肠道病毒71型3C蛋白酶在大肠杆菌中的表达及活性研究

本研究利用大肠杆菌表达系统构建肠道病毒71型3C蛋白酶,并进行纯化,对其酶活性进行研究。首先,将3C蛋白酶基因克隆至pET28a载体,在大肠杆菌BL21(DE3)中表达,Ni-NTA柱亲和层析纯化获得3C蛋白酶,经肠激酶酶切去除N端His标签后获得无His标签的3C蛋白酶,再以荧光多肽为底物进行酶活性研究。经过双酶切鉴定和测序证实,重组表达质粒pET28a-3C构建正确,表达的重组3C蛋白酶相对分子质量约22kD;纯化后有无His标签的3C蛋白酶均能催化荧光底物3B-3C,并且两者的酶动力学数据无显著差异,含有His标签的3C蛋白酶Km、Vmax、Kcat分别为22μM、434nM.Min-1、0.0669 Min-1;其最适反应pH为7.0,最佳反应温度为30℃～37℃。本实验成功表达并纯化了重组3C蛋白酶,该酶具有良好的活力,为抗病毒抑制剂、结构蛋白组装、疫苗开发及3C蛋白酶检测方法的研发奠定了基础。     

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.     

Functional Comparison of SCARB2 and PSGL1 as Receptors for Enterovirus 71

Human scavenger receptor class B, member 2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL1) have been identified to be the cellular receptors for enterovirus 71 (EV71). We compared the EV71 infection efficiencies of mouse L cells that expressed SCARB2 (L-SCARB2) and PSGL1 (L-PSGL1) and the abilities of SCARB2 and PSGL1 to bind to the virus. L-SCARB2 cells bound a reduced amount of EV71 compared to L-PSGL1 cells. However, EV71 could infect L-SCARB2 cells more efficiently than L-PSGL1 cells. The results suggested that the difference in the binding capacities of the two receptors was not the sole determinant of the infection efficiency and that SCARB2 plays an essential role after attaching to virions. Therefore, we examined the viral entry into L-SCARB2 cells and L-PSGL1 cells by immunofluorescence microscopy. In both cells, we detected internalized EV71 virions that colocalized with an early endosome marker. We then performed a sucrose density gradient centrifugation analysis to evaluate viral uncoating. After incubating the EV71 virion with L-SCARB2 cells or soluble SCARB2 under acidic conditions below pH 6.0, we observed that part of the native virion was converted into an empty capsid that lacked both genomic RNA and VP4 capsid proteins. The results suggested that the uncoating of EV71 requires both SCARB2 and an acidic environment and occurs after the internalization of the virus-receptor complex into endosomes. However, the empty capsid formation was not observed after incubation with L-PSGL1 cells or soluble PSGL1 under any of the tested pH conditions. These results indicated that SCARB2 is capable of viral binding, viral internalization, and viral uncoating and that the low infection efficiency of L-PSGL1 cells is due to the inability of PSGL1 to induce viral uncoating. The characterization of SCARB2 as an uncoating receptor greatly contributes to the understanding of the early steps of EV71 infection.     

Human SCARB2 Transgenic Mice as an Infectious Animal Model for Enterovirus 71

Enterovirus 71 (EV71) and coxsackievirus (CVA) are the most common causative factors for hand, foot, and mouth disease (HFMD) and neurological disorders in children. Lack of a reliable animal model is an issue in investigating EV71-induced disease manifestation in humans, and the current clinical therapies are symptomatic. We generated a novel EV71-infectious model with hSCARB2-transgenic mice expressing the discovered receptor human SCARB2 (hSCARB2). The challenge of hSCARB2-transgenic mice with clinical isolates of EV71 and CVA16 resulted in HFMD-like and neurological syndromes caused by E59 (B4) and N2838 (B5) strains, and lethal paralysis caused by 5746 (C2), N3340 (C4), and CVA16. EV71 viral loads were evident in the tissues and CNS accompanied the upregulated pro-inflammatory mediators (CXCL10, CCL3, TNF-α, and IL-6), correlating to recruitment of the infiltrated T lymphocytes that result in severe diseases. Transgenic mice pre-immunized with live E59 or the FI-E59 vaccine was able to resist the subsequent lethal challenge with EV71. These results indicate that hSCARB2-transgenic mice are a useful model for assessing anti-EV71 medications and for studying the pathogenesis induced by EV71.