Special Topic : Infection and Immunity New targets for controlling Ebola virus disease

埃博拉病毒病是由纤丝病毒科(filoviridae)的埃博拉病毒(Ebola virus，EBOV)所引起的一种急性出血性传染病，该病病死率极高，达50%-90%, 是人类最致命的病毒性传染病之一。世界卫生组织统计数据显示，在2013年底至2015年西非埃博拉疫情大爆发中超过22500人感染埃博拉病毒，因感染丧生的人数达9000人。不论是感染病例数量、死亡人数，还是受影响地区范围，都达到了该病毒1976年被发现以来的最大规模。2014年8 月8 日，EBOV 的爆发被正式做为国际高度关注的突发公共卫生事件。目前，世界各国都没有有效控制埃博拉疫情的药物和治疗方法。 
 
该文结合埃博拉病毒生命周期，从病毒入侵、宿主释放可溶性蛋白等角度分析了25-羟基胆固醇（25-hydroxyl cholesterol，25HC）等天然免疫抗病毒小分子化合物对埃博拉病毒的潜在抑制作用，从预防埃博拉病毒感染角度阐述了新的药物靶点的研究战略进展，运用系统生物学方法阐述了病原体-宿主之间的相互作用。 
 
文章首先介绍了靶向埃博拉病毒入侵宿主细胞过程的小分子抑制剂的研究进展。不同于其他包膜病毒，埃博拉病毒是通过诱导细胞启动微胞饮（Macropinocytosis）作用而侵入细胞内部。最近研究显示，two-pore channel(TPC)蛋白家族在埃博拉病毒入侵宿主细胞的过程中扮演着重要角色，而小分子化合物Tetrandrine能够潜在特异性地抑制TPC通道，从而阻碍病毒的入侵过程。因此，类似于TPCs，靶向病毒入侵的其他小分子抑制药物还有待进一步研究发现。同时，宿主编码的干扰素诱导ISG蛋白具有强烈的抗病毒感染天然免疫效应功能。作者实验室研究结果显示胆固醇-25 羟化酶（Cholesterol-25-hydroxylase，CH25H）能够将胆固醇转化成一种可溶性因子-25 羟基胆固醇（25HC）。在P-4生物安全等级研究中证明25HC可显著降低野生型EBOV（Zaire Strain，扎伊尔株）对人体细胞的感染，显示其具有作为新型抗埃博拉病毒感染的紧急治疗药物的巨大潜力。 
 
除了靶向病毒入侵过程，文章还描述了靶向病毒出芽从宿主细胞脱落、感染细胞释放可溶性蛋白过程的药物研究进展。其中，埃博拉病毒GP蛋白，无论是覆盖在病毒表面，还是在感染过程中从受感染细胞脱落，都可以参与多种免疫应答反应，在病毒生命周期以及宿主-病原体相互作用过程中扮演重要角色，是抑制埃博拉病毒侵染、复制、发病过程的潜在有效靶点。 
 
目前，为国家抗击EBOV 病毒侵染、传播的小分子药物应急战略储备，研制和制备高效、安全且能快速效治疗埃博拉病毒的药物迫在眉睫。针对不同靶点的小分子抑制药物，进一步阐明埃博拉病毒病的发病机理还有待进一步研究。（


