Defective Influenza Viral Ribonucleoproteins Cause Interference

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.     

The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.     

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.     

携带TAP标签的重组甲型流感病毒的构建与鉴定

【目的】将TAP标签构建到WSN病毒基因组上,得到含有TAP标签的重组流感病毒,以便进行后续的病毒追踪。【方法】利用反向遗传学技术,对甲型流感病毒A/WSN/33(H1N1)的PA片段进行改造来插入TAP(tandemaffinitypurification)标签序列。通过病毒拯救得到表达外源标签TAP的重组流感病毒WSNPA-TAP,并对拯救出的重组病毒进行生物学鉴定。【结果】成功拯救出重组流感病毒并命名为WSN PA-TAP。重组病毒基因组测序表明重组病毒的序列正确,利用RNA银染技术观察到重组病毒的全基因组片段。重组流感病毒WSN PA-TAP在MDCK细胞上测定生长曲线,发现该重组病毒的复制能力比野生型WSN弱;Westernblotting检测到PA-TAP融合蛋白的表达,其分子质量为96 kDa。【结论】成功拯救出能够表达外源标签TAP的重组流感病毒WSN PA-TAP,为筛选与甲型流感病毒聚合酶有关的宿主蛋白的研究提供了新思路,同时也为以甲型流感病毒为载体携带外源基因的探索提供了重要依据。     

Genetic analysis of influenza virus NS1 gene: a temperature-sensitive mutant shows defective formation of virus particles

To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 mutants was generated by PCR-mediated mutagenesis. A collection of mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus mutant library by a novel RNP competition virus rescue procedure. Several temperature-sensitive (ts) mutant viruses were obtained by screening of the mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

High-efficiency formation of influenza virus transfectants.

cDNA-derived RNAs were introduced into the genomes of influenza viruses by using an improved ribonucleoprotein (RNP) transfection protocol. Up to 10(5) viral transfectants with a novel neuraminidase gene could be obtained by using a 35-mm dish (10(6) cells) for RNP transfection. In addition to genes coding for surface proteins (hemagglutinin and neuraminidase), we also exchanged a gene coding for nonsurface proteins. The cDNA-derived influenza A/PR/8/34 virus NS gene was introduced into a temperature-sensitive mutant with a defect in this gene. We suggest that the term influenza virus transfectant be used for those viruses which are made by RNP transfection with cDNA-derived RNA.     

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.     

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。     

The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.     

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40°C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.     

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．     

Attenuated influenza virus construct with enhanced hemagglutinin protein expression

Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.     

Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses

The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.     

Attenuating mutations of the matrix gene of influenza A/WSN/33 virus

The matrix protein (M1) of influenza virus plays an essential role in viral replication. Our previous studies have shown that basic amino acids 101RKLKR105 of M1 are involved in RNP binding and nuclear localization. For the present work, the functions of 101RKLKR105 were studied by introducing mutations into the M gene of influenza virus A/WSN/33 by reverse genetic methods. Individual substitution, R101S or R105S, had a minimal effect on viral replication. In contrast, the double mutation R101S-R105S was synergistic and resulted in temperature sensitivity reflected by reduced viral replication at a restrictive temperature. To investigate the in vivo effect on infection, BALB/c mice were infected with either A/WSN/33 wild-type (Wt) or mutant viruses and assessed for signs of illness, viral replication in the lungs, and survival rates. The results from mouse studies indicated that the R101S-R105S double mutant virus was strongly attenuated, while single mutant viruses R101S and R105S were minimally attenuated compared to A/WSN33 Wt under the same conditions. In challenge studies, mice immunized by infection with R101S-R105S were fully protected from lethal challenge with A/WSN/33. The replication and attenuating properties of R101S-R105S suggest its potential in development of live influenza virus vaccines.     

Functional replacement of the carboxy-terminal two-thirds of the influenza A virus NS1 protein with short heterologous dimerization domains

The NS1 protein of influenza A/WSN/33 virus is a 230-amino-acid-long protein which functions as an interferon alpha/beta (IFN-alpha/beta) antagonist by preventing the synthesis of IFN during viral infection. In tissue culture, the IFN inhibitory function of the NS1 protein has been mapped to the RNA binding domain, the first 73 amino acids. Nevertheless, influenza viruses expressing carboxy-terminally truncated NS1 proteins are attenuated in mice. Dimerization of the NS1 protein has previously been shown to be essential for its RNA binding activity. We have explored the ability of heterologous dimerization domains to functionally substitute in vivo for the carboxy-terminal domains of the NS1 protein. Recombinant influenza viruses were generated that expressed truncated NS1 proteins of 126 amino acids, fused to 28 or 24 amino acids derived from the dimerization domains of either the Saccharomyces cerevisiae PUT3 or the Drosophila melanogaster Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that the attenuation was mainly due to an inability to inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure.