早期的病毒进入测定的鉴定和抗病毒化合物的评价

Summary

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Abstract

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Introduction

Viral infections are a constant threat to the public health and a significant cause of epidemic diseases, morbidity, and deaths worldwide. Specific modes of control against viral infections include vaccine development and antiviral therapies. While vaccine efforts have proven successful in immunizing against several viruses, many viral pathogens remain without a protective vaccine including dengue virus (DENV), human cytomegalovirus (HCMV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV)^1-5. Antivirals, on the other hand, play an important role for the management of these viral infections when prophylactic vaccines are unavailable. However, to date, only few licensed and cost-effective antiviral drugs are available compared to the number of viral pathogens that threatens the public health. More importantly, due to an increase in global travel and rapid urbanization, the situation is aggravated by risks of epidemic outbreaks from emerging/re-emerging viral infections that are being introduced into non-indigenous areas⁶. Recent outbreaks caused by severe acute respiratory syndrome (SARS) virus, influenza viruses (H1N1, H5N1, H3N2, and H7N9), DENV, West Nile virus (WNV), measles virus (MV), Middle East Respiratory Syndrome (MERS) virus, and Ebola virus^6-12 are among the examples reflecting the need for antivirals development when immunization and/or therapeutics are unavailable. In addition, there is always a potential risk of generating drug-resistant infections with currently used antivirals. Thus, the continuous development and expansion of the scope of antivirals to these emerging/re-emerging viral infections are necessary to provide better management strategies and safeguard the public health.

Most antiviral therapies consist of direct acting antivirals (DAAs) which target a specific viral protein or cofactor that mediates important steps in the viral life cycle. For example, the nucleoside analogue Acyclovir inhibits herpesvirus DNA polymerase, protease inhibitors Boceprevir and Telaprevir antagonize the HCV NS3, and Oseltamivir and Zanamivir are neuraminidase inhibitors that block the release of influenza virus particles from infected cells^13-15. There are however very few licensed viral entry inhibitors including Enfuvirtide, which targets HIV gp41 to prevent fusion, and Maraviroc, which blocks the HIV co-receptor CCR5, thereby preventing viral entry¹⁶. Exploring novel antagonists/inhibitors to viral entry could help provide additional therapeutics for prophylactic or therapeutic use, such as in combination with other antivirals with a different mechanism of action to better manage viral infections^17-19.

Identification of antivirals can involve structure-guided drug design and candidate drug screening-based strategy. Methods for assessing antiviral activity of test agents include biochemical assays of enzymatic activity and evaluation by cell-based systems^20-23. In cell-based systems, the viral life cycle can be dissected into distinct stages of infection, such as entry events (attachment, fusion, uncoating), the replication phase (viral genome replication and protein translation), and virion egress (assembly, maturation, and release). Since the assays can be adapted to investigate each specific stage using various tools and methods, this approach allows identification/examination of potential candidate antivirals with a specific mechanism of action targeting the distinct stage analyzed. For instance, to analyze drug effect specifically on the free virus particles prior to binding to the host cell, a ‘viral inactivation assay’ can be performed. In this assay, the virus is allowed to incubate with the test drug and then diluted to titrate out the drug before infecting a cell monolayer. Additional steps such as viral attachment and entry/fusion stages can also be analyzed individually, by shifting the temperature during the infection. For many enveloped viruses, viral entry/fusion at the host cell membrane is greatly facilitated at 37 °C, but is typically precluded at 4 °C which does not affect virus binding^24-29. Finally, the use of reporter viruses or cell systems could facilitate these studies and permit a high-throughput analysis.

We previously employed the cell-based approach and dissected the early entry of various enveloped viruses for the examination of antiviral compounds that potentially antagonize viral entry^30,31. Herein, the various methods used, including viral inactivation, viral attachment, and viral entry/fusion assays, are described.

