Caspase Activation and Specific Cleavage of Substrates after Coxsackievirus B3-Induced Cytopathic Effect in HeLa Cells

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.     

Human Astrocytic Cells Support Persistent Coxsackievirus B3 Infection

Enteroviruses can frequently target the human central nervous system to induce a variety of neurological diseases. Although enteroviruses are highly cytolytic, emerging evidence has shown that these viruses can establish persistent infections both in vivo and in vitro. Here, we investigated the susceptibility of three human brain cell lines, CCF-STTG1, T98G, and SK-N-SH, to infection with three enterovirus serotypes: coxsackievirus B3 (CVB3), enterovirus 71, and coxsackievirus A9. Persistent infection was observed in CVB3-infected CCF-STTG1 cells, as evidenced by prolonged detection of infectious virions, viral RNA, and viral antigens. Of note, infected CCF-STTG1 cells expressed the nonfunctional canonical viral receptors coxsackievirus-adenovirus receptor and decay-accelerating factor, while removal of cell surface chondroitin sulfate from CCF-STTG1 cells inhibited the replication of CVB3, suggesting that receptor usage was one of the major limiting factors in CVB3 persistence. In addition, CVB3 curtailed the induction of beta interferon in infected CCF-STTG1 cells, which likely contributed to the initiation of persistence. Furthermore, proinflammatory chemokines and cytokines, such as vascular cell adhesion molecule 1, interleukin-8 (IL-8), and IL-6, were upregulated in CVB3-infected CCF-STTG1 cells and human progenitor-derived astrocytes. Our data together demonstrate the potential of CCF-STTG1 cells to be a novel cell model for studying CVB3-central nervous system interactions, providing the basis toward a better understanding of CVB3-induced chronic neuropathogenesis.     

细胞自噬促进柯萨奇病毒B3复制的研究

本研究探索柯萨奇病毒B3(Coxsackievirus B3,CVB3)感染引起的自噬与病毒复制之间的关系。CVB3感染HeLa细胞,并在病毒感染后6 h、8 h和10 h时检测LC3-Ⅰ蛋白、LC3-Ⅱ蛋白和p62蛋白的表达水平。结果显示CVB3病毒感染促使LC3-Ⅱ/LC3-Ⅰ比值升高,同时降低p62蛋白的表达。分别将自噬诱导剂雷帕霉素(Rapamy-cin)、自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3MA)或溶酶体抑制剂阿洛司他丁(Aloxistatin,E46D)预处理HeLa细胞2 h,CVB3感染药物处理细胞并在病毒感染6 h后收集细胞、检测CVB3病毒VP1蛋白的表达。结果显示雷帕霉素和E64D促使CVB3病毒VP1蛋白表达增加,而3MA降低CVB3病毒VP1蛋白的表达。本研究得出结论 CVB3病毒感染诱导自噬进而促进病毒复制。     

小檗碱通过JNK通路调控柯萨奇病毒B3感染心肌细胞凋亡的作用

小檗碱是具有细胞保护作用的生物碱,能够在柯萨奇病毒B3(CVB3)感染引起的病毒性心肌炎小鼠中发挥心肌保护作用,但具体的机制未阐明。在内皮细胞中,小檗碱通过c-Jun氨基末端激酶(JNK)通路抑制细胞凋亡,因此本研究将分析小檗碱通过JNK通路调控CVB3感染心肌细胞凋亡的作用。H9c2心肌细胞分为对照组(不含药物的DMEM处理)、模型组(含CVB3的DMEM处理)、小檗碱组(含CVB3及小檗碱的DMEM处理)、小檗碱+JNK质粒组(含CVB3、小檗碱、JNK质粒的DMEM处理),检测细胞凋亡率、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、活性氧(ROS)、丙二醛(MDA)的含量、p-JNK、cleaved caspase-3、bax、bcl-2的表达量。结果显示,模型组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量高于对照组,bcl-2的表达量低于对照组(P<0.05);小檗碱组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量低于模型组,bcl-2的表达量高于模型组(P<0.05);小檗碱+JNK质粒组的细胞凋亡率、TNF-α、IL-6、ROS、MDA的含量、p-JNK、cleaved caspase-3、bax的表达量高于小檗碱组,bcl-2的表达量低于小檗碱组(P<0.05)。以上结果表明小檗碱对CVB3感染心肌细胞的凋亡具有抑制作用,抑制JNK通路是介导这一作用可能的分子机制。     

Expression and Purification of Recombinant 3C Proteinase of Coxsackievirus B3

We have cloned various lengths of coxsackievirus B3 cDNA encompassing the region encoding the 3C proteinase, which is essential to the viral replication cycle. Such viral cDNAs were fused in frame to the 5′terminal portion of the lacZ’ gene carried on the vector pUCl18 to express mature 3C proteinase in Escherichia coli. In the E. coli cells containing pCXB108 or pCXB117, constructed for this study, a large amount of 23-kDa protein was synthesized in the presence of IPTG. This protein was purified and was shown to be intact 3C proteinase. These data suggest that 3C proteinase, expressed as a part of a fusion protein, was active in E. coli and released itself from the precursor fusion protein by autocatalytic cleavage.     

