Paper intensive reading (十一)：Nuclear localization of Zika virus NS5 contributes to suppression

论文题目：Nuclear localization of Zika virus NS5 contributes to suppression of type I interferon production and response

scholar 引用：0

页数：10

发表时间：20 December 2019

发表刊物：JOURNAL OF GENERAL VIROLOGY，IF 2.809

作者：Zikai Zhao​, Mengying Tao, Wei Han​, Zijing Fan, Muhammad Imran​, Shengbo Cao, Jing Ye，华中农业大学

摘要：

Zika virus (ZIKV,寨卡病毒) is an emerging mosquito-borne flavivirus, which caused an unprecedented epidemic in Latin America. Among all viral non-structural proteins in flavivirus, NS5 is the most highly conserved and has multiple crucial functions, including participating in viral RNA replication and suppressing host innate immunity. Although ZIKV NS5 prominently localizes in the nucleus during infection, its specific nuclear localization signal (NLS), and its role in viral replication and pathogenesis(发病机制) remain controversial. Here, we identified aa 11–90 and aa 370–406 regions that contain NLSs, which are critical for nuclear localization of ZIKV NS5. Further experiments demonstrated that nuclear localization of ZIKV NS5 predominantly participates in suppression of interferon(干扰素) regulatory factor 3 (IRF3)-mediated activation of type I IFN (IFN-I) transcription and inhibition of IFN-I downstream response independent of its effect on signal transducers and activators of transcription 2 (STAT2) degradation. These results suggest that subcellular localization of NS5 is important for its function on innate immune suppression, which provides new insight into ZIKV pathogenesis.

Keywords
Zika virus (ZIKV), NS5, nuclear localization signal (NLS), type I interferon (IFN-I), immune evasion

本篇paper中涉及到的缩写简称：

Cter18, C-terminal 18 amino acids;

DAPI, 4',6-Diamidino-2-Phenylindole;

DENV, dengue virus; 登革热病毒

EGFP, enhanced green fluorescent protein; 增强型绿色荧光蛋白

GST, Glutathione S-transferase; 谷胱甘肽s-转移酶

HCV, Hepatitis C virus; 丙型肝炎病毒

IFNAR1, interferon alpha and beta receptor subunit 1;

IFN-I, type I interferon;

IRF3, interferon regulatory factor 3;

JEV, Japanese encephalitis virus; 日本脑炎病毒

MTase, methyltransferase; 甲基转移酶

NES, nuclear export signal;

NLS, nuclear localization signal;

RdRP, RNA-dependent RNA polymerase; 聚合酶

RT, room temperature; 室温

SeV, Sendai virus; 仙台病毒，乙型副流感病毒

STAT2, signal transducers and activators of transcription 2;