Protocol

注意：确保涉及细胞培养和病毒感染的所有程序认证的生物安全罩恰当的样品的生物安全水平正在处理进行的。用于说明协议的目的，Gaussia萤光素酶报道标记的HCV被用作模型病毒32。在代表性的结果的情况下，该化合物chebulagic酸（华侨衣馆联合会）和punicalagin（PUG）被用作候选抗病毒剂靶向早期病毒进入过程中与细胞表面糖胺聚糖病毒糖蛋白的相互作用的步骤31。肝素，这是众所周知的干扰的许多病毒30,31,33,34的条目，被用作在这样的上下文阳性对照治疗。对于基本背景上病毒学技术，病毒传播，测定病毒滴度，并感染剂量在噬斑形成单位（PFU）的表达，集中形成单位（FFU），或感染（MOI）的多重性，读者重新ferred参考35。对于现有的例子和用于在代表性的结果示出病毒优化的条件下，读者可参考表1，图1A和图2A列出引用30-32,36-39以及细节。 1.细胞培养，复方制剂，复方细胞毒性生长的相应细胞系的病毒感染的系统要分析（ 表1）。用于HCV，生长的Huh-7.5细胞在Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），200U / ml的青霉素G，200微克/ ml链霉素，和0.5微克/毫升两性霉素B 使用他们各自的溶剂中制备试验化合物和对照：例如，溶解华侨衣馆联合会和PUG在二甲亚砜（DMSO）;在无菌双蒸水准备肝素。对于所有后续稀释，用培养基。 注意：最终concentDMSO在受试化合物的治疗比是在实验中小于1％; 1％的DMSO被包括作为比较，在测定法的阴性对照治疗。 通过使用细胞存活力测定试剂如XTT测定对细胞的病毒感染的试验化合物（如，华侨衣馆联合会和PUG）的细胞毒性（2,3-双[2-甲氧基-4-硝基-5-磺苯基] -5 – 苯基氨基）羰基] -2H-四唑氢氧化物）： 用于HCV，种子的Huh-7.5细胞在96孔板（每孔1×10 4个细胞），并在37℃下在5％CO 2培养箱O / N，得到的单层。 申请DMSO对照（1％）或试验化合物华侨衣馆联合会和PUG增加浓度（例如，0，10，50，100，和500微米），以在培养孔，一式三份。 孵育在37℃下72小时，然后丢弃在板中的培养基，并用200μl磷酸盐缓冲盐水（PBS）洗细胞两次。 加入100μlassayin的从XTT基于体外毒理学检测试剂盒克溶液至每孔，并孵育所述板在37℃另外3小时，以允许XTT甲臜的生产。 确定用酶标仪在492nm处测试波长和690纳米的参照波长的吸光度。 计算使用下面的公式存活细胞的百分数：细胞活力（％）=在/正如×100％，其中“在”和“作为”指的是试验化合物的吸光度和溶剂对照（如1％的DMSO。 ）治疗，分别。确定试验化合物的50％细胞毒性（CC 50）从分析软件的浓度如棱镜的GraphPad根据制造商的协议。 2.读出病毒感染注意：病毒感染的读出取决于所使用的病毒系统上，并且可以涉及方法，如噬菌斑测定法或MEA从记者标记病毒suring记者的信号。的方法，用于检测基于所述萤光素酶报道分子活性的报道子 – 丙型肝炎病毒感染进行说明。 收集从被感染的孔中的上清液，并在17000×g离心在微量澄清5分钟，在4℃。 混合20微升测试上清液至50μl从Gaussia萤光素酶测定试剂盒萤光素酶底物和用发光根据制造商的说明直接测量。 表达的HCV的感染性的相对光单位（RLU），用于确定病毒抑制率（％）的日志10和计算试验化合物根据制造商的方案使用的算法从的GraphPad Prism软件50％有效浓度（EC 50）抗丙型肝炎病毒感染。 3.病毒灭活试验注：潜伏期的例子和病毒的剂量对各种病毒一再列在图1A中 。较高浓度的病毒，也可以通过增加的MOI / PFU测试。 种子的Huh-7.5细胞在96孔板（每孔1×10 4个细胞），并在5％CO 2培养箱在37℃下O / N，得到的单层。 孵育试验化合物或对照（最终浓度为：华侨衣馆联合会= 50μM; PUG = 50μM;肝素= 1,000微克/毫升; DMSO = 1％）与HCV颗粒在37℃（图1A，'长期' ）以1：1的比例。