基于CRISPR/Cas9技术的高通量筛选平台:发掘病毒复制相关宿主分子的新途径

病毒作为严格的细胞内寄生生物,需要多种宿主蛋白辅助其完成生命周期。寻找与病毒复制相关的宿主因子并揭示其作用机制,将有助于阐明病毒的感染机制,为疫病的防治提供新靶标。与RNA干扰技术相比,近年来兴起的CRISPR/Cas9技术能更特异、高效、准确地实现基因组编辑,因而在功能基因研究中得到更广泛应用。而基于CRISPR/Cas9系统的宿主全基因组sgRNA文库高通量筛选技术平台,可快速发现参与病毒侵入、复制等生物学过程的关键宿主因子,通过明确病毒-宿主分子相互作用进而揭示病毒的生命周期,为分子病毒学和免疫学提供了强大的研究工具。本文主要总结了基于CRISPR/Cas9技术的高通量筛选平台的具体筛选流程,归纳和讨论了该平台在筛选调控病毒复制相关宿主因子中的应用现状和发展前景。     

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．     

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.     

猪肠道冠状病毒与入侵受体氨基肽酶N的相互作用

猪肠道冠状病毒是目前危害养猪产业的重要病原。目前已发现能够感染猪肠道的致病性冠状病毒有4种：猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪丁型冠状病毒和猪肠道甲型冠状病毒。冠状病毒感染宿主的第一步是识别宿主细胞膜受体分子并与之结合,随后启动入侵及膜融合进而使病毒基因组进入宿主细胞内部。因此,冠状病毒受体是决定其宿主范围及组织嗜性的关键因素。确定冠状病毒受体及病毒与受体的结合机制对预防新发病毒及开发冠状病毒治疗性药物具有重要意义。猪传染性胃肠炎病毒利用猪氨基肽酶N（aminopeptidase N,APN）作为感染宿主的功能性受体,并利用唾液酸作为辅助结合因子。猪APN最初也被鉴定为猪流行性腹泻病毒的功能性受体,但近年的研究结果与前面的报道存在较大的差异,产生了较大的争议。最近的研究认为,猪丁型冠状病毒的功能性受体也是APN,并且猪丁型冠状病毒能够利用多个物种的APN作为功能性受体,这与其跨物种传播具有密切关系。最新发现的猪肠道甲型冠状病毒则不使用APN作为其入侵受体。本文综述了前面3种猪肠道病毒感染宿主细胞的受体及结合机制的研究进展,并比较分析了猪APN及唾液酸在不同猪肠道冠状病毒入侵宿主过程中结合方式的异同,为进一步研究新发猪肠道冠状病毒受体提供参考。     

疱疹病毒膜融合的分子机制

囊膜病毒与宿主细胞的膜融合是病毒入侵宿主细胞的重要过程,这一过程涉及到病毒囊膜表面糖蛋白与宿主细胞表面受体之间的相互作用和构象变化．疱疹病毒有多个糖蛋白及不同类型的细胞作用受体,相应的受体-糖蛋白复合体构成方式也有多种,其引致的膜融合机制被认为是目前病毒融合机制研究中最复杂的,近年来被广泛研究并取得突破性进展．从病毒糖蛋白与相应受体的结构与功能、受体-糖蛋白复合体的形成与入侵途径,以及膜融合模式几个方面,全面综述疱疹病毒膜融合的分子机制,并展望了未来研究趋势．     