TBEV, tick-borne encephalitis virus; 蜱传脑炎病毒

WNV, West Nile virus; 西尼罗河病毒

YFV, yellow fever virus; 黄热病毒

ZIKV, Zika virus.寨卡病毒

Introduction：

• 寨卡病毒是一种黄热病毒，寨卡病毒感染跟很多神经系统症状有关, 如格林·巴利综合征和头小畸形。
• NS5 is the largest and the most conserved protein of flavivirus.
• During flavivirus infection, a replication complex constituted with NS5 and other non-structural proteins is assembled on the cytoplasmic side of the endoplasmic reticulum (ER) membrane with the help of host proteins 在黄病毒感染过程中，在宿主蛋白的帮助下，由NS5等非结构蛋白组成的复制复合体在内质网(ER)膜的胞质侧组装
• NS5 unusually localizes in the nucleus with no clear role in viral replication and pathogenesis
• IL-8是什么？白细胞介素-8是巨噬细胞和上皮细胞等分泌的细胞因子。白细胞介素-8结合趋化因子受体白细胞介素-8受体α和白细胞介素-8受体β而对嗜中性粒细胞有细胞趋化作用而实现对炎症反应的调节。白细胞介素-8还有很强的促血管生成作用。白细胞介素-8在小支气管炎和囊形纤维化的发病中起重要作用。
• Previous research has demonstrated that DENV NS5 nuclear accumulation affects the production of virus and IL-8, and can also interact with host nuclear active spliceosomes to enhance its replication 登革热病毒的NS5核积累作用已经有了一些研究，但是寨卡病毒在这方面的研究尚不充足。
• kDa是什么单位？生物学中蛋白质的分子量单位kDa也简称为kD 1kDa=1000摩尔质量单位
• It has been reported that proteins, which are larger than 45 kDa, require intrinsic targeting signals called nuclear localization signals (NLSs) to mediate its shuttle between the nucleus and cytoplasm 研究表明，大于45kda的蛋白质需要称为核定位信号(NLSs)的内在目标信号来调节其在细胞核和细胞质之间的穿梭
• CRM1是什么？Chromosome maintenance protein 1 (CRM1) 染色体维持蛋白1
• antagonist 拮抗剂,拮抗剂（antagonist）与受体结合后本身不引起生物学效应，但阻断该受体激动剂介导的作用。根据是否可逆性地与结合到受体的激动剂发生竞争，拮抗剂可以分为两类。竞争性拮抗剂和非竞争性拮抗剂。
• During flavivirus infection, NS5 has been reported as a key antagonist at different IFN-I signalling cascade stage by diverse strategies. 在黄病毒感染过程中，NS5被报道为不同IFN-I信号级联阶段的关键拮抗剂。
• 干扰素具有抗病毒作用，拮抗剂会抑制干扰素的生成，那拮抗剂是对病毒有利的吗？NS5是病毒，自然是帮助病毒复制的呀。懂了~ 参考：蜱传脑炎病毒感染引发细胞信号转导的研究进展
• it is still unclear whether the mechanisms are associated with the subcellular localization. 目前的问题
• In this study, we show the aa 370–406, a conserved NLS in other flaviviruses, rather than the C-terminal 18 residues, which was previously demonstrated as a NLS in DENV NS5, is sufficient to lead the nuclear localization of ZIKV NS5.在DENV病毒中， C-terminal 18 residues；ZIKV病毒不同，是aa 370–406
• Furthermore, a new NLS-containing region aa 11–90 was identified in ZIKV NS5. 在ZIKV中，还发现了一个新的NLS区
• Our results also reveal the relevance of NS5 subcellular localization with its function on modulating IFN-I response, which provides a new understanding on the mechanism of ZIKV immune evasion. 另外，NS5 subcellular localization的功能，也在本研究中获得结果。NS5亚细胞定位与其调节IFN-I应答功能的相关性

