丙型肝炎病毒非结构蛋白NS3／4A的研究进展

丙型肝炎病毒（HCV）非结构蛋白NS3共有631个氨基酸组成，具有丝氨酸蛋白酶、三磷酸核苷酶（NTpase）和螺旋酶（Helicase）的功能，在HCV多聚蛋白的成熟和病毒复制过程中发挥着重要作用．非结构蛋白NS4A，其主要功能就是作为NS3丝氨酸蛋白酶的辅助因子，在HCV多氨酸成熟过程中发挥着不可替代的调节作用．此外，NS3／4A还参与NS5A的超磷酸化修饰过程．     

日本乙型脑炎病毒对干扰素诱导基因ISG15表达的影响

为研究机体干扰素诱导基因对日本乙型脑炎(JEV)感染的作用,通过IFN-α和JEV刺激猪睾丸(ST)细胞,实时荧光定量PCR检测Mx1、Mx2、OAS1、OAS2、PKR和干扰素诱导基因-15(ISG15)表达情况,发现除PKR外,其他基因相对mRNA量都有不同程度上升,其中ISG15上升最为显著。ISG15在ST细胞中过表达,检测病毒基因组和滴度,结果表明:ISG15可显著抑制JEV增殖;shRNA靶向干扰ISG15,JEV病毒量上升,说明ISG15在体外对JEV的增殖有抑制作用。将JEV的C、M、E、NS1、NS2A、NS2B、NS3、NS4A、NS4B基因克隆入pcDNA3.1真核表达载体,转染ST细胞后E和NS3基因引起ISG15相对mRNA量显著上升,说明JEV的E和NS3基因参与诱导ISG15的表达。     

Efficient initiation of HCV RNA replication in cell culture

Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.     

Regulation of influenza virus infection by microRNAs

MicroRNAs(miRNAs) are small noncoding RNAs of 18–25 nucleotides(nt) in length that represent key regulators of many normal cellular functions through the inhibition of mRNA translation and mRNA degradation. To date, over 2 500 mature miRNAs have been identified in plants, animals and several types of viruses. Influenza A virus(IAV), which is a negativesense, single-stranded RNA virus, does not encode viral miRNA. However, IAV infection can alter the expression of host miRNAs, either in cell culture or in host. In turn, host miRNAs regulate IAV life cycle through directly binding to IAV genome or indirectly targeting host factors associated with viral replication. In this review, we briefly summarized the role and significance of miRNA in relation to IAV pathogenesis. Understanding the role of cellular miRNAs during viral infection may be beneficial to the identification of novel therapeutic strategies to block IAV replication.     

PI3K/Akt信号转导通路在ALV-J感染中作用的初步研究

 【目的】探讨ALV-J在宿主细胞中复制与PI3K/Akt信号转导通路的关系。【方法】将血管瘤病变型ALV-J毒株HN06和骨髓瘤病变型ALV-J毒株NX0101分别感染DF-1细胞,通过Western blot、Real-time PCR、IFA和ELISA等方法,观察细胞Akt蛋白磷酸化水平、病毒RNA表达水平和病毒蛋白表达水平等指标。【结果】HN06株和NX0101株在体外细胞中复制水平有差异。HN06株的早期感染可引起Akt转导通路的活化,病毒引起的Akt磷酸化具有病毒滴度依赖性,而且能被PI3K特异性抑制剂LY294002所抑制,表明HN06株诱导的Akt活化是PI3K途径依赖的。LY294002可在病毒感染早期呈剂量依赖性地显著降低受染细胞中HN06 RNA水平、囊膜蛋白水平和细胞培养物上清中的病毒粒子含量。【结论】PI3K/Akt信号转导通路活化对HN06株在细胞感染早期具有重要的作用,该结果与已报道的有关细胞PI3K/Akt信号转导通路参与NX0101株的早期感染的结论一致。本研究为进一步阐明ALV-J入侵宿主细胞和复制的精确机制等研究奠定了基础。     

Complete replication of hepatitis C virus in cell culture

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.     

