Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A^pro), but not the mutant 2A^pro (2A^pro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A^pro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.     

3株肠道病毒71型毒株的全基因组序列分析

目的：对获得的3株肠道病毒71（EV71）型毒株进行全基因组序列测定,并对其进化特点及分型进行初步分析。方法：提取病毒RNA,反转录得到eDNA,PCR分段扩增覆盖病毒全长序列的6个重叠片段（不包括多聚腺苷酸尾）;用软件将3株EV71的备片段序列进行拼接、编辑和校正,随后进行氨基酸翻译及序列比较;用MEGA4.1软件构建系统进化树。结果：获得了3株EV71的全长序列：GDV103株基因组全长7404 nt,包括741bp的5’端非编码区（UTR）、6582bp的病毒基因组编码区（ORF）及81bp的3’UTR;安徽株（Anhui2007）基因组全长7405nt,包括742bp的5＇UTR、6582bp的ORF及81bp的3＇UTR;VR1432株基因组全长7408nt,包括743bp的5’UTR、6582bp的ORF及83bp的3’UTR长。经同源性比对和进化树分析,证实GDV103和安徽株EV71属于C基因型的C4基因亚型。而VR1432株则属于C基因型的C2基因亚型。结论：获得了3株EV71的全长基因组序列,并进一步探讨了其型别,为下一步的干扰素保护宴,哈重定了基础．     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.     

靶向ICP4的小干扰RNA对HSV-1病毒复制能力的影响

HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。     

肠道病毒71型安徽、河南株的分离与VP1序列进化分析

旨在研究手足口病患者中肠道病毒71型分离株的病毒基因型特征。采集手足口病患者的粪便标本,进行病毒分离和逆转录-聚合酶链式反应(RT-PCR)特异性扩增进行鉴定,同时选取其中9株EV71分离株,对其抗原决定簇部位VP1区进行核酸序列测定,并参考EV71 A、B、C各基因型的参考株和以往中国EV71的分离株进行同源分析和构建系统发生树。结果显示,所分析的9株病毒株均为C4亚型,3株安徽株H7、H8和H9的VP1序列相似度很高(≥98.8%,其中H7、H9的相似度为100.0%),4株河南株H3、H4、H5和H6相似度较高(≥98.4%,H3、H4和H5≥99.6%,其中H3、H4的相似度为100.0%),它们同河南株H1、H5的相似度也较高(≥97.2%),河南株H2虽然与其他河南株具有较高的序列相似度,但进化分析表明,其与安徽株同源性较高。结果表明,安徽株H7、H8和H9株变异速率明显加快,这可能导致了手足口病在安徽省的率先爆发与大流行,河南株H2最初可能由安徽传入河南。     

HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication

EV71 (enterovirus 71) RNA contains an internal ribosomal entry site (IRES) that directs cap-independent initiation of translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs) from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2) as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.     

颗粒体病毒离体复制

迄今 ,尚未建立稳定的颗粒体离体复制系统 ,颗粒体病毒在离体复制中的研究仍处于探索阶段。就颗粒体病毒离体复制的研究进展进行了综述 ,主要包括影响颗粒体病毒离体复制的细胞类型 ,培养温度以及颗粒体病毒离体复制的作用机理和目前仍存在的问题。     

Mitochondria Redistribution in Enterovirus A71 Infected Cells and Its Effect on Virus Replication

Enterovirus A71(EV-A71) is one of the main causative agents of hand, foot and mouth disease(HFMD) and it also causes severe neurologic complications in infected children. The interactions between some viruses and the host mitochondria are crucial for virus replication and pathogenicity. In this study, it was observed that EV-A71 infection resulted in a perinuclear redistribution of the mitochondria. The mitochondria rearrangement was found to require the microtubule network, the dynein complex and a low cytosolic calcium concentration. Subsequently, the EV-A71 non-structural protein 2 BC was identified as the viral protein capable of inducing mitochondria clustering. The protein was found localized on mitochondria and interacted with the mitochondrial Rho GTPase 1(RHOT1) that is a key protein required for attachment between the mitochondria and the motor proteins, which are responsible for the control of mitochondria movement.Additionally, suppressing mitochondria clustering by treating cells with nocodazole, EHNA, thapsigargin or A23187 consistently inhibited EV-A71 replication, indicating that mitochondria recruitment played a crucial role in the EV-A71 life cycle. This study identified a novel function of the EV-A71 2 BC protein and provided a potential model for the regulation of mitochondrial motility in EV-A71 infection.     