正文组织架构：

1. Introduction

2. Methods

2.1 Cells lines, viruses and antibodies 细胞系，病毒和抗体

2.2 Plasmid construction 质粒构建

2.3 Immunofluorescence analysis 免疫荧光分析

2.4 dual-luciferase reporter assay 双重荧光素酶报告基因检测

2.5 RnA extraction and quantitative real-time PCR RNA提取和实时定量PCR

2.6 Immunoprecipitation and Western blotting 免疫沉淀和蛋白质印迹

2.7 Statistical analysis 统计分析

3. Results

3.1 Both aa 1-320 and aa 321-903 of ZIKV NS5 accumulate in the cell nucleus

3.2 Both aa 11-90 and aa 370-406 of ZIKV NS5 contain NLSs

3.3 Residues of ZIKV NS5 are essential for interaction with importin α

3.4 ZIKV NS5 nuclear localization contributes to the suppression of IFn-I production

3.5 ZIKV NS5 nuclear localization is associated with inhibition of IFn-I response

4. Discussion

正文部分内容摘录：

• nuclear localization signal (NLS, 核定位信号) 
• 核定位是什么？核定位序列(Nuclear localization sequence)或者核定位信号——蛋白质的一个结构域，通常为一短的氨基酸序列，它能与入核载体相互作用，使蛋白能被运进细胞核。
• IRF3，干扰素调节因子是什么？干扰素调节因子（Interferon regulatory factors, IRFs）是调节干扰素（interferon, IFN）、干扰素刺激应答基因（interferon stimulated genes, ISG）及其他相关基因表达的重要转录因子，通过调节干扰素、干扰素刺激应答基因和其它密切相关基因表达而发挥多种生物学效应。
• 干扰素是单个核细胞产生的一个糖蛋白类同源细胞因子家族，称为IFN家族，现已经基因重组技术生产，并被广泛应用于治疗白血病、淋巴瘤、实体瘤及病毒、原虫、寄生虫和真菌感染，是一类具有抗病毒、抑制细胞生长和调节细胞免疫学功能的细胞因子，IFN基因表达受干扰素调节因子调节，并经干扰素调节因子及干扰素刺激性应答基因产物介导，激活相关信号转导途径而发挥其生物学效应。
• IFN-α/β 一种干扰素的名称
• 干扰素的性质及类型：干扰素是由多种细胞产生的具有广泛的抗病毒、抗肿瘤和免疫调节作用的可溶性糖蛋白。干扰素在整体上不是均一的分子，可根据产生细胞分为3种类型：白细胞产生的为α型；成纤维细胞产生的为β型；T细胞产生的为ω型。根据干扰素的产生细胞、受体和活性等综合因素将其分为2种类型：Ⅰ型和Ⅱ型。
• Ⅰ型干扰素又称为抗病毒干扰素,其生物活性以抗病毒为主.Ⅰ型干扰素有3种形式：IFNα、IFNβ和IFNω
• Ⅰ型干扰素的主要诱生剂是病毒及人工合成的双链RNA，此外某些细菌和原虫感染及某些细胞因子也能够诱导Ⅰ型干扰素的产生。IFNα和IFNω的表达细胞非常局限，以白细胞为主；但IFNβ则可由几乎所有的有核细胞产生。
• 干扰素的生物活性有较严格的种属特异性，即某一种属细胞产生的干扰素，只能作用于相同种属的细胞。Ⅰ型干扰素的抗病毒作用较强。
• IRF-3又是什么？IRF-3、IRF-5、IRF-7都是病毒诱导产生I类IFN必需的转录因子，在抗病毒感染性免疫反应中发挥重要作用。缺乏IRF-3的大鼠对病毒感染敏感，而缺乏IRF-3的EFS(embryonic fibroblasts胚胎纤维原细胞)不仅病毒诱导的IFN-α/β的基因表达水平下降，表达的亚类也改变。
• 同时缺乏IRF-3/IRF-7的EFS细胞受病毒感染后无IFN-α/β的产生，但经IRF-3/IRF-7共转染的EFS细胞则可以产生正常的IFN-α/β. 表明IRF-3和IRF-7介导病毒诱导性IFN-α/β产生并确保了细胞遭受病毒感染时IFN-α/转录的效率和多样性。
• IRF-3是病毒诱导IFN-β产生所必需的转录因子，具有广泛的组织表达谱，但基因组中呈单拷贝，编码50kD蛋白质，但在病毒感染或IFN处理的细胞中，IRF-3的mRNA水平未见升高。

2. Methods

2.1 Cells lines, viruses and antibodies 细胞系，病毒和抗体

• 交待了细胞、病毒、抗体的来源
• cell lines：human embryonic kidney (HEK 293T) ，Vero，C6/36 ，HeLa 
• viruses：ZIKV SZ01 strain中国科学院武汉病毒研究所的张博博士提供，并在C6 / 36 /细胞中繁殖；Sendai virus (SeV) 华中农业大学农业微生物学国家重点实验室的赵玲博士提供
• antibodies：

1. Monoclonal mouse anti-ZIKV NS5 was generated in our lab. 自己实验室
2. Antibodies against IRF3 (A11118, 1 : 1000), GAPDH (AC002,1 : 3000), Flag (AE005, 1 : 1000) and GFP (AE011, 1 : 1000) were purchased from ABclonal Technology. 买的
3. Antibody against STAT2 was from Cell Signaling Technology (72 604S, 1 : 1000).买的

2.2 Plasmid construction 质粒构建

• 什么是质粒构建？质粒构建是分子生物学研究中最常用的实验技术。原理依赖于限制性核酸内切酶，DNA连接酶和其他修饰酶的作用，分别对目的基因和载体DNA进行适当切割和修饰后，将二者连接在一起，再导入宿主细胞，实现目的基因在宿主细胞内的正确表达。
• All ZIKV NS5 mutants mentioned in the article were generated based on NS5-WT by using overlap extension PCR.
• Briefly, NS5-WT construct was used as template for overlap extension PCR, and the PCR products were digested and cloned into pcDNA4.0 as described above.
• The truncated NS5 fragments including aa 1–320, aa 1–406, aa 321–903 and aa 407–903 were digested with XhoI and KpnI and cloned into pre-cut pEGFP-C1 vector (Clontech). 看起来，aa 1–320， aa 321–903就是全部的NS5吗？
• EGFP-GST fluorescence reporter constructs: 看起来挺复杂的
• Dual-luciferase reporter plasmids储存在实验室中

