MEK/ERK activation plays a decisive role in Zika virus morphogenesis and release

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.


Introduction
In recent years, Brazil has experienced an increase in flavivirus outbreaks. Between 2015 and 2019, there were close to four million cases of dengue virus (DENV) infection, the introduction of Zika virus (ZIKV) caused over 3 000 cases 47 Page 2 of 11 of congenital defects in children, and the reemergence of yellow fever virus (YFV) caused nearly 700 deaths [1]. These viral infections have in common early symptoms like fever, myalgia, arthralgia, and headache, but their outcomes are different. DENV has a high incidence, and severe cases can lead to death by internal hemorrhage and shock [2]. ZIKV morbidity has been associated with Guillain-Barré syndrome in adults and several neurological defects in newborn children, including microcephaly [3]. YFV mortality is 20-50% in severe cases due to liver failure and hemorrhage; vaccination protects efficiently against YFV but lacks full coverage [4].
Flavivirus are enveloped viruses around 50 nm in diameter with an 11-kb single-stranded positive-sense RNA genome [5]. This RNA is translated as a single polyprotein that is cleaved into three structural proteins (C, prM/M, and E) and seven non-structural proteins that are involved in genome replication, particle assembly, and host-virus interactions (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [6]. During infection, the virus is internalized by receptormediated endocytosis, and inside the cell, its envelope fuses with the endosome membrane and viral RNA is released into the cytoplasm. Subsequently, RNA translation, genome replication, and viral assembly take place in the modified endoplasmic reticulum membrane. Finally, viruses are transported and mature through the trans-Golgi network and are released by exocytosis [7]. Antiviral compounds can be classified into those that target the virus and those that target the host, including cellular pathways that are important for host-virus interactions [8]. The mitogen-activated protein kinase (MAPK) pathway consists of a family of serine/threonine kinases present in all eukaryotic cells that form a signaling system that is highly conserved from yeasts to humans. These kinases participate in various cellular processes and can be divided in three major pathways depending on the kinase that is activated: the extracellular signal-regulated kinase (ERK), which responds to mitogenic signals, and the c-Jun N-terminal kinase (JNK) and p38 MAPK, which regulate inflammatory responses [9]. Inactive ERK is present in the cytoplasm and is activated by phosphorylation by mitogen-activated protein kinase (MEK), after which phospho-ERK (pERK) targets substrates in the cytoplasm and the cell nucleus [9].
Our group has already reported the importance of the MEK/ERK pathway in flavivirus replication. Mice infected with DENV2 and treated with the MEK inhibitor AZD6244 had reduced viral titers in serum and were protected from lethal viral challenge [10]. Using another MEK inhibitor, U0126, YFV replication was reduced in mice, while DENV2, DENV3, and Saint Louis encephalitis virus (SLEV) showed a significant titer reduction in cell culture [11]. More recently, we showed that ZIKV titers were also reduced by treatment with two MEK inhibitors in vitro [1]: selumetinib, which is being tested as a drug for treating uveal melanoma in phase 3 clinical trials [12], and trametinib, which has already been approved by the FDA for the treatment of BRAF-mutated melanoma [13]. In this study, we further evaluated the role of the MEK/ERK pathway and the effect of trametinib on Asian and African strains of ZIKV, addressing viral genome replication, morphogenesis, and release.

Viruses
The Asian ZIKV strain PE243/2015 [14], the prototype African ZIKV strain MR766, the DENV3 genotype I strain MG20/2004 [15], and the American/Asian genotype strain DENV2 PI59/2006 [16] were propagated in C6/36 cells. The YFV 17DD vaccine strain was propagated in Vero cells [11]. Briefly, cells were infected at a multiplicity of infection (MOI) of 0.01 in supplemented medium, and after 5-7 days, virus was recovered from the supernatant. Mockinfected cells were treated following the same procedure with medium alone.

