Human lung epithelial cells (A549) were cultured in DMEM, Madin-Darby canine kidney epithelial cells (MDCK II) in MEM and human bronchial epithelial cells (Calu-3) in Ham's F-12/DMEM. Media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS). Human IAV strains A/Puerto Rico/8/34 (H1N1, PR8M) and A/WSN/33 were obtained from the strain collection of the Institute of Virology Muenster (Germany). The avian IAV strain A/FPV/Bratislava/79 (H7N7, FPV) was originally taken from the strain repository of the Institute of Virology, Giessen, Germany. Mouse-adapted A/Seal/Massachusetts/1/80 (H7N7, SC35M) (described in Gabriel et al., 2005), and A/Anhui/1/2013 (H7N9) were a kind gift from Thorsten Wolff, Robert-Koch-Institute (Berlin, Germany). All experiments and handling of samples containing H7N9 or H7N7 FPV infectious particles were performed in a biological safety level 3 containment. All viruses were propagated on MDCK II cells and infections were performed as described previously (Hrincius et al., 2011).

Vemurafenib (PLX-4032, Zelboraf™) was purchased from Active Biochem and U0126 was taken from the inhibitor stock of the Institute of Virology, Muenster (Germany). SB202190 and SP600125 were purchased from Calbiochem.

For stimulation with human epidermal growth factor (EGF) or human tumor necrosis factor alpha (TNFα), A549 cells were pretreated with Vemurafenib (25 μM) or DMSO in DMEM for 60 min. Subsequently, EGF (R&D Systems) (30 ng/ml) or TNFα (Sigma-Aldrich) (5 ng/ml) were added to the medium and cells were lysed after 5 min (EGF) or 30 min (TNFα), respectively.

Apoptosis was induced by treatment of A549 cells with 1 μM Staurosporine (Sigma-Aldrich) for 5 h, with human TNFα (5 ng/ml) (Sigma-Aldrich) or with human SuperKillerTRAIL™ (50 ng/ml) (Enzo Life Sciences GmbH) for 4 h.

For viral RNA stimulation, total RNA was isolated from mock-infected (cellular RNA, cRNA) or FPV-infected (MOI 5; viral RNA, vRNA) A549 cells 8 hours post infection (hpi) using peqGOLD TriFast™ (Peqlab) reagent as described previously (Börgeling et al., 2014).

For co-treatment of A549 cells with U0126 and Vemurafenib, cells were pretreated with U0126 (50 μM) for 30 min before Vemurafenib (25 μM) was added. After 60 min, cells were transfected with 500 ng vRNA or cRNA per 12-well using Lipofectamine® 2000 (Thermo Fisher Scientific). Six hours post transfection, cell lysates were subjected to reverse transcription followed by qRT-PCR.

Plaque titration was performed as described earlier (Seyer et al., 2012). Plaques were visualized by neutral red staining and progeny virus titers are depicted as plaque forming units per milliliter (PFU/ml).

A549 cells were grown in 96-well plates and cultured in presence of Vemurafenib or DMSO. Staurosporine (Sigma-Aldrich) (2 μM) was added to the medium as positive control. At the times indicated, WST-1 (water soluble tetrazolium)-based cell proliferation assay (Roche) was performed according to the manufacturer's instructions. Absorbance was measured at 450 nm.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) as described by the manufacturer. Subsequently, 500 ng total RNA was reverse transcribed with RevertAid H Minus ReverseTranscriptase (Thermo Fisher Scientific) and oligo(dT) primers (MWG-Biotech AG) according to the manufacturer's protocol. The cDNA was subjected to qRT-PCR, which was performed using a Roche LightCycler 480 and Brilliant SYBR Green III Mastermix (Agilent) according to the manufacturer's instructions. The following primers were used: human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward (5′-gcaaattccatggcaccgt-3′) and reverse (5′-gccccacttgatttggagg-3′), IAV polymerase basic protein 1 (PB1) forward (5′-catacagaagaccagtcgggat-3′) and reverse (5′-gtctgagctcttcaatgtggtgga-3′); IAV matrix protein 1 (M1) forward (5′-tgcaaaaacatcttcaagtctctg-3′) and reverse (5′-agatgagtcttctaaccgaggtcg-3′), IAV non-structural protein (NS) forward (5′-gaggacttgaatggaatgataaca-3′) and reverse (5′-gtctcaattcttcaatcaatcaaccatc-3′), human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) forward (5′-gtctctctgtgtggctgtaacttacg-3′) and reverse (5′-aaacaagcaatgccacttttgg-3′) and human tumor necrosis factor alpha (TNFα) forward (5′-atgagcactgaaagcatgatc-3′) and reverse (5′-gagggctgattagagagaggt-3′). Relative changes in expression levels (n-fold) were determined by the 2^ΔΔCt method (Livak and Schmittgen, 2001).

