A型流感病毒NS1与突触后密度蛋白-95的相互作用

目的 A型流感病毒NS1蛋白是一种多功能的致病因子,能够与被感染细胞中的多种蛋白相互结合,影响并干扰宿主细胞内的信号转导、蛋白质合成及抗病毒反应。突触后密度蛋白(Postsynaptic density protein95,PSD-95)主要存在于神经元及SH-SY-5Y等神经来源的细胞株中。假设NS1能够与PSD-95结合,则更有利于了解A型流感病毒对神经元及相关细胞的作用机制。方法通过酵母双杂交,GST-pull down及免疫荧光技术分别从体外和体内两方面检测NS1与PSD-95的相互作用。结果酵母双杂交表明,仅转染PGAD-NS51/PGBK-PSD-95的QDO有菌落生长,且α-半乳糖苷酶活性显著高于阳性对照;而转染PGAD-NS32/PGBK-PSD-95的QDO无菌落生长;GST-pull down表明仅NS51与PSD-95孵育后,能够被Western-blot检测到;免疫荧光表明NS51与PSD-95可能存在共定位,而NS32与PSD-95则不存在共定位。结论 H5N1(A/chicken/Guangdong/1/2005)的NS1能够与PSD-95结合;反之,H3N2(A/Shantou/602/06)的NS1则不能。     

中国“人-猪-禽”基因重排H1N2亚型猪流感病毒全基因克隆及遗传进化的研究

[目的]为了研究2006年从广西病猪肺组织中分离的H1N2亚型猪流感病毒(SIV)A/Swine/Guangxi/13/2006(H1N2)(Sw/Gx/13/06)的遗传学特性和8个基因的来源.[方法]运用RT PCR方法对其全基因进行了克隆并运用分子生物学软件对其基因序列进行了遗传进化分析.[结果]血凝素(HA)、核蛋白(NP)、基质蛋白(M)和非结构蛋白(NS)基因来源于猪古典H1N1亚型流感病毒;神经氨酸酶(NA)和聚合酶蛋白(PB1)基因来源于人的H3N2亚型流感病毒;聚合酶蛋白(PA)和聚合酶蛋白(PB2)基因来自于禽流感病毒.[结论]可见Sw/GX/13/06是一株"人-猪-禽"三源基因重排H1N2亚型SIV且与美国(1999-2001年)和韩国(2002年)分离到该型病毒的有明显的亲缘关系.据我们所知,这是中国首次报道含有禽流感病毒基因片段的重排H1N2 SIV,该病毒是否对养猪业和人类公共卫生健康具有潜在的威胁,有待于进一步研究.     

No Serological Evidence of Influenza A H1N1pdm09 Virus Infection as a Contributing Factor in Childhood Narcolepsy after Pandemrix Vaccination Campaign in Finland

Background

Narcolepsy cataplexy syndrome, characterised by excessive daytime sleepiness and cataplexy, is strongly associated with a genetic marker, human leukocyte antigen (HLA) DQB1*06:02. A sudden increase in the incidence of childhood narcolepsy was observed after vaccination with AS03-adjuvanted Pandemrix influenza vaccine in Finland at the beginning of 2010. Here, we analysed whether the coinciding influenza A H1N1pdm pandemic contributed, together with the Pandemrix vaccination, to the increased incidence of childhood narcolepsy in 2010. The analysis was based on the presence or absence of antibody response against non-structural protein 1 (NS1) from H1N1pdm09 virus, which was not a component of Pandemrix vaccine.

Methods

Non-structural (NS) 1 proteins from recombinant influenza A/Udorn/72 (H3N2) and influenza A/Finland/554/09 (H1N1pdm09) viruses were purified and used in Western blot analysis to determine specific antibody responses in human sera. The sera were obtained from 45 patients who fell ill with narcolepsy after vaccination with AS03-adjuvanted Pandemrix at the end of 2009, and from controls.

Findings

Based on quantitative Western blot analysis, only two of the 45 (4.4%) Pandemrix-vaccinated narcoleptic patients showed specific antibody response against the NS1 protein from the H1N1pdm09 virus, indicating past infection with the H1N1pdm09 virus. Instead, paired serum samples from patients, who suffered from a laboratory confirmed H1N1pdm09 infection, showed high levels or diagnostic rises (96%) in H1N1pdm virus NS1-specific antibodies and very high cross-reactivity to H3N2 subtype influenza A virus NS1 protein.