Enterovirus 71 Induces Mitochondrial Reactive Oxygen Species Generation That is Required for Efficient Replication

Redox homeostasis is an important host factor determining the outcome of infectious disease. Enterovirus 71 (EV71) infection has become an important endemic disease in Southeast Asia and China. We have previously shown that oxidative stress promotes viral replication, and progeny virus induces oxidative stress in host cells. The detailed mechanism for reactive oxygen species (ROS) generation in infected cells remains elusive. In the current study, we demonstrate that mitochondria were a major ROS source in EV71-infected cells. Mitochondria in productively infected cells underwent morphologic changes and exhibited functional anomalies, such as a decrease in mitochondrial electrochemical potential ΔΨ_m and an increase in oligomycin-insensitive oxygen consumption. Respiratory control ratio of mitochondria from infected cells was significantly lower than that of normal cells. The total adenine nucleotide pool and ATP content of EV71-infected cells significantly diminished. However, there appeared to be a compensatory increase in mitochondrial mass. Treatment with mito-TEMPO reduced eIF2α phosphorylation and viral replication, suggesting that mitochondrial ROS act to promote viral replication. It is plausible that EV71 infection induces mitochondrial ROS generation, which is essential to viral replication, at the sacrifice of efficient energy production, and that infected cells up-regulate biogenesis of mitochondria to compensate for their functional defect.     

Design and In Vitro Evaluation of Layer by Layer siRNA Nanovectors Targeting Breast Tumor Initiating Cells

Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44⁺/CD24⁻/EpCAM⁺ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs in vitro.     

Conformational Plasticity of the 2A Proteinase from Enterovirus 71

The 2A proteinase (2A^pro) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A^pro from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A^pro was comprised of an N-terminal domain of a four-stranded antiparallel β sheet and a C-terminal domain of a six-stranded antiparallel β barrel with a tightly bound zinc atom. Unlike in other 2A^pro structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A^pro of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII β strands. This was supported by molecular dynamic simulation. The structural variation among different 2A^pro structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.     

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

mRNA Decay Factor AUF1 Binds the Internal Ribosomal Entry Site of Enterovirus 71 and Inhibits Virus Replication

AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.     

EV71小鼠适应株制备及体内外感染特点

目的用1日龄ICR小鼠传代制备EV71小鼠适应株,研究EV71亲代株与小鼠适应株的体内外感染特点,建立EV71感染ICR小鼠动物模型,为病毒疫苗和抗病毒药物的研究提供实用的动物评价工具。方法用1日龄ICR小鼠进行EV71病毒(Fuyang-0805)的传代,得到小鼠传代株。以一定浓度亲代株和传代株病毒分别接种RD、Vero、SY5Y、Caco-2四种细胞,定量方法检测各时间点不同毒株在四种细胞上的复制数量,CCK8方法测定各时间点细胞的存活率;同时,两毒株分别腹腔注射感染1日龄小鼠,定期安乐死动物,采集肺、小肠、骨骼肌、大脑四种器官组织,进行动物体内病毒半定量和定量分析,同时进行各器官组织病理学观察、免疫组织化学鉴定。结果与亲代毒株相比较,小鼠传代株(EV71-MMP4)表现出更强的肌肉来源细胞嗜性与毒性;同时,两毒株腹腔注射感染1日龄小鼠后,EV71-MMP4感染的小鼠体重增长较正常小鼠体重增长缓慢;半定量和定量RT-PCR显示,在小鼠肌肉中的病毒载量于感染后1d和5d达到高峰。EV71-MMP4感染组感染率较高、病毒组织分布较广、感染持续性较好、病毒载量较高,高剂量病毒感染后小鼠小肠、心肌和骨骼肌可观察到细胞空泡变性、淋巴细胞浸润等病理变化。免疫组织化学显示感染后小鼠骨骼肌有EV71病毒特异分布。结论阜阳EV71小鼠适应株表现出较亲代毒株更好的小鼠易感性、细胞毒性,所建立的动物模型可用于EV71病毒致病机制、感染特点的研究和病毒疫苗及药物的评价。     

Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 μM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.