For western blot analysis, cells were lysed in radioimmunoprecipitation assay buffer (RIPA) as described earlier (Seyer et al., 2012). Protein lysates were cleared by centrifugation, mixed with 5x Laemmli buffer, separated by SDS-PAGE and blotted onto nitrocellulose membranes.

Antisera directed against ERK2 (C-14; #sc-154) and IAV PB1 (vK-20; #sc-17601) were purchased from Santa Cruz Biotechnology and α-Tubulin antibodies (#T6199) from Sigma-Aldrich. Antiserum against IAV PB2 protein was a kind gift of E. Fodor (Sir William Dunn School of Pathology, Oxford, UK; Carr et al., 2006). Mouse monoclonal antibodies against IAV NS1 were developed at the Institute of Virology Muenster (Germany) and can be purchased from Santa Cruz Biotechnology (#sc-130568). IAV M1 (#MCA-401) antibodies were obtained from AbD Serotec, while antibodies directed against IAV M2 (#GTX125951) and NS2/NEP (#GTX125953) were purchased from Genetex.

Antibodies directed against the phospho-sites of Akt (Ser473; #9271), ATF2 (Thr71; #9221), ERK1/2 (p44/42 MAPK, Thr202/Tyr204; #9106), MK2 (Thr222; #3316), MEK1/2 (Ser217/221, 41G9; #9154), 4E-BP1 (Thr37/46, 236B4; #2855), p-p70S6K (Thr389, 108D2; #9234), S6 Rib. Prot. (Ser235/236, D57.2.2E; #4858), MKK3/MKK6 (Ser189/Ser207, 22A8; #9236) as well as antibodies for detection of Caspase 3 (#9662), Caspase 8 (cleaved, 18C8, Asp391; #9496), Caspase 8 (D35G2; #4790), Caspase 9 (cleaved, Asp315; #9505), and Caspase 9 (C9; #9508) were obtained from Cell Signaling Technology. Phospho-specific antibodies directed against JNK (Thr183/Tyr185; #612541) and p38 MAPK (Thr180/Tyr182; #612281) and antiserum directed against PARP (#611039) were purchased from BD Bioscience.

A549 cells infected with FPV (MOI 5) were fixed with 4% formaldehyde for 15 min at 4°C at the indicated time points. After washing, permeabilization was performed with 0.1% TritonX-100 for 15 min at RT. Nucleoprotein (NP) localization was detected using anti-influenza A virus NP primary antibodies (AbD Serotec, #MCA-400) and Alexa Fluor® 488 chicken anti-mouse IgG secondary antibodies (Thermo Fisher Scientific, #A21200). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). For quantification, two random areas were analyzed using the fluorescence microscope BIOZERO (Keyence). Quantification of nuclear localization was performed using FIJI Software's Cell Counter plugin (Schindelin et al., 2012).

Statistical significance was calculated with GraphPad Prism software versions 5 and 6 using the indicated statistical tests. P-values are indicated by asterisks ^*p = 0.01–0.05; ^**p = 0.001–0.01; ^***p = 0.0001–0.001; ^****p < 0.0001.