基于包膜蛋白和Tat蛋白筛选HIV-1细胞融合抑制剂的高效方法

旨在建立一个细胞-细胞融合系统,高效筛选对HIV-1病毒细胞-细胞间传播有抑制作用的药物。构建了p EGFP-Tat质粒,将p EGFP-Tat质粒和HIV-1包膜质粒共转染HEK-293T细胞,成为表达Tat蛋白和包膜蛋白的效应细胞,然后与表达CD4及辅助受体和β-半乳糖苷酶、荧光素酶双报告基因的靶细胞TZM-bl融合,建立了细胞-细胞融合系统,并进行条件优化,确定了最佳的融合体系。用阳性融合抑制剂maraviroc以及没有融合抑制作用的AZT和raltegravir作用于该体系,证明该系统可以特异性有效筛选具有细胞融合抑制作用的药物。用该系统测试了8个样本,发现两种样品对融合有一定的抑制作用。该方法背景值低,特异性强,可用来高效筛选具有切断HIV-1病毒细胞-细胞间传播作用的抗病毒药物。     

p53基因表达沉默的稳定Vero细胞系的建立及特性分析

利用慢病毒介导的RNA干扰技术,建立稳定沉默p53基因的Vero细胞模型。设计针对p53基因的shRNA序列,构建p53shRNA慢病毒载体并感染Vero细胞;嘌呤霉素筛选阳性细胞,局部消化法挑选细胞克隆;利用RT-PCR和West-ernblot检测p53的表达水平;利用虫荧光素酶报告系统,分析p53的转录激活功能。RT-PCR和Westernblot结果显示,慢病毒介导的shRNA有效地沉默p53基因表达;与对照组细胞相比,干扰组细胞的p53的转录激活功能明显降低。慢病毒介导的shRNA高效、稳定地沉默了p53基因的表达,同时,有效地降低了p53的转录激活功能,为研究p53的生物学功能提供了有利的工具。     

HIV-1进攻靶细胞的机制及相应环节抑制剂

HIV－1是导致获得性免疫缺陷综合症（AIDS）的流行最广、破坏力最强的病毒。HIV－1分两个步骤特异性地进攻CD4^ 细胞：一是利用表面糖蛋白gp120和靶细胞膜上的受体结合;二是通过跨膜糖蛋白gp41使病毒的包膜和靶细胞的质膜发生融合,经过上述步骤,病毒的核心蛋白和遗传物质得以进入人体,然其中进行复制,遇时,细胞膜的稳定性被破坏,细胞的内外环境失去平衡,最终导致细胞死亡。HIV－1进攻靶细胞的机制研究所取得的成就为研制安全有效的抗HIV／AIDS药物提供了新的思路和方向。     

高通量筛选孤儿受体和膜结合配体的MACS/FACS方法的建立

本文报道了一种用于分离、筛选孤儿受体和膜结合配体的高通量的表达克隆系统(expression cloning system).该系统是以高效表达的逆转录病毒载体为核心,以高通量的磁力分离法（magnetic cell sorter,MACS）和高精确度分选型流式细胞仪（FACS）相结合的独特的筛选方法.具体方法是将配体通过生物反应对固定在磁粒上 （如biotin/streptavidin, Fc/protein A等）.该磁粒的特点是非常微小,相当一个病毒颗粒.当配体蛋白结合到表达受体的BaF3细胞上时,该细胞将停留在MACS磁场中,从而与非受体表达细胞分开.MACS的优点是效率高,可同时操作上亿细胞.这些细胞是无法直接在分选型流式细胞仪上操作的.当这些阳性细胞增殖后,再次用同一配体染色,用更精确的分选型流式细胞仪分离,直至完全纯化.受体基因将用PCR方法,用载体引物克隆.利用该系统可以从1000 000个细胞中筛选出低至2个表达配体/受体的阳性细胞.为验证该系统的可行性,应用该系统在相应的cDNA文库中筛选到2个已知基因的细胞受体.应用诱饵B7-1从人T淋巴细胞cDNA文库中筛选其功能受体CD28.应用B7-H2作为诱饵,从活化的人T淋巴细胞cDNA文库中筛选到ICOS.最终,从纯化的受体表达细胞中分离出受体的cDNA编码序列.这些结果表明,改进的表达克隆系统适合于大规模分离孤儿受体和膜结合配体,以及在后基因组时代用于研究新蛋白的功能.     