2.3 Immunofluorescence analysis 免疫荧光分析

• 什么是免疫荧光技术？免疫荧光技术又称荧光抗体技术，是标记免疫技术中发展最早的一种。它是在免疫学、生物化学和显微镜技术的基础上建立起来的一项技术。很早以来就有一些学者试图将抗体分子与一些示踪物质结合，利用抗原抗体反应进行组织或细胞内抗原物质的定位。
• 感觉像protocol的一些内容

2.4 dual-luciferase reporter assay 双荧光素酶报告基因检测

• 什么是双荧光素酶报告基因检测？
• 荧光素酶报告基因是以荧光素为底物来检测萤火虫荧光素酶活性的一种报告系统。荧光素酶可以催化荧光素氧化成氧化荧光素，在荧光素氧化的过程中，会发出生物荧光。荧光素和荧光素酶这一生物发光系统，可以极其灵敏、高效地检测基因的表达。是检测转录因子与目的基因启动子区DNA相互作用的一种检测方法。
• 生物体内DNA转录成RNA是基因表达的关键过程，基因表达调控主要发生在转录水平。基因分子生物学研究领域的趋势之一是逐渐从基因结构和功能分析转到基因顺式作用元件和转录因子及其转录调控机制上，对基因调控的研究将是今后相当长一段时间内功能基因组学研究的热点之一。双荧光素酶报告基因检测已经成为研究转录因子参与基因调控的有效手段，通过对启动子DNA片段的分析，验证启动子结合元件的反式激活能力，探讨转录因子在信号转导中的分子机制。
• 跟百度百科上的技术流程基本类似。
• cells were harvested and lysed(细胞溶解) for measuring firefly and Renilla luciferase activities by Dual-Luciferase Reporter Assay System (Promega).

2.5 RnA extraction and quantitative real-time PCR RNA提取和实时定量PCR

• 实时荧光定量PCR （Quantitative Real-time PCR）是一种在DNA扩增反应中，以荧光化学物质测每次聚合酶链式反应（PCR）循环后产物总量的方法。通过内参或者外参法对待测样品中的特定DNA序列进行定量分析的方法。
• Total RNA in treated cells was extracted using TRIzol Reagent (Invitrogen), and 1 µg RNA was used to synthesize cDNA using a first-strand cDNA synthesis kit (TOYOBO).
• Quantitative real-time PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems) and SYBR Green PCR Master Mix (TOYOBO).
• Data were normalized to the level of β-actin expression in each sample. 

2.6 Immunoprecipitation and Western blotting 免疫沉淀印迹分析

• IP-Western分析仍作为一种热门的技术来鉴定蛋白-蛋白互作，以及多蛋白复合物中的未知蛋白质。其步骤包括细胞裂解、抗体-抗原（免疫）复合物的形成、免疫复合物的沉淀和Western印迹分析
• 感觉也是很protocol的内容

2.7 Statistical analysis 统计分析

• All results are presented as the mean ± sem.
• Statistical significance was determined by Student’s t-test, and a P-value<0.05 was considered to be statistically significant. t检验
• Analyses were conducted using the PRISM v7.0 software program (GraphPad Software).专业的医学绘图软件

