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.

Importance: The 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.

FIG 1

RIG-I-dependent IFN production in PAMs induced by virion-derived vRNA. (A) PAMs were infected with SIV/Sk02 and its isogenic NS1 mutant virus. SIV/Sk02 NS1 1-99 at a multiplicity of infection of 1 for 4 h. The levels of IFN-α/β mRNA were quantified by qRT-PCR. (B) PAMs were transfected with increasing amounts of virion-derived vRNA from three IAV H1N1 strains (SIV/Sk02, Halifax210, and Tx91). IFN-α production was determined at 24 h p.t. via an ELISA. The integrity and identity of these vRNA were examined on 2.8% polyacrylamide–7 M urea gels and visualized by silver staining. (C) PAMs were transfected with virion-derived vRNA from PR8 (50 ng) with or without CIAP treatment. IFN-α production was determined at 24 h p.t. The vRNA integrity was examined on a 2.8% denaturing gel. (D) ATPase activity of purified hRIG-I was determined in the presence of increasing amounts of either untreated or CIAP-treated PR8 vRNA. (E) PAMs were left untreated or pretreated with 10 μM or 20 μM chloroquine for 1 h, followed by transfection with PR8 vRNA (50 ng) or poly(I·C) for 8 h in the presence of inhibitor. IFN-α production was determined in an ELISA. (F) PAMs pretreated with chloroquine were stimulated with LPS (1 μg/ml) for 8 h, and IL-1β production was determined by ELISA. (G) PAMs were transfected with 20 nM off-target siRNA (siOT) or siRNA against porcine RIG-I, TLR3, or TLR7 for 24 h, followed by transfection with PR8 vRNA (50 ng) for 24 h. IFN-α production was determined in an ELISA. (H) The knockdown efficiency of siRNA was examined by qRT-PCR. Data were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase, and expression was calculated by using the ΔΔC_T method relative to the result with siOT.

FIG 2

The IFN stimulatory region lies within the vRNA noncoding region. (A) Schematic diagram of the RNA Pol I-based RNP reconstitution system used to synthesize the structurally defined panhandle vRNA in vivo. The viral coding region (CR, in an antisense orientation) flanked by the 5′ and 3′ NCR of the IAV segment was inserted between the RNA Pol I promoter and terminator. Detailed nucleotide composition of the panhandle structure is illustrated according to a previous report (25). Symbols for base pairs: solid line, Watson-Crick base pair; broken line, mismatch; dot, wobble base pair. (B) PAMs were transfected with RNA (100 ng) extracted from reconstituted vRNP representing each of the eight IAV segments for 24 h. Reconstituted RNA from the Pol I vector (vec) and poly(I·C) were used as negative and positive controls, respectively. IFN-α production was determined in an ELISA. (C) PAMs were transfected with 20 nM off-target siRNA (siOT) or siRNA against porcine RIG-I, TLR3, or TLR7 for 24 h, followed by transfection with reconstituted RNA from the NP segment for 24 h. IFN-α production was determined in an ELISA. (D) PAMs were transfected with either total or fractionated (>200 nt) reconstituted RNA (100 ng) derived from Tx91-NA constructs containing different lengths of the coding region, FL, loop810, or loop96, for 24 h. IFN-α production was determined in an ELISA.

FIG 3

The panhandle structures bind to and activate RIG-I in vitro. (A) In silico-predicted hairpin WT panhandle structure and its variants. Transition and deletion/insertion mutations in the Complete and debulged variants are highlighted in yellow and green, respectively. (B) Representative gels showing EMSA results for hRIG-I with the WT and mutant panhandle structures without ATP. (C) Summary of EMSA results, fitted into the specific binding with Hill slope function (one site). (D) Effects of WT and mutant panhandle RNA binding on the ATPase activity of hRIG-I. Data fitting was performed with the Michaelis-Menten function. (E) Stimulation of the chicken IFN-β promoter in DF-1 cells by the WT and mutant panhandle RNA with or without duck RIG-I (dRIG-I) cotransfection. A synthetic 19-mer 5′ppp-dsRNA and its control dsRNA (5′OH-dsRNA) were used as controls. Data were normalized to the internal Renilla luciferase activity, and statistical significance was determined relative to the WT panhandle.

FIG 4

Panhandle-stabilizing mutations promote IFN stimulatory activity in the context of full-length vRNA in PAMs. (A) The PP7CP pulldown fraction was examined by Western blotting against viral NP and PB2 proteins. (B) The relative levels of vRNA, cRNA, and mRNA in the PP7CP pulldown fraction from the A/WSN/33 NA segment were quantified by qRT-PCR. Data were normalized to 18S rRNA and are expressed based on the ΔΔC_T method relative to the result with the Pol I vector control. (C) RNA extracted from the PP7CP pulldown fraction was resolved on a 4% polyacrylamide–7 M urea gel and visualized by SYBR Gold staining. Virion-derived PR8 vRNA was loaded in parallel as a size marker. Of note, the NA segments of WSN33 and PR8 are of similar sizes (1,409 versus 1,413 nt). (D) PAMs were transfected with PP7CP pulldown vRNA or PR8 vRNA (50 ng) for 24 h. IFN-α production was determined in an ELISA. (E) PAMs were transfected with an equal molar amount (3 × 10⁷ copies) of purified vRNA harboring panhandle-stabilizing mutations for 24 h. IFN-α production was determined in an ELISA and is expressed as the fold change over the WT vRNA-induced level.

FIG 5

Panhandle-stabilizing mutations do not impair viral replication. (A) Firefly luciferase expression from the NP-LUC constructs was measured directly from reconstituted HEK293T cells and normalized to internal Renilla luciferase activity. (B, C, and D) The absolute levels of viral mimetic mRNA (B), vRNA (C), and cRNA (D) in total reconstituted RNA from the Complete and debulged panhandle variants were quantified by strand-specific qRT-PCR. Of note, the number of copies of vRNA template produced from cellular RNA Pol I transcription was determined by omitting the PB2 subunit in reconstitution. This number was subtracted from the total RNA copy number.

FIG 6

Minor contribution of the distal segment-specific region to vRNA-induced IFN production. (A) In silico-predicted WT panhandle structure. The segment-specific region is boxed, and sequential introduction of transversion mutations is illustrated by an arrow. (B, C, and D) The absolute levels of viral mimetic vRNA (B), cRNA (C), and mRNA (D) in total reconstituted RNA from panhandle variants carrying sequential segment-specific mutations were quantified by strand-specific qRT-PCR. (E and F) PAMs were transfected with an equal molar amount of vRNA (4 × 10⁷ copies) (E) or combined vRNA and cRNA (total, 4 × 10⁷ copies) (F) from panhandle variants carrying sequential segment-specific mutations for 24 h. IFN-α production was determined in an ELISA, and results are expressed as the fold change over the WT panhandle-induced level.