口蹄疫病毒入侵宿主细胞研究进展

口蹄疫病毒(FMDV)是小RNA病毒科,口蹄疫病毒属的典型成员,是一种基因组大约含有8 400个核苷酸的无囊膜单股正链RNA病毒。大量研究发现识别细胞表面受体并侵入细胞是FMDV感染宿主细胞非常重要的环节;对FMDV而言,利用哪种受体就决定了利用哪种內吞路径。近年来在口蹄疫病毒入侵宿主细胞方面进行了大量研究,在一定程度上解释了口蹄疫病毒感染机制方面的问题,为解决实际生产问题提供了重要依据。对前期工作进行阶段性总结,为后期深入研究口蹄疫病毒致病机制和探索更有效的防治措施提供参考。     

Efficient Infection Mediated by Viral Receptors Incorporated into Retroviral Particles

Many host cell surface proteins, including viral receptors, are incorporated into enveloped viruses. To address the functional significance of these host proteins, murine leukemia viruses containing the cellular receptors for Rous sarcoma virus (Tva) or ecotropic murine leukemia virus (MCAT-1) were produced. These receptor-pseudotyped viruses efficiently infect cells expressing the cognate viral envelope glycoproteins, with titers of up to 10⁵ infectious units per milliliter for the Tva pseudotypes. Receptor and viral glycoprotein specificity and functional requirements are maintained, suggesting that receptor pseudotype infection recapitulates events of normal viral entry. The ability of the Tva and MCAT-1 pseudotypes to infect cells efficiently suggests that, in contrast to human immunodeficiency virus type 1 entry, neither of these retroviral receptors requires a coreceptor for membrane fusion. In addition, the ability of receptor pseudotypes to target infected cells suggests that they may be useful therapeutic reagents for directing infection of viral vectors. Receptor-pseudotyped viruses may be useful for identifying new viral receptors or for defining functional requirements of known receptors. Moreover, this work suggests that the production of receptor pseudotypes in vivo could provide a mechanism for expanded viral tropism with potential effects on the pathogenesis and evolution of the virus.     

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.     

Dissection of gammaretroviral receptor function by using type III phosphate transporters as models

Gammaretroviruses that enter cells via binding to a surface receptor use one of two fundamental mechanisms. In the first, binding of the virus particle to its cognate receptor is followed by fusion and internalization. The second, less common mechanism requires the addition of an accessory protein in order to achieve fusion and entry into the target cells; this protein is usually the soluble form of the envelope protein containing the receptor-binding domain (RBD). For some viruses, such as amphotropic murine leukemia virus (A-MLV), particles with fusion-defective envelope proteins can enter cells in the presence of their own RBD or that of another viral envelope, regardless of its cognate receptor, suggesting that these viruses share a common entry mechanism. A notable exception is gibbon ape leukemia virus (GALV). Fusion-impaired GALV envelope mutants can be trans-activated for infectivity only by GALV RBDs. Using dually functional GALV/A-MLV receptors, we examined the role of receptor with respect to which RBD could overcome fusion impaired virus entry.     

The three faces of paramyxovirus attachment proteins

Enveloped viruses encode membrane-associated glycoproteins that direct the initial stages of virus infection. These usually oligomeric structures bind virions to cell surface receptors and, subsequently, direct fusion of viral membranes with cellular membranes. These structures are also the primary targets of neutralizing antibody as well as potential targets for antiviral agents. In several systems, solving the structure of a virus surface glycoprotein has been enormously valuable to our understanding of virus entry and the mechanisms of entry inhibition. The recent report of the structure of a paramyxovirus attachment protein should clarify the mechanism of cell entry by these viruses.     

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.     

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.     

