Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS, USA.
J Virol. 2012 Jul;86(14):7520-9. doi: 10.1128/JVI.07204-11. Epub 2012 May 2.
Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.
汉坦病毒糖蛋白前体（GPC）在翻译后被切割成两种糖蛋白，Gn 和 Gc。转染表达 GPC 或 Gn 和 Gc 的质粒的细胞显示 Gn 被翻译后降解。用自噬抑制剂 3-甲基腺嘌呤、LY-294002 或 Wortmanin 处理细胞可挽救 Gn 降解，表明 Gn 被宿主自噬机制降解。共聚焦显微镜成像显示 Gn 通过未知机制靶向自噬体进行降解。自噬标记物 LC3-I 和 LC3-II 的检测表明，Gn 表达和辛诺柏 hantavirus（SNV）感染均诱导细胞发生自噬。为了阐明自噬的诱导和 Gn 的清除是否在病毒复制周期中起作用，我们使用小干扰 RNA（siRNA）下调自噬基因 BCLN-1 和 ATG7，并随时间监测病毒复制。这些研究表明，抑制宿主自噬机制可抑制细胞中的辛诺柏病毒复制，表明 Gn 的自噬清除对于病毒的有效复制是必需的。我们的研究为病毒发病机制提供了机制上的见解，并揭示了 SNV 利用宿主自噬机制降低了一种对宿主细胞中有效复制至关重要的病毒成分的固有稳态水平。
