Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure–activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase–activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Zika virus is a positive-sense, single-stranded RNA virus in the Flavivirus genus. Other viruses in this genus include West Nile virus, dengue virus, yellow fever virus, and tick-borne encephalitis (1). Most people who contract Zika virus are asymptomatic; however, in a subset of patients, infection with Zika virus can have catastrophic consequences. When infection occurs during pregnancy, the virus may cross the placental barrier and infect the fetus, resulting in congenital abnormalities, most notably microcephaly (2). Additionally, an increased risk of Guillain–Barré syndrome is associated with Zika virus infection (3). Some patients develop an encephalitis or myelitis (3). Although significant advances in the field have occurred since the South American outbreak of 2015/2016 (4), no drugs or vaccines are approved for the treatment of, or protection from, Zika virus. Additionally, no small molecules have advanced to clinical trials. The global at risk population continues to expand; the first local transmission of Zika virus in Europe was reported in October of 2019 (5). Due to the potential for serious outcomes, optimized treatment strategies and prophylactic measures are needed.Flaviviruses require proteolytic processing of polyprotein precursors to yield a functional viral particle. These cleavages are catalyzed by both host and virally encoded proteases (6). Inhibition of the virus-encoded protease is a strategy for drug development that has proven effective for other viruses. Several small-molecule inhibitors of the Zika virus protease were discovered over the past 3 y (7); however, a combination of factors make the pursuit of additional, specific inhibitors of the Zika virus protease a necessary undertaking. A split-luciferase complementation-based assay identified erythrosin B as an allosteric inhibitor of the Zika virus protease. Erythrosin B inhibited Zika virus replication in human placental and neural progenitor cells (8); however, it is a red food coloring dye (FD&C Red No. 3), and at concentrations higher than the acceptable daily intake (0.1 mg/kg per day) can prevent dopamine transport (9). Separately, an in silico screen predicted that novobiocin, an off-market antibacterial agent, would be a competitive inhibitor of the Zika virus protease. Novobiocin inhibited Zika virus infection in Vero cells, Huh-7 cells, and immunosuppressed mice (10); however, several properties of novobiocin make it unfavorable for the treatment of Zika virus. Novobiocin treatment can result in gastrointestinal discomfort, various forms of skin rashes, and leucopenia, a rare but potentially serious complication (11). Furthermore, serum concentrations are variable following oral administration (11), its penetration into the cerebrospinal fluid is poor (12), and treatment of infants with novobiocin caused inhibition of bilirubin metabolism, resulting in jaundice (13).Flavivirus polyprotein processing occurs in the endoplasmic reticulum by the two-component protease, which consists of the small hydrophobic integral membrane protein, NS2B, and the cytosolic NS3 protein. The importance of the NS2B domain in the formation of an active protease complex was first shown for yellow fever virus and dengue virus (14), and subsequently for Zika virus (15). Using fluorogenic or chromogenic peptide substrates, in vitro assays were established by connecting the NS2B hydrophilic cofactor peptide and NS3 protease domain by a noncleavable, flexible polypeptide linker. Alternatively, the NS2B and NS3 proteins can be expressed on different promoters and allowed to complex in solution. Whether one construct should be used in preference to the other for drug discovery is an open question. Increased activity of the unlinked construct has been observed (15), however, one inhibitor exhibited equivalent IC₅₀ values (16).We report the use of both the linked and unlinked protease constructs in a high-throughput drug screening effort. An artificial intelligence (AI)-driven virtual screen was utilized to increase the inhibitor hit rate. Compounds were then validated in several orthogonal cell-based infection assays. We identified two compounds as potent in vitro Zika virus inhibitors, MK-591 and JNJ-40418677. A third compound, the tetracycline antibiotic methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent Zika virus mouse model.     

Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.

