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

Abstract

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.

Mir‐302c mediates influenza A virus‐induced IFNβ expression by targeting NF‐κB inducing kinase

“…RIG-I and MDA5 can bind mitochondrial Cardif (also known as MAVS, IPS-1, and VISA) through caspase recruitment domains, initiating downstream signaling that leads to IFN induction [20]. In the case of IAV infections, RIG-I and TLR3 are identified as two major PRRs induced by IAV infection [21,22]. RIG-I senses the accumulation of IAV 5 0 -triphosphate RNA, which leads to the activation of type-I IFN [21].…”

“…In the case of IAV infections, RIG-I and TLR3 are identified as two major PRRs induced by IAV infection [21,22]. RIG-I senses the accumulation of IAV 5 0 -triphosphate RNA, which leads to the activation of type-I IFN [21]. In contrast, TLR3 activates proinflammatory cytokines in response to IAV infection [22].…”

To Conquer the Host, Influenza Virus Is Packing It In: Interferon-Antagonistic Strategies beyond NS1

“…RIG-I possesses two N-terminal caspase recruitment domains (CARDs), a central RNA helicase domain of the DExD/H box type, and a C-terminal domain (CTD) that is important for RNA ligand binding (9). RIG-I responds strongly to 5=-end-triphosphorylated dsRNA structures (5=-ppp-dsRNA), like the "panhandle," which can be formed by complementary sequences of the 5= and 3= termini of the FLUAV genome (10)(11)(12). The binding of RIG-I to the FLUAV panhandle occurs immediately after the RNPs enter the host cell and can impose a direct antiviral effect via the disassembling of the RdRP complex (13).…”

RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression

“…One study suggested that influenza A virus (IAV) RNA synthesis and nuclear export but not viral replication trigger IFN gene expression (12). Other studies have proposed that RIG-I can recognize the incoming negativesense RNA virus via its 5=-triphosphorylated genomic RNA, even though it is bound by NP proteins and the viral RNA polymerase complex (13)(14)(15). It has also been suggested that the incoming influenza A virus carries certain viral structures that inhibit RIG-I activation and IFN induction, even though the viral RNA is recognized by RIG-I at the time of entry into the cytosol (16).…”