Complete Genome Sequence of a Coxsackievirus B3 Isolated from a Sichuan Snub-Nosed Monkey

Coxsackievirus B3 (CVB3) is an enterovirus in the family Picornaviridae that is significant to human health, being associated with myocarditis, aseptic meningitis, and pancreatitis, among other conditions. In addition to humans, Sichuan snub-nosed monkeys can be infected and killed by CVB3. Here, we report the first complete genome sequence of a novel coxsackievirus B3 strain, SSM-CVB3, which was isolated from a deceased Sichuan snub-nosed monkey with severe myocarditis. Our findings may aid in understanding the evolutionary characteristics and molecular pathogenesis of this virus.     

SAR studies of 9-norbornylpurines as Coxsackievirus B3 inhibitors

Coxsackievirus and related enteroviruses are important human pathogens that cause various diseases with clinical manifestations ranging from trivial flu-like syndromes to dangerous or even fatal diseases such as myocarditis, meningitis and encephalitis. Here, we report on our continuous SAR study focused on 9-(bicyclo[2.2.1]hept-2-yl)-9H-purines as anti-enteroviral inhibitors. The purine moiety was modified at positions 2, 6 and 8. Several analogues inhibited Coxsackievirus B3 as well as other enteroviruses at low-micromolar concentrations. The 6-chloropurine derivative was confirmed as the most active compound in this series.     

Expression and purification of recombinant 3C proteinase of Coxsackievirus B3.

We have cloned various lengths of coxsackievirus B3 cDNA encompassing the region encoding the 3C proteinase, which is essential to the viral replication cycle. Such viral cDNAs were fused in frame to the 5'terminal portion of the lacZ' gene carried on the vector pUC118 to express mature 3C proteinase in Escherichia coli. In the E. coli cells containing pCXB108 or pCXB117, constructed for this study, a large amount of 23-kDa protein was synthesized in the presence of IPTG. This protein was purified and was shown to be intact 3C proteinase. These data suggest that 3C proteinase, expressed as a part of a fusion protein, was active in E. coli and released itself from the precursor fusion protein by autocatalytic cleavage.     

柯萨奇病毒B组3型基因组剪切片段的序列分析

柯萨奇病毒B组(Coxsackievirus B,CVB)感染细胞时其基因组RNA存在不稳定现象,但产生机制尚不清楚。本研究将柯萨奇病毒B组3型(CVB3)感染细胞后,利用5′ cDNA末端快速扩增技术(5′ rapid amplification of cDNA ends,5′ RACE)扩增并克隆细胞内CVB3基因组片段,并对每条序列及其5′端的二级结构进行分析。结果获得的20条CVB3基因组片段,长度为 2 067～5 547 bp,片段断端主要分布于2Apro和2C编码区。RNAfold分析显示,这些片段多数在5′断点端形成二级茎-环结构。本研究显示,CVB在宿主细胞感染时可形成大量不完整基因组RNA片段,这些片段可在5′断点端形成局部双链结构,提示片段不是随机产生,可能是RNA酶剪切产物。此发现有助于理解CVB基因组不稳定的机制。     

