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.     

Caveolar Endocytosis Is Required for Human PSGL-1-Mediated Enterovirus 71 Infection

Enterovirus 71 (EV71) causes hand, foot, and mouth disease and severe neurological disorders in children. Human scavenger receptor class B member 2 (hSCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) are identified as receptors for EV71. The underling mechanism of PSGL-1-mediated EV71 entry remains unclear. The endocytosis required for EV71 entry were investigated in Jurkat T and mouse L929 cells constitutively expressing human PSGL-1 (PSGL-1-L929) or human rhabdomyosarcoma (RD) cells displaying high SCARB2 but no PSGL-1 by treatment of specific inhibitors or siRNA. We found that disruption of clathrin-dependent endocytosis prevented EV71 infection in RD cells, while there was no influence in Jurkat T and PSGL-1-L929 cells. Disturbing caveolar endocytosis by specific inhibitor or caveolin-1 siRNA in Jurkat T and PSGL-1-L929 cells significantly blocked EV71 infection, whereas it had no effect on EV71 infection in RD cells. Confocal immunofluorescence demonstrated caveola, and EV71 was directly colocalized. pH-dependent endosomal acidification and intact membrane cholesterol were important for EV71 infection, as judged by the pretreatment of inhibitors that abrogated the infection. A receptor-dominated endocytosis of EV71 infection was observed: PSGL-1 initiates caveola-dependent endocytosis and hSCARB2 activates clathrin-dependent endocytosis.     

Human SCARB2-mediated entry and endocytosis of EV71

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.     

miR-545 promoted enterovirus 71 replication via directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6

Enterovirus 71 (EV71) is a small, nonenveloped icosahedral RNA virus and is the predominant causative pathogen of hand-foot-and-mouth disease. Recently, microRNAs (miRNAs) are reported to play important roles in the pathogenesis of EV71 replication. This study investigated the role of miR-545 in the EV71 replication and explored the underlying molecular mechanisms. We showed that miR-545 was upregulated in the EV71-infected human embryonic kidney (HEK) 293 cells and rhabdomyosarcoma (RD) cells. Overexpression of miR-545 promoted the viral replication of EV71 and attenuated the inhibitory effects of EV71 on cell viability in HEK293 and RD cells; while knockdown of miR-545 significantly suppressed the EV71 replication in these two cell lines. Bioinformatics analysis and luciferase reporter assay showed that miR-545 directly targeted the 3′untranslated region of phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in HEK293 cells. Furthermore, miR-545 negatively regulated the messenger RNA (mRNA) and protein expression of PTEN and TRAF6. The mRNA and protein expression of PTEN and TRAF6 was also suppressed by EV71 infection, which was attenuated by miR-545 knockdown in HEK293 cells. Overexpression of PTEN and TRAF6 both suppressed the EV71 replication in HKE293 cells, and also attenuated the enhanced effects of miR-545 overexpression on the EV71 replication in HEK293 cells. Collectively, our study for the first time showed that miR-545 had an enhanced effect on the EV71 replication in HEK293 and RD cells. Further mechanistic results indicated that miR-545 promoted EV71 replication at least partly via targeting PTEN and TRAF6.     

MEK1/2抑制剂U0126抑制肠道病毒71型的复制

为了揭示肠道病毒71型(enterovirus71,EV71)的复制与宿主细胞Raf/MEK/ERK信号通路(简称ERK通路)的相互关系,本研究应用临床诊断为手足口病的患儿疱疹液,通过易感细胞分离培养、RT-PCR及序列测定,以及Western印迹技术等方法,成功分离到EV71临床株.进一步用该分离株感染易感细胞,通过观察宿主细胞p-ERK1/2蛋白磷酸化水平、病毒特异性衣壳蛋白VP1水平、病毒半数组织培养感染量(50%tissue culture infectious dose,TCID50),以及感染细胞的CPE等指标,以期揭示ERK通路在EV71复制的作用.结果表明,EV71的复制可引起细胞ERK通路的活化;而用MEK1/2特异性的抑制剂U0126预先抑制ERK通路的活化,可显著地降低受染细胞上清液中的病毒的感染滴度(以TCID50表示)、受染细胞中EV71VP1蛋白水平、受染细胞中EV71核酸水平,以及受染细胞的细胞病变效应(cytopathic effect,CPE).提示ERK信号通路的活化对EV71的复制具有重要的作用.本研究为进一步阐明EV71在宿主细胞内的复制机制、寻找新型抗病毒靶标等研究奠定了良好的基础.     

新型病毒衣壳结合剂哌嗪新衍生物抑制肠道病毒71型的复制

目的 研究哌嗪新衍生物体外对肠道病毒71型（EV71）的抑制作用。方法 培养EV71感染的人横纹肌肉瘤RD细胞,建立体外病毒感染模型,将哌嗪新衍生物作用于RD细胞,通过Western blot、Real-time PCR和病毒滴度检测细胞内EV71病毒蛋白和mRNA的表达水平及培养基上清中子代病毒颗粒的数量。并且观察细胞病变效应（CPE）,采用CCK-8法检测哌嗪新衍生物对细胞活性的影响。结果 哌嗪新衍生物VP1-4浓度为5 μg/mL能够显著抑制RD细胞中EV71 VP1蛋白的表达,EV71 mRNA水平降低了（93.8±3.1）%,IC50约为0.016 μg/mL,培养基上清病毒滴度降低了（51.1±0.8）%。而且VP1-4能够减缓EV71病毒感染引起的CPE。此外,化合物VP1-4 CC50>200 μg/mL,说明细胞毒性低,安全性较高。结论 本研究证实VP1-4能有效抑制EV71的复制,可以作为先导化合物开展进一步研究。     

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.     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth

Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5'-untranslated region. Virus-host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.     

靶向IRE1/XBP1信号通路的细胞水平高通量筛选模型建立

目的:建立基于细胞水平的inositol-requiring 1/X-box-binding protein 1 (IRE1/XBP1)信号通路高通量筛选模型,用于发现新型IRE1/XBP1信号通路抑制剂。方法:构建pCAX-F-XBP1△DBD-luciferase质粒,并与pcDNA3.1质粒共转人胚肾细胞HEK293,G418抗性筛选获得多个稳定表达荧光素酶的单克隆。结果:首先利用内质网应激诱导剂衣霉素(tunicamycin,TM)考察单克隆对内质网应激反应的敏感性,确定6#单克隆用于后续研究;其次对细胞接种量、溶剂DMSO终浓度和TM的作用浓度与孵育时间等条件进行优化,最终确定高通量筛选模型条件, Z'因子达到0.62;最后对包含多个激酶抑制剂在内的449个化合物进行筛选,发现27个潜在的IRE1/XBP1抑制剂,其中MG132、Sunitinib和Staurosporine的IC50分别为6.61(±1.51)μmol/L、6.25(±0.36)μmol/L和48(±8)nmol/L。结论:成功建立有效靶向IRE1/XBP1信号通路的高通量药物筛选模型,为基于IRE1/XBP1信号通路为靶点的药物发现奠定坚实基础。