Yannick Simonin

Zika Virus Strains Potentially Display Different Infectious Profiles in Human Neural Cells.

The recent Zika virus (ZIKV) epidemic has highlighted the poor knowledge on its physiopathology. Recent studies showed that ZIKV of the Asian lineage, responsible for this international outbreak, causes neuropathology in vitro and in vivo. However, two African lineages exist and the virus is currently found circulating in Africa. The original African strain was also suggested to be neurovirulent but its laboratory usage has been criticized due to its multiple passages. In this study, we compared the French Polynesian (Asian) ZIKV strain to an African strain isolated in Central African Republic and show a difference in infectivity and cellular response between both strains in human neural stem cells and astrocytes. Consistently, this African strain led to a higher infection rate and viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology and predict neurological impairment associated with African ZIKV.

Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursors.

BACKGROUND & AIMS Apicobasal polarity, which is essential for epithelial structure and function, is targeted by several tumour-related pathogens and is generally perturbed in the course of carcinogenesis. Hepatitis C virus (HCV) infection is associated with a strong risk of hepatocellular carcinoma, typically preceded by dysplastic alterations of cell morphology. We investigated the molecular mechanisms and the functional consequences of HCV-driven perturbations of epithelial polarity. METHODS We used biochemical, genetic, and cell biology approaches to assess the impact of hepatitis C viral protein NS5A on the polarity and function of hepatocytes and hepatic progenitors. Transgenic animals and xenograft models served for in vivo validation of the results obtained in cell culture. RESULTS We found that expression of HCV-NS5A in primary hepatic precursors and in immortalized hepatocyte cell lines gave rise to profound modifications of cell polarity, leading to epithelial to mesenchymal transition (EMT). NS5A, either alone or in the context of the full complement of viral proteins in the course of infection, acted through activating Twist2, a transcriptional regulator of EMT. The effects of NS5A were additive to those of TGF-β, a cytokine abundant in diseased liver and highly relevant to HCV-related pathology. Moreover, NS5A cooperates with oncogenic Ras, giving rise to transformed, invasive cells that are highly tumorigenic in vivo. CONCLUSIONS Our data suggest that in the context of HCV infection, NS5A favors formation of preneoplastic lesions by disrupting cell polarity and additional oncogenic events cooperate with the viral protein to give rise to motile and invasive tumour cells.

Poliovirus-Induced Apoptosis Is Reduced in Cells Expressing a Mutant CD155 Selected during Persistent Poliovirus Infection in Neuroblastoma Cells

ABSTRACT Poliovirus (PV) can establish persistent infections in human neuroblastoma IMR-32 cells. We previously showed that during persistent infection, specific mutations were selected in the first extracellular domain of the PV receptor (CD155) of these cells (N. Pavio, T. Couderc, S. Girard, J. Y. Sgro, B. Blondel, and F. Colbère-Garapin, Virology 274:331-342, 2000). These mutations included the Ala 67 → Thr substitution, corresponding to a previously described allelic form of the PV receptor. The mutated CD155Thr67 and the nonmutated IMR-32 CD155 (CD155IMR) were expressed independently in murine LM cells lacking the CD155 gene. Following infection of the cells with PV, we analyzed the death of cells expressing these two forms of CD155. Levels of DNA fragmentation, caspase activity, and cytochrome c release were lower in LM-CD155Thr67 cells than in LM-CD155IMR cells. Thus, the level of apoptosis was lower in cells expressing mutated CD155 selected during persistent PV infection in IMR-32 than in cells expressing the wild-type receptor.

Calpain activation by hepatitis C virus proteins inhibits the extrinsic apoptotic signaling pathway

An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down‐regulate expression of BH3‐only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas‐mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down‐regulated in patients in the context of noncirrhotic HCV‐linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor–mediated apoptotic signal. Finally, human HCV‐related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains. Conclusion: Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune‐mediated elimination of HCV‐infected cells. (Hepatology 2009.)

Lymphotoxin Signaling Is Initiated by the Viral Polymerase in HCV-linked Tumorigenesis

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.