Inhibition of the Raf/MEK/ERK signaling cascade has been shown to limit the replication of various influenza viruses based on the retention of viral ribonucleoprotein complexes in the nucleus (Pleschka et al., 2001). But, so far, nothing is known about the potential influence of the clinically approved B-Raf^V600E inhibitor Vemurafenib on viral propagation. Therefore, wildtype BRAF-containing human lung epithelial A549 cells were treated with the indicated concentrations of Vemurafenib and were subsequently infected with the IAV strain A/FPV/Bratislava/79 (H7N7, FPV). The effective concentration 50% (EC₅₀) was analyzed by standard plaque assays 24 hpi. Here, Vemurafenib efficiently inhibited viral replication already at low micromolar concentrations (EC₅₀ = 3.8 μM; Figure 1A). Furthermore, strain-specific differences in EC₅₀ were observed, with the compound being most effective in SC35M infection [SC35M (H7N7): 0.22 μM; Figure S1A]. Of note, Vemurafenib was also active against the recently emerged viruses of the H7N9 subtype with a quite low EC₅₀ [A/Anhui (H7N9): 0.64 μM; Figure S1B]. Nevertheless, for all strains tested, the most efficient decrease in progeny virus titers was observed with concentrations higher than 20 μM, leading to a reduction of viral replication of more than one log step. Next, the question arises as to whether this effect is specific for avian influenza viruses or whether the presence of Vemurafenib decreases viral replication of IAV strains of human subtypes as well. As expected, the antiviral activity of Vemurafenib against avian IAV was confirmed for human IAV. The human H1N1 strains A/Puerto Rico/8/34 (PR8M) and A/WSN/33 (WSN) were also highly sensitive to Vemurafenib (Figure 1B), indicating a broad antiviral activity against various IAV strains of different origin.

The Raf/MEK/ERK signaling cascade is known to transmit signals from mitogen- and growth factor-receptors to regulate cell proliferation and apoptosis (Mebratu and Tesfaigzi, 2009), cellular processes that have been shown to be involved in efficient IAV replication (Wurzer et al., 2003; He et al., 2010). To exclude that the described effect of Vemurafenib on viral propagation is due to impaired cell viability, WST-1-based proliferation assays were performed. As cleavage of the substrate WST-1 only occurs in living and metabolically active cells, cellular proliferation and viability can be determined. The results shown in Figure 1C clearly indicate that the inhibitor did not significantly affect cellular proliferation and viability. Thus, the observed impact of Vemurafenib on viral replication occurs independent of modulated cell viability. A549 cells have been shown to possess special features, e.g. a distinct mode of apoptosis induction (Hasegawa et al., 2007). To exclude that Vemurafenib-mediated effects on viral replication are based on cell line specific features, other susceptible cells, such as the human bronchial epithelial Calu-3 cells, were infected with the IAV strain SC35M and subsequently treated with Vemurafenib. Also here, Vemurafenib efficiently inhibited viral replication (Figure 1D), ruling out that the observed effect is cell line specific.

In summary, Vemurafenib possesses a prominent and broad antiviral activity, significantly impairing replication of different IAV subtypes and strains that is independent of cytostatic or toxic activities.

Vemurafenib inhibits B-Raf kinase activity in cells carrying BRAF^V600E, nevertheless, it was described to hyperactivate the Raf/MEK/ERK signaling cascade in BRAF^WT cells (Halaban et al., 2010; Vin et al., 2013). Furthermore, Vemurafenib has been shown to possess high levels of off-target inhibition of various kinases in exposure of ex-vivo melanoma tumor lysates (Tahiri et al., 2013). Interestingly, here, the kinase substrates that distinguished between BRAF^WT and BRAF^V600E tumors represented kinases mainly involved in the phosphatidylinositide 3-kinase (PI3K) and MAPK signaling network. Importantly, these signal transduction pathways were described to be necessary for efficient IAV replication (Pleschka et al., 2001; Ehrhardt et al., 2006; Nencioni et al., 2009; Nacken et al., 2012).

To decipher signaling cascades involved in Vemurafenib-mediated inhibition of viral replication, the impact of the compound on IAV-induced cellular signaling cascades was analyzed in BRAF^WT-carrying A549 cells. Indeed, Vemurafenib caused a hyperinduction of the Raf/MEK/ERK signaling cascade as shown by increased phosphorylation of ERK1/2 as well as MEK1/2 (Figure 2A). In contrast, IAV-induced activation of all other cellular signaling cascades analyzed, such as MAPKs JNK and p38 but also the PI3K/Akt pathway, were strongly inhibited by Vemurafenib treatment (Figure 2A).

