Alphacoronavirus protein 7 modulates host innate immune response

Innate immune response is the first line of antiviral defense resulting, in most cases, in pathogen clearance with minimal clinical consequences. Viruses have developed diverse strategies to subvert host defense mechanisms and increase their survival. In the transmissible gastroenteritis virus (TGEV) as a model, we previously reported that accessorygene 7 counteracts the host antiviral response by associating with the catalytic subunit of protein phosphatase 1 (PP1c). In the present work, the effect of the absence of gene 7on the host cell, during infection, was further analyzed by transcriptomic analysis. The pattern of gene expression of cells infected with a recombinant mutant TGEV, lacking gene7 expression (rTGEV-δ7), was compared to that of cells infected with the parental virus (rTGEV-wt). Genes involved in the immune response, the interferon response, and inflammation were upregulated during TGEV infection in the absence of gene 7. An exacerbated innate immune response during infection with rTGEV-δ7 virus was observed both in vitro and invivo. An increase in macrophage recruitment and activation in lung tissues infected with rTGEV-δ7 virus was observed compared to cells infected with the parental virus. In summary, the absence of protein 7 both in vitro and in vivo led to increased proinflammatory responses and acute tissue damage after infection. In a porcine animal model, which is immunologically similar to humans, we present a novel example of how viral proteins counteract host antiviral pathways to determine the infection outcome and pathogenesis

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress

AIMS: Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. METHODS AND RESULTS: Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. CONCLUSION: ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.This work was supported by the Ministerio de Ciencia e Innovación and Fondo Europeo de Desarrollo Regional (FEDER)/FSE (SAF2016-80305P; SAF2017-88089-R; SAF2016-79151-R; RTI2018-099246-B-I00), Ministerio de Innovación, Cultura y Deportes (PGC2018-097019-B-I00), Instituto de Salud Carlos III (ISCIII; FIS PI18/0919); Comunidad de Madrid (CM) (AORTASANA B2017/BMD-3676) FEDER-a way to build Europe, Bayer AG (2019-09-2433), CM-Universidad Autónoma de Madrid (SI1-PJI-2019-00321), and British Heart Foundation (CH/12/4/29762; RE//18/6/34217). M.G.-A. was supported by an FPI-UAM fellowship, R.R.-D. by a Juan de la Cierva contract (IJCI-2017-31399), and A.C.M. by a Walton Fellowship, University of Glasgow. The CNIC is supported by ISCIII, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)

Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus

Nucleic acids encoding prrsv gp5-ecto domain and m protein

The present invention relates to nucleic acids comprising: (a) a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:I (sequence ofthe GP5 ecto-domain ofthe European strain ofPRRSV) or a sequence having at least 85% identity to SEQ ID NO: 1, which sequence includes at least one amino acid capable offorming a disulfide bridge; and (b) a sequence encoding a polypeptide comprising the amino acid sequence SEQ ID NO: 2 (sequence of the GP5 M protein of the European strain of PRRSV) or a sequence having at least 85% identity to SEQ ID NO: 2; (c) wherein the nucleic acid does not comprise a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (sequence ofthe GP5 protein except the ecto-domain ofthe European strain of PRRSV) or a sequence having at least 80% identity to SEQ ID NO: 3. The present invention further provides respective nucleic acids from other strains of PRRSV and their use as a vaccine for the treatment of animals.Peer reviewedConsejo Superior de Investigaciones Científicas (España), PFIZER OLOT, S.L.U.A1 Solicitud de patente con informe sobre el estado de la técnic

Coronavirus nsp14 modulates the innate immune response

Trabajo presentado en el XIV International Nidovirus Symposium 2017, celebrado en Kansas City, Missouri (Estados Unidos), del 4 al 9 de junio de 2017Coronavirus (CoV) non-structural protein 14 (nsp14) is encoded by the replicase gene and it is part of the replication-transcription complex. Nsp14 protein is a bifunctional enzyme bearing 3'-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single- and double-stranded RNAs and is part of a proofreading system responsible for the high fidelity of CoV replication. Nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon (IFN) and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but to date the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described. A set of point mutants affecting different motifs within the ExoN domain of nsp14 protein was generated, using transmissible gastroenteritis virus (TGEV) as a model. A specific mutation within zinc finger 1 (ZF-C) led to a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant rTGEVZF-C caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times post-infection. Consequently, the mutant virus triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of IFN-beta, TNF, and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced, when compared to the parental virus. Moreover, the mutant virus decreased the antiviral pathway activation produced by the addition of exogenous dsRNA or by the wild-type virus infection, suggesting that the mutant virus actively reduced dsRNA levels in these situations. Overall, our data revealed a novel role for CoV nsp14 ExoN domain in modulation of the innate immune response. In addition to CoVs, only arenaviruses encode a protein with exonuclease activity that, similarly to CoV nsp14, counteracts the innate immune response. Therefore, this enzymatic function could be a novel target for the development of broad-spectrum antivirals against these relevant pathogens

Coronavirus Nsp14 modulates the innate immune response

Trabajo presentado en el 6th European Congress of Virology, celebrado en Hamburgo (España), del 19 al 22 de octubre de 2016Coronavirus (CoV) non-structural protein 14 (nsp14) is encoded by the replicase gene and it is part of the replication-transcription complex. Nsp14 protein is a bifunctional enzyme bearing 3´-5´ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single- and double-stranded RNAs and is part of a proofreading system responsible for the high fidelity of CoV replication. Nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon (IFN) and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but to date the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described. A set of point mutants affecting different motifs within the ExoN domain of nsp14 protein was generated, using transmissible gastroenteritis virus (TGEV) as a model. A specific mutation within zinc finger 1 (ZF-C) led to a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant rTGEVZF-C caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times post-infection. Consequently, the mutant virus triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of IFN-β, TNF, and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced, when compared to the parental virus. Moreover, the mutant virus decreased the antiviral pathway activation produced by the addition of exogenous dsRNA or by the wild-type virus infection, suggesting that the mutant virus actively reduced dsRNA levels in these situations. Overall, our data revealed a novel potential role for CoV nsp14 in modulation of the innate immune response

Mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response

Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication- transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as >non-self> by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus. Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response.This work was supported by a grant from the Government of Spain (BIO2013-42869-R), a U.S. National Institutes of Health (NIH) project grant (2P01AI060699-06A1), IMI and the European Commission, and in-kind contributions from EFPIA partners (ZAPI project, IMI grant agreement no. 115760). M.B. and S.Z. received a contract from the Government of Spain and the NI

Reprint of: Coronavirus reverse genetic systems: Infectious clones and replicons

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).This work was supported by grants from the Ministry of Science and Innovation of Spain (MCINN) (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” (HEALTH-F3-2009-223498), and the National Institute of Health (NIH) of USA (2P01AI060699-06A1)