The K186E amino acid substitution in the canine influenza virus H3N8 NS1 protein restores its ability to inhibit host gene expression

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2. Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. Contrastingly, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE: Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses of equine and avian origin, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, that contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen

The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2. Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. Contrastingly, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE: Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses of equine and avian origin, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, that contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1

Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral non-structural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host-species barrier. We characterised the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionary distinct NS1s counteract the IFN response using different and mutually exclusive mechanisms: while the NS1s of early EIVs block general gene expression by binding to the cellular polyadenylation specific factor 30 (CPSF30), NS1s from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and became rapidly selected during EIV evolution, highlighting the importance of evolutionary processes on immune evasion mechanisms during IAV adaptation

Regulation of PURA gene transcription by three promoters generating distinctly spliced 5-prime leaders: a novel means of fine control over tissue specificity and viral signals

<p>Abstract</p> <p>Background</p> <p>Purα is an evolutionarily conserved cellular protein participating in processes of DNA replication, transcription, and RNA transport; all involving binding to nucleic acids and altering conformation and physical positioning. The distinct but related roles of Purα suggest a need for expression regulated differently depending on intracellular and external signals.</p> <p>Results</p> <p>Here we report that human <it>PURA </it>(<it>hPURA</it>) transcription is regulated from three distinct and widely-separated transcription start sites (TSS). Each of these TSS is strongly homologous to a similar site in mouse chromosomal DNA. Transcripts from TSS I and II are characterized by the presence of large and overlapping 5'-UTR introns terminated at the same splice receptor site. Transfection of lung carcinoma cells with wild-type or mutated <it>hPURA </it>5' upstream sequences identifies different regulatory elements. TSS III, located within 80 bp of the translational start codon, is upregulated by E2F1, CAAT and NF-Y binding elements. Transcription at TSS II is downregulated through the presence of adjacent consensus binding elements for interferon regulatory factors (IRFs). Chromatin immunoprecipitation reveals that IRF-3 protein binds <it>hPURA </it>promoter sequences at TSS II in vivo. By co-transfecting <it>hPURA </it>reporter plasmids with expression plasmids for IRF proteins we demonstrate that several IRFs, including IRF-3, down-regulate <it>PURA </it>transcription. Infection of NIH 3T3 cells with mouse cytomegalovirus results in a rapid decrease in levels of <it>mPURA </it>mRNA and Purα protein. The viral infection alters the degree of splicing of the 5'-UTR introns of TSS II transcripts.</p> <p>Conclusions</p> <p>Results provide evidence for a novel mechanism of transcriptional control by multiple promoters used differently in various tissues and cells. Viral infection alters not only the use of <it>PURA </it>promoters but also the generation of different non-coding RNAs from 5'-UTRs of the resulting transcripts.</p

Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen

AGL2017-82570-RReverse genetics approaches for the development of new vaccines against influenza A virus infections

Influenza A viruses (IAVs) represent a serious concern globally because they are capable of rapid spread and cause severe disease in humans and other animals. The development and implementation of plasmid-based reverse genetics approaches have allowed the manipulation and recovery of recombinant IAVs from complementary DNA copies of the viral genome. Furthermore, IAV reverse genetics have provided researchers an efficient and powerful platform to introduce specific changes in the viral genome with the final goal of studying IAV biology, designing more effective vaccine strategies, and to reduce the rates of incidence and mortality associated with viral infections. In this review, we briefly discuss IAV reverse genetics and their applications to prevent IAV infections.Influenza research in LM-S laboratory has been partially funded by the New York Influenza Center of Excellence (NYICE, NIH 272201400005C), a member of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract No. HHSN272201400005C (NYICE), the NIH/NIAID 1R01AI145332, and the Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP) grant W81XWH-18-1-0460. Research in M.L.D laboratory was supported with funds from Comunidad de Madrid, Spain, reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018-094213-A-I00). Research in A.N laboratory was partially funded with the ‘Ramon y Cajal’ Fellowship RYC-2017 from Spanish Ministry of Economy, Industry and Competitiveness and the Spanish Ministry of Science, Innovation and Universities (AGL2017-82570-R)

Novel Functions of IFI44L as a Feedback Regulator of Host Antiviral Responses

We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) gene in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of double-stranded RNA (dsRNA) or by IFN treatment. The mechanism likely involves the interaction of IFI44L with cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as inhibitor of nuclear factor kappa B (IκB) kinase alpha (IKKα), IKKβ, and IKKε. Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3)-mediated and nuclear factor kappa-B (NF-κB) inhibitor (IκBα)-mediated phosphorylation by IKKε and IKKβ, respectively. According to these results, IFI44L is a good target for treatment of diseases associated with excessive IFN levels and/or proinflammatory responses and for reduction of viral replication.This project was funded with federal funds from the National Institute of Allergy andInfectious Diseases (NIAID), National Institutes of Health (NIH), Department of Health and Human Services, under CEIRS contract HHSN272201400005C to D.J.T.; through a University of Rochester Research Award to M.L.D.; and with funds from Comunidad de Madrid, Madrid, Spain (reference 2017-T1/BMD-5155), to M.L.D