A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.Virus infections depend on complex interactions between viral and cellular proteins. Consequently, the nature of these interactions has important implications for viral cell type specificity, tissue tropism, and pathogenesis. Group B coxsackieviruses (CVB1 to CVB6), members of the genus Enterovirus within the family of Picornaviridae, are human pathogens that cause a broad spectrum of diseases, ranging from mild upper respiratory illnesses to more severe infections of the central nervous system, heart, and pancreas (61). These viruses have also been associated with certain chronic muscle diseases and myocardial infarction (2, 3, 12, 13, 22).The positive single-stranded RNA genome (approximately 7,500 nucleotides in length) of CVBs is encapsidated within a small T=1, icosahedral shell (30 nm in diameter) comprised of repeating identical subunits made up of four structural proteins (VP1 to VP4). Parts of VP1, VP2, and VP3 are exposed on the outer surface of the capsid, whereas VP4 is positioned on the interior. The virion morphology is characterized by a star-shaped mesa at each 5-fold icosahedral symmetry axis, surrounded by a narrow depression referred to as the “canyon” (69). All six serotypes of CVB can use the coxsackie and adenovirus receptor (CAR) for cell attachment and entry (9, 55, 82). Some strains of CVB1, -3, and -5 also use decay accelerating factor ([DAF] CD55) for initial attachment to the host cell; however, binding to DAF alone is insufficient to permit entry into the cell (10, 54, 76).Picornaviruses are generally characterized by their cytolytic nature in cell culture. However, several in vivo and in vitro studies have shown that some picornaviruses, e.g., poliovirus, Theiler''s murine encephalomyelitis virus, foot-and-mouth disease virus, CVB3, CVB4, and CVB5, may also establish persistent, noncytolytic infections (4, 29, 35, 39, 62, 74). Recently, it has been shown that the diverse outcomes of picornaviral infections may depend on interactions between the virus and the apoptotic machinery of the infected cell (14, 30, 71). Several picornaviral proteins have been identified as inducers of an apoptotic response, including viral capsid proteins VP1, VP2, and VP3, as well as nonstructural proteins 2A and 3C (7, 20, 32, 33, 42, 50, 63). In addition, antiapoptotic activity has been assigned to the nonstructural proteins 2B and 3A (16, 59).Picornaviruses have the potential to adapt rapidly to new host environments. Virus features affecting adaptability include high mutation rates, short replication times, large populations, and frequent incidences of recombination (25-27, 53). Consequently, picornaviruses exist as genetically heterogenous populations, referred to as viral quasispecies (25, 26).Previously, the CVB2 prototype strain Ohio-1 (CVB2O) was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells (66). Two amino acid changes were identified in the capsid-coding region, and one was identified in the 2C-coding region of the adapted virus. Further characterization of the virus-host interaction showed that the infection was not affected by anti-DAF antibodies, indicating the use of an alternative receptor.In this study, the amino acid substitutions associated with the adaptation of CVB2O to cytolytic infection of RD cells were evaluated. Site-directed mutagenesis studies showed that a single amino acid change in the VP1 capsid protein was responsible for the cytolytic RD phenotype. In addition, as indicated by caspase activation and DNA degradation, the apoptotic pathway was activated in RD cells infected by the cytolytic virus.     

H9c2细胞对B组柯萨奇病毒3型重组株的易感性

H9c2细胞是来源于大鼠胚胎心脏组织的成肌细胞系,B组柯萨奇病毒(group B Coxsackievirus,CVB)是心肌炎和扩张型心肌病的主要病原.本研究观察了CVB3在H9c2细胞中的感染性,探讨H9c2细胞是否可用于CVB致心肌疾病的实验研究.用整合了增强型绿色荧光蛋白(EGFP)或海肾荧光素酶(RLuc)的...     

失配双链核糖核酸抑制柯萨奇B3病毒感染细胞的研究

通过药物抑制病毒致细胞病变效应实验和药物对病毒空斑形成抑制实验,研究低毒性失配双链核糖核酸（Poly I：C12u）和已知的抗病毒药物双链核糖核酸聚肌胞（Poly I：C）在细胞水平对柯萨奇病毒感染的抑制作用比较,证明Poly I：C12U的抗病毒作用。实验结果表明Poly I：C12U和Poly I：C均可有效地抑制病毒空斑的形成,且量效关系已确定。     

受miRNA-205调控的重组柯萨奇病毒B3的构建及其复制

本研究在柯萨奇病毒B3(coxsackievirus B3,CVB3)基因组P1编码区与P2编码区之间插入一段has-miRNA-205-3p和has-miRNA-205-5p(简称miR-205)的靶序列,得到重组病毒v205T,并比较分析了它在人宫颈癌细胞系HeLa细胞(miR-205低水平表达)和非小细胞肺癌细胞系A549细胞(miR-205高水平表达)中的复制情况。结果表明,插入的miR-205靶序列不影响病毒在HeLa细胞中的复制水平,但抑制了病毒在A549细胞中的复制,病毒滴度为对照的1%以下。为探讨v205T在2株细胞中复制差异的原因,进一步加入miR-205的类似物和抑制物。miR-205类似物可抑制v205T在HeLa细胞中复制和杀伤细胞的水平,而miR-205抑制物可提高v205T在A549细胞中的复制和杀伤细胞的水平。结果表明,v205T的复制确实受miR-205的调控。本研究为开发基于CVB3载体的溶瘤病毒和针对CVB3的减毒活疫苗提供了依据。