Zika virus (ZIKV), a mosquito-borne enveloped RNA virus that belongs to the Flaviviridae family, has gained notoriety recently, due to its explosive outbreaks and association with serious clinical diseases, such as Guillain-Barré syndrome in adults and microcephaly in newborns (1–4). Currently, no ZIKV-specific therapies or prophylactic vaccines are available (5). ZIKV genome is a positive-sense, single-stranded RNA [ssRNA(+)] (6). The viral replication occurs on the surface of the endoplasmic reticulum (ER), where the double-strand RNA (dsRNA) is synthesized from viral genomic ssRNA(+) and transcribed into new proteins (7, 8). The viral genome is translated into a polyprotein, which is proteolytically processed into three structural proteins (capsid [C], precursor membrane [prM], and envelop [Env]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), by both host and viral proteases (9, 10). ZIKV infection can rewire cellular components to establish viral replication complexes (RCs), which increase the local concentration of viral and cellular factors for efficient viral replication (11–13). However, the function of rewired cytoskeleton network remains elusive in ZIKV infection.Vimentin is the most abundant intermediate filaments (IFs), which generally surrounds the nucleus and extends throughout the cytoplasm, providing help to important biological processes, such as organelle positioning, cell migration, and cell signaling (14). Except for acting as an integrator of cellular mechanical functions, the highly dynamic nature of vimentin filaments enables it to respond rapidly to pathological stimuli (15). Several studies have observed the phenomenon of vimentin network rearrangement in viral infections and proposed that the role of intact vimentin scaffold could contribute to the viral life cycle (16–21). However, evidence for the dynamic changes of vimentin IFs during ZIKV infection and its contribution to RCs integrity and stability remain understudied.In addition to providing a structural scaffold, there is evidence indicating that cytoskeletal proteins also regulate translational apparatus (22). For example, ribosomes can physically associate with microtubules (MTs) and F-actin in different cells (23, 24). Disorganization of F-actin by cytochalasin D impairs local protein synthesis in isolated axoplasmic nerve fibers (25). The interaction between keratin IFs and the Y subunit of eukaryotic elongation factor-1 (eEF1BY) plays an essential role in protein synthesis (26). MTs can bind to the cytoplasmic tail of RRBP1 and take part in ER organization and neuronal polarity (27, 28). However, information on the spatial and functional relationship between vimentin IFs and the translational machinery, especially in the context of virus infection, remain completely unknown.In this study, we investigated the function of vimentin IFs in ZIKV infection. By monitoring spatial-temporal responses of the cellular vimentin network throughout various steps of the ZIKV life cycle, we demonstrated that ZIKV infection induces massive rearrangements of cytoplasmic vimentin. When vimentin protein was genetically depleted from cells, distribution of viral proteins is scattered within infected cells, indicating its “organizer” role. Viral RNA replication, protein synthesis, and virion release are subsequently reduced. Using mass spectrometry (MS) and RNA-sequencing (RNA-seq) analysis, we discovered many intimate interactions between vimentin and RNA-binding proteins (RBPs), and that vimentin facilitates ZIKV RNA replication by interacting with and regulating the level and subcellular distribution of RRBP1, indicating its “regulator” role. Thus, our work establishes important connections among vimentin filaments dynamics, ZIKV RCs, and cellular RBPs in highly effective infection.     

Dengue,chikungunya and Zika virus infections among Dutch travellers to Suriname: a prospective study during the introduction of chikungunya and Zika virus, 2014 to 2017

BackgroundSuriname, a country endemic for dengue virus (DENV), is a popular destination for Dutch travellers visiting friends and relatives and tourist travellers. Chikungunya and Zika virus (CHIKV, ZIKV) were introduced in 2014 and 2015, respectively. Data on infection risks among travellers are limited.AimWe aimed to prospectively study incidence rate (IR) and determinants for DENV, ZIKV and CHIKV infection in adult travellers to Suriname from 2014 through 2017.MethodsParticipants kept a travel diary and were tested for anti-DENV, anti-ZIKV and anti-CHIKV IgG antibodies (Euroimmun). Selected samples were subjected to an in-house DENV and ZIKV PRNT50. The IR (infections/1,000 person-months of travel) and IR ratio and determinants for infection were calculated.ResultsTravel-acquired infections were found in 21 of 481 participants: 18 DENV, four ZIKV and two CHIKV, yielding an IR_DENV of 47.0 (95% CI: 29.6–74.6), IR_ZIKV of 11.6 (95% CI: 4.4–31.0) and IR_CHIKV of 5.6 (95% CI: 1.4–22.2)/1,000 person-months. In nine DENV and three ZIKV infected participants, infections were PRNT50-confirmed, yielding a lower IR_DENV of 23.3 (95% CI: 12.1–44.8) and an IR_ZIKV of 8.4 (95% CI: 2.7–26.1) per 1,000 person-months. Tourist travel was associated with DENV infection. ZIKV and CHIKV infections occurred soon after their reported introductions.ConclusionsDespite an overestimation of serologically confirmed infections, Dutch travellers to Suriname, especially tourists, are at substantial risk of DENV infection. As expected, the risk of contracting ZIKV and CHIKV was highest during outbreaks. Cross-reaction and potential cross-protection of anti-DENV and -ZIKV antibodies should be further explored.     

In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70

To gain insights into the functions of a viral RNA replicase, we have assembled in vitro and entirely from nonplant sources, a fully functional replicase complex of Tomato bushy stunt virus (TBSV). The formation of the TBSV replicase required two purified recombinant TBSV replication proteins, which were obtained from E. coli, the viral RNA replicon, rATP, rGTP, and a yeast cell-free extract. The in vitro assembly of the replicase took place in the membraneous fraction of the yeast extract, in which the viral replicase-RNA complex became RNase- and proteinase-resistant. The assembly of the replicase complex required the heat shock protein 70 (Hsp70 = yeast Ssa1/2p) present in the soluble fraction of the yeast cell-free extract. The assembled TBSV replicase performed a complete replication cycle, synthesizing RNA complementary to the provided RNA replicon and using the complementary RNA as template to synthesize new TBSV replicon RNA.     