To exclude that the reduced activation of cellular signaling cascades in presence of Vemurafenib is only secondary due to altered viral replication, induction of signal transduction was analyzed in a virus-free approach. A549 cells were pretreated with Vemurafenib and subsequently stimulated with epidermal growth factor (EGF), a well-known inducer of the Raf/MEK/ERK signaling cascade (Figure 2B). Here, Vemurafenib effectively inhibited EGF-induced PI3K signaling as demonstrated by a decreased phosphorylation of the Akt kinase, while still hyperactivating the Raf/MEK/ERK pathway. To analyze whether Vemurafenib also inhibits proinflammatory and stress-related signaling, A549 cells were stimulated with tumor necrosis factor alpha (TNFα). Interestingly, Vemurafenib treatment significantly inhibited TNFα-activated JNK and p38 MAPK signaling cascades resulting in a decreased phosphorylation of downstream transcription factor ATF2 (Figure 2C), highlighting that Vemurafenib directly limits the activation of various cellular signaling cascades while inducing a hyperactivation of the Raf/MEK/ERK signaling cascade. This broad interference with the activity of cellular kinases is not a secondary effect caused by impaired virus replication and might contribute to the antiviral potential of Vemurafenib.

Besides a hyperactivation of the Raf/MEK/ERK signaling cascade and decreased cellular kinase activation, Vemurafenib has been shown to suppress apoptosis as a result of reduced MAPK JNK signaling in vitro and in vivo (Vin et al., 2013). Upon IAV infection, apoptosis is initiated by multiple viral factors resulting in the expression of apoptosis-inducing cytokines such as TRAIL (TNF-related apoptosis-inducing ligand) (Lowy, 2003), which lead to the cleavage of initiator caspases 8 and 9 and activate downstream executioner caspase 3 (Igney and Krammer, 2002). Importantly, proper timely regulation of apoptosis was shown to be crucial for efficient IAV replication by contributing to the nuclear export of progeny vRNPs (Wurzer et al., 2003; Muhlbauer et al., 2015).

To analyze the effects of Vemurafenib on apoptosis induction, A549 cells were treated with Vemurafenib or DMSO and were subsequently stimulated with Staurosporine, a potent inducer of apoptosis. As marker for apoptosis progression, cleavage of caspases 8, 9, and 3 as well as of the downstream acting poly (ADP-ribose) polymerase (PARP) was investigated by western blot analysis. As expected, Vemurafenib readily inhibited Staurosporine-induced apoptosis as demonstrated by decreased PARP cleavage (Figure 3A). Remarkably, not only activation of caspases 3 and 8 belonging to the TRAIL-induced so-called extrinsic pathway were inhibited, but also activation of caspase 9 of the intrinsic pathway was strongly decreased. These results highlight an early inhibition of Staurosporine-mediated apoptosis induction by Vemurafenib occurring upstream of caspase activation.

It was previously shown that IAV initiates apoptosis in late phases of viral replication by induction of different cellular signaling cascades leading to the expression of TRAIL, TNFα, or Fas ligand (Wurzer et al., 2004). Furthermore, it has been described that MAPKs such as JNK as well as p38 are involved in the IAV-induced expression of apoptosis-inducing cytokines (Maruoka et al., 2003; Lee et al., 2005). Both are signaling cascades that are efficiently inhibited by Vemurafenib. To answer the question as to whether the compound interferes with IAV-mediated expression of apoptosis-inducing cytokines, A549 cells were pretreated with Vemurafenib or DMSO and were subsequently infected with FPV. TRAIL mRNA expression was analyzed by qRT-PCR. As expected, IAV infection resulted in the expression of TRAIL mRNA starting at 6 hpi, which was efficiently inhibited by Vemurafenib treatment (Figure 3B). To conclude, Vemurafenib inhibits IAV-mediated apoptosis already early at the level of apoptosis-inducing cytokine expression.

