Interactions between the Influenza A Virus RNA Polymerase Components and Retinoic Acid-Inducible Gene I

Abstract

The influenza A virus genome possesses eight negative-strand RNA segments in the form of viral ribonucleoprotein particles (vRNPs) in association with the three viral RNA polymerase subunits (PB2, PB1, and PA) and the nucleoprotein (NP). Through interactions with multiple host factors, the RNP subunits play vital roles in replication, host adaptation, interspecies transmission, and pathogenicity. In order to gain insight into the potential roles of RNP subunits in the modulation of the host's innate immune response, the interactions of each RNP subunit with retinoic acid-inducible gene I protein (RIG-I) from mammalian and avian species were investigated. Studies using coimmunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFc), and colocalization using confocal microscopy provided direct evidence for the RNA-independent binding of PB2, PB1, and PA with RIG-I from various hosts (human, swine, mouse, and duck). In contrast, the binding of NP with RIG-I was found to be RNA dependent. Influenza A virus is a member of the Orthomyxoviridae family and contains eight segments of negative-sense genomic RNA. Within the virus particle, viral RNAs (vRNAs) are wrapped with multiple copies of the nucleoprotein (NP) and are bound to a heterotrimeric polymerase complex composed of basic polymerases 1 and 2 (PB1 and PB2, respectively) and the acidic polymerase (PA) to collectively form viral ribonucleoprotein particles (vRNPs). Upon entry and uncoating, the vRNPs travel to the nucleus through interactions with importin-like factors and components of the nuclear pore complex. The vRNPs form the minimal units for transcription and replication of viral RNAs. PB2 recognizes and binds the methylated cap of cellular pre-mRNAs (1), and then PA cleaves the cap and generates 5=-capped RNA fragments (2), which are then used to prime viral mRNA transcription by PB1 (3). The accumulation of NP during infection is thought to favor the switching of viral RNA from transcription to replication (4) and promote the nuclear export of progeny vRNPs (in association with the viral proteins M1 and NEP and cellular factors) (5). The protein components of vRNPs are also involved in virulence, host adaptation, and interspecies transmission of influenza A virus, presumably due to interactions with numerous host factors (6).The innate immune response, especially the interferon (IFN)-mediated response, makes up the first line of defense against the invasion of pathogens. The production of type I IFN is primarily induced by Toll-like receptors (TLRs) located in the endosome or cellular membrane and RIG-I like receptors (RLRs) distributed mainly in the cytoplasm. RIG-I has been identified as the major cellular sensor for IFN induction upon influenza virus infection (7,8). RIG-I recognizes the 5= triphosphate (5=ppp) in viral genomic RNAs (9, 10) or double-stranded RNA (dsRNA) intermediates synthesized during virus replication (7, 11). Recently, RIG-I was shown to preferentially associate with short subgenomic viral segments in infe...

Influenza Virus Adaptation PB2-627K Modulates Nucleocapsid Inhibition by the Pathogen Sensor RIG-I

“…However, the virus remains vulnerable during the transit to the nucleus, since at this stage of infection neither the classic IFN antagonist NS1 (Hale, 2014) nor the host response suppressors PA-X (Jagger et al, 2012) or PB1-F2 (Varga et al, 2011) would be expressed. Therefore, it appears plausible that the nucleocapsids themselfes have to cope with the pathogen recognition system, an assumption that is in agreement with our data and also with previous reports that FLUAV polymerase subunits can prevent IFN induction (Marcus et al, 2005; Perez-Cidoncha et al, 2014) and interact with RIG-I and MAVS (Graef et al, 2010; Iwai et al, 2010; Li et al, 2014; Liedmann et al, 2014). Moreover, for influenza B virus it was demonstrated that nucleocapsids were sufficient to induce IFN, whereas for FLUAV RNA synthesis was required (Osterlund et al, 2012).…”

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

“…This recognition might be coupled with viral transcription and replication that involve active viral polymerase and other host factors. This is of particular interest since RIG-I has been observed to partially relocalize into the cell nucleus during late stages of infection (62); however, whether this relocalization event correlates with IFN induction requires further investigation.…”

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

“…Besides its structural role, NP also diminishes PRR activation by recruiting the cellular RNA helicases UAP56 and URH49, supposedly by unwinding any dsRNA that arises during genome replication (19). Moreover, two studies found that RNPs can interact with RIG-I or sequester it to the nucleus (22,23). PB1, PB2, and PA also interact with the host cell RNA polymerase II repressor DR1, which downregulates the expression of FLUAV-relevant ISGs, like RIG-I, MDA5, MX1, and IFITM (24).…”