Conclusion

Based on our findings, it is unlikely that H1N1pdm09 virus infection contributed to a sudden increase in the incidence of childhood narcolepsy observed in Finland in 2010 after AS03-adjuvanted Pandemrix vaccination.     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

A型流感病毒NS1蛋白羧基端PL结构域影响NS1在细胞核内的定位

A型流感病毒NS1蛋白羧基端4个氨基酸可以与PDZ结构域(the domain of PSD95,Dig and ZO-1)相结合,称为PL结构域(PDZ ligand domain)．对不同亚型或毒株的流感病毒而言,其NS1蛋白PL结构域的组成存在比较大的差异．有研究发现这种差异能够影响NS1与宿主细胞蛋白的相互作用进而影响病毒的致病力．为进一步探讨PL结构域对NS1蛋白生物学特性的影响,首先构建出4种不同亚型流感病毒(H1N1、H3N2、H5N1、H9N2)来源的NS1绿色荧光蛋白表达质粒．在此基础上,对野生型H3N2病毒NS1表达质粒进行人工改造,将其PL结构域缺失或者替换为其他亚型流感病毒的PL结构域,制备出4种重组NS1蛋白表达质粒．通过比较上述不同NS1蛋白在HeLa细胞中的定位情况发现,只有野生型H3N2病毒的NS1蛋白可以定位于核仁当中,而野生型H1N1、H5N1、H9N2病毒的NS1蛋白以及PL结构域缺失或替代的H3N2病毒NS1蛋白都不能定位于核仁．而通过比较上述NS1蛋白在流感病毒易感的MDCK细胞中的定位,进一步发现所有这些蛋白均不定位于核仁．上述结果表明：PL结构域的不同可以明显影响NS1蛋白在HeLa细胞核内的定位和分布,这有可能造成其生物学功能的差异．同时,NS1蛋白在细胞核内的定位还与宿主细胞的来源有着密切关系．     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

H7N9亚型禽流感病毒非结构蛋白1(NS1)基因序列分析及2013上海分离株NS1的原核表达

目的:对2013年3月发生的感染人的新型H7N9亚型禽流感病毒的非结构蛋白1(NS1)基因序列进行同源性分析,构建NS1重组质粒并表达。方法:从GenBank获得2006~2013年不同来源的H7N9亚型病毒NS1序列,并进行同源性比较;利用PCR方法从H7N9亚型禽流感病毒株A/Shanghai/4664T/2013(H7N9)基因组cDNA中扩增得到全长NS1基因,并将该片段定向克隆到原核表达载体pET28a上,构建重组质粒pET28a-NS1,经酶切鉴定,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞后,IPTG诱导表达,且进行Western印迹分析。结果:经序列分析,2013年暴发的H7N9型禽流感病毒的NS1基因核苷酸序列同源性为95%~100%,与之前暴发的H7N9型流感病毒NS1基因序列的同源性为86.4%~90.7%,表明2次暴发的该型流感分离株属于不同的进化分支;PCR扩增得到约680 bp的NS1基因序列,所克隆的NS1基因在原核细胞中的表达产物主要以包涵体形式存在,SDS-PAGE检测结果表明重组蛋白相对分子质量为25×103,Western印迹分析证实表达产物为H7N9禽流感病毒NS1蛋白。结论:为进一步研究H7N9亚型流感病毒NS1蛋白功能及基于NS1蛋白的抗病毒药物奠定了基础。     

Modification of nonstructural protein 1 of influenza A virus by SUMO1

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.     

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in China should be given a high priority.     

Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.     

Mutations in the C-terminal tail of NS1 protein facilitate the replication of classical swine H1N1 influenza A virus in mice

The NS1 protein of classical swine H1N1 influenza A virus evolved dynamically during the past 80 years, most notable changes happened in the four C-terminal sequences and the C-terminal truncation of 11 amino acids. However, the role of these changes on the virulence of classical swine H1N1 influenza A virus remains unknown. Using reverse genetics, three NS1 mutant viruses (RSEV, GSEI, and EPEV) and a wild-type virus (PEQK) were generated from A/Swine/Shanghai/1/2005 virus and the pathogenicity of the viruses was determined in mice. The results showed that RSEV and PEQK viruses could not infect the mice. By contrast, GSEI and EPEV viruses could replicate in the lungs of mice without prior adaptation. The viral titers in lungs from GSEI and EPEV virus-infected mice were 2,300 and 7 pfu/g at fourth-day post-infection, respectively. Mild-to-moderate alveolitis was observed in the histopathological test of lungs from GSEI and EPEV virus-infected mice. The results indicated that C-terminal GSEI and EPEV motifs of NS1 protein involved in viral virulence and facilitated the A/Swine/Shanghai/1/2005 virus crossing the species barrier from swine to mice.     

Analysis of cellular proteome alterations in porcine alveolar macrophage cells infected with 2009 (H1N1) and classical swine H1N1 influenza viruses

The H1N1/2009 influenza virus has the potential to cause a human pandemic, and sporadic cases of human-to-pig transmission have been reported. In this study, two influenza viruses were isolated from pigs. A phylogenetic analysis showed that the A/swine/NanChang/F9/2010(H1N1) (F9/10) strain shared a high degree of homology with the pandemic H1N1/2009 virus, and A/swine/GuangDong/34/2006 (H1N1) (34/06) strains was a classical swine influenza virus. A proteomic analysis was performed to investigate possible alterations of protein expression in porcine alveolar macrophage (PAM) cells infected by the F9/10 and 34/06 viruses over different time courses. Using 2-DE in association with MALDI-TOF MS/MS, we identified 13 up-regulated and 21 down-regulated protein spots, including cytoskeleton proteins, cellular signal transduction proteins, molecular biosynthesis proteins and heat shock proteins. The most significant changes in the infected cells were associated with molecular biosynthesis proteins and heat shock proteins. We analysed the biological characteristics of the F9/10 and 34/06 viruses in vivo and in vitro. The F9/10 virus showed greater pathogenicity than the 34/06 virus in PAM cells and mice. This study provides insights into the biologic characteristics, potential virulence alteration and cross-species transmission mechanisms of the pandemic H1N1/2009.     

Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.     

禽流感病毒最新研究进展

本文针对2004年爆发的禽流感疫病,回顾了2004年至2005年期间禽流感病毒的研究进展。逆转录聚合酶链式反应技术为禽流感病毒的分型提供了一种快速、可靠、准确的方法。对H5N1禽流感病毒致病机制的研究发现,其强致病性在于它可以躲避人类抗病毒细胞因子的作用,NS1基因编码蛋白的92位谷氨酸在其中发挥了关键作用。由于禽流感疾病多引起结膜炎,并与病毒细胞受体的研究结果相结合,有科学家认为眼部特异性是禽流感病毒的一个总体特征。社会普遍关注禽流感疫苗的研制,人类和禽类流感A型病毒M2蛋白胞外区域的序列比对工作为疫苗研制提供了一条新的思路,依据神经氨酸酶抑制剂抑制病毒的出芽繁殖原理的疫苗正在研制过程中,而利用siRNA预防和治疗禽流感也是很有潜力的一种方法。禽流感病毒研究的另一个热点是病毒基因节段的重配问题。     

Effects of the C-terminal truncation in NS1 protein of the 2009 pandemic H1N1 influenza virus on host gene expression

The 2009 pandemic H1N1 influenza virus encodes an NS1 protein with 11 amino acids (aa) truncation at the C-terminus. The C-terminal tail of influenza virus NS1 protein constitutes a nucleolar localization signal (NoLS) and is the binding domain of the cellular pre-mRNA processing protein, poly(A)-binding protein II (PABII). Here, our studies showed that the C-terminal-truncated NS1 of the 2009 pandemic virus was inefficient at blocking host gene expression, extension of the truncated NS1 to its full length increased the inhibition of host gene expression. Mechanistically, this increased inhibition of host gene expression by the full-length NS1 was not associated with nucleolar localization, but was due to the restoration of NS1's binding capacity to PABII. Furthermore, in vitro and in vivo characterization of two recombinant viruses encoding either the C-terminal 11-aa truncated or full-length NS1 of the 2009 pandemic virus showed that the C-terminal 11-aa truncation in NS1 did not significantly alter virus replication, but increased virus pathogenicity in mice.     

不同亚型流感病毒NS1蛋白的细胞定位差异分析

NS1蛋白是流感病毒编码的一种小分子多功能蛋白,可在病毒的复制过程中抑制宿主细胞的抗病毒免疫应答。为研究不同亚型流感病毒的NS1蛋白在细胞内的定位差异,分别用H1N1亚型WSN、PR8和CA04毒株,H9N2亚型SD毒株及H7N9亚型AH01毒株感染A549、MDCK细胞系以及构建的可表达不同亚型流感病毒NS1蛋白的p CMV-Myc-NS1质粒转染293T细胞,用激光共聚焦显微镜观察发现不同亚型流感病毒在不同细胞系和时间点的定位差异,感染后24 h时WSN和PR8毒株的NS1主要定位于细胞质中,而CA04和SD毒株主要定位于细胞核内。另外,观察过表达的WSN、SD和AH01毒株NS1的细胞定位,转染后24 h时WSN毒株NS1定位于细胞质中,而SD和AH01毒株主要定位于细胞核中。经氨基酸序列比对,对WSN毒株NS1蛋白进行关键氨基酸点突变,结果显示单一位点的改变未导致NS1蛋白细胞定位的改变,其细胞定位的差异不是由单一位点决定的。综上所述,分析不同亚型中的NS1的定位差异,这对进一步了解NS1蛋白同宿主细胞不同区域的蛋白的相互作用、流感病毒的调节机制以及病毒感染细胞中天然免疫反应具有一定的指导意义。     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

人H5N1亚型禽流感病毒安徽株NS1基因的克隆及在原核系统的表达

利用RT-PCR方法,从人H5N1亚型禽流感病毒安徽株扩增到了NS1基因,对其进行了克隆、序列测定和分析,并在原核系统高效表达和纯化了NS1蛋白。进化分析表明,A/Anhui/01/2005毒株与近些年国内分离的水禽H5N1病毒进化关系更为接近。NS1与福建、湖南分离的禽流感病毒同源性最高,分别达到99.1%和98.2%。序列分析表明,与病毒的致病性相关的92位氨基酸为Asp,与病毒的细胞因子抗性相关的80~84位氨基酸发生缺失,与断裂/多聚腺苷酸化特异性因子结合的基序改变为GFEWN,和病毒致死性相关的PL基序为ESEV。随后在大肠杆菌高效表达并纯化了NS1蛋白。NS1基因及其编码产物的特性分析以及在原核系统的表达,为进一步研究NS1的致病机制和抗病毒药物研制奠定了基础。     

The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells

The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.