Viral infection and treatment
Confluent Vero cells were infected at the designated MOI for 1 hour and then washed and incubated in supplemented MEM in 5% CO 2 at 37 °C for the times indicted for each experiment. When specified, cells were incubated with the kinase inhibitors for different time intervals. Selumetinib, trametinib, JNK inhibitor VIII, and SB203580 were dissolved in DMSO and used at the concentrations indicated for each experiment. In most experiments, virus was collected from the supernatant, but to evaluate intracellular replication, virus was also recovered separately from cell layers. Supernatants from 24-well plates were collected and stored at -80 °C, while cell layers were washed four times with cold PBS, after which 200 μl of cold supplemented MEM was added to cover each well. Plates were then frozen at -80 °C and thawed at room temperature three times. Cell layers were collected by scraping, harvested, and clarified by centrifugation at 2500 g and 4 °C for 5 min. Supernatants were stored at -80 °C.

Virus titration
Viral suspensions were serially diluted in supplemented MEM and incubated with Vero cells for 1 hour for adsorption. Cells were then overlaid with 1.5% carboxymethylcellulose (CMC) (Synth, São Paulo, Brazil) in supplemented MEM and incubated in 5% CO 2 at 37 °C for 4-5 days. Cells were then fixed with 3.7% formaldehyde, and viral plaques were made visible by staining with 1% crystal violet solution. For YFV, we used 1% CMC, and for DENV, we used BHK-21 cells with 0.8% CMC.

Transmission electron microscopy
Vero cells were grown in 25-cm 2 culture flasks and infected with ZIKV PE243 at an MOI of 1 for 1 hour. Cells were then incubated with DMSO or 20 µM of trametinib in supplemented MEM for 3 days. Mock-infected cells were treated in the same way and used as controls. The cells were then washed with MEM, fixed with 2.5% glutaraldehyde (grade I, Electron Microscopy Sciences, Germany) at room temperature for 1 hour, and processed as described previously [17]. Cells were examined using a Tecnai G2-Spirit FEI 2006 transmission electron microscope operating at 80 kV at the Microscopy Center of the Universidade Federal de Minas Gerais, MG, Brazil.

RNA extraction, cDNA synthesis, and quantitative PCR
Infected Vero cells in 24-well plates were washed three times with cold PBS, and total RNA was extracted from cell layers using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was obtained using 1 μg of total RNA, 0.5 μg of random hexamers, and M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed with primers targeting the ZIKV envelope gene (forward, 5′-CCG CTG CCC AAC ACAAG-3′; reverse, 5′-CCA CTA ACG TTC TTT TGC AGA CAT -3′) [18], 3 μl of cDNA and SYBR Green PCR Master Mix (Promega, Madison, WI, USA) in a StepONE Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Experiments were done in triplicate, and data were normalized to the Hprt gene (forward, 5-AGC CCT GGC GTC GTG ATT A-3; reverse, 5-TCT CGA GCA AGA CGT TCA GT-3) and presented in arbitrary units. Absolute quantitation was done using a standard curve of serial dilutions of a plasmid containing the ZIKV amplicon. The viral genome was quantified as genomic copies per μg of total RNA.

Statistical analysis
All experiments were repeated at least three times, and the data are presented as the mean ± SD. Results were analyzed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). Comparison between groups was made using Student's t-test, considering p < 0.05 to be significant.  and viral titers were determined by plaque assay (PFU/ml). (C) Vero cells were infected with ZIKV PE243 or MR766 at an MOI of 0.1 and then treated with DMSO or with increasing concentrations of JNK inhibitor VIII or the p38 MAPK inhibitor SB203580 for 24 hours. Viral titers were determined by plaque assay (PFU/ml). The data are the average of three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to DMSO, using a two-tailed t-test

