草鱼AHZC-88细胞抗出血病病原病毒机制初探

本文通过细胞表面病毒受体分析,病毒感染细胞的电镜观察以及细胞核和细胞质可溶性蛋白组分的电泳分析,对AHZC-88细胞的抗病毒机制进行了初步的探讨。结果表明,病毒不仅能与该细胞表面结合,而且能够进入细胞,但是不能在细胞内复制增殖。电泳分析显示该细胞的胞质可溶性蛋白组分比对病毒敏感的ZC—7901细胞多了一条区带。这些结果提示AHZC-88细胞对病毒的抗性可能是由于病毒在细胞内的脱衣壳过程受到了抑制所致。     

Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

正链RNA病毒基因组的复制

许多正链RNA病毒是严重危害人类健康的病原体,是造成经济植物动物死亡的致病因子.正链RNA病毒的基因组为正链RNA,其复制酶是依赖RNA的RNA聚合酶,非编码区是病毒基因组复制的主要调控位点,3’非编码区是复制酶的第一结合位点,正链RNA病毒基因组大多可能按copy-back模型进行复制.瘟病毒基因组的复制过程出现正链复制本的数量大于负链复制本的数量,这可能是以RF中间体的负链RNA为模板、正链RNA被置换的形式进行复制的结果.本文概述了HCV细胞培养系统的研究进展.     

宿主细胞泛素系统与病毒相互作用的研究

真核细胞中,泛素系统执行对大多数短周期寿命蛋白的选择性降解,且已发现由泛素介导的对蛋白的降解调控在细胞的多种生命过程中起重要作用。病毒侵染宿主细胞后,细胞的泛素系统与一些重要的病毒蛋白相互作用,参与调节病毒的生活周期,如病毒迁移,核酸复制,免疫逃避,出芽等过程。     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Box C/D RNA protein complexes (RNPs) direct site-specific 2'-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-? resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide-substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.     

Myristic acid is coupled to a structural protein of polyoma virus and SV40

In the lytic cycle of papova viruses, both uncoating of the viral genome after infection and assembly of functional virions take place in the cell nucleus. The mechanisms by which newly internalized virions are targeted to the nucleus and viral DNA encapsidated into particles are poorly understood. Although the major capsid protein VP1 is involved in endocytosis, and largely defines virion structure, the functions of the minor proteins VP2 and VP3 have remained obscure. Here we show that VP2 from both polyoma virus and simian virus 40 (SV40) is covalently linked to myristic acid; this is the first report of a myristylated protein in the nucleus and of a fatty acid being important in the structure of a nonenveloped virus. We consider the implications of this unusual modification on encapsidation and suggest that VP2 may be a scaffolding protein for virion assembly.     

Structural changes of envelope proteins during alphavirus fusion

Alphaviruses are enveloped RNA viruses that have a diameter of about 700?? and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.     

双链RNA技术在果树病毒研究中的应用

含ＲＮＡ基因组的植物病毒在复制时会产生双链ＲＮＡ（ｄｓＲＮＡ）。本文介绍了用ＣＦ－１１纤维素粉提取ｄｓＲＮＡ的步骤,并列举了ｄｓＲＮＡ在果树病毒研究中的应用,其主要应用有：１）果树病毒病及病原鉴定;２）病毒分化株系的鉴定;３）以ｄｓＲＮＡ为中介对病毒核酸序列进行测定;４）探针制备和ｃＤＮＡ克隆的获得;５）用于检测病毒卫星ＲＮＡ和亚基因组ＲＮＡ;６）侵染性测定和制备抗血清等方面的研究。     

RNA interference screen for human genes associated with West Nile virus infection

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.     

An interferon-induced cellular enzyme is incorporated into virions

The mechanisms by which interferon inhibits viral growth are only partially understood. Several enzymatic activities increase in cells shortly after treatment with interferon. One of these enzymes, oligo-isoadenylate synthetase, synthesizes (2'-5') isoadenylate oligomers which strongly stimulate the activity of a cellular ribonuclease, RNase F (ref. 7). Interferon also significantly increases the activity of a protein kinase which phosphorylates the initiation factor eIF-2 and can inhibit in vitro protein synthesis. Such interferon-induced enzymes, which affect RNA and protein metabolism, might be responsible for many of its effects on viruses. Indeed, inhibition of viral protein and RNA synthesis appears to have a major role in the antiviral state. We have now investigated possible interactions of the two enzymes with viral constituents during the course of infection and found that in two different membrane-coated RNA viruses, vesicular stomatitis virus (VSV) and Moloney murine leukaemia virus (M-MuLV), there is an accumulation of the 2'-5') oligo-isoadenylate synthetase (E) in the virions. Most of the enzyme is bound to the virion ribonucleoprotein core. The incorporation of E into the virions suggests a direct involvement of the enzyme in regulation of virus functions.     

