Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina.
Centro de Virología Animal (CEVAN), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires C1440FFX, Argentina.
Viruses. 2018 Apr 17;10(4):199. doi: 10.3390/v10040199.
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.
我们之前已经表明，感染细胞培养物中的沙粒病毒（Junín（JUNV）、Tacaribe（TCRV）和 Pichindé）会促进丝裂原活化蛋白激酶（MAPKs）细胞外信号调节激酶 1 和 2（ERK1/2）的磷酸化，并且这种激活对于实现有效的感染是必需的。在这里，我们研究了 ERK1/2 在 JUNV 和 TCRV 增殖的早期步骤中的贡献。用 U0126（ERK1/2 信号通路的抑制剂）处理培养细胞不会影响 JUNV 的吸附、内化和脱壳。相比之下，U0126 导致病毒蛋白表达和 RNA 合成明显减少，而 ERK1/2 通路的激活剂存在时，JUNV RNA 合成显著增加。此外，U0126 削弱了基于 TCRV 的复制子系统中的报告基因表达，证实了该化合物阻碍沙粒病毒大分子合成的能力。通过使用基于细胞的测定法，我们确定该抑制剂不影响合成的 TCRV 样 mRNA 的翻译。在 U0126 处理的细胞中未发现翻译因子 eIF2α的磷酸化模式发生变化。我们的结果表明，U0126 会损害病毒 RNA 合成，从而导致随后病毒蛋白表达减少。因此，我们得出结论，ERK1/2 信号转导的激活对于有效的沙粒病毒 RNA 合成是必需的。