ZIKV induces ERK phosphorylation and may require it for viral replication
To evaluate whether the MEK/ERK pathway was activated by ZIKV infection, Vero cells were starved and then were either mock infected or infected with ZIKV PE243 at an MOI of 2 for 6, 12, 18, or 24 hours. Cell extracts were collected, subjected to Western blot (WB), and immunoprobed with anti-phospho-ERK and anti-β-actin. As shown in Fig. 1, ZIKV stimulated ERK1/2 phosphorylation above the basal levels at 12 and 18 hpi. Repeating the experiment with an MOI of 1 resulted in better definition at 18 hpi.
Next, to investigate whether ERK phosphorylation is important for ZIKV replication, Vero cells were infected with ZIKV PE243 or ZIKV MR766 at an MOI of 0.1, and after 1 hour of adsorption, the supernatant was removed and cells were incubated with DMSO alone or with 5, 10, 20, or 40 μM selumetinib or trametinib up to 36 hours. Culture supernatants were collected and viral titers were determined by plaque assay. The replication of the Asian and African strains decreased up to 2-log 10 units (p < 0.001) after treatment with each of these inhibitors, and the antiviral effect of trametinib was clearly dose-dependent ( Fig. 2A). We also examined the inhibitory effect of selumetinib and trametinib on other flaviviruses. Table 1 summarizes the effect on ZIKV and YFV at 36 hours and on DENV at 48 hours because it has a longer replication cycle in Vero cells. Trametinib efficiently inhibited ZIKV PE243, ZIKV MR766, YFV 17DD, DENV2, and DENV3; selumetinib also inhibited all of the viruses but inhibited YFV 17DD and DENV3 to a lesser degree. Overall, trametinib showed a more efficacious pan-antiviral effect than selumetinib.
Next, we evaluated this effect on multiple cycles of ZIKV replication. Vero cells were infected with PE243 or MR766 at an MOI of 0.1 and then treated with either DMSO or 20 μM trametinib for 4 days. Culture supernatants were collected at different times, and viral titers were determined by plaque assay. Both ZIKV PE243 and MR766 had an average decrease of 2-log 10 units when treated with trametinib (Fig. 2B). The inhibition of ERK1/2 phosphorylation during the four days of treatment was confirmed by WB (data not shown). The cytotoxicity of selumetinib and trametinib in Vero cells was evaluated by staining with MTT and crystal violet at 36 hours, and for 1 to 4 days, cell viability was above 70% at all of the concentrations that were tested (Supplementary Fig.  S1A, Supplementary Methods). We followed cell proliferation, counting live and dead cells over time, and found mostly a cytostatic effect. To rule out a virucidal effect, we incubated each virus with 20 μM trametinib for 1 hour at 37 °C. No difference in titer was observed between DMSO-and trametinib-treated virus ( Supplementary Fig.  S1B). Selumetinib also showed no virucidal effect (data not shown).
We also investigated whether other MAPK pathways could be involved in ZIKV replication by treating infected cells with an inhibitor of JNK (JNK inhibitor VIII) or p38 MAPK (SB203580) for 24 hours. ZIKV PE243 replication decreased after treatment with each of these inhibitors in a dose-dependent manner, but ZIKV MR766 replication was reduced only when SB203580 was used at a concentration of 40 μM (Fig. 2C).

Trametinib may block ZIKV morphogenesis
Next, we used transmission electron microscopy (TEM) to evaluate the effect of trametinib on viral morphogenesis. Vero cells were infected with ZIKV PE243 at an MOI of 1 and treated with DMSO or 20 μM trametinib for 3 days. Cells were then fixed and prepared for TEM. Infected cells treated with DMSO induced vesicle production and endoplasmic reticulum hypertrophy. Viral particles were observed as small geometrical structures within the distended cisternae of the rough endoplasmic reticulum (Fig. 3A left, white arrows). At higher magnification, the geometrical form and the less electron-dense envelope could be distinguished (Fig. 3A right, white arrows). At the bottom of the image, two less electron-dense structures similar in size and shape to virus particles, one of which was visibly still in contact with the endoplasmic membrane (Fig. 3A right, black arrows) might represent viral particles in the process of formation. In contrast, infected and trametinibtreated cells showed no evident sign of viral infection, and no viral particles were distinguishable (Fig. 3B). It is possible that viral morphogenesis was blocked by treatment with trametinib, since no structures similar to viral particles were observed. The experiment was carried out at least  three times with similar results, and representative images were chosen from at least 100 fields. Quantitation of viral genomic RNA in the cell layers by qPCR showed no effect of trametinib treatment on genomic RNA replication at 12, 18, and 24 hpi (Fig. 4). Thus, the antiviral effect of trametinib was probably exerted at the level of ZIKV morphogenesis.