A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.     

siRNA对乙型脑炎病毒复制的抑制效果

以JEV的NSl基因mRNA为靶序列,设计合成了4个siRNA序列,构建了各自的表达质粒pS-NSlA、pS-NSlB、pS-NSlC和pS-NSlD.通过间接免疫荧光、Western blot检测、RT-PCR和病毒空斑检测等方法对这些siRNAs抑制JEV复制的效果进行了评价.结果表明4个siRNA表达质粒均对JEV在细胞中的复制具有不同程度的抑制作用,其中pS-NSlC、pS-NSlD有显著的抑制作用.说明针对NSl基因的siRNAs可以有效抑制JEV的复制,并有望成为应对JEV感染的治疗方法.     

Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase

NS3, an essential helicase for replication of hepatitis C virus, is a model enzyme for investigating helicase function. Using single-molecule fluorescence analysis, we showed that NS3 unwinds DNA in discrete steps of about three base pairs (bp). Dwell time analysis indicated that about three hidden steps are required before a 3-bp step is taken. Taking into account the available structural data, we propose a spring-loaded mechanism in which several steps of one nucleotide per adenosine triphosphate molecule accumulate tension on the protein-DNA complex, which is relieved periodically via a burst of 3-bp unwinding. NS3 appears to shelter the displaced strand during unwinding, and, upon encountering a barrier or after unwinding >18 bp, it snaps or slips backward rapidly and repeats unwinding many times in succession. Such repetitive unwinding behavior over a short stretch of duplex may help to keep secondary structures resolved during viral genome replication.     

HCV persistence and immune evasion in the absence of memory T cell help

Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.     

Molecular modeling of the HIV-1 protease and its substrate binding site

The human immunodeficiency virus (HIV-1) encodes a protease that is essential for viral replication and is a member of the aspartic protease family. The recently determined three-dimensional structure of the related protease from Rous sarcoma virus has been used to model the smaller HIV-1 dimer. The active site has been analyzed by comparison to the structure of the aspartic protease, rhizopuspepsin, complexed with a peptide inhibitor. The HIV-1 protease is predicted to interact with seven residues of the protein substrate. This information can be used to design protease inhibitors and possible antiviral drugs.     

Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase

Nonhexameric helicases use adenosine triphosphate (ATP) to unzip base pairs in double-stranded nucleic acids (dsNAs). Studies have suggested that these helicases unzip dsNAs in single-base pair increments, consuming one ATP molecule per base pair, but direct evidence for this mechanism is lacking. We used optical tweezers to follow the unwinding of double-stranded RNA by the hepatitis C virus NS3 helicase. Single-base pair steps by NS3 were observed, along with nascent nucleotide release that was asynchronous with base pair opening. Asynchronous release of nascent nucleotides rationalizes various observations of its dsNA unwinding and may be used to coordinate the translocation speed of NS3 along the RNA during viral replication.     

Effect of actinomycin D on cellular nucleic acid synthesis and virus production

Actinomycin D inhibits the synthesis of ribonucleic acid in L cells and the yield of vaccinia virus containing deoxyribonucleic acid, but it does not inhibit cellular deoxyribonucleic acid synthesis or the multiplication of Mengo virus containing ribonucleic acid. These observations serve to distinguish the replication of viral ribonucleic from ribonucleic acid synthesis which is controlled by viral or cellular deoxyribonucleic acid.     

禽呼肠孤病毒σNS和σC基因shRNA载体构建及其干扰效果测定

禽呼肠孤病毒σC基因是病毒粘附蛋白,σNS基因具有结合单链RNA活性。根据siRNA靶序列设计原则,设计并合成siRNA模板并克隆到shRNA表达载体pSilencer-CMV 4.1 neo。分别构建了针对σC基因的shRNA载体C1、C2、C3和针对σNS基因的shRNA载体NS1、NS2、NS3。将构建的shRNA载体和阴性对照分别与表达σC和σNS基因的融合蛋白的真核表达载体pEGFP-σC及pEGFP-σNS共转染DF-1细胞。荧光显微镜观察结果表明,6个shRNA片段不同程度地抑制各自融合蛋白的表达。Real-time PCR检测结果表明,C3和NS1体外干扰病毒复制的效果最佳。     