3. Results

3.1 Both aa 1-320 and aa 321-903 of ZIKV NS5 accumulate in the cell nucleus

• As shown in Fig. 1a, ZIKV NS5 localized in the nucleus and formed punctuated NS5 nuclear bodies in all types of cells during infection, suggesting the prominent nuclear localization of ZIKV NS5 is independent of cell type.
• 
• 什么是亚细胞定位？亚细胞定位是指某种蛋白或表达产物在细胞内的具体存在部位。例如在核内、胞质内或者细胞膜上存在。GFP是绿色荧光蛋白，在扫描共聚焦显微镜的激光照射下会发出绿色荧光，从而可以精确地定位蛋白质的位置。
• (b). Diagram of EGFP-tagged constructs
• 
• To further explore which region in ZIKV NS5 contributes to its nuclear localization, plasmids encoding EGFP fused gene segments of residues 1–320, 1–406, 321–903 or 407–903 were constructed (Fig. 1c)
• 
•  the residues 320–405 are known as a conserved NLS in flavivirus
• As expected, we found that EGFP-NS5 (1–406) and EGFP-NS5 (321–903), both of which contain aa 321–406, strongly accumulated in the nucleus, while EGFP-NS5 (407–903) showed predominantly cytoplasmic localization (Fig. 1d), suggesting that aa 321–406 may contain the NLS of ZIKV NS5.
• 
• 新发现：Interestingly, we found that EGFP-NS5 (1–320) also accumulated in the nucleus, implying a possible new functional NLS within this region.

3.2 Both aa 11-90 and aa 370-406 of ZIKV NS5 contain NLSs

• To further determine the sequences contributing to the ZIKV NS5 nuclear localization, a fluorescence reporter plasmid was generated by fusing GST to the C-terminal of EGFP (EGFP-GST) (Fig. 2a). 
• As shown in Fig. 2a, the EGFP-GST-2×SV40 fusion protein prominently localized in the nucleus, while the EGFP-GST mostly accumulated in the cytoplasm. 
• 
• The molecular weight of EGFP-GST fusion protein (~53 kDa) exceeds the exclusion limit of nuclear pore complex (NPC), so the nuclear direction ability of any predicted NLS can be validated by inserting it into this reporter plasmid(质粒).
• nuclear pore complex (NPC)核孔复合体是镶嵌在内外核膜上的篮状复合体结构，主要由胞质环、核质环、核篮等结构组成。核孔复合体可以看做一种特殊的跨膜运输蛋白复合体，是一个双功能、双向性的亲水性核质交换通道，控制物质进出细胞核。
• We found that EGFP-GST-(370-406) strongly accumulated in the nucleus, while EGFP-GST-(321-369) mainly accumulated in the cytoplasm, and EGFP-GST-(321-406) showed comparatively balanced distribution between the nucleus and the cytoplasm (Fig. 2b), suggesting aa 370–406 of ZIKV NS5 is a potential NLS，原来就是这样确定NLS的，好像挺简单直观的。
• 
• As expected, cytoplasmic accumulation of Flag-NS5Δ370–406 was observed, which demonstrates that residues 370–406 are responsible for nuclear localization of ZIKV NS5 (Fig. 2b). 缺少了370-406以后，观察到没有聚集在细胞核了，所以更进一步验证了那个想法：residues 370–406 are responsible for nuclear localization of ZIKV NS5
• 接下来的 aa 11-90的验证方法应该是类似的
• step1：aa 1–320 of ZIKV NS5 was divided into three fragments (aa 1–100, aa 101–200 and aa 201–320)
• As shown in Fig. 2c, only EGFP-GST-(1-100) predominantly expressed in the nucleus, implying a possible NLS located within aa 1–100. cNLS Mapper was subsequently used to predict NLS within aa 1–100
• 
• no typical monopartite or bipartite NLS was identified如何鉴定typical NLS？
• step2：the fluorescence distribution of chimeric EGFP-GST proteins fused with different C-terminal or N-terminal truncations of aa 1–100 was analysed (Fig. 2d).
• 
• Only EGFP-GST-(1-90) and EGFP-GST-(11-90) showed the comparable quantity of nuclear accumulation with EGFP-GST-(1-100), while EGFP-GST-(1-70) and EGFP-GST-(21-90) were observed predominantly in cytoplasm, which suggests that aa 11–20 and aa 71–90 are essential for subcellular localization of EGFP-GST-(1-100) (Fig. 2e).
• 
• step3：To investigate whether aa 11–20 and aa 71–90 contain the sequence elements responsible for nuclear localization, residues 1–10, 1–20, 1–30, 61–90, 71–90 and 81–90 were fused with EGFP-GST individually. 见图d
• However, none of them could mediate nuclear transportation of EGFP-GST (Fig. 2f), 这一次没有发现聚集在细胞核的段indicating that no full-length NLS was included in these regions, and multiple stretches of amino acids in aa 11–90 may be responsible for nuclear localization. 
• 
• The critical role of aa 11–90 on nuclear localization of ZIKV NS5 was further confirmed by cytoplasmic accumulation of ZIKV NS5 mutant lacking of aa 11–90 (Flag-NS5Δ11–90) (Fig. 2g). 
• 
• Taken together, these results demonstrate that both aa 370–406 and aa 11–90 can determine the nuclear localization of ZIKV NS5.
• step4：Inconsistent with findings of DENV2, Flag-NS5ΔCter18 of ZIKV showed a similar subcellular distribution with full-length NS5 (Fig. 2h). 
• 
• These findings demonstrated that Cter18 of ZIKV NS5 are not essential for nuclear localization of ZIKV NS5.