例如，到一个加入100μl病毒接种含有1×10 4 FFU，添加100微升100μM的华侨衣馆联合会工作稀释的;这产生华侨衣馆联合会治疗以50μM的终浓度。 稀释病毒 – 药物混合物以“亚治疗”（无效）的试验化合物的浓度。例如，华侨衣馆联合会和PUG针对HCV的无效浓度为1μM的31;因此这需要稀释50倍，可实现9.8 ml基础培养基（细胞培养用培养基有2％FBS）的病毒 – 药物混合物。 注意：稀释至低于治疗浓度可防止显著相互作用的试验化合物和在宿主细胞表面之间，并且允许检查对无细胞病毒颗粒的治疗效果。请注意，此稀释依赖于试验化合物对特定病毒感染的抗病毒的剂量响应，并先于执行此特定测定31来确定。 为了比较，混合病毒用测试化合物，并立即稀释（无潜伏期），以亚治疗浓度在感染前（图1A，'短期'）。 加入100μl稀释的HCV药物的混合物上的Huh-7.5细胞单层（病毒的量现在是在1×10 2 FFU;终MOI = 0.01），并孵育3小时，在37℃，以允许病毒吸附。 继感染，去除稀释接种，并轻轻地用200微升PBS洗涤孔两次。 注：轻轻地执行清洗，以避免解除细胞。 应用100微升培养基的各孔中，在37℃下72小时。 通过如'2中所述测定上清液的荧光素酶活性分析所得的感染。病毒感染的读数“。 4.病毒附着试验注意：潜伏期和病毒剂量的各种病毒的例子被列在图2A中 ，'附件“。较高浓度的病毒，也可以通过增加的MOI / PFU测试。 种子的Huh-7.5细胞在96孔板（每孔1×10 4个细胞），并在5％CO 2培养箱在37℃下O / N，得到的单层。 预冷板中的单层细胞在4℃FO，R 1小时。 共同处理细胞HCV的接种物（MOI = 0.01）和测试化合物或对照物（终浓度如下：华侨衣馆联合会= 50μM; PUG = 50μM;肝素= 1,000微克/毫升; DMSO = 1％），在4℃下3小时。例如，到90微升病毒接种含有1×10 2 FFU，加入10微升500μM的华侨衣馆联合会工作稀释的;这产生华侨衣馆联合会治疗以50μM的终浓度，并以MOI对细胞单层的感染丙型肝炎病毒= 0.01。 注意：是进行实验在4℃下重要的，因为它允许病毒结合，但排除了其中最有效地发生在37℃的条目。执行在冰上加入病毒和试验化合物，并在4℃冰箱中随后的温育，以确保温度保持在4℃。 除去上清液并轻轻洗涤细胞单层用200μl冰冷的PBS两次。 注：轻轻地执行清洗，以避免解除细胞<。/ LI> 应用100微升培养基的各孔中，在37℃下72小时。 通过如'2中所述测定上清液的荧光素酶活性分析所得的感染。病毒感染的读数“。 5.病毒进入/融合试验注意：潜伏期和病毒剂量为各种病毒的实施例被列在图2A'进入/融合“。较高浓度的病毒，也可以通过增加的MOI / PFU测试。 种子的Huh-7.5细胞在96孔板（每孔1×10 4个细胞），并在5％CO 2培养箱在37℃下O / N，得到的单层。 预冷板中的单层细胞在4℃下1小时。 感染细胞HCV的（MOI = 0.01），在4℃下进行3小时。例如，使用100微升病毒接种含1×10 2 FFU。 注：执行ADDI病毒接种物在冰上并在4℃冰箱中随后的温育和灰保持在4℃下，其允许病毒结合但不条目的温度。 除去上清液，轻轻用200μl冰冷的PBS洗涤细胞单层两次。 注：轻轻地执行清洗，以避免解除细胞。 对待孔用测试化合物或对照（最终浓度为：华侨衣馆联合会= 50μM; PUG = 50μM;肝素= 1,000微克/毫升; DMSO = 1％），并在37℃下进行3小时。例如，加入10微升500微米华侨衣馆联合会的工作稀释到90微升的媒体，混合，把井;这产生华侨衣馆联合会治疗以50μM的终浓度。 注意：从4℃的移位到37℃现在促进病毒进入/融合事件，因此允许测试化合物“在本具体步骤效果的评估。 吸出含有药物的上清并除去非内化胞病毒的任一洗涤用200μl柠檬酸缓冲液（50mM柠檬酸钠，4mM的氯化钾，pH值3.0）或PBS中。在37℃下温育72小时前，应用100微升基础培养基。 通过如'2中所述测定上清液的荧光素酶活性分析所得的感染。病毒感染的读数“。

