Identification of RNA Helicase A as a Cellular Factor That Interacts with Influenza A Virus NS1 Protein and Its Role in the Virus Life Cycle

Influenza but not wildtype virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCEInfluenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense.

Section: Discussionsupporting

confidence: 77%

Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

Tawaratsumida

Phan

Hrincius

et al. 2014

“…1D). Most of the cellular proteins had been previously identified as influenza virus-related host proteins (4,28,42,43). However, we noticed that MOV10, an RNA helicase, had not been previously implicated in the replication of IAV.…”

Section: Isolation Of Influenza a Virus Vrnp Complex And Identificatimentioning

confidence: 86%

Host Protein Moloney Leukemia Virus 10 (MOV10) Acts as a Restriction Factor of Influenza A Virus by Inhibiting the Nuclear Import of the Viral Nucleoprotein

Zhang

Huang

Tan

et al. 2016

The viral ribonucleoprotein (vRNP) complex of influenza A viruses (IAVs) contains an RNA-dependent RNA polymerase complex (RdRp) and nucleoprotein (NP) and is the functional unit for viral RNA transcription and replication. The vRNP complex is an important determinant of virus pathogenicity and host adaptation, implying that its function can be affected by host factors. In our study, we identified host protein Moloney leukemia virus 10 (MOV10) as an inhibitor of IAV replication, since depletion of MOV10 resulted in a significant increase in virus yield. MOV10 inhibited the polymerase activity in a minigenome system through RNA-mediated interaction with the NP subunit of vRNP complex. Importantly, we found that the interaction between MOV10 and NP prevented the binding of NP to importin-␣, resulting in the retention of NP in the cytoplasm. Both the binding of MOV10 to NP and its inhibitory effect on polymerase activity were independent of its helicase activity. These results suggest that MOV10 acts as an anti-influenza virus factor through specifically inhibiting the nuclear transportation of NP and subsequently inhibiting the function of the vRNP complex. IMPORTANCEThe interaction between the influenza virus vRNP complex and host factors is a major determinant of viral tropism and pathogenicity. Our study identified MOV10 as a novel host restriction factor for the influenza virus life cycle since it inhibited the viral growth rate. Conversely, importin-␣ has been shown as a determinant for influenza tropism and a positive regulator for viral polymerase activity in mammalian cells but not in avian cells. MOV10 disrupted the interaction between NP and importin-␣, suggesting that MOV10 could also be an important host factor for influenza virus transmission and pathogenicity. Importantly, as an interferon (IFN)-inducible protein, MOV10 exerted a novel mechanism for IFNs to inhibit the replication of influenza viruses. Furthermore, our study potentially provides a new drug design strategy, the use of molecules that mimic the antiviral mechanism of MOV10.

“…The set of pPOLI plasmids (in a pDZ vector) carrying each of the eight RNA segments of Tx91 was generously provided by Adolfo García-Sastre (Mount Sinai School of Medicine, New York, NY). The pPOLI-NP-LUC plasmid was constructed by insertion of the firefly luciferase gene flanking the NP-carrying region in an antisense orientation between the human RNA polymerase I promoter and terminator (28). All panhandle derivatives were constructed in the pPOLI-NP-LUC backbone by site-directed mutagenesis (Stratagene) and confirmed by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Influenza A Virus Panhandle Structure Is Directly Involved in RIG-I Activation and Interferon Induction

Liu¹,

Park²,

Pyo

et al. 2015

Self Cite

110

Retinoic acid-inducible gene I (RIG-I) is an important innate immune sensor that recognizes viral RNA in the cytoplasm. Its nonself recognition largely depends on the unique RNA structures imposed by viral RNA. The panhandle structure residing in the influenza A virus (IAV) genome, whose primary function is to serve as the viral promoter for transcription and replication, has been proposed to be a RIG-I agonist. However, this has never been proved experimentally. Here, we employed multiple approaches to determine if the IAV panhandle structure is directly involved in RIG-I activation and type I interferon (IFN) induction. First, in porcine alveolar macrophages, we demonstrated that the viral genomic coding region is dispensable for RIG-I-dependent IFN induction. Second, using in vitro-synthesized hairpin RNA, we showed that the IAV panhandle structure could directly bind to RIG-I and stimulate IFN production. Furthermore, we investigated the contributions of the wobble base pairs, mismatch, and unpaired nucleotides within the wild-type panhandle structure to RIG-I activation. Elimination of these destabilizing elements within the panhandle structure promoted RIG-I activation and IFN induction. Given the function of the panhandle structure as the viral promoter, we further monitored the promoter activity of these panhandle variants and found that viral replication was moderately affected, whereas viral transcription was impaired dramatically. In all, our results indicate that the IAV panhandle promoter region adopts a nucleotide composition that is optimal for balanced viral RNA synthesis and suboptimal for RIG-I activation. IMPORTANCEThe IAV genomic panhandle structure has been proposed to be an RIG-I agonist due to its partial complementarity; however, this has not been experimentally confirmed. Here, we provide direct evidence that the IAV panhandle structure is competent in, and sufficient for, RIG-I activation and IFN induction. By constructing panhandle variants with increased complementarity, we demonstrated that the wild-type panhandle structure could be modified to enhance RIG-I activation and IFN induction. These panhandle variants posed moderate influence on viral replication but dramatic impairment of viral transcription. These results indicate that the IAV panhandle promoter region adopts a nucleotide composition to achieve optimal balance of viral RNA synthesis and suboptimal RIG-I activation. Our results highlight the multifunctional role of the IAV panhandle promoter region in the virus life cycle and offer novel insights into the development of antiviral agents aiming to boost RIG-I signaling or virus attenuation by manipulating this conserved region.