Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA

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.

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

110

“…We assume that vRNP formation stabilizes nascent vRNA in a similar fashion. For the majority of vRNAs and cRNAs, different genome segments show similar levels throughout an infection (21,31). Hence, we do not explicitly distinguish between individual segments but rather consider their total number per cell.…”

Section: Methodsmentioning

confidence: 99%

“…Besides the differential regulation of mRNA and cRNA synthesis during the early phase of infection, both species cease to accumulate toward the late phase. In contrast, vRNAs are continuously synthesized (21,31,53). This implies distinct control mechanisms for positive-and negativestrand RNA synthesis.…”

mentioning

confidence: 99%

Modeling the Intracellular Dynamics of Influenza Virus Replication To Understand the Control of Viral RNA Synthesis

Heldt

Frensing

Reichl

2012

bInfluenza viruses transcribe and replicate their negative-sense RNA genome inside the nucleus of host cells via three viral RNA species. In the course of an infection, these RNAs show distinct dynamics, suggesting that differential regulation takes place. To investigate this regulation in a systematic way, we developed a mathematical model of influenza virus infection at the level of a single mammalian cell. It accounts for key steps of the viral life cycle, from virus entry to progeny virion release, while focusing in particular on the molecular mechanisms that control viral transcription and replication. We therefore explicitly consider the nuclear export of viral genome copies (vRNPs) and a recent hypothesis proposing that replicative intermediates (cRNA) are stabilized by the viral polymerase complex and the nucleoprotein (NP). Together, both mechanisms allow the model to capture a variety of published data sets at an unprecedented level of detail. Our findings provide theoretical support for an early regulation of replication by cRNA stabilization. However, they also suggest that the matrix protein 1 (M1) controls viral RNA levels in the late phase of infection as part of its role during the nuclear export of viral genome copies. Moreover, simulations show an accumulation of viral proteins and RNA toward the end of infection, indicating that transport processes or budding limits virion release. Thus, our mathematical model provides an ideal platform for a systematic and quantitative evaluation of influenza virus replication and its complex regulation.

“…3A). VA9 or VN36 cells were infected with A/Memphis/7/01 (MOI of 0.01), and strand-specific RT-PCR was used to determine the levels of matrix and NP gRNA (28). Eight hours after infection, influenza virus matrix and NP gRNA levels were approximately 10 1.5 -fold lower in VA9 cells than in VN36 cells (P Ͻ 0.0001) (Fig.…”

Section: Ifn-␣-inducedmentioning

confidence: 98%

Myxovirus Resistance Gene A (MxA) Expression Suppresses Influenza A Virus Replication in Alpha Interferon-Treated Primate Cells

Matzinger

Carroll

Dutra

et al. 2013