PDF(Pubmed)
Abstract：
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
摘要：
在我们之前的调查中,我们阐明了在非结构蛋白NSsW61Y中第61位的色氨酸到酪氨酸取代在减少非结构蛋白（NSs）和核蛋白（NP）之间的相互作用中的作用，阻碍病毒复制。在这项研究中,我们重点研究了NS通过自噬体的调节参与复制。最初，我们检查了NP表达水平的影响，复制的标记，在HeLa细胞感染严重发热血小板减少综合征病毒（SFTSV）后，有或没有抑制NP结合。Western印迹分析显示在NSsW61Y表达条件下NP水平降低。此外，在表达NSsW61Y的HeLa细胞中，经典自噬体标志物p62和LC3的表达水平降低，揭示单个病毒蛋白参与自噬。随后的实验证实，NSsW61Y干扰自噬通量，如氯喹治疗后LC3B和P62水平降低所证明的，自噬体-溶酶体融合的抑制剂。LysoTracker染色表明,与表达野生型NS的细胞相比,表达NS突变体的细胞中的溶酶体减少。我们进一步探索了mTOR相关的调节途径，受NS突变体表达影响的关键调节因子。观察到的复制抑制可能与NSs的构象变化有关，损害它们与NP的结合并改变mTOR调节，自噬中一个重要的上游信号组件。这些发现阐明了NSsW61Y与抑制宿主自噬机制之间的复杂相互作用，这对于促进病毒复制的自噬体的产生至关重要。