寨卡病毒E蛋白及第三结构域的原核表达和多克隆抗体制备

目的 在大肠杆菌中克隆表达和纯化寨卡病毒(Zika Virus,ZIKV)包膜糖蛋白(Envelope Protein,E)及第三结构域(Envelope DomainⅢ,EDⅢ),并制备两种免疫原的鼠多克隆抗体。方法 通过Vero-E6细胞培养扩增ZIKV,提取病毒总RNA并反转录为cDNA,利用E和EDⅢ基因的cDNA序列构建原核表达载体pET32a/E和pET28a/EDⅢ,转入E.coli BL21(DE3),IPTG诱导表达,采用Ni⁺柱亲和层析法纯化蛋白。以纯化的重组蛋白为抗原免疫BALB/c小鼠,采集抗血清,间接ELISA法测定效价,Western Blot检测特异性。结果 成功表达并纯化重组蛋白E和EDⅢ,获得的多克隆抗体效价均达到1:409 600,Western Blot检测多克隆抗体可特异性识别重组E蛋白和EDⅢ以及天然E蛋白。结论 成功制备出特异性抗寨卡病毒E蛋白和EDⅢ的鼠源多克隆抗体,为深入探索寨卡病毒致病机制、检测方法和免疫策略奠定了研究基础。     

L612K变异MxA蛋白体外抑制水泡性口膜炎病毒复制的研究

目的研究L612K变异MxA蛋白抑制水泡性口膜炎病毒（VsV）复制活性。方法将野生型MxA蛋白重组表达载体pcDNA3.1MxA（wT）和L612K变异MxA蛋白载体pcDNA3.1-MxA（L612K）分别瞬时转染Wish细胞,24h后VsV感染细胞,感染后48hMTT检测各组细胞增殖数;另取wish细胞转染MxA载体和对照DNA,24h后加入VsV,感染24h后收集细胞,RT-PCR检测VSV mRNA水平,Western blot检测MxA蛋白的表达水平。结果野生型和L612K变异MxA蛋白均在wish细胞有较好表达;MTT检测L612K变异组细胞增殖数明显低于野生型（t=1.13,P〈0.01）,RT-PCR检测L612K变异组VsVmRNA水平明显高于野生型组（t=0.13,P〈0.01）。结论L612K变异可能使MxA蛋白降低了对VsV的抑制活性。     

Accuracy of the Hammersmith infant neurological examination for the early detection of neurological changes in infants exposed to Zika virus: A case-cohort study

The Hammersmith infant neurological examination (HINE) is a highly predictive tool for the easy and low-cost detection of cerebral palsy. Between 2015 and 2016, the rapid spread of the Zika virus (ZIKV) in Brazil was responsible for an increase in microcephaly cases. This study aimed to verify the accuracy of the HINE for the early detection of neurological problems in Brazilian babies exposed to ZIKV.This was a cross sectional case-control study of children exposed to ZIKV. This study was part of the Jundiaí ZIKV Cohort. Of a total sample of 782 children, 98 were evaluated (26 in the exposed group and 63 in the control group). We included late preterm infants and term infants who were exposed to the ZIKV and were participants in the ZIKV Cohort study. Student''s t-test and stepwise multivariate logistic regression were used to compare groups.Of the 26 items evaluated in the five scored categories of the HINE (cranial nerve function, posture, movements, tone, reflexes, and reactions), only the difference in ankle dorsiflexion between the exposed and the control groups was statistically significant. However, some items showed a significant trend in relation to the control group.Our results demonstrated the importance of early neurological assessment of infants exposed to ZIKV, even in those without a microcephaly diagnosis.     

SEC14L2, a lipid-binding protein,regulates HCV replication in culture with inter- and intra-genotype variations

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商陆抗病毒蛋白体外对HepG2.2.15细胞HBV复制的影响

目的　探讨商陆抗病毒蛋白 (pokeweedantiviralprotein ,PAP)体外抗乙型肝炎病毒 (HBV)的活性。方法　以不同浓度的PAP S作用于培养的HepG2 .2 .15细胞 ,采用ELISA、荧光定量PCR法分别检测细胞上清液中的HBsAg、HBeAg及HBVDNA水平的变化 ,并用MTT比色法检测细胞成活率。结果　随着PAP S浓度的增加 ,对HBsAg、HBVDNA的抑制作用亦逐渐增强 ,不同药物剂量组之间差异十分显著 (P <0 .0 1)。PAP S处理后 96h ,PAP S对HBsAg和HBeAg的半数抑制浓度 (ID50 )分别为 6.9μg/ml和 3 .2 μg/ml ,半数中毒浓度 (CD50 )为 2 9.3 μg/ml,治疗指数 (TI) =ID50 /CD50 分别为 4.3和9.3。MTT试验结果显示 ,药物的细胞毒性也呈剂量依赖型 ,倒置显微镜下观察在 5 0 μg/ml～ 10 0 μg/ml剂量组出现部分细胞脱壁和死亡现象。结论　PAP S在体外有直接的抗HBV作用。。     

Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide

Summary.  The role of the p38 mitogen-activated protein kinase (MAPK) pathway in hepatitis B virus (HBV) replication was investigated in this study. After transient transfection with HBV plasmid, p38 MAPK, but not JNK or ERK1/2, was significantly phosphorylated in human hepatoma cell Huh7. Interestingly, HBV proteins and RNA synthesis were significantly inhibited by a specific inhibitor of p38 MAPK, SB203580, in a dose-dependent manner. Intracellular core-associated DNA, extracellular virion-associated DNA and covalently closed circular DNA were also significantly inhibited by SB203580. Further results showed the antiviral role of nitric oxide (NO) on the suppression of HBV replication and downregulation of p38 MAPK phosphorylation. In conclusion, these results suggested that suppression of phosphorylation of p38 MAPK by inhibitor or NO could inhibit intracellular HBV replication.     

B基因型乙型肝炎病毒感染性克隆的构建及其复制能力评价

目的 构建在亚太地区广泛流行的B基因型HBV可复制性克降,为该基因型相关研究奠定基础.方法 采用分子克隆的方法在体外分别扩增HBV基因组的相应片段,并进行连接,构建含1.3倍HBV基因组的可复制性克隆.体外转染Huh7细胞系以评价其抗原分泌和病毒复制的情况,使用高压尾静脉注射建立急性感染小鼠模型以评价体内病毒分泌情况以及肝内抗原表达的情况.结果 所构建的克隆体外转染Huh7细胞后可分泌高浓度的HBsAg和HBeAg,并可以检测到明显的转录子和复制中间体.高压尾静脉注射小鼠后肝组织内可检测到HBcAg表达,血清中可以检测到HBV DNA,其水平随时间发生动态变化.结论 所构建的克隆在体外及体内均可进行复制,具有良好的复制能力.     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P＜0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P＜0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.     

Correlation of hepatitis B core antigen and beta-catenin expression on hepatocytes in chronic hepatitis B virus infection: relevance to the severity of liver damage and viral replication

BACKGROUND AND AIMS: The topographical distribution of hepatitis B core antigen (HBcAg) is related to the pathogenesis of liver damage caused by hepatitis B virus (HBV) infection. beta-catenin plays an important role in both intracellular adhesion and Wnt signaling transduction pathways. This study investigated the intrahepatic expression of HBcAg and beta-catenin in chronic HBV infection, and correlated the results with the degree of liver damage and viral replication. METHOD: Liver sections from 73 patients with chronic HBV infection were examined immunohistochemically for HBcAg and beta-catenin. RESULTS: The distribution of HBcAg could be classified into four types: only nucleus (C-1), both nucleus and cytoplasm (C-2), only cytoplasm (C-3) and all negative for nucleus and cytoplasm (C-4). Significant differences in serum aminotransferase level, HBV DNA and necroinflammatory score were observed among the different distribution types, and as the distribution of HBcAg changed from C-1 to C-4, fibrosis stage and hepatitis B e antigen (HBeAg) negative/anti-HBe positive rate increased concurrently. The distribution of beta-catenin could be classified into two types: only membrane (B-1) and membrane with nucleus or cytoplasm (B-2). B-2 showed higher serum aminotransferase level and necroinflammatory score than B-1. Between B-1 and B-2, there was no significant difference in serum HBV DNA level or fibrosis stage. CONCLUSIONS: In chronic HBV infection, HBcAg distribution may change from C-1 to C-4 gradually, and in correlation with serum aminotransferase, and HBV DNA and HBeAg negative/anti-HBe positive rate. Nuclear or cytoplasmic distribution of beta-catenin, compared with exclusive membranous distribution of beta-catenin, is related to active hepatitis, but not viral replication.     

The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy

The hepatitis C virus (HCV) is a major causative agent of transfusion-acquired and sporadic non-A, non-B hepatitis worldwide. Infections most often persist and lead, in 50% of all patients, to chronic liver disease. As is characteristic for a member of the family Flaviviridae , HCV has a plus-strand RNA genome encoding a polyprotein, which is cleaved co- and post-translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin-like serine proteinase located in the N-terminal domain of non-structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54-residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X-ray crystal structure and current attempts towards development of efficient inhibitors.     

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.