Inhibition of Ongoing Influenza A Virus Replication Reveals Different Mechanisms of RIG-I Activation

Abstract

Pattern recognition receptors provide essential nonself immune surveillance within distinct cellular compartments. Retinoic acid-inducible gene I (RIG-I) is one of the primary cytosolic RNA sensors, with an emerging role in the nucleus. It is involved in the spatiotemporal sensing of influenza A virus (IAV) replication, leading to the induction of type I interferons (IFNs). Nonetheless, the physiological viral ligands activating RIG-I during IAV infection remain underexplored. Other than full-length viral genomes, cellular constraints that impede ongoing viral replication likely potentiate an erroneous viral polymerase generating aberrant viral RNA species with RIG-I-activating potential. Here, we investigate the origins of RIG-I-activating viral RNA under two such constraints. Using chemical inhibitors that inhibit continuous viral protein synthesis, we identify the incoming, but notde novo-synthesized, viral defective interfering (DI) genomes contributing to RIG-I activation. In comparison, deprivation of viral nucleoprotein (NP), the key RNA chain elongation factor for the viral polymerase, leads to the production of aberrant viral RNA species activating RIG-I; however, their nature is likely to be distinct from that of DI RNA. Moreover, RIG-I activation in response to NP deprivation is not adversely affected by expression of the nuclear export protein (NEP), which diminishes the generation of a major subset of aberrant viral RNA but facilitates the accumulation of small viral RNA (svRNA). Overall, our results indicate the existence of fundamentally different mechanisms of RIG-I activation under cellular constraints that impede ongoing IAV replication.IMPORTANCEThe induction of an IFN response by IAV is mainly mediated by the RNA sensor RIG-I. The physiological RIG-I ligands produced during IAV infection are not fully elucidated. Cellular constraints leading to the inhibition of ongoing viral replication likely potentiate an erroneous viral polymerase producing aberrant viral RNA species activating RIG-I. Here, we demonstrate that RIG-I activation during chemical inhibition of continuous viral protein synthesis is attributable to the incoming DI genomes. Erroneous viral replication driven by NP deprivation promotes the generation of RIG-I-activating aberrant viral RNA, but their nature is likely to be distinct from that of DI RNA. Our results thus reveal distinct mechanisms of RIG-I activation by IAV under cellular constraints impeding ongoing viral replication. A better understanding of RIG-I sensing of IAV infection provides insight into the development of novel interventions to combat influenza virus infection.

Structure and Function of the Influenza Virus Transcription and Replication Machinery

“…In particular, it has been shown that svRNA synthesis can influence vRNA synthesis by binding to the viral RNA polymerase (Perez et al 2012). The viral nonstructural protein 2 (NS2/NEP) may be involved in regulating svRNA expression (Perez et al 2010;Liu et al 2019).…”

Cytoplasm and Beyond: Dynamic Innate Immune Sensing of Influenza A Virus by RIG-I

“…They retain the 5= and 3= genomic noncoding regions and thus the same panhandle structure activating RIG-I. While several lines of evidence argue against IFN induction by incoming vRNPs (41,55,56), chemical inhibition of ongoing viral replication reveals the direct contribution of incoming DI RNA as contained in the IAV stocks to RIG-I activation and IFN induction (Fig. 2, step 1) (56).…”

“…While several lines of evidence argue against IFN induction by incoming vRNPs (41,55,56), chemical inhibition of ongoing viral replication reveals the direct contribution of incoming DI RNA as contained in the IAV stocks to RIG-I activation and IFN induction (Fig. 2, step 1) (56). The mechanism for the preferential RIG-I sensing of incoming DI RNA over vRNP is currently unknown but likely lies in the level of exposure of the panhandle structure and/or its cellular distribution.…”

“…As the key RNA chain elongation factor, viral NP regulates the synthesis of full-length viral genomes and abortive replication products (59). Using an NP-free condition that abolished full-length viral genome synthesis, it was recently demonstrated that the viral polymerase produces aberrant viral RNAs activating nuclear RIG-I (56). Two distinct aberrant RNAs representing abortive replication products of negative polarity were revealed whose accumulation was sensitive to expression of the viral nuclear export protein (NEP).…”

“…Both being generated under NP-deficient conditions, it also remains obscure whether the abortive replication products are equivalent to mvRNA. The immunostimulatory activity of mvRNA, like DI RNA, is resistant to RNA extraction, whereas that of abortive replication products is not, indicating a distinct origin (56,58). A difference in the cellular distribution of the two classes of aberrant RNA may also exist that contributes to differential activation of cytoplasmic versus nuclear RIG-I (Fig.…”