Novel Insights into Cell Entry of Emerging Human Pathogenic Arenaviruses

Viral hemorrhagic fevers caused by emerging RNA viruses of the Arenavirus family are among the most devastating human diseases. Climate change, global trade, and increasing urbanization promote the emergence and re-emergence of these human pathogenic viruses. Emerging pathogenic arenaviruses are of zoonotic origin and reservoir-to-human transmission is crucial for spillover into human populations. Host cell attachment and entry are the first and most fundamental steps of every virus infection and represent major barriers for zoonotic transmission. During host cell invasion, viruses critically depend on cellular factors, including receptors, co-receptors, and regulatory proteins of endocytosis. An in-depth understanding of the complex interaction of a virus with cellular factors implicated in host cell entry is therefore crucial to predict the risk of zoonotic transmission, define the tissue tropism, and assess disease potential. Over the past years, investigation of the molecular and cellular mechanisms underlying host cell invasion of human pathogenic arenaviruses uncovered remarkable viral strategies and provided novel insights into viral adaptation and virus–host co-evolution that will be covered in the present review.     

Human T-cell leukemia virus type 1 receptor expression among syncytium-resistant cell lines revealed by a novel surface glycoprotein-immunoadhesin

The envelope glycoproteins of human T-cell leukemia virus type 1 (HTLV-1) perform functions that are crucial for virus entry into cells. The surface glycoprotein (SU) is responsible for viral recognition of, and binding to, target cells through its interaction with an unknown cell surface receptor. To facilitate molecular analysis of the receptor-binding properties of SU and to characterize the cellular receptor employed by HTLV-1, we have expressed a recombinant SU fused to the Fc domain of human immunoglobulin G. Here, we demonstrate that this novel SU-immunoadhesin retains both the biochemical properties of Fc and the receptor-binding specificity of the HTLV-1 SU. We use this SU-immunoadhesin to demonstrate, by direct cell surface binding assays, that the receptor used by HTLV-1 has been conserved through vertebrate evolution. Moreover, using murine-human somatic cell hybrids we provide data that do not support the previously assigned location for the HTLV-1 receptor on human chromosome 17. Most importantly, we show that many cell lines that are resistant to HTLV-1 envelope-mediated infection and syncytium formation express functional receptors that are recognized by the HTLV-1 SU. Based on our results, we suggest that for some HTLV-1-resistant cell lines the block to viral entry occurs at a late post-receptor-binding step of the entry process. Our findings will be of value in developing new strategies to identify the cellular receptor used by HTLV-1.     

The role of cellular adhesion molecules in virus attachment and entry

As obligate intracellular parasites, viruses must traverse the host-cell plasma membrane to initiate infection. This presents a formidable barrier, which they have evolved diverse strategies to overcome. Common to all entry pathways, however, is a mechanism of specific attachment to cell-surface macromolecules or ‘receptors’. Receptor usage frequently defines viral tropism, and consequently, the evolutionary changes in receptor specificity can lead to emergence of new strains exhibiting altered pathogenicity or host range. Several classes of molecules are exploited as receptors by diverse groups of viruses, including, for example, sialic acid moieties and integrins. In particular, many cell-adhesion molecules that belong to the immunoglobulin-like superfamily of proteins (IgSF CAMs) have been identified as viral receptors. Structural analysis of the interactions between viruses and IgSF CAM receptors has not shown binding to specific features, implying that the Ig-like fold may not be key. Both proteinaceous and enveloped viruses exploit these proteins, however, suggesting convergent evolution of this trait. Their use is surprising given the usually occluded position of CAMs on the cell surface, such as at tight junctions. Nonetheless, the reason for their widespread involvement in virus entry most probably originates in their functional rather than structural characteristics.     

Stochastic entry of enveloped viruses: fusion versus endocytosis

Infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or "spikes," on the viral membrane. The initial entry mechanism is usually classified as fusogenic or endocytotic. However, binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. Both fusion and internalization have been observed to be viable pathways for many viruses. We develop a stochastic model for viral entry that incorporates a competition between receptor-mediated fusion and endocytosis. The relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. We find different parameter regimes where the entry pathway probabilities can be analytically expressed. Experimental tests of our mechanistic hypotheses are proposed and discussed.