3.3 Residues of ZIKV NS5 are essential for interaction with importin α

• As reported previously, a conservative motif KRPR in NS5 of flaviviruses is crucial for interaction with importins [20], we construct a series of ZIKV NS5 mutants by introducing alanine substitutions in this motif, and their subcellular distribution was analysed (Fig. 3a). 
• 
• Construct NS5-E containing alanine substitutions of all residues is the only mutant whose nuclear localization was completely impaired (Fig. 3b).
• 
•  And when these mutations were introduced into EGFP-GST-(370-406), its fluorescence distribution relocated to the cytoplasm (Fig. 3b), suggesting these residues are critical for nuclear localization of ZIKV NS5.
• To further examine whether this motif in ZIKV NS5 is responsible for interaction with importins, co-immunoprecipitation assay was performed with cells co-transfected with plasmid encoding NS5-WT or NS5-E and importin α2, which has been reported to direct the nuclear transport of ZIKV NS5
• The result showed that importin α2 was co-immunoprecipitated with NS5-WT but not NS5-E, suggesting as key residues involved in interaction of ZIKV NS5 with importin α.
• 500 ng empty vector, NS5-WT or NS5-E
• 
• the importin α2-Myc是什么？

3.4 ZIKV NS5 nuclear localization contributes to the suppression of IFn-I production

• To investigate whether this ability is associated with its subcellular localization, HeLa cells were transfected with plasmid encoding NS5-WT or NS5-E followed by poly(I:C) stimulation, and the induction of IFN-β was measured by qRT-PCR. 
• As shown in Fig. 4a, NS5-WT significantly suppressed the expression of IFN-β, but NS5-E showed no obvious effect. 
• 
• Consistently, IFN-β promoter activity determined by luciferase reporter system was also inhibited by NS5-WT but not NS5-E (Fig. 4b). 
• 
• These results suggest that nuclear localization of ZIKV NS5 contributes to its inhibitory ability on IFN-I transcription.
• To elucidate whether nuclear localization of ZIKV NS5 plays a role in inhibiting the transcriptional activity of IRF3 or NF-κB. 
• The IRF-3-responsive or NF-κB-responsive IFN-β promoter luciferase assay was performed with cells expressing NS5-WT or NS5-E. 
• Compared with NS5-WT, NS5-E showed a similar inhibitory effect on NF-κB activation while an alleviated capability on IRF3 inhibition (Fig. 4c), suggesting that mutations on NLS of ZIKV NS5 interfere with its function on restraining IRF3 activation. 
• 
• it is important to clarify whether the mutations would compromise their interaction or affected the nucleus-cytoplasmic shuttling of IRF3.
• As shown in Fig. 4d, both NS5-WT and NS5-E could interact with endogenous IRF3 either under the stimulation with SeV or not. 
• 
• The immunofluorescence assay revealed a strong co-localization of NS5-WT and IRF3 in cell nucleus after SeV stimulation (Fig. 4e).
• 
• Interestingly, in the case of NS5-E, a cytoplasmic co-localization with IRF3 was observed in unstimulated cells without any impact on nuclear translocation of IRF3 after stimulation, suggesting that mutations on NS5 did not impair the interaction with IRF3.
• These findings indicate the possibility that nuclear interaction of NS5 and IRF3 is required for inhibition of IRF3-mediated IFN-I transcription.这些结果表明NS5和IRF3的核相互作用可能是抑制IRF3介导的IFN-I转录的必要条件。