The activity of MAPKs is known to be closely related to the regulation of apoptosis and, in context of IAV infection, pro- as well as anti-apoptotic functions have been described (Wada and Penninger, 2004). Particularly, the Raf/MEK/ERK signaling cascade, which is hyperactivated by Vemurafenib treatment, has been shown to exert anti-apoptotic as well as proliferation promoting functions (Mebratu and Tesfaigzi, 2009). To analyze whether the Vemurafenib-induced inhibition of apoptosis during IAV infection results from the hyperactivation of the Raf/MEK/ERK signaling cascade, ERK1/2 activation was inhibited by pretreatment with the MEK inhibitor U0126. To avoid differences in apoptosis progression due to altered viral replication, a non-dynamic stimulus mimicking viral infection was used. The main pathogen-associated molecular pattern (PAMP), that is sensed by different pattern-recognition receptors (PRRs) to induce cellular signaling cascades in viral infections, is viral RNA. Particularly, detection of the 5′-triphosphate structure in viral RNAs by the cytoplasmic helicase RIG-I plays an important role in IAV infection (Pichlmair et al., 2006). Therefore, A549 cells were transfected with total RNA isolated from virus-infected cells (vRNA) or from uninfected cells (cRNA) as a control. Cleavage of PARP was used as indicator for onset of apoptosis. Interestingly, transfection of RNA led to an induction of ERK phosphorylation, which was efficiently blocked by the MEK inhibitor U0126 (Figure 3C). Furthermore, presence of Vemurafenib enhanced ERK1/2 activation while there was no phosphorylation detectable in combinational treatment with U0126. Interestingly, while the stimulation with vRNA- induced apoptosis, which was not affected by MEK inhibition, Vemurafenib significantly inhibited PARP cleavage even in presence of U0126. Thus, Vemurafenib-induced hyperactivation of the Raf/MEK/ERK signaling cascade is not responsible for the interference with IAV-induced apoptosis. Furthermore, these results were verified on the Vemurafenib-mediated inhibition of IAV-induced TRAIL as well as TNFα mRNA expression, showing no significant effects of inhibition of ERK1/2 hyperactivation (Figure S2). Therefore, Vemurafenib inhibits IAV-mediated apoptosis-inducing cytokine expression independent of ERK1/2 hyperactivation, suggesting an important role of the inhibition of different stress kinase pathways by Vemurafenib resulting in the decreased expression of apoptosis-inducing cytokines.

It has been shown previously that induction of apoptosis in infected cells is required for efficient viral replication, and that stimulation with TRAIL consequently enhances viral propagation (Wurzer et al., 2004). To analyze whether the anti-viral effect of Vemurafenib is mediated by inhibition of cytokine-induced apoptosis, A549 cells were infected with SC35M and were treated with Vemurafenib or DMSO. Subsequently, cells were stimulated with recombinant TRAIL or TNFα and progeny virus titers were analyzed 8 hpi. As expected, treatment with Vemurafenib resulted in reduced progeny virus titers (Figure 3D). Furthermore, stimulation with TRAIL as well as with TNFα showed the tendency to enhance viral replication. Surprisingly, this tendency was also observed in Vemurafenib-treated cells in presence of TRAIL, but not with TNFα stimulation. Nevertheless, TRAIL stimulation was not sufficient to completely rescue viral replication.

In summary, Vemurafenib strongly impairs viral replication, an effect that is partially attributed to the inhibition of IAV-induced apoptosis mediated by reduced TRAIL expression.

While Vemurafenib targets the Raf/MEK/ERK signaling cascade causing its hyperactivation, IAV-induced p38 and JNK MAPK signaling as well as apoptosis onset were efficiently inhibited. These Vemurafenib-affected pathways were all described to have an impact on viral propagation (Ludwig et al., 2006; Ehrhardt and Ludwig, 2009). To gain further insights in Vemurafenib-mediated inhibition of viral replication, different steps in viral life cycle progression were analyzed.

Inhibition of the Raf/MEK/ERK signaling cascade has been shown to impair viral replication by nuclear retention of progeny vRNPs (Pleschka et al., 2001). Furthermore, Nencioni and colleagues described the same retention of vRNPs when p38 MAPK was inhibited in Madin-Darby canine kidney cells, and IAV-induced apoptosis progression has been shown to be required for RNP export in late stages of the replication cycle as well (Wurzer et al., 2003; Muhlbauer et al., 2015). So far, nothing is known about the interplay of these different pathways in nuclear RNP export and whether this plays a role in Vemurafenib-mediated interference with viral replication.

For this reason, intracellular localization of vRNPs was analyzed by detection of nucleoprotein (NP), which is the major component of vRNP complexes. Importantly, Vemurafenib did not alter the rate of infected cells as the amount of NP positive cells did not change due to Vemurafenib treatment (% NP+, open bars), indicating that there is no obvious impact of the kinase inhibitor on virus entry (Figure 4A). While Vemurafenib slightly and transiently delayed nuclear vRNP export at 5 hpi (Figure 4A, % nuclear NP, checkered bars and Figure 4B), progeny vRNPs were readily exported from the nucleus at later time points compared to DMSO-treated cells. These results indicate that in presence of Vemurafenib, active nuclear vRNP export mediated by the hyperactivated Raf/MEK/ERK signaling cascade might compensate for the decreased passive export attributed to apoptosis inhibition.