Representative Results

在图1中，“病毒灭活法”进行检查两个特定的天然化合物华侨衣馆联合会和PUG是否能灭活在无细胞状态的不同包膜病毒和防止后续感染。这些化合物的细胞毒性和抗病毒剂量反应之前已经执行机理研究31来确定。该病毒进行预处理，试验化合物，然后将病毒药物的混合物稀释到亚治疗浓度接种前到各个细胞单层对每种病毒系统。 如图1，两个华侨衣馆联合会和PUG似乎与无细胞的病毒粒子相互作用，导致该受保护的细胞单层从后续感染不可逆作用。两个试验化合物实现了接近100％的抑制抗HCMV，丙型肝炎病毒，和DENV-2，而一个60％ – 80％的块，观察抗MV和RSV。这些结果suggEST是华侨衣馆联合会和PUG必须通过他们的失活和中和他们感染上这些免费的病毒颗粒直接的影响。 在图2中，附着和进入/融合测定法进行了探索对来自人巨细胞病毒，丙型肝炎病毒，DENV-2，MV和RSV这些早期病毒进入相关的事件华侨衣馆联合会和PUG的效果。既华侨衣馆联合会和PUG有效地防止所研究的病毒到相应的宿主细胞结合作为对所得病毒感染（图2，'附件'：浅灰色条）所示的抑制。由两种化合物对病毒附着的抑制效果是相似的针对HCMV的（图2B），丙型肝炎病毒（图2C），DENV-2（图2D）和RSV（图2F），范围从90 – 100％。另一方面，PUG似乎比华侨衣馆联合会抗MV 结合 （图2E）更有效，与来自总重量的抑制率Ø化合物50变 – 80％。对照处理肝素，这是众所周知的方框条目的许多病毒，也抑制的HCMV附着，DENV-2，RSV，广告的MV，但针对HCV效率较低。随后的“病毒进入/融合法”研究华侨衣馆联合会和PUG是否保留在病毒进入/融合阶段的活动（ 图2，“进入/融合”：深灰色条）。同样，两种华侨衣馆联合会和PUG观察到有效地削弱的检查的病毒（图2B – F），该病毒进入/融合步骤，得到50 -在相应的细胞单层的90％的保护作用。肝素也有效抑制进入/融合DENV-2和呼吸道合胞病毒感染，但对人巨细胞病毒，丙型肝炎病毒，和MV（<40％的抑制率平均）少有效。 病毒电池类型巨细胞病毒 <TD> HEL 丙型肝炎病毒咦 – 7.5 DENV-2 维罗 MV CHO-SLAM RSV 喉癌Hep-2 表1：宿主细胞类型为病毒感染用于在代表性的结果中描述的每个病毒感染系统的小区类型指示。关于细胞的其他细节可参考31中找到。 图1.灭活的病毒感染由试验化合物华侨衣馆联合会和PUG的不同病毒，用测试化合物长时间处理 。（保温1.5 -滴定前3小时）;（浅灰色条）或短时间（立即稀释;深灰色稀释至低于治疗concentra前巴）在37℃下灰和感染在各自的宿主细胞随后的分析。实验（左图所示）与最终的病毒浓度的（A）的示意图（PFU /孔或MOI），长期病毒药潜伏期（ⅰ），和随后的温育时间（ⅱ）表示在右边的表中的每个病毒。分析为（B）中的HCMV，（C）的丙型肝炎病毒，（D）的DENV-2，（E）的MV， 和 （F）的RSV中指示每个附加面板。结果绘制针对DMSO阴性对照治疗病毒感染和示出的数据是平均值±来自三个独立实验的平均值（SEM）的标准误差。这个数字已经被修改的参考31。 请点击此处查看该图的放大版本。 试验化合物华侨衣馆联合会和PUG针对病毒附着和进入/融合的抗病毒活性。图2.评价。（A）中的实验程序，病毒浓度（PFU /孔或MOI），和加法和治疗用测试化合物的时间（ⅰ，ⅱ，ⅲ）呈现在原理图和关联的表的每个病毒。在病毒吸附分析（浅灰色条），不同类型的细胞的单层被预先冷却，在4℃保持1小时，然后共同处理与相应的病毒和试验化合物，在4℃（1.5 – 3小时;ⅰ）洗掉接种物和试验化合物用于随后的孵育前（37℃; ii）和检查病毒感染。在病毒进入/融合分析（深灰色条），去籽细胞单层被预冷，在4℃保持1小时，然后挑战与各自病毒在4℃下进行1.5 – 3小时（一）。然后将细胞洗涤并用（二），在此期间温度转换至37℃以促进病毒进入/融合事件试验化合物额外潜伏期处理。在温育结束时，细胞外病毒是由任一柠檬酸盐缓冲液（pH3.0）或PBS洗涤除去细胞进一步培养（ⅲ）进行病毒感染的分析。结果（B）中的HCMV，（C）的丙型肝炎病毒，（D）的DENV-2，（E）的MV，和（F）的RSV中指示每个附加面板。数据绘制对病毒感染的DMSO阴性对照处理和表示为平均值±SEM来自三个独立实验。这个数字已经被修改的参考31。 请点击此处查看该图的放大版本。

