A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

Loss of function of the influenza A virus NS1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (PI3K)

A panel of influenza A viruses encoding mutant NS1 proteins was created in which a number of NS1 functions, including interactions with dsRNA, PI3K, CPSF30 and PKR, were inhibited. Surprisingly, given previous reports that NS1 activates PI3K to prevent apoptosis, the mutant viruses rUd-Y89F and rUd-P164/7A that fail to activate PI3K did not induce any more apoptosis than wild-type virus in MRC-5 and A549 cells, even though these cells are highly sensitive to inducers of apoptosis. Induction of cell death by the apoptogenic rUd-184-8(P) virus could not be prevented by serum-mediated activation of PI3K/Akt. Neither infection of MRC-5 or A549 cells with wild-type virus nor constitutive expression of NS1 prevented cell death caused by apoptosis inducers, suggesting that NS1 is not directly anti-apoptotic. Our data suggest that the loss of a functionally intact NS1 protein promotes apoptosis, but this is not due to an inability to activate PI3K.     

Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus

The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3′ processing

AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 3′ end formation, the molecular basis for CPSF–AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30–RNA interaction is essential for mRNA 3′ processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 3′ processing. Our data suggest that AAUAAA recognition in mammalian mRNA 3′ processing is more complex than previously thought and involves multiple protein–RNA interactions.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.     

Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.     

Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides

An influenza A virus, A/turkey/Oregon/71, was shown by protein gel analysis to code for an NS1 protein approximately half the size of those of other influenza A viruses. Sequence analysis of the NS gene of this virus revealed a 10 nucleotide deletion resulting in an NS1 protein of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by indirect immunofluorescence analysis of virus infected cells. Also, A/turkey/Oregon/71 virus grew to high titer in embryonated chicken eggs comparable to other influenza A viruses. We also identified a laboratory variant of an influenza B virus, clone 201, which codes for a truncated NS1 protein. Sequence analysis revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids as compared to other influenza B virus NS1 proteins possessing a length of 281 amino acids. Again as shown for the NS1 proteins of other influenza B viruses the NS1 polypeptide of B virus clone 201 was found to localize in the nucleus of infected cells. It appears that large deletions in the carboxyl terminus of the NS1 proteins of influenza A and B viruses can be tolerated without affecting the functional integrity of the NS1 polypeptide.     

反义硫代修饰寡核苷酸体外抗甲型流感病毒感染的效应研究

目的 在感染甲型流感病毒(influenza A virus,IFAV)的人气道上皮细胞(9HTEO)中观察与IFAV PB1、M2、NS、PB2、HA基因瓦补的反义硫代修饰寡核苷酸(ASODN)的抗病毒活性.方法 细胞外体系观察没计的ASODN效率,筛选对IFAV有效的3个ASODN进行9HTEO的抗病毒效应.倒置显微镜下观察IFAV的敛细胞病变作用和ASODN的细胞保护作用.MTT方法测细胞存活率,空斑试验、RT-PCR、Westem blot、免疫荧光检测ASODN特异性抗病毒效应,MTT法检测ASODN对细胞的保护作用.结果 IFAV感染复数(MOI)在0.1以上时,培养5 d后IFAV感染的9HTEO细胞全部死亡.设计的ASODN能够提高感染细胞的存活率,且呈量效依赖关系.空斑试验、RT-PCR、Western blot、免疫荧光试验证实在细胞水平、基因转录水平、蛋白水平针对IFAV PB1、M2、NS基因mRNA转录起始点保守序列没计的ASODN具有特异性抗IFAV作用.结论 ASODN具有较明显的抗病毒活性,为进一步研究和开发新型抗IFAV药物提供了实验依据.为高致病性禽流感(H5N1)的基因治疗提供了新的思路和理论依据.     