Since impaired nuclear vRNP export does not seem to be the cause for reduced viral replication in Vemurafenib-treated cells, the mechanistic background of Vemurafenib action was further explored by addressing other important steps in the viral life cycle, namely transcription and translation. A549 cells were pretreated with Vemurafenib or DMSO and subsequently infected with FPV. After cell lysis, viral protein expression was investigated by western blot analysis (Figure 4C). Here, expression of several viral proteins was reduced in presence of Vemurafenib already early upon infection, notably affecting proteins expressed from spliced RNAs such as M2 and NEP. Interestingly, treatment with Vemurafenib did not significantly alter the expression of viral PB1, NS, or M1 m/cRNAs at all time points analyzed (Figure S3), indicating a direct impact of Vemurafenib on viral protein translation.

Since expression of viral proteins depends on the host cell translation machinery, several important cellular proteins involved in translation were analyzed for their activation. Here, analysis was focused on downstream targets of the signaling pathways that have been previously shown to be inhibited by Vemurafenib. MAPK-activated protein kinase-2 (MAPKAPK2, MK2) has been shown to be activated by MAPK p38 in IAV infection and to play a role in the inhibition of protein kinase R (PKR) (Luig et al., 2010), a kinase that represses cellular protein translation thereby limiting viral replication. As expected, IAV-induced MK2 phosphorylation was strongly inhibited by Vemurafenib (Figure 4D). On the contrary, the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) was affected in Vemurafenib-treated cells only 4 hpi. 4E-BP1 is a repressor of translation initiation that gets inactivated by phosphorylation, which is induced by the PI3K pathway (Gingras et al., 1998). Furthermore, there were no considerable differences in the activation of ribosomal protein S6 kinase beta-1 (p70S6K) and its downstream target ribosomal protein S6 (S6 Rib. Prot.) at all time points analyzed. The latter proteins are described to be activated by PI3K signaling leading to an induction of protein synthesis (Chung et al., 1994). Together, these results indicate that viral protein synthesis but not viral transcription is affected by Vemurafenib, possibly due to an inhibition of MK2 activation leading to a more pronounced PKR-mediated translation inhibition.

Taken together, Vemurafenib-induced inhibition of viral replication is based on impaired MAPK-mediated cellular functions such as induction of apoptosis and protein expression. To analyze whether MAPKs p38 and JNK are involved in the observed antiviral effects of Vemurafenib, A549 cells were infected with the IAV strain SC35M and were subsequently treated with the p38 inhibitor SB202190, the JNK inhibitor SP600125 or with a combination of both in presence or absence of Vemurafenib (Figure 4E). Interestingly, even though the single inhibition of these MAPK pathways had more or less pronounced effects on viral progeny, a combination of the inhibitors led to a significant reduction in viral titers. Importantly, the inhibition of p38 and JNK in presence of Vemurafenib did not alter Vemurafenib-mediated interference with viral replication, emphasizing that Vemurafenib indeed acts antiviral via inactivating these MAPK pathways. Furthermore, the Vemurafenib-induced reduction in viral propagation was more pronounced compared to a combined p38 and JNK inhibition, suggesting the involvement of additional targets leading to efficient restriction of viral progeny by Vemurafenib.

Finally, we addressed the question as to whether the Vemurafenib-mediated inhibition of viral replication is based on a decelerated viral life cycle progression or whether Vemurafenib decreases the total number of infectious progeny viral particles produced over time. To discriminate between these two scenarios, A549 cells were infected with the IAV strains FPV or SC35M and were subsequently treated with Vemurafenib or DMSO to assess multi-cycle replication capability by standard plaque assays. Interestingly, Vemurafenib significantly limited viral replication of both viruses resulting in a prominent reduction of viral titers up to three orders of a magnitude compared to DMSO-treated control cells (Figure 4F).

In summary, Vemurafenib affects viral protein expression and delays nuclear export of progeny vRNPs leading to an efficient inhibition of viral replication at least in part by diminishing the activity of MAPK pathways. These results suggest a distinct interplay of Vemurafenib with various cellular signaling pathways resulting in a broad antiviral activity of the compound.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.