Determination of the ZIKV replication cycle
To gain insight into the ZIKV replication cycle, we quantified both the viral genome and infectious viral particles at different times. For genome quantitation, Vero cells were infected with ZIKV PE243 at an MOI of 2, and cultured for 1, 2, 3, 4, 5, 8, or 12 hours, and total RNA was then extracted from the cell layers and quantified by RT-qPCR. Quantitation of intracellular viral RNA showed a decrease from 1-3 hpi, and later, an increase from 8-12 hpi (Fig. 5A). Titers of intra-or extracellular virions were determined to monitor viral assembly and release. Vero cells were infected with PE243 at an MOI of 1 and cultured for 18 hours. Culture supernatants and cell layers were collected separately every two hours, and viral titers were determined by plaque assay. Intracellular virions were detected at 12 hpi, and extracellular virions increased exponentially starting at 14 hpi (Fig. 5B). The stages of viral replication are represented in

Trametinib affects ZIKV morphogenesis and release
Finally, to determine which specific stage of viral replication is affected by treatment with trametinib, Vero cells were infected with ZIKV PE243 at an MOI of 0.1 and treated with 20 μM trametinib for different intervals. Infection was allowed to proceed for 24 hours, and after that, supernatants or cell layers were collected and viral titers were determined by plaque assay. Pretreatment with trametinib for 1 hour did not have a significant antiviral effect, but postinfection treatment had a time-dependent antiviral effect, as demonstrated by a reduction in the number of extracellular virions compared to the control (DMSO-treated cells) (Fig. 6A). Moreover, treatment at 6-hour intervals revealed a more pronounced reduction in viral replication from 12-18 and 18-24 hpi when compared to the control (DMSOtreated cells) (Fig. 6B). Thus, trametinib treatment may have particularly affected the late viral replication stages. Furthermore, the reduction in the number of intracellular ZIKV PE243 virions suggests an effect on morphogenesis (Fig. 6C), and the higher reduction of extracellular virions of ZIKV PE243 may indicate a cumulative effect on viral release (Fig. 6D). ZIKV MR766 showed a reduction in intracellular virions, but it was less pronounced for extracellular virions (Fig. 6E and F).