Influenza A virus interacts extensively with the cellular SUMOylation system during infection

SUMOylation, the post-translational conjugation of the Small Ubiquitin-like MOdifier (SUMO) to a target protein, regulates a wide array of cellular processes and plays important roles for numerous viruses during infection. However, the relevance of the cellular SUMOylation system for influenza virus infection remains mostly unexplored. We previously reported that the non-structural protein of influenza A virus NS1 is a bona fide SUMO target. Here we determine that at least four additional influenza virus proteins, namely PB1, NP, M1, and NS2, are also authentic SUMO targets, and provide data supporting that PB1, NP, and M1 are SUMOylated during viral infection. The functional relevance of SUMOylation for these proteins is supported by the observation that, despite no apparent changes in the cellular levels of the E1 and E2 SUMO enzymes, influenza viral infection leads to a global increase in cellular SUMOylation. This increase, characterized by the appearance of two new SUMOylated proteins of ～70kDa and ～52kDa of molecular weight, is dependent upon viral replication and cannot be recreated by interferon stimulation alone. Altogether, these observations indicate that influenza A virus interacts extensively with the cellular SUMOylation system during infection and suggest that SUMOylation plays an important role during influenza virus infection, potentially contributing to the functional diversity exhibited by influenza viral proteins.     

The Y3** ncRNA promotes the 3′ end processing of histone mRNAs

We demonstrate that the Y3/Y3** noncoding RNAs (ncRNAs) bind to the CPSF (cleavage and polyadenylation specificity factor) and that Y3** associates with the 3′ untranslated region (UTR) of histone pre-mRNAs. The depletion of Y3** impairs the 3′ end processing of histone pre-mRNAs as well as the formation and protein dynamics of histone locus bodies (HLBs), the site of histone mRNA synthesis and processing. HLB morphology is also disturbed by knockdown of the CPSF but not the U7-snRNP components. In conclusion, we propose that the Y3** ncRNA promotes the 3′ end processing of histone pre-mRNAs by enhancing the recruitment of the CPSF to histone pre-mRNAs at HLBs.     

Deglycosylation of the NS1 protein of dengue 2 virus,strain 16681: Construction and characterization of mutant viruses

Summary. The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK₂ cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK₂ cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37°C and 39°C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.     

Key role of Asp16 in proteolysis of influenza A NP protein by caspases in infected cells

The main nucleocapsid protein NP (molecular weight--56 kD) of human influenza A virus (IAV) was found to be subject to the N-terminal proteolysis in position Asp16 with production of aNP (molecular weight--53 kD) in the infected cells' apoptosis. It was assumed that NP of avian and animal influenza viruses was not subject to proteolysis since it has Gly16. To verify the above assumption the NP chimeric gene of human influenza virus was developed; Asp16 was replaced by Gly by means of "site-oriented" mutagenesis in the above gene, after that, the A/WSN/33 (H1N1) mutant of human influenza virus with "avian" NP and with point mutation (Gly16) was developed by using the method of "reverse genetics". The "human" influenza virus with "avian" chimeric NP/Gly16 turned out to be viable but had a lower replication velocity versus its wild-nature counterpart. It is noteworthy, that the mutant virus caused the cellular apoptosis in the remote infection period the way the wild virus did; however, NP of the former was found to be resistant to cellular caspasas and was not subject to proteolysis in infected cells. The conclusion is that Asp16 in NP molecule of human IAV is involved into the regulation process of virus replication and is the key element in NP proteolysis by cellular caspasas in cells' apoptosis.     

A single point mutation (Y89F) within the non-structural protein 1 of influenza A viruses limits epithelial cell tropism and virulence in mice

The nonstructural protein 1 (A/NS1) of influenza A viruses (IAV) harbors several src homology (SH)-binding motifs (bm) that mediate interactions with cellular proteins. In contrast to the sequence variability of the second SH3bm, tyrosine 89, within the SH2bm is a highly conserved residue among IAV strains. This prompted us to evaluate the necessity of this SH2bm for IAV virulence. In an in vivo mouse model, we observed drastic reductions in weight loss, mortality, and virus titers in lung and bronchoalveolar lavage fluid after infection with the mutant virus PR8 A/NS1-Y89F (PR8 Y89F) when compared with wild-type virus (PR8 wt). Concomitantly, we observed decreased inflammation and less severe pathologic changes, reflecting reduced levels of virus titers. At histologic analysis, lungs infected with PR8 wt virus showed widespread destruction of the bronchiolar epithelium, with extensive distribution of virus antigen within tracheal, bronchial, bronchiolar, and alveolar epithelium. In marked contrast, the bronchiolar epithelium after infection with the mutant PR8 Y89F virus was entirely intact, and the severity and extent of viral infection was reduced and strongly restricted to alveoli. These findings demonstrate that change of a single residue of the highly conserved SH2bm within the A/NS1 results in restricted virus spread in mouse lung and strongly reduced virulence, which illustrates the necessity of the SH2bm for IAV-induced pathogenicity.