丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性。实验结果表明:在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的。总之,实验结果可提示HCVNS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应。NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中。     

Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.     

Endoplasmic reticulum stress is induced and modulated by enterovirus 71

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER‐resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2α, but infection with UV‐inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2α was dependent on PKR: eIF2α phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR‐eIF2α pathway. Pulse‐chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus‐dependent, ATF6‐independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1‐mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71‐induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.