Expression of AIDS virus envelope gene in recombinant vaccinia viruses

Acquired immune deficiency syndrome (AIDS) is an infectious disease characterized by severe impairment of the patient's cell-mediated immune system. Several lines of evidence have indicated that the aetiological agent of AIDS is a group of T-lymphotropic retroviruses, variously known as lymphadenopathy-associated virus (LAV), human T-lymphotropic virus type III (HTLV-III) and AIDS-associated retrovirus (ARV). Serological surveys have indicated that as many as one million people in the United States may have been infected by LAV/HTLV-III, and the spread of AIDS has become a global concern. The need for a better understanding of the viral immunology and for a vaccine against AIDS is self-evident. To this end, we have constructed recombinant vaccinia viruses containing the envelope (env) gene of LAV, and demonstrate here that cells infected with these viruses express immunoreactive proteins similar to those present on LAV virions. Experimental animals infected with these recombinant viruses elicited antibodies that specifically recognized LAV envelope proteins.     

人和动物的多瘤病毒

多瘤病毒分类于乳多空病毒科，ＤＮＡ肿瘤病毒。病毒无囊膜，直径４０ｎｍ～４５ｎｍ，有３种～４种衣壳蛋白，基因组为约５０００对核苷酸组成的双链闭合环状ＤＮＡ。病毒在自然界分布广泛，目前已从人、兔子、小牛、鸟类、啮齿类和灵长类等动物分离到多种多瘤病毒。各病毒内部有共同的属特异性抗原，但大多数病毒表面蛋白无血清学交叉反应。病毒在容许细胞中增殖良好，能使非容许细胞发生转化，在转化细胞中病毒ＤＮＡ以整合到宿主染色体的方式存在。多瘤病毒感染有严格的种特异性，在自然宿主内大多数病毒呈隐性感染，但人和虎皮鹦鹉多瘤病毒对宿主有一定致病性。本文就有关多瘤病毒的感染和生物学特性研究进展作一概述     

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

Viruses have developed diverse non-immune strategies to counteract host-mediated mechanisms that confer resistance to infection. The Vif (virion infectivity factor) proteins are encoded by primate immunodeficiency viruses, most notably human immunodeficiency virus-1 (HIV-1). These proteins are potent regulators of virus infection and replication and are consequently essential for pathogenic infections in vivo. HIV-1 Vif seems to be required during the late stages of virus production for the suppression of an innate antiviral phenotype that resides in human T lymphocytes. Thus, in the absence of Vif, expression of this phenotype renders progeny virions non-infectious. Here, we describe a unique cellular gene, CEM15, whose transient or stable expression in cells that do not normally express CEM15 recreates this phenotype, but whose antiviral action is overcome by the presence of Vif. Because the Vif:CEM15 regulatory circuit is critical for HIV-1 replication, perturbing the circuit may be a promising target for future HIV/AIDS therapies.     

Biologically diverse molecular variants within a single HIV-1 isolate

AIDS is a disorder characterized by a slow progressive impairment of immune function and by infection of human immunodeficiency viruses (HIV-1, HIV-2). Our knowledge of how these viruses cause disease in man, or how the related lentiviruses (visna and equine infectious anaemia virus) cause disease in animals, is still fragmentary. In particular, the significance of genetic variation in HIV-1, occurring within populations, within individuals and over periods of time, and the mechanisms of viral persistence remain unclear. To address these issues we prepared a series of proviral clones of HIV-1 originating from a single patient and compared their biological properties. Here we show that hybrid genomes (in which the envelope region of six viral clones were separately substituted into a prototype HIV-1 genome) generated viruses with widely differing capacity to grow in human T cells, cell lines and monocytoid cultures. These data suggest that extensive biological variation exists in vivo within an infected individual and is in part determined at the level of the viral envelope.     

Bombyx mori bidensovirus: The type species of the new genus Bidensovirus in the new family Bidnaviridae

Bombyx mori bidensovirus （BmBDV）, which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut columnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence sug- gests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies.     

质型多角体病毒在家蚕体内入侵与复制研究

采用在家蚕活体内研究家蚕质型多角体病毒(BmCPV)的入侵增殖复制过程,探讨BmCPV的入侵过程和增殖复制机制。实验用健康的三龄起蚕经口接种BmCPV后不同时间取中肠后部固定,制作电镜样品,在透射电子显微镜下观察,结果发现BmCPV病毒被食下1.5 h后,在中肠上皮组织圆筒形细胞微绒毛之间有病毒粒子存在,6 h后病毒进入微绒毛内,并向细胞质移动,9 h后观察到圆筒形细胞质中有病毒粒子。作者认为病毒入侵是以整个病毒粒子穿越中肠围食膜,吸附并进入微绒毛,感染圆筒形细胞,与前人病毒入侵只是髓核进入细胞,而病毒衣壳仍留在细胞外的推测不同。随后病毒核心物质进入细胞核,启动复制循环。经过隐潜期后病毒复制,在感染24 h后,细胞质中形成病毒发生基质,子代病毒开始形成,逐渐增多。感染48 h后,圆筒形细胞质中的多角体蛋白逐渐沉积并将病毒粒子包埋,最终形成新的多角体。     

A role for the nuclear envelope in controlling DNA replication within the cell cycle

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.