Hepatitis C Virus Controls Interferon Production through PKR Activation

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection

Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response

Enhanced Zika virus susceptibility of globally invasive Aedes aegypti populations

The drivers and patterns of zoonotic virus emergence in the human population are poorly understood. The mosquito Aedes aegypti is a major arbovirus vector native to Africa that invaded most of the world’s tropical belt over the past four centuries, after the evolution of a “domestic” form that specialized in biting humans and breeding in water storage containers. Here, we show that human specialization and subsequent spread of A. aegypti out of Africa were accompanied by an increase in its intrinsic ability to acquire and transmit the emerging human pathogen Zika virus. Thus, the recent evolution and global expansion of A. aegypti promoted arbovirus emergence not solely through increased vector–host contact but also as a result of enhanced vector susceptibility

dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

viruse

A potential robust antiviral defense state in the common vampire bat: Expression, induction and molecular characterization of the three interferon-stimulated genes -OAS1, ADAR1 and PKR

International audienceBats are known to harbor many zoonotic viruses, some of which are pathogenic to other mammals while they seem to be harmless in bats. As the interferon (IFN) response represents the first line of defense against viral infections in mammals, it is hypothesized that activation of the IFN system is one of the mechanisms enabling bats to co-exist with viruses. We have previously reported induction of type I IFN in a cell line from the common vampire bat, Desmodus rotundus, upon polyinosinic:polycytidylic acid (poly(I:C)) stimulation. To deepen our knowledge on D. rotundus’ IFN-I antiviral response, we molecularly characterized three interferon-stimulated genes (ISGs), OAS1, PKR and ADAR1, closely implicated in the IFN-I antiviral response, and tested their functionality in our cellular model. We first found that D. rotundus encoded two OAS1 paralogs, OAS1a and OAS1b, and that the functional domains of the four ISGs characterized were highly conserved with those of other mammals. Despite their significant transcription level in the absence of stimulation, the transcription of the four ISGs characterized was enhanced by poly(I:C). In addition, the transcription of OAS1a and OAS1b appears to be differentially regulated. These findings demonstrate an active ISG antiviral response in D. rotundus in which OAS1b may play an important role

Mosquito‐bacteria interactions during larval development trigger metabolic changes with carry‐over effects on adult fitness

International audienceIn animals with distinct life stages such as holometabolous insects, adult phenotypic variation is often shaped by the environment of immature stages, including their interactions with microbes colonizing larval habitats. Such carry-over effects were previously observed for several adult traits of the mosquito Aedes aegypti after larval exposure to different bacteria, but the mechanistic underpinnings are unknown. Here, we investigated the molecular changes triggered by gnotobiotic larval exposure to different bacteria in Ae. aegypti. We initially screened a panel of 16 bacterial isolates from natural mosquito breeding sites to determine their ability to influence adult life-history traits. We subsequently focused on four bacterial isolates (belonging to Flavobacterium, Lysobacter, Paenibacillus, and Enterobacteriaceae) with significant carry-over effects on adult survival and found that they were associated with distinct transcriptomic profiles throughout mosquito development. Moreover, we detected carry-over effects at the level of gene expression for the Flavobacterium and Paenibacillus isolates. The most prominent transcriptomic changes in gnotobiotic larvae reflected a profound remodeling of lipid metabolism, which translated into phenotypic differences in lipid storage and starvation resistance at the adult stage. Together, our findings indicate that larval exposure to environmental bacteria trigger substantial physiological changes that impact adult fitness, uncovering a possible mechanism underlying carry-over effects of mosquito-bacteria interactions during larval development

Risk of arbovirus emergence via bridge vectors: case study of the sylvatic mosquito Aedes malayensis in the Nakai district, Laos

International audienceMany emerging arboviruses of global public health importance, such as dengue virus (DENV) and yellow fever virus (YFV), originated in sylvatic transmission cycles involving wild animals and forest-dwelling mosquitoes. Arbovirus emergence in the human population typically results from spillover transmission via bridge vectors, which are competent mosquitoes feeding on both humans and wild animals. Another related, but less studied concern, is the risk of 'spillback' transmission from humans into novel sylvatic cycles. We colonized a sylvatic population of Aedes malayensis from a forested area of the Nakai district in Laos to evaluate its potential as an arbovirus bridge vector. We found that this Ae. malayensis population was overall less competent for DENV and YFV than an urban population of Aedes aegypti. Olfactometer experiments showed that our Ae. malayensis colony did not display any detectable attraction to human scent in laboratory conditions. The relatively modest vector competence for DENV and YFV, combined with a lack of detectable attraction to human odor, indicate a low potential for this sylvatic Ae. malayensis population to act as an arbovirus bridge vector. However, we caution that opportunistic blood feeding on humans by sylvatic Ae. malayensis may occasionally contribute to bridge sylvatic and human transmission cycles

Development of molecular and cellular tools to decipher the type I IFN pathway of the common vampire bat

International audienceThough the common vampire bat, Desmodus rotundus, is known as the main rabies virus reservoir in Latin America, no tools are available to investigate its antiviral innate immune system. To characterize the IFN-I pathway, we established an immortalized cell line from a D. rotundus fetal lung named FLuDero. Then we molecularly characterized some of the Toll-like receptors (TLR3, 7, 8 and 9), the three RIG-I-like receptor members, as well as IFNa1 and IFNb. Challenging the FLuDero cell line with poly (I:C) resulted in an up-regulation of both IFNa1 and IFNb and the induction of expression of the different pattern recognition receptors characterized. These findings provide evidence of the intact dsRNA recognition machinery and the IFN-I signaling pathway in our cellular model. Herein, we generated a sum of insightful specific molecular and cellular tools that will serve as a useful model to study virusehost interactions of the common vampire bat

Enhanced mosquito vectorial capacity underlies the Cape Verde Zika epidemic

International audienceThe explosive emergence of Zika virus (ZIKV) across the Pacific and Americas since 2007 was associated with hundreds of thousands of human cases and severe outcomes, including congenital microcephaly caused by ZIKV infection during pregnancy. Although ZIKV was first isolated in Uganda, Africa has so far been exempt from large-scale ZIKV epidemics, despite widespread susceptibility among African human populations. A possible explanation for this pattern is natural variation among populations of the primary vector of ZIKV, the mosquito Aedes aegypti . Globally invasive populations of Ae . aegypti outside of Africa are considered effective ZIKV vectors because they are human specialists with high intrinsic ZIKV susceptibility, whereas African populations of Ae . aegypti across the species’ native range are predominantly generalists with low intrinsic ZIKV susceptibility, making them less likely to spread viruses in the human population. We test this idea by studying a notable exception to the patterns observed across most of Africa: Cape Verde experienced a large ZIKV outbreak in 2015 to 2016. We find that local Ae . aegypti in Cape Verde have substantial human-specialist ancestry, show a robust behavioral preference for human hosts, and exhibit increased susceptibility to ZIKV infection, consistent with a key role for variation among mosquito populations in ZIKV epidemiology. These findings suggest that similar human-specialist populations of Ae . aegypti in the nearby Sahel region of West Africa, which may be expanding in response to rapid urbanization, could serve as effective vectors for ZIKV in the future