's disease virus (MDV), the Mardivirus prototype; it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159-162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens.

It is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek's disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC. RESEARCH HIGHLIGHTS Use of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host-pathogen interactions. pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons. pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

Atrophy of primary lymphoid organs induced by Marek's disease virus during early infection is associated with increased apoptosis, inhibition of cell proliferation and a severe B-lymphopenia.

Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells.

Induction of DNA Damages upon Marek's Disease Virus Infection: Implication in Viral Replication and Pathogenesis.

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis.IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

Keratinocytes derived from chicken embryonic stem cells support Marek's disease virus infection: a highly differentiated cell model to study viral replication and morphogenesis.

BACKGROUND: Marek's disease is a virus disease with worldwide distribution that causes major losses to poultry production. Vaccines against Marek's disease virus, an oncogenic alphaherpesvirus, reduce tumour formation but have no effect on virus shedding. Successful horizontal virus transmission is linked to the active viral replication in feather follicle epithelial cells of infected chickens, from which infectious viral particles are shed into the environment. The feather follicle epithelium is the sole tissue in which those infectious particles are produced and no in vitro cell-systems can support this highly efficient morphogenesis. We previously characterized embryonic stem-cell-derived keratinocytes, showing they display a marker-gene profile similar to skin keratinocytes, and therefore we tested their susceptibility to Marek's disease virus infection. FINDINGS: We show herein that keratinocytes derived from chicken embryonic stem-cells are fully permissive to the replication of either non-pathogenic or pathogenic Marek's disease viruses. All viruses replicated on all three keratinocyte lines and kinetics of viral production as well as viral loads were similar to those obtained on primary cells. Morphogenesis studies were conducted on infected keratinocytes and on corneocytes, showing that all types of capsids/virions were present inside the cells, but extracellular viruses were absent. CONCLUSIONS: The keratinocyte lines are the first epithelial cell-line showing ectodermal specific markers supporting Marek's disease virus replication. In this in vitro model the replication lead to the production of cell-associated viral progeny. Further work will be devoted to the study of relationship between 3D differentiation of keratinocytes and Marek's disease virus replication.

Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus.

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.

Derivation of keratinocytes from chicken embryonic stem cells: establishment and characterization of differentiated proliferative cell populations.

A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

Cell cycle modulation by Marek's disease virus: the tegument protein VP22 triggers S-phase arrest and DNA damage in proliferating cells.

Marek's disease is one of the most common viral diseases of poultry affecting chicken flocks worldwide. The disease is caused by an alphaherpesvirus, the Marek's disease virus (MDV), and is characterized by the rapid onset of multifocal aggressive T-cell lymphoma in the chicken host. Although several viral oncogenes have been identified, the detailed mechanisms underlying MDV-induced lymphomagenesis are still poorly understood. Many viruses modulate cell cycle progression to enhance their replication and persistence in the host cell, in the case of some oncogenic viruses ultimately leading to cellular transformation and oncogenesis. In the present study, we found that MDV, like other viruses, is able to subvert the cell cycle progression by triggering the proliferation of low proliferating chicken cells and a subsequent delay of the cell cycle progression into S-phase. We further identified the tegument protein VP22 (pUL49) as a major MDV-encoded cell cycle regulator, as its vector-driven overexpression in cells lead to a dramatic cell cycle arrest in S-phase. This striking functional feature of VP22 appears to depend on its ability to associate with histones in the nucleus. Finally, we established that VP22 expression triggers the induction of massive and severe DNA damages in cells, which might cause the observed intra S-phase arrest. Taken together, our results provide the first evidence for a hitherto unknown function of the VP22 tegument protein in herpesviral reprogramming of the cell cycle of the host cell and its potential implication in the generation of DNA damages.

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells.

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.