PERSPECTIVES MICROBIOLOGY Special Topic: Infection and Immunity
New targets for controlling Ebola virus disease F. Xiao-Feng Qin 1, * , Cheng-Yu Jiang 2,3 , Taijiao Jiang 1,4 and Genhong Cheng 1,5, * EBOV infects multiple different cell types and replicates rapidly in vivo [1].Due to its long filamentous structure, EBOV is internalized by macropinocytosis after cell surface receptor binding by the viral glycoprotein (GP).Unlike many other enveloped viruses, EBOV entry process involves multiple steps of traversing of the virion through endosomal vesicle route before its nuclear core is released into cell cytoplasm for replication [2].In an attempt to systematically delineate the complex process how EBOV enters host cells, Sakurai et al. has recently revealed that two previous unknown calcium channels belonging to two-pore channel (TPC) family play the central role in the late steps of EBOV entry process [3].TPCs are the major calcium channels activated by NAADP (nicotinic acid adenine dinucleotide phosphate) as well as phosphatidylinositol 3,5-biphosphate (PI(3,5)P2) in the late endosomes required for their acidification and maturation.In the absence of TPCs or when their channel activity is blocked, even though EBOV virion can still travel to the late endosomal/lysosomal compartment, it cannot penetrate into the cytoplasm.Interestingly, this impairment is not due to the failure of cleavage of EBOV GP by cathepsin B/L or trafficking of the GPcleaved viral particles to the NPC1+ endosome compartments, but is caused by blockage of viral-host membrane fusion [3].One of the most significant findings of this work is that small molecule compound Tetrandrine, originally from Chinese herb medicine (such as Stephania tetrandra) can potently and selectively block EBOV entry through its inhibition on TPC channels, with IC50 as low as 55 nM [3].Thus, the lengthy entry process is EBOV's Achilles' heel; like TPCs, other host factors required for this process are good candidates to be targeted by small molecule inhibitors; however, further tests need to be performed in animal models infected with wild-type Ebola viruses.
As oppose to the pro-viral host factors, a number of proteins produced by host cells can strongly antagonize EBOV infection.Interferon-stimulated gene (ISG) encoded host factors are well known for their role in antiviral function [4].Among them, IFITM1,2,3 are highly effective in inhibiting EBOV entry by blocking membrane fusion in late endosome/ lysosome [2].
Cholesterol-25-hydroxylase (CH25H) is another ISG protein that can potently inhibit EBOV infection, which was initially identified by our high-throughput overexpression screening [5].CH25H encodes an enzyme catalyzing the hydroxylation of cholesterol at position 25 in host cells, thus forming and releasing 25-hydroxyl cholesterol (25HC) into the circulation [4,6].
Importantly, our work shows that it is 25HC responsible for the antiviral activity of CH25H [5].Indeed, other oxysterols (hydroxylated cholesterols) can also effectively inhibit EBOV infection.In fact, antiviral activity of some synthetic 25HC oxysterol is even much stronger that the naturally existing 25HC [5,6].Mechanistic studies further show that 25HC also operates at viral entry step, blocking the viral-host membrane fusion [6].It will be interesting to see whether 25HC can act synergistically with IFITM1,2,3 in antagonizing EBOV entry in late endosome/lysosome compartment, as both affect the curving and synapse formation in the final step of membrane fusion.No doubt, better understanding of the molecule events that control viral-host membrane fusion would aid the development of more potent and specific drugs inhibiting EBOV entry.
Apart from targeting entry, blocking viral particle budding and release from host cells also should also be a valid strategy for controlling EBOV infection and pathogenesis [1,2].In this regard, another ISG protein, Tetherin, has been shown to effectively inhibit EBOV budding.Intriguingly, EBOV GP can antagonize this function.GP interacts with Tetherin in the viral budding raft, and such interaction prevents the formation of multimeric complex of Tetherin, which is required for blocking EBOV virion release [7,8].Thus, it is feasible that compounds targeting GP-Tetherin interaction can boost up Tetherin's anti-EBOV activity.
In addition to the intracellular functions, soluble forms of EBOV GP released from the infected cells can thwart the neutralization activity of the antiviral antibodies and dampen adaptive immune responses in general [2].More recently, work by Sheng et al. has revealed another danger nexus of EBOV GP in blood vessel pathogenesis [9].The study shows that GP can induce high-level expression of mi-RNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p in endothelial cells, which in turn downregulates protein levels of adhesion-related molecules tissue factor pathway inhibitor (TFPI), dystroglycan1 (DAG1) and the caspase 8 and FADD-like apoptosis regulator (CFLAR) [9].Such alteration leads to severe apoptosis of endothelial cells and thus damage of blood vessels, which is associated with hemorrhagic fever syndrome.
As discussed above, EBOV GP clearly plays a central role in viral life cycle and host-pathogen interaction, and therefore is a high-value target for disease intervention.Indeed, our recent computational modeling work has put this view in another perspective.We show that GP forms a major node of comutation network for EBOV evolution, one of the key contributing factors to its case fatality rate, i.e. the pathogenicity power of the virus [10].Furthermore, the study has also revealed a high degree of connectivity of GP with the viral NP and L proteins in the comutation network, which is entirely unexpected, suggesting potentially important functional relevance [10].Experiments are under the way in testing whether paired comutations are involved in physical interactions and whether such interactions can be exploited for drug targeting to control viral infection, replication or pathogenesis.In summary, as EBOV can infect and replicate rapidly once inside the human body, a systems approach should be implemented to identify multiple drug targeting sites to hit this deadly virus from head to toe all around its way (Fig. 1).Recent progress in the field has shown that such systematic research effort is highly promising and will lead to the development of effective therapeutics to intervene Ebola viral disease in near future.

Figure 1 .
Figure1.A systems approach to identify host factors which can serve as novel drug targets for Ebola viral disease intervention.Genome wide or focused overexpression or RNAi/CRISPR gene knockdown/knockout screenings are carried out in vitro using EBOV reporter virus systems.Both anti-or pro-EBOV host factors can be identified through such functional screenings.Genes encoding anti-or pro-EBOV host factors will be further characterized by host-pathogen protein-protein interaction mapping, structure-function analysis and computational modeling to reveal potential drug targets.We anticipate the drug targets will include host endosomal proteins (such as TPCs), ISGs (like IFITMs) and miRNAs, which play pivotal roles in controlling the key steps of EBOV life cycle.