Influenza A virus (IAV) infections are still a major global threat for humans, especially for the risk groups of young children and the elderly. Annual epidemics and sporadically occurring pandemics highlight the necessity of effective antivirals that can limit viral replication. The currently licensed antiviral drugs target viral factors and are prone to provoke viral resistance. In infected host cells IAV induces various cellular signaling cascades. The Raf/MEK/ERK signaling cascade is indispensable for IAV replication because it triggers the nuclear export of newly assembled viral ribonucleoproteins (vRNPs). Inhibition of this cascade limits viral replication. Thus, next to their potential in anti-tumor therapy, inhibitors targeting the Raf/MEK/ERK signaling cascade came into focus as potential antiviral drugs. The first licensed MEK inhibitor Trametinib (GSK-1120212) is used for treatment of malignant melanoma, being highly selective and having a promising side effect profile. Since Trametinib may be qualified for a repurposing approach that would significantly shorten development time for an anti-flu use, we evaluated its antiviral potency and mode of action. In this study, we describe that Trametinib efficiently blocks replication of different IAV subtypes in vitro and in vivo. The broad antiviral activity against various IAV strains was due to its ability to interfere with export of progeny vRNPs from the nucleus. The compound also limited hyper-expression of several cytokines. Thus, we show for the first time that a clinically approved MEK inhibitor acts as a potent anti-influenza agent.

SummaryPhosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non‐structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post‐translational events yet. Here, we show that the newly identified phospho‐site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double‐stranded RNA and TRIM25 as well as complex formation with RIG‐I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon‐antagonistic activity are needed.

Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis-inducing cytokines and led to hampered viral protein expression most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action.

Fig 1. Susceptibility of the coronavirus TGEV to low-pH treatment. (A) Treatment at pH 4.0 and 25°C in an IVIG solution. Stability controls (virus spiked into IVIG solution at neutral pH [pH 7.0] and held at process temperature) are shown in light gray. Medium stability controls (virus spiked into titration medium and held at process temperature) are shown in blue. Limits of detection (LOD) are shown for 21-day test samples (based on cumulated volume of samples with no residual infectivity) and controls only. (B) Treatment at pH 4.0 and 37°C in an IVIG intermediate in presence of PS80. Stability controls (virus spiked into IVIG intermediate at neutral pH [pH 7.0] and held at process temperature) are shown in light gray. LOD is shown for 540-minute test samples. (A, B) Results of two independent replicates are shown (Run A and Run B). Open symbols indicate when the virus titer has reached the detection limit (no residual infectivity detected).[Color figure can be viewed at wileyonlinelibrary.com]