Differential virulence between Asian and African lineages of Zika virus

Zika virus (ZIKV) is a small enveloped positive-sense single-stranded RNA virus belonging to the genus Flavivirus of the Flaviviridae family that has reemerged in recent years as a human pathogen with epidemic potential. To date, the ongoing epidemic in the Americas has led to tens of thousands of confirmed cases in Brazil alone (Pan American Health Organization Zika-Epidemiological Report, 2 March 2017). More concerning are the reports of severe neurological disorders such as Guillain-Barré syndrome and congenital Zika syndrome (microcephaly and other neurodevelopmental defects), which led the World Health Organization to declare the ZIKV epidemic as a Public Health Emergency of International Concern in 2016. Phylogenetic analyses show that ZIKV can be classified into 2 main lineages: the African lineage and the Asian lineage, the latter of which is responsible for the recent epidemics. Because recent ZIKV infections were associated with the development of congenital and neurological disorders, a key question was raised as to whether Asian-lineage ZIKV strains were phenotypically different from the African lineage strains. It is well described that mutations acquired during Flavivirus evolution can alter their virulence and/or tropism [1]. ZIKV is primarily transmitted by the mosquito species Aedes aegypti and Aedes albopictus. Several studies show that vector transmission can differ between ZIKV strains, as the overall ZIKV infection prevalence and transmission rates of African strains may be higher in A. aegypti than Asian strains [2], suggesting that viral adaptation may have occurred—similar to a single mutation in the chikungunya virus envelope protein that affected vector specificity and epidemic potential. During the recent outbreak, it became clear that ZIKV is sometimes able to cause a prolonged infection. Numerous studies showed that Asian-lineage strains isolated in South America replicate at low levels in tissues months after the initial infection. For example, viral genome can be found in stillborn babies who were infected early during gestation [3], weeks after initial infection in sperm, and in rhesus monkeys, ZIKV can be found in the cerebral spinal fluid (CSF) 42 days after infection, weeks after the virus was cleared from the blood [4]. However, to date, no data are available on the ability of African-lineage strains to cause prolonged infections, but early studies in 2016 suggested that an African ZIKV strain was highly pathogenic and led to cell death; when the first reports on a potential link between microcephaly and ZIKV emerged, laboratories started to study the effect of ZIKV on human neural precursor cells (hNPCs)/human neural stem cells (hNSCs) with MR766, the most available strain at the time (Fig 1 and Table 1). These first studies showed strong tropism and deleterious effects on NSC homeostasis and growth (e.g., [5]). However, some inconsistencies between this rather strong virulence and the long-term developmental effects associated with ZIKV infections led researchers to question the biological validity of this strain, which has been

Phenotypic differences between Asian and African lineage Zika viruses in human neural progenitor cells

The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells—which are important target cells in the development of congenital microcephaly—less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly. ABSTRACT Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). IMPORTANCE The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells—which are important target cells in the development of congenital microcephaly—less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.

NOD1 Participates in the Innate Immune Response Triggered by Hepatitis C Virus Polymerase

ABSTRACT Hepatitis C virus (HCV) triggers innate immunity signaling in the infected cell. Replication of the viral genome is dispensable for this phenotype, and we along with others have recently shown that NS5B, the viral RNA-dependent RNA polymerase, synthesizes double-stranded RNA (dsRNA) from cellular templates, thus eliciting an inflammatory response, notably via activation of type I interferon and lymphotoxin β. Here, we investigated intracellular signal transduction pathways involved in this process. Using HepaRG cells, a model that largely recapitulates the in vivo complexities of the innate immunity receptor signaling, we have confirmed that NS5B triggered increased expression of the canonical pattern recognition receptors (PRRs) specific for dsRNA, namely, RIG-I, MDA5, and Toll-like receptor 3 (TLR3). Unexpectedly, intracellular dsRNA also led to accumulation of NOD1, a receptor classically involved in recognition of bacterial peptidoglycans. NOD1 activation, confirmed by analysis of its downstream targets, was likely due to its interaction with dsRNA and was independent of RIG-I and mitochondrial antiviral signaling protein (MAVS/IPS-1/Cardif/VISA) signaling. It is likely to have a functional significance in the cellular response in the context of HCV infection since interference with the NOD1 pathway severely reduced the inflammatory response elicited by NS5B. IMPORTANCE In this study, we show that NOD1, a PRR that normally senses bacterial peptidoglycans, is activated by HCV viral polymerase, probably through an interaction with dsRNA, suggesting that NOD1 acts as an RNA ligand recognition receptor. In consequence, interference with NOD1-mediated signaling significantly weakens the inflammatory response to dsRNA. These results add a new level of complexity to the understanding of the cross talk between different classes of pattern recognition receptors and may be related to certain complications of chronic hepatitis C virus infection.

Modulation of Oxidative Stress by Twist Oncoproteins

Expression of developmental genes Twist1 and Twist2 is reactivated in many human tumors. Among their oncogenic activities, induction of epithelial to mesenchymal transition is believed to increase cell motility and invasiveness and may be related to acquisition of cancer stem cell phenotype. In addition, Twist proteins promote malignant conversion by overriding two oncogene-induced failsafe programs: senescence and apoptosis. Reactive oxygen species (ROS) are also important mediators of apoptosis, senescence and motility and are tightly linked to disease, notably to cancer. We report here that Twist factors and ROS are functionally linked. In wild type cells both Twist1 and Twist2 exhibit antioxidant properties. We show that Twist-driven modulation of oncogene-induced apoptosis is linked to its effects on oxidative stress. Finally, we identify several targets that mediate Twist antioxidant activity. These findings unveil a new function of Twist factors that could be important in explaining their pleiotropic role during carcinogenesis.

Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016

Infection with Usutu virus (USUV) has been recently associated with neurologic disorders, such as encephalitis or meningoencephalitis, in humans. These findings indicate that USUV is a potential health threat. We report an acute human infection with USUV in France putatively associated with a clinical diagnosis of idiopathic facial paralysis.