Discussion
The increased incidence of flavivirus infections and the grave neonatal complications seen with Zika in Brazil [1] reveal the urgent need for antiviral treatments. Inhibitors that target cellular pathways and consequently affect viral replication in an indirect manner give us two advantages: less selection of resistant viral strains and the possibility to target closely related viruses [8]. The MEK/ERK pathway has been reported to play an important role in the replication of flaviviruses [10,11,[19][20][21], including ZIKV [1] and the closely related hepatitis C virus (HCV) [22]. In this study, we evaluated the antiviral effect of trametinib and the correlation between the MEK/ERK pathway and the ZIKV replication cycle. ZIKV infection induced ERK phosphorylation at 12 and 18 hpi, but we could not see this at 6 and 24 hpi because our Vero cells had a high basal level of pERK. Recently, another group observed pERK induction by ZIKV infection at early times, from 5 to 30 min, in A549 cells, concomitantly with EGFR activation and internalization [23]. Thus, ZIKV might exploit the MEK/ERK pathway as described for other viruses [24]. Trametinib showed a sustained antiviral effect over time, inhibiting both ZIKV Asian and African strains for at least four days. Although trametinib is primarily used for cancer treatment [13], these results strengthen the argument for its use against ZIKV. Trametinib has also been reported to exert an antiviral effect against human immunodeficiency virus (HIV) [25] and influenza virus [26]. As a note, in this study we showed that JNK and p38 MAPK pathways may also be important for ZIKV replication, especially for the Asian strain PE243, but more studies are required to understand their role.
Next, we used TEM to investigate which stage of viral replication is affected by trametinib. We observed viral particles at the third day of infection, but no viral particles were seen when cells were infected with ZIKV PE243 and treated with trametinib. This may be due to a strong antiviral effect of trametinib on ZIKV PE243 replication, since the same results were obtained in three independent experiments with at least 100 fields analyzed in each. However, we cannot completely exclude the possibility that treated infected cells were somehow lost during sample preparation. The infection plus the drug treatment might have rendered these cells more susceptible to loss during the fixation process. Finally, we were only able to perform infections at an MOI of 1 due to the low titer of the viral stock. More studies are required to evaluate the impact of treatment on viral morphogenesis, and we encourage researchers to reproduce these analyses We tried another approach by determining the time range of each stage of ZIKV PE243 replication in Vero cells. For ZIKV PE243, the number of RNA copies showed a tendency to increase at around 8 hpi, indicating genome replication. Other studies have produced similar results; the beginning of genome replication of ZIKV MR766 can be observed between 6 and 12 hpi in BJ cells and microglial cell lines [27], and for ZIKV PRVABC59 (Puerto Rico) and ZIKV FLR (Colombia), genome replication begins at between 8 and 12 hpi in HUVEC cells [28]. We detected the first intracellular ZIKV PE243 virions at 12 hpi and viral release at 14 hpi. Similarly, ZIKV PF-25013-18 (French Polynesia) progeny were first detected at 12 hpi in A549 cells [29]. These data are consistent with our observation that ZIKV PE243 Asian strain can complete one cycle in Vero cells within 14 hours.
Trametinib pretreatment for 1 hour (Fig. 6A) and ZIKV genome quantitation by qPCR (Fig. 4) indicated that neither viral adsorption nor viral genome replication were affected by trametinib. Sabino et al. [23] obtained similar results with PD98059, a MEK inhibitor that was found to impair ZIKV H/PF/2013 (French Polynesia) and ZIKV 976 (Uganda) infection in A549 cells but did not target viral entry or genome replication. They demonstrated the importance of EGFR for ZIKV entry but also suggested a possible role of ERK at other stages of infection [23]. Treatment for various lengths of time followed by quantification of intracellular and extracellular virions showed that trametinib affected morphogenesis and viral release of ZIKV PE243 and MR766. Since viral replication is a continuous process, ZIKV morphogenesis and release should be very active between 12 and 18 hpi, which correlates with the induction of ERK phosphorylation and the antiviral effect of trametinib. Other MEK inhibitors have been shown to affect the morphogenesis of DENV3 [10], YFV [11], and HCV [22]. Thus, it is probable that ERK phosphorylation stimulated by ZIKV is important for ZIKV morphogenesis and release.
In conclusion, we provide evidence that ZIKV stimulates ERK phosphorylation during viral morphogenesis and release and that trametinib especially blocks these stages of ZIKV replication. Our results demonstrate that trametinib can inhibit Asian and African ZIKV strains over time. Additional studies are still needed to visualize the impaired morphogenesis, and also to evaluate the antiviral efficiency of this compound in animal models. ERK is an important kinase for replication of ZIKV and other flaviviruses, but it may not act directly on viral proteins. During infection, viruses exploit cellular pathways, and it is possible that ZIKV hijacks the MEK/ERK mitogenic signal for its own benefit. ERK activation is responsible for the transition from the G0/G1 phase to the S phase [30], providing a proliferative environment during ZIKV infection, while trametinib causes the cell to remain in the G1 phase [31], negating this effect. On the other hand, the ZIKV E protein has been reported to block the cell at the G2/M phase [32]. Apparently, some viruses induce a proliferative state in the cell but without necessarily completing mitosis [33]. Thus, there might be a complex host-virus relationship between the cell cycle and ZIKV replication. The next steps should be to identify the link, i.e., the downstream target of ERK -a protein or RNA transcript that is associated with the cell cycle -that participates directly in ZIKV replication.