羊口疮病毒在单层细胞上繁殖特性的研究

本文采用奶山羊睾丸细胞对羊口疮病毒的繁殖特性进行了研究。从来自皇城、灵台和阿沿沟牧场的病羊痂皮组织中分到HC、LT和AT三株口疮病毒。比较了吸附一、混合一、改进混合三种方法分离培养口疮病毒的效果。进一步选用第15代HC分离物分别测定了吸附时间、接种浓度同细胞致病作用(CPE)、以及病毒复制、CPE与培养时间的关系,对比了细胞毒与痂皮毒的理化特性。结果表明,1—10代CPE很不规则,且有显著的毒株差异,而10代以后CPE趋于稳定。病毒复制于感染后42小时达到高峰,效价为10~6TCID_50/0.2ml,50—60％CPE为其标指;芽生释放的病毒具有一层外膜结构。还发现具有2或3个核衣壳的病毒粒子。     

非洲猪瘟病毒MGF110-5L-6L蛋白诱导宿主细胞翻译阻滞和应激颗粒形成的作用机制

【背景】非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）感染引发的一种猪烈性传染病,是全球公认的养猪业“头号杀手”,至今尚无安全有效的疫苗和药物。病毒作为专性细胞内寄生物,必须通过“劫持”宿主翻译系统为病毒蛋白合成服务。其中翻译起始因子eIF2α作为翻译调控的核心节点,控制细胞应激反应和翻译重编程走向,对病毒毒力、嗜性、致病性及免疫逃逸等具有重要影响,eIF2α磷酸化调控无疑是病毒与宿主细胞竞争翻译资源的重要阵地之一。然而,关于ASFV编码蛋白与eIF2α磷酸化作用关系的认知极度匮乏。【目的】探究非洲猪瘟病毒MGF110-5L-6L蛋白对宿主细胞翻译阻滞和促进应激颗粒形成的作用机制,为深入揭示非洲猪瘟病毒的致病机制研究提供科学依据。【方法】在前期利用荧光素酶报告基因载体和绿色荧光报告载体,筛选发现外源表达MGF110-5L-6L极显著上调eIF2α磷酸化水平的基础上。选择猪肺泡巨噬细胞3D4/21和猪肾细胞PK-15作为研究用细胞系,利用质粒转染和特异性化学药物处理等方法,结合免疫印迹和激光共聚焦...     

A brief review of microRNA and its role in PRRSV infection and replication

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded RNA virus, mainly infects cells of monocyte/macrophage lineage. Recently, host microRNAs were shown to be capable of modulating PRRSV infection and replication by multiple ways such as targeting viral genomic RNA, targeting viral receptor and inducing antiviral response. MicroRNAs are small RNAs and have emerged as important regulators of virus-host cell interactions. In this review, we discuss the identified functions of host microRNAs in relation to PRRSV infection and propose that cellular microRNAs may have a substantial effect on cell or tissue tropism of PRRSV.     

Aggresomes and autophagy generate sites for virus replication

The replication of many viruses is associated with specific intracellular compartments called virus factories or virioplasm. These are thought to provide a physical scaffold to concentrate viral components and thereby increase the efficiency of replication. The formation of virus replication sites often results in rearrangement of cellular membranes and reorganization of the cytoskeleton. Similar rearrangements are seen in cells in response to protein aggregation, where aggresomes and autophagosomes are produced to facilitate protein degradation. Here I review the evidence that some viruses induce aggresomes and autophagosomes to generate sites of replication.     

Calcium modulation activates Epstein-Barr virus genome in latently infected cells

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.     

猪日本乙型脑炎病毒NS5原核表达和多克隆抗体制备

提取猪日本乙型脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,扩增JEV-NS5基因片段,亚克隆到原核表达载体pET-28(a)上,构建重组原核表达质粒pET-28(a)-JEV-NS5,转化大肠埃希氏菌BL21(DE3)菌株,IPTG诱导表达重组JEV-NS5蛋白,获得分子质量为103 ku的重组蛋白.该蛋白主要以包涵体形式表达,表达量占总菌体蛋白的35%以上,His-band Ni+纯化后,获得高纯度重组蛋白占总蛋白的比例达75%以上.以纯化的蛋白免疫小鼠,制备小鼠抗JEV-NS5抗体,抗体效价达到3×104,并能与病毒感染的样本反应,证明该蛋白具有较好的特异性.