Laboratory of Molecular Virology, International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
Laboratory of Molecular Virology, International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste, Italy.
J Virol. 2014 Jun;88(12):6611-22. doi: 10.1128/JVI.03736-13. Epub 2014 Apr 2.
Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA-binding proteins involved in the repression of initiation of translation of cellular mRNAs and in the formation of stress granules. We show that TIA-1 and TIAR interact with viral RNA in TBEV-infected cells. During TBEV infection, cytoplasmic TIA-1 and TIAR are recruited at sites of viral replication with concomitant depletion from stress granules. This effect is specific, since G3BP1, another component of these cytoplasmic structures, remains localized to stress granules. Moreover, heat shock induction of stress granules containing TIA-1, but not G3BP1, is inhibited in TBEV-infected cells. Infection of cells depleted of TIA-1 or TIAR by small interfering RNA (siRNA) or TIA-1(-/-) mouse fibroblasts, leads to a significant increase in TBEV extracellular infectivity. Interestingly, TIAR(-/-) fibroblasts show the opposite effect on TBEV infection, and this phenotype appears to be related to an excess of TIA-1 in these cells. Taking advantage of a TBE-luciferase replicon system, we also observed increased luciferase activity in TIA-1(-/-) mouse fibroblasts at early time points, consistent with TIA-1-mediated inhibition at the level of the first round of viral translation. These results indicate that, in response to TBEV infection, TIA-1 is recruited to sites of virus replication to bind TBEV RNA and modulate viral translation independently of stress granule (SG) formation.
This study (i) extends previous work that showed TIA-1/TIAR recruitment at sites of flavivirus replication, (ii) demonstrates that TIAR behaves like TIA-1 as an inhibitor of viral replication using an RNA interference (RNAi) approach in human cells that contradicts the previous hypothesis based on mouse embryonic fibroblast (MEF) knockouts only, (iii) demonstrates that tick-borne encephalitis virus (TBEV) is capable of inducing bona fide G3BP1/eIF3/eIF4B-positive stress granules, (iv) demonstrates a differential phenotype of stress response proteins following viral infection, and (v) implicates TIA-1 in viral translation and as a modulator of TBEV replication.
黄病毒是全球人类和动物疾病的主要病因。蜱传脑炎病毒（TBEV）是在欧洲流行的最重要的节肢动物传播黄病毒，也是蜱传脑炎的病原体，这是一种潜在致命的中枢神经系统感染。然而，宿主蛋白在 TBEV 感染过程中的作用还知之甚少。在这项工作中，我们研究了细胞蛋白 TIA-1 和其伴侣因子 TIAR，它们是应激诱导的 RNA 结合蛋白，参与细胞 mRNA 翻译起始的抑制和应激颗粒的形成。我们表明，TIA-1 和 TIAR 在 TBEV 感染细胞中与病毒 RNA 相互作用。在 TBEV 感染期间，细胞质 TIA-1 和 TIAR 被募集到病毒复制部位，同时从应激颗粒中耗竭。这种效应是特异性的，因为这些细胞质结构的另一个组成部分 G3BP1 仍然定位于应激颗粒中。此外，热休克诱导包含 TIA-1 但不包含 G3BP1 的应激颗粒的形成在 TBEV 感染细胞中受到抑制。用小干扰 RNA（siRNA）或 TIA-1(-/-) 小鼠成纤维细胞转染耗尽 TIA-1 或 TIAR 的细胞，会导致 TBEV 细胞外感染力显著增加。有趣的是，TIAR(-/-) 成纤维细胞对 TBEV 感染表现出相反的影响，这种表型似乎与这些细胞中 TIA-1 过量有关。利用 TBE 荧光素酶复制子系统，我们还观察到 TIA-1(-/-) 小鼠成纤维细胞在早期时间点的荧光素酶活性增加，这与 TIA-1 在翻译起始的第一轮中对病毒翻译的抑制一致。这些结果表明，TIA-1 在响应 TBEV 感染时被募集到病毒复制部位，与 TBEV RNA 结合，并调节病毒翻译，而不依赖于应激颗粒（SG）的形成。
本研究（i）扩展了先前的工作，表明 TIA-1/TIAR 在黄病毒复制部位被募集，（ii）利用人类细胞的 RNA 干扰（RNAi）方法证明，TIAR 像 TIA-1 一样是病毒复制的抑制剂，这与仅基于小鼠胚胎成纤维细胞（MEF）敲除的先前假设相矛盾，（iii）表明 tick-borne encephalitis virus（TBEV）能够诱导真正的 G3BP1/eIF3/eIF4B 阳性应激颗粒，（iv）表明病毒感染后应激反应蛋白表现出不同的表型，（v）表明 TIA-1 参与病毒翻译，并作为 TBEV 复制的调节剂。
