Role of the ERK1/2 Signaling Pathway in the Replication of Junín and Tacaribe Viruses

Abstract

We have previously shown that the infection of cell cultures with the arenaviruses Junín (JUNV), Tacaribe (TCRV), and Pichindé promotes the phosphorylation of mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (ERK1/2) and that this activation is required for the achievement of a productive infection. Here we examined the contribution of ERK1/2 in early steps of JUNV and TCRV multiplication. JUNV adsorption, internalization, and uncoating were not affected by treatment of cultured cells with U0126, an inhibitor of the ERK1/2 signaling pathway. In contrast, U0126 caused a marked reduction in viral protein expression and RNA synthesis, while JUNV RNA synthesis was significantly augmented in the presence of an activator of the ERK1/2 pathway. Moreover, U0126 impaired the expression of a reporter gene in a TCRV-based replicon system, confirming the ability of the compound to hinder arenavirus macromolecular synthesis. By using a cell-based assay, we determined that the inhibitor did not affect the translation of a synthetic TCRV-like mRNA. No changes in the phosphorylation pattern of the translation factor eIF2α were found in U0126-treated cells. Our results indicate that U0126 impairs viral RNA synthesis, thereby leading to a subsequent reduction in viral protein expression. Thus, we conclude that ERK1/2 signaling activation is required for an efficient arenavirus RNA synthesis.

Keywords: ERK; Junín virus; Tacaribe virus; arenavirus; cell signaling; replication.

Figure 1

Inhibition of the ERK1/2 pathway does not affect Junín virus (JUNV) entry or uncoating. (a) Vero cells were mock-treated or treated with U0126 (15 µM) for 1 h, and then cells were infected with JUNV (m.o.i. = 1) in presence or absence of U0126 for 1 h at 4 °C. Following extensive washing, cells were lysed and viral infectivity was quantified by plaque assay. Data are mean values ± standard deviation (SD); (b) Vero cells were mock-treated or treated with U0126 (15 µM) or CZ (40 µM) for 30 min and then infected with JUNV for 1 h at 4 °C. Next, cultures were incubated at 37 °C in the presence or absence of the inhibitors. At different time points, internalized virions were quantified by plaque assay. Data are mean values ± SD. ANOVA (Tukey post-hoc test): ** p < 0.01, *** p < 0.001; (c) Vero cells infected with JUNV for 1 h at 4 °C were incubated at 37 °C and at the indicated time points U0126 (15 µM) or NH₄Cl (50 mM) was added. After 3 h of treatment, uncoated viral particles were quantified by plaque assay. Data are mean values ± SD. ANOVA (Tukey post-hoc test): ** p < 0.01, *** p < 0.001.

Figure 2

U0126 inhibits N protein expression and viral production. Vero cells were infected with JUNV (m.o.i. = 1) and at different time points U0126 (15 µM) was added. At 14 h p.i. cells were lysed and the levels of expression of N and p-ERK were analyzed by Western blot using ERK as loading control (a), and virus production was determined by plaque assay (b). Data are mean values ± SD. ANOVA (Dunnett post-hoc test): ** p < 0.01, *** p < 0.001.

Figure 3

U0126 inhibits viral glycoprotein expression. (a) Cells were infected with JUNV (m.o.i. = 1) and U0126 (15 µM) was added 2 h later. At 24 h p.i. total and membrane G1 expression was analyzed by immunofluorescence (IF) assay. Scale bars: 10 µm; (b) The percentage of fluorescent cells was calculated by counting 20 random selected fields; data represent mean values ± SD. ANOVA (Tukey post-hoc test): *** p < 0.001; (c) Vero cells were infected with JUNV (m.o.i. = 1) and U0126 (15 µM) was added at the indicated time points. At 14 h p.i. cells were treated for 30 min with culture medium at pH 5.0. Ten hours later, cells were fixed, stained with Giemsa and visualized using an optic microscope. Scale bars: 10 µm; The size (d) and number (e) of syncytia were calculated by counting 20 random selected fields (400× magnification); data represent mean values ± SD. ANOVA (Dunnett post-hoc test): * p < 0.05.

Figure 4

U0126 inhibits viral RNA synthesis. (a) Schematic representation of the S RNA transcription/replication processes. The genome S RNA (gSRNA) and the full-length antigenome S RNA (agSRNA), N mRNA, glycoprotein precursor (GPC) mRNA are depicted. IGR: intergenic region. The primers used for detection of gSRNA by Q RT-PCR are indicated with arrows; (b) Total RNA was extracted at 14 h p.i. from JUNV infected Vero cells treated with U0126 (15 µM), ribavirin (100 µM), PMA (100 nM), or left untreated, and gSRNA was detected by Q RT-PCR. Data represent mean values ± SD. ANOVA (Dunnett post-hoc test): *** p < 0.001; (c) Schematic representation of Tacaribe virus (TCRV) S RNA analog MG-FLUC (MG- firefly luciferase), which contains (5′ to 3′) the S genome 5′ UTR, followed by a linker sequence corresponding to the 3′ terminal region of the GPC open reading frame (ORF) (shadowed box); the complete S IGR, then the FLUC ORF in an antisense orientation, and the complete S genome 3′ UTR; (d) BSR cells were plasmid-transfected to co-express TCRV N and L proteins along with the MG-FLUC RNA. Transfection mixes, which included a plasmid expressing RLUC to control for transfection efficiency, were removed after 4 h and cells were further incubated in the presence of U0126. Cytoplasmic extracts, obtained at 20 h post-transfection, were assayed for FLUC and RLUC activities and FLUC activity data were normalized against the corresponding RLUC levels. Mean normalized FLUC values ± SD from two independent experiments (each performed in triplicate) are shown as a percentage of data corresponding to untreated cultures taken as 100%. 2-tailed paired Student’s t test: *** p < 0.001; (e) Western blot analysis of N protein and p-ERK levels in samples obtained from BSR cells transfected with MG-FLUC, expressing or not N and L in the presence or absence of U0126, as described in d. ERK detection was used as loading control.

Figure 5

U0126 does not affect viral mRNA translation. (a) Schematic representation of capped synthetic transcripts used in this study; (b) BSR cells were transfected with virus- or cell-like mRNA, along with a transcript (RLUC), expressing RLUC in a cap-independent manner, as an internal control (Materials and methods). Three h post-transfection, cells were treated or not with U0126 for 4 h, followed by the determination of FLUC and RLUC activities in cell lysates. Mean normalized FLUC values (FLUC/RLUC ± SD) from two independent experiments (each performed in triplicate) are shown. ANOVA (Tukey post-hoc test): *** p < 0.001.

Figure 6

U0126 does not affect eukaryotic initiation factor 2α (eIF2α) phosphorylation. (a) Uninfected and JUNV infected (m.o.i. = 1) Vero cells were treated with U0126 during 12 h or were left untreated. Then cells were lysed and the level of eIF2α and ERK phosphorylation was assessed by Western blot; (b) Quantification of p-eIF2α, data represent mean values of relative intensity with respect to ERK ± SD.