Induction and evasion of type I interferon responses by influenza viruses

f As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broadspectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para-and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

Yan

Krumm

Sun

et al. 2013

“…Downregulation of IFNAR1 is critical for efficient IAV replication. Since IFNAR1 is essential for triggering type I IFN-mediated antiviral responses (12)(13)(14), IFNAR1 degradation by IAV could be beneficial to virus replication. It was also reported that inhibition of ubiquitin-proteasome degradation pathway represses IAV infection and propagation (57).…”

Section: Iav Induces the Degradation Of Ifnar1 Proteinmentioning

confidence: 99%

“…Therefore, type I IFNs play an important role in the host defense system against viruses, including IAV (12)(13)(14). Influenza viral RNAs with a 5=ppp trigger the retinoic acid-inducible gene 1 (RIG-I)-mediated signaling pathway (15).…”

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confidence: 99%

Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1

Xia

Vijayan

Pritzl

et al. 2016

114

Influenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses, particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and how IAV regulates the type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces the degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced antiviral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed antiviral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the endoplasmic reticulum (ER) stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated antiviral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity. IMPORTANCE Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN Influenza virus infection causes seasonal and pandemic influenza with significant morbidity and mortality in humans (1). Outbreaks of avian influenza by highly pathogenic H5N1 and H7N9 viruses have raised the risk for the occurrence of another influenza pandemic (2-4). The genome of influenza A virus (IAV) encodes at least 11 proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins (NS1 and NS2), polymerase proteins (PA, PB1, and PB2), and PB1-F2 (5, 6). Antiviral drugs against influenza that block the function of viral proteins such as NA and M2 were developed to treat the infection. However, because of the high mutability, several strains of seasonal influenza and avian influenza viruses were shown to be resistant to the current antiviral drugs (6-8). Therefore, designing new therapeutics and identifying cellular targets of the infection are important to effectively control influenza.Type I interferons (IFNs), which include multiple IFN-␣ subtypes and IFN-␤, induce the expression of numerous interferonstimulated genes (ISGs) that establish antiviral states (9-11). Therefore, type I IFNs play an important role in the host defense system against viruses, including IAV (12)(13)(14). Influenza v...

“…When host cells are invaded by viruses, the cells immediately respond by synthesizing and secreting alpha and/or beta interferon (IFN-␣/␤) (IFN pathway), which subsequently binds to its cognate receptors on the cell surface (8,9). The engagement of IFN-␣/␤ and its receptors activates JAK1 and TYK2 to phosphorylate the STAT1 and STAT2 signal transducers (JAK-STAT pathway).…”

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confidence: 99%

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

Hsu

Jeng

et al. 2015

Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-␤) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV)replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection.