植物病毒病媒介昆虫的传毒特性和机制研究进展

植物病毒病是农作物的“癌症”, 至今缺少有效的防治方法。目前已知80%的植物病毒病依赖于媒介昆虫传播, 而媒介昆虫对植物病毒的传播是一个昆虫、 病毒、 寄主植物互作的过程, 历经获毒、 持毒和传毒等多个阶段, 昆虫体内一系列病毒受体或蛋白参与了这个过程。昆虫传播病毒的方式有口针携带式、 前肠保留式和体内循环式3类, 它们各自对应的持久性为非持久性、 半持久性和持久性, 不同昆虫获取这3类病毒的获毒时间、 在体内存留位置和传毒时间也各不相同。 这个过程受到媒介昆虫的性别及龄期、 寄主植物、 环境条件、 昆虫体内共生菌等多种因素的影响。与之相关的蛋白主要有病毒衣壳蛋白(CP)、 次要衣壳蛋白（CPm）、 GroEL蛋白、 辅助因子(HC)和下颚口针蛋白等。近年来对植物病毒基因组的研究也取得了很大的进展, 对昆虫传毒机制的研究正受到越来越广泛的关注。本文综述了近年来该领域内的相关研究进展, 包括昆虫传播植物病毒的传毒方式、 影响传毒效率的因素、 传毒机制特别是昆虫体内与病毒传播可能相关的受体等。     

病毒的细胞进入研究进展及其应用前景

病毒感染的初期事件包括病毒与细胞表面受体的相互作用和进入细胞的过程,而病毒的宿主细胞专一性很大程度上取决于这一阶段的专一识别特征和特殊要求。人乳头状瘤病毒、人免疫缺陷病毒和单纯疱疹病毒是感染人类的几种常见病原物,该文简要综述和讨论了与人体健康关系密切的这三种重要病毒表面的蛋白组分、宿主细胞表面受体及其相互作用和病毒的细胞进入的研究进展,以及在以病毒的细胞进入过程为靶点的抗病毒药物研发中的应用前景。     

麻疹病毒受体与病毒侵入

麻疹病毒是一种具囊膜的负链RNA病毒,两种主要的囊膜蛋白血凝素蛋白(H)和膜融合蛋白(F)表达在膜表面负责病毒侵入过程中与宿主受体的结合和膜融合过程.病毒囊膜蛋白与受体的相互作用是病毒侵入宿主的关键步骤,决定了病毒感染能力、种属和组织嗜性.因此,囊膜病毒与受体的结合位点往往成为重要的抗病毒药物的靶点.目前已发现的3种麻疹病毒受体包括CD46、SLAM和Nectin-4.以下综述了麻疹病毒受体的特征及在病毒侵入中的作用、麻疹病毒H蛋白与受体的相互作用机制,为抗病毒药物设计及麻疹病毒作为肿瘤治疗性载体的应用提供理论依据.     

唾液酸受体并非流感病毒各亚型在雪貂组织中播散分布的决定因子

目的探讨流感病毒在雪貂组织中的分布与唾液酸受体的关系。方法用病毒分离的方法分析流感病毒H5N1（SZ406H,A/VN/1203/04）,SH1N1,H3N2（Brisbane/09,HK/09）在雪貂各组织中分布,用直接免疫荧光法分析雪貂各组织的唾液酸受体的分布,并通过体外实验证实活病毒与组织上受体的结合。结果 H5N1（SZ）和H5N1（A/VN/1203/04）在雪貂的肝、脾、肺、肠中有分布,H5N1（A/VN/1203/04）在脑组织中也有分布,而SH1N1、H3N2（Brisbane/09,HK/09）只分布于肠组织。而唾液酸受体SAα2,6Gal和SAα2,3Gal的I型受体分布于脾、心、肺、肠、脑组织中,和SAα2,3Gal II型受体分布于肝、脾、心、肺、肠、脑组织。SH1N1病毒与SAα2,6Gal能结合,而H5N1与SAα2,3Gal结合。结论 H5N1能在雪貂的多器官组织组织中分布和繁殖,而H3N2和SH1N1仅能在肠组织中分布繁殖。SAα2,6Gal和SAα2,3Gal受体在雪貂多器官组织中均有表达,说明唾液酸受体是病毒进入的门户,但不是病毒分布的决定因子。     

高效薄层层析-病毒覆盖结合法比较细胞表面神经节苷脂与不同动物源性新城疫病毒结合特性

【目的】神经节苷脂是新城疫病毒入侵宿主细胞的受体,但不同动物源性的新城疫病毒利用受体的特性是否存在差异尚不明确。以鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株为研究对象,比较两株病毒受体结合特性的差异。【方法】提取鸡胚和鹅胚成纤维细胞新城疫病毒受体成分——神经节苷脂,用高效薄层层析法比较两种细胞所含神经节苷脂的类型和含量;通过高效薄层层析-病毒覆盖结合法比较两种病毒与不同细胞神经节苷脂的结合特性,最后通过病毒红细胞吸附抑制试验进一步验证神经节苷脂与病毒之间的相互作用关系。【结果】鸡胚和鹅胚成纤维细胞所含的神经节苷脂成分存在明显差异;高效薄层层析-病毒覆盖结合实验结果显示NA-1和F48E9与神经节苷脂的结合模式不同,NA-1主要与神经节苷脂GD1a结合,即包含双SAα2,3Gal末端的神经节苷脂,而F48E9可与更多类型的神经节苷脂结合,从单唾液酸到三唾液酸形式的神经节苷脂如GM1、GD1a、GD1b、GT1b等。【结论】鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株在入侵靶细胞时优先选择利用的受体不同。     

New insights into viral structure and virus–cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus–cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV–cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.     

高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

综述: 病毒与宿主细胞的糖基化修饰及相关功能

病毒的复制和对宿主的入侵与自身结构蛋白的糖基化修饰密切相关．对于宿主而言,在病毒感染宿主和宿主抗病毒的过程中,宿主的糖基化过程一方面可抑制病毒的复制和入侵,另一方面可促进病毒对宿主的感染,抑制宿主糖苷酶可抑制病毒的复制．从病毒方面来看,由于病毒自身缺乏糖基化修饰系统,病毒的糖基化过程是借宿主细胞内的合成系统对自身进行糖基化修饰．病毒的糖基化过程对病毒蛋白的折叠与稳定、病毒的感染和入侵、参与识别宿主细胞受体和参与病毒的免疫逃逸等过程起着重要的作用．随着糖基化研究技术的发展,以糖基化为基础的功能应用也越来越深入：如新型病毒疫苗和新型抗病毒药物的研制,以糖蛋白质组学研究为基础的质谱技术和生物信息学方法的发展,以及利用糖基化对病毒性疾病的诊断和治疗等,这些均为糖基化深入研究发展奠定了基础．本文就病毒与宿主细胞糖基化过程、相关功能以及研究应用等进展作一综述．     

甲型流感病毒感染过程中干扰素介导的天然免疫应答机制

甲型流感病毒作为引起人类和动物急性呼吸道传染病的一个主要病原体,在世界范围内广泛流行。研究表明,甲型流感病毒感染宿主后会诱导宿主的天然免疫应答。甲型流感病毒感染可引起Toll样受体(Toll like receptors,TLRs)和RIG-Ⅰ样受体(RIG-Ⅰ like receptors,RLRs)等宿主模式识别受体介导的抗病毒信号通路的活化,并在多种机制调控下诱导干扰素和其他细胞因子的表达,如Ⅰ型干扰素、Ⅲ型干扰素等,从而启动干扰素刺激基因(Interferon stimulated genes,ISGs)的转录及其抗病毒蛋白的表达,进而实现抗病毒作用。本文就甲型流感病毒感染与干扰素介导的天然免疫应答相关的信号通路和调控机制进行综述。     

Role of receptor binding specificity in influenza A virus transmission and pathogenesis

The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glycans present on the apical surface of host cells can function as receptors for the influenza virus hemagglutinin (HA) protein. Avian and human influenza viruses typically have a different sialic acid (SA)‐binding preference and only few amino acid changes in the HA protein can cause a switch from avian to human receptor specificity. Recent experiments using glycan arrays, virus histochemistry, animal models, and structural analyses of HA have added a wealth of knowledge on receptor binding specificity. Here, we review recent data on the interaction between influenza virus HA and SA receptors of the host, and the impact on virus host range, pathogenesis, and transmission. Remaining challenges and future research priorities are also discussed.     

A simple macroscopic model for the diffusion and adsorption kinetics of r-adenovirus

The diffusion of viruses toward cells is a limiting step of the infection process. To be modeled correctly, this step must be evaluated in combination with the adsorption of the virus to the cell surface, which is a rapid but reversible step. In this paper, the recombinant adenovirus (rAd) diffusion and its adsorption to 293S cells in suspension were both measured and modeled. First, equilibrium experiments permitted to determine the number of receptors on the surface of 293S (R(T) = 3,500 cell(-1)) and the association constant (K(A) = 1.9 x 10(11) M(-1)) for rAd on these cells based on a simple monovalent adsorption model. Non-specific binding of the virus to the cell surface was not found to be significant. Second, total virus particle degradation rates between 5.2 x 10(-3) and 4.0 x 10(-2) min(-1) were measured at 37 degrees C in culture medium, but no significant virus degradation was observed at 4 degrees C. Third, free viral particle disappearance rates from a mixed suspension of virus and cells were measured at different virus concentrations. Experimental data were compared to a phenomenological dynamic model comprising both the diffusion and the adsorption steps. The diffusion to adsorption ratio, a fitted parameter, confirmed that the contact process of a virus with a cell is indeed diffusion controlled. However, the characteristic diffusion time constants obtained, based on a reversible adsorption model, were eightfolds smaller than those reported in the literature, based on diffusion models that assume irreversible adsorption.     

The Mechanism of Henipavirus Fusion： Examining the Relationships between the Attachment and Fusion Glycoproteins

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.     

Emerging complexity and new roles for the RIG-I-like receptors in innate antiviral immunity

Innate immunity is critical for the control of virus infection and operates to restrict viral susceptibility and direct antiviral immunity for protection from acute or chronic viral-associated diseases including cancer. RIG-I like receptors (RLRs) are cytosolic RNA helicases that function as pathogen recognition receptors to detect RNA pathogen associated molecular patterns (PAMPs) of virus infection. The RLRs include RIG-I, MDA5, and LGP2. They function to recognize and bind to PAMP motifs within viral RNA in a process that directs the RLR to trigger downstream signaling cascades that induce innate immunity that controls viral replication and spread. Products of RLR signaling also serve to modulate the adaptive immune response to infection. Recent studies have additionally connected RLRs to signaling cascades that impart inflammatory and apoptotic responses to virus infection. Viral evasion of RLR signaling supports viral outgrowth and pathogenesis, including the onset of viral-associated cancer.     

Putative dengue virus receptors from mosquito cells

Dengue viruses are arthropod-borne, single-stranded RNA viruses. Aëdes aegypti and Aëdes albopictus are the principal vectors. In order to understand the molecular basis of dengue virus infections we explored the biochemical identity of dengue-2 (DEN-2) virus receptors in the Aëdes albopictus-derived cell line C6/36. We show here that DEN-2 interacts with two major polypeptides of 80 and 67 kDa. Polyclonal anti-C6/36 membrane antibodies block DEN-2 binding to intact C6/36 monolayers as well as to membrane extracts. Our results strongly suggest that the identified polypeptides are part of the DEN-2 receptors.     

流感病毒识别糖链受体分子机制的研究进展

近年来,由于流感病毒(influenza virus)不可预测的局部流行和有可能引发全球大流行,其一直是研究的热点课题之一．流感病毒表面糖蛋白血凝素(hemagglutinin,HA)特异识别宿主细胞表面的糖链受体是流感病毒感染宿主、进而复制并继续传播的生物学基础．影响流感病毒宿主特异性的两个主要因素是HA自身的变化(包括基因突变、重组、糖基化位点数量和糖基化位置的变化)和宿主细胞表面糖链受体的变化(包括糖链受体的类型、分布和分子构象的改变)等．因此准确掌握这些信息有助于人们进一步加强对流感病毒的防控．本文主要从糖组学角度概述了流感病毒识别糖链受体的分子机制,重点介绍流感病毒宿主细胞表面糖链受体的研究进展．     

Putative hepatitis C virus cell receptors

Virus entry into a host cell comprises the first step of the viral life cycle. Blockage of this process can stop or prevent the rise of the infection. Development of compounds exhibiting directed blocking activity requires information about host cell and viral molecules, which are involved into reciprocal recognition resulting in the virus entry into the cell. This review is devoted to the problems of the identification of plasma membrane molecules, involved in binding of hepatitis C virus and its subsequent transfer inside the cells. The putative role of these molecules as hepatitis C virus receptors and co-receptors in the beginning and development of the infection is discussed.     

Human trachea primary epithelial cells express both sialyl(α2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(α2-6)Gal receptor for human influenza viruses

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-α2-3(6)-Galβ1-3(4)-GlcNAcβ1-. In addition, the Siaα2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaα2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in “The Receptors' [Conn M Ed] 2, 131–219 (1985); Suzuki Y, Prog Lipid Res 33, 429–57 (1994); Ito T, J Virol 73, 6743–51 (2000); Suzuki Y, J Virol 74, 11825–31 (2000); Suzuki T, J. Virol 75, 4604–4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399–408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (α2-3 and α2-6), using fluorescence-activated cell-sorting analysis. The results showed that both α2-3- and α2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have α2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaα2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaα2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both α2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and α2-6-linked Sias recognized by human influenza virus receptors.     

Binding affinity of GM3 lactone for influenza virus

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.     

Analysis of the Glycyrrhizic Acid-Binding Sites with Phage Display

Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.     

Rice Ragged Stunt Oryzavirus: role of the viral spike protein in transmission by the insect vector

A 39 kDa protein, known as the viral spike protein or one of the protein components forming the viral spike, encoded by genomic segment 9 (S9) of Rice Ragged Stunt Oryzavirus (RRSV) was obtained by enzymatic cleavage of a fusion protein expressed by S9 cDNA in bacteria with proteinase factor Xa. The feeding of an insect vector — the rice brown planthopper (Nilaparvata lugens) on purified expressed 39 kDa protein before the inoculation of the insects on diseased rice plants could completely inhibit the vector transmission ability of the insect. The presence of a 32 kDa insect cell membrane protein which could bind to 39 kDa viral spike protein indicated that the inhibition might be resulted from the competition in the interactions of 39 kDa protein and intact virus with the virus receptors on the insect cells. These results suggest that the spike proteins of the plant reoviruses are essential for the virus infection in the interactions of virus, insect vectors and host plants. These results are also useful in the practical applications.