3.5 ZIKV NS5 nuclear localization is associated with inhibition of IFn-I response

• To investigate if this function of ZIKV NS5 is dependent on its subcellular localization, the activity of IFN-stimulated response element (ISRE) promoter was determined in cells transfected with plasmid encoding NS5-WT or NS5-E. 
• As shown in Fig. 5a, although both NS5-WT and NS5-E significantly inhibited the activation of ISRE promoter, NS5-E showed relatively weaker inhibitory effect, indicating that ZIKV NS5 nuclear localization is partially involved in inhibition of IFN-I downstream signalling.
• 
• To further determine whether NS5 nuclear localization contributes to STAT2 degradation, HeLa cells were transfected with plasmid expressing with NS5 WT or NS5-E, followed by IFN-β stimulation. The results showed that both NS5-WT and NS5-E degraded endogenous STAT2 (Fig. 5b, c), suggesting that ZIKV NS5 mediated degradation of STAT2 is independent on its nuclear accumulation.
• 
• 
• Taken together, these data demonstrate that ZIKV NS5 nuclear localization participates in inhibition of IFN-I response through a STAT2 degradation-independent mechanism.

4. Discussion

• 三个主要结论

1. both aa 11–90 and aa 370–406 of ZIKV NS5 are responsible for the nuclear localization of ZIKV NS5,
2. and are critical residues for interaction of NS5 with importin α.
3. Furthermore, we found that NS5 nuclear localization is associated with its inhibitory effect on IFN-I production and response.

• 补充figure，真的没找到。。。
•  aa 370–406 of ZIKV NS5, a highly conserved sequence with aa 369–405 of DENV and other flaviviruses (Fig. S1a, available in the online version of this article), is also sufficient to direct the protein into the nucleus
• However, no infectious viral particle could be rescued after mutation, suggesting that mutation of aa 390–393 in ZIKV NS5 is lethal to virus replication. 一个results中没有的侧面证明
• Inconsistently, our results revealed that Cter18 of ZIKV NS5 is not required for its nuclear localization. This might be due to the low similarity of the motif KRFR, which was considered as a class 2 NLS responsible for the nuclear localization of DENV2 NS5 (Fig. S1b) 关于ZIKV和DENV不一致性的分析
• Unfortunately, no NLSs specific to the importin α/β pathway was predicted by cNLS Mapper within this region, implying a non-classical NLS in aa 1–100. 非经典NLS的鉴定方法，没有pathway？
• Therefore, it is difficult to determine the key residues within aa 11–90 that contribute to NS5 subcellular localization by using site-directed mutagenesis. 因为不是full-length的NLS
• Deletion of either aa 370–406 or aa 11–90 in NS5 altered its subcellular localization (Fig. 2b, g). This may be associated with the protein structure of NS5. However, the underlaying mechanism need to be further explored. 对于results的解释，也许跟蛋白质结构有关，相关机制需要深入探讨。
• Our study demonstrated for the first time that nuclear accumulation of ZIKV NS5 contributes to its function on blocking of IRF3 activation and IFN-I production. 本文的创新性？
• These results raise the possibility that only ZIKV NS5, which interacts with IRF3 in the nucleus, could inhibit IFN-I production.
• Our findings also reveal that nuclear localization of ZIKV NS5 contributes to its function on inhibiting the ISRE activation without affecting STAT2 degradation. ISRE是什么来着？
• It is incomprehensive to evaluate the activation of IFN-I downstream signalling simply by measuring STAT2 degradation. 本文的不足之处？
• These results imply a potential STAT2-independent mechanism involved in ZIKV NS5-mediate inhibition of IFN-I response.
• Taken together,

1. our study defined two regions of ZIKV NS5 (aa 11–90 and aa 370–406) are responsible for nuclear localization.
2. In addition, we found that the nuclear localization of ZIKV NS5 is involved in its inhibitory effect on type I IFN production and response.

• These findings may provide new insight into the mechanisms for innate immune evasion and pathogenesis of ZIKV.