SARS-CoV-2 challenges health care systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed.

The phosphatidylinositol-3-kinase (PI3K) was identified to be activated upon influenza A virus (IAV) infection. An early and transient induction of PI3K signalling is caused by viral attachment to cells and promotes virus entry. In later phases of infection the kinase is activated by the viral NS1 protein to prevent premature apoptosis. Besides these virus supporting functions, it was suggested that PI3K signalling is involved in dsRNA and IAV induced antiviral responses by enhancing the activity of interferon regulatory factor-3 (IRF-3). However, molecular mechanisms of activation remained obscure. Here we show that accumulation of vRNA in cells infected with influenza A or B viruses results in PI3K activation. Furthermore, expression of the RNA receptors Rig-I and MDA5 was increased upon stimulation with virion extracted vRNA or IAV infection. Using siRNA approaches, Rig-I was identified as pathogen receptor necessary for influenza virus vRNA sensing and subsequent PI3K activation in a TRIM25 and MAVS signalling dependent manner. Rig-I induced PI3K signalling was further shown to be essential for complete IRF-3 activation and consequently induction of the type I interferon response. These data identify PI3K as factor that is activated as part of the Rig-I mediated anti-pathogen response to enhance expression of type I interferons.

The non-structural protein 1 (A/NS1) of influenza A viruses (IAV) harbours several src-homology domain (SH) binding motifs that are required for interaction with cellular proteins. The SH3 binding motif at aa212-217 [PPLPPK] of A/NS1 was shown to be essential for binding to the cellular adaptor proteins CRK and CRKL. Both regulate diverse cellular effector pathways, including activation of the MAP-kinase JNK that in turn mediates antiviral responses to IAV infection. By studying functional consequences of A/NS1–CRK interaction we show here that A/NS1 binding to CRK contributes to suppression of the antiviral-acting JNK–ATF2 pathway. However, only IAV that encode an A/NS1-protein harbouring the CRK/CRKL SH3 binding motif PPLPPK were attenuated upon downregulation of CRKI/II and CRKL, but not of CRKII alone. The PPLPPK site-harbouring candidate strains could be discriminated from other strains by a pronounced viral activation of the JNK–ATF2 signalling module that was even further boosted upon knock-down of CRKI/II. Interestingly, this enhanced JNK activation did not alter type-I IFN-expression, but rather resulted in increased levels of virus-induced cell death. Our results imply that binding capacity of A/NS1 to CRK/CRKL has evolved in virus strains that over-induce the antiviral acting JNK–ATF2 signalling module and helps to suppress the detrimental apoptosis promoting action of this pathway.

Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associatedStaphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureussuper-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV andS. aureus.