Discussion

In this report the methods to identify and evaluate antiviral compounds based on a mechanistic approach of dissecting the early viral entry events were described. Specifically, the assays allowed us to examine the effect of test compounds on free virus particles, viral attachment, and viral entry/fusion. Critical steps were implemented to distinctly evaluate the drug effect on the specific stage of early viral entry. For instance, in the ‘viral inactivation assay’, the dilution of the virus-drug mixture to sub-therapeutic concentration prevents significant interaction between the test compound and the host cell surface by ‘titrating out’ the drug. This ensures that the inhibitory effect observed on the subsequent infection of the host cell is due to a direct interaction between the test compound and the cell-free virions, rather than an effect from the test compound on host cell membrane or membrane-associated molecules, including viral receptors³⁰. Similarly, the shift in temperature between 4 °C (which allows for virus binding but not entry) and 37 °C (which facilitates virus entry/fusion) in the ‘viral attachment assay’ and ‘viral entry/fusion assay’ are crucial to determine the test compound’s effect on each of these specific events. This is feasible due to the temperature sensitivity of enveloped viruses during these steps in the infection^24-29. It is therefore important that the assays are performed at the indicated temperature to ensure the accuracy of the results; for example, by carrying out the experiment on ice to maintain at 4 °C and by placing the sample directly in a 37 °C incubator for the temperature shift. In addition, the use of negative (ex. DMSO solvent for drug preparation) and positive (ex. heparin treatment) controls also help further establish the assays’ accuracy. The utility and applicability of such methods have been demonstrated in many antiviral studies^{26,30,31,40,41}. Note that while heparin is included as a control for all three assays in the context of the representative results, it typically blocks the initial virus binding rather than the ensuing fusion/entry step (as reflected by the data in Figure 2). Additional controls could also be used, such as neutralizing antibodies directed against the virus (for viral inactivation assay), antibodies that mask the cell surface receptors for the virus (for viral attachment assay), and membrane fusion inhibitors (for viral fusion/entry assay).

The assays described in this report, which are specific to the early stages of the viral infection, are useful in terms of application as secondary tests to characterize the mechanism of action of candidate drugs from primary screens which typically target the viral infection more broadly. Alternatively, they could also be incorporated in primary screens if one is specifically looking for inhibitors of early viral entry, including virus inactivating agents, viral attachment antagonists, and inhibitors to viral entry/fusion. In this case, their use allows a more focused and precise screen analysis for the identification of mechanism-specific antiviral candidates, which, in turn, would expedite downstream drug development.

The use of cell-based assays in identifying antiviral agents provides several important advantages compared to biochemical assays, including revealing potential off-target effects (such as cytotoxicity) and adding physiological relevance to the bioactivity of the test agents⁴². These issues are important considerations for deciding whether a candidate agent is of value for continuation in subsequent phases of drug development. Similarly, the early viral entry-specific assays described in this report allow examination of the drug effect on the distinct viral entry stage at the cellular level, and more specifically in the context of an authentic viral infection in vitro. The results obtained from such assays would therefore help better predict the antiviral efficacy of the test compounds and also identify potentially unwanted off-target effects against the host cell. One potential limitation though, is that an in vitro cell-based assay may not completely reflect the actual in vivo entry step in the context of a natural viral infection. Nonetheless, the assays presented in this protocol do serve as an analytical platform for mechanism-based identification and evaluation of novel antiviral agents.

The development of reporter viruses or reporter cell systems to quantitate the amount of viral infection has greatly facilitated cell-based screening and evaluation of antiviral compounds. Examples include the use of recombinant viruses carrying a reporter gene or by means of recombinant human cell lines containing a reporter gene driven by the specific virus promoter^31,43. In this report, the infection from luciferase-tagged HCV can be easily monitored by quantitating the reporter signal, thus facilitating data analysis. By incorporating these useful reporter-based tools, the early viral entry assays described here can essentially be adapted into high-throughput format for mechanism-based screening of small molecule libraries.

In conclusion, a protocol was described for assays dissecting the early viral entry as a means of identifying and evaluating mechanism-specific antiviral compounds. Such assays would be useful for discovering novel antagonists/inhibitors to viral entry and help expand the scope of antiviral agents for development as prophylactic and/or therapeutic treatments.

Materials

DMEM	GIBCO	11995-040
FBS	GIBCO	26140-079
Penicillin-Streptomycin	GIBCO	15070-063
Amphotericin B	GIBCO	15290-018
DMSO	Sigma	D5879
In vitro toxicology assay kit, XTT-based	Sigma	TOX2
PBS pH 7.4	GIBCO	10010023
Microplate reader	Bio-Tek Instrument, Inc.	ELx800
Microcentrifuge	Thermo Scientific	75002420
BioLux Gaussia luciferase assay kit	New England Biolabs	E3300L
Luminometer	Promega	GloMax-20/20
Sodium citrate, dihydrate	Sigma	71402
Potassium chloride	Sigma	P5405