Pandemic influenza virus, pH1N1, induces asthmatic symptoms via activation of innate lymphoid cells.

BACKGROUND: The pandemic strain of the influenza A virus (pH1N1) in 2009 caused many complications in patients. In this study, we introduce asthmatic symptoms as a complication of pH1N1 infection in children, not having a relationship with asthma history. The aim of this study was to quantify asthmatic symptoms in pH1N1-infected children and elucidate the underlying mechanisms of airway hyper-responsiveness (AHR) induced in a murine model of pH1N1 infection. METHODS: As a retrospective study, pH1N1-infected children who were hospitalized with moderate to severe acute asthmatic symptoms were enrolled and administered a methacholine challenge test (MCT) at 3 months post-discharge. Additionally, the induction of AHR by pH1N1 infection was measured by MCT in wild-type and Rag1(-/-) mice. The effect of the innate immune response on the development of AHR following pH1N1 infection was investigated. RESULTS: More than 70% of the pH1N1-infected children without a pre-infection diagnosis of asthma had a negative response on the MCT. None of these children had recurrent wheezing or asthma during the 3 years following pH1N1 infection. The development of AHR in pH1N1-infected mice was associated with an elevation in IL-33 and innate lymphoid cells 2 (ILC2). CONCLUSIONS: This study demonstrates that pH1N1 infection directly induces transient asthmatic symptoms in patients regardless of their medical history. pH1N1 infection was shown to stimulate the rapid development of AHR and Th2-type cytokine secretion in mice via the activation of ILC2; it may be activated independently of adaptive immunity.

Anti-influenza virus activity of green tea by-products in vitro and efficacy against influenza virus infection in chickens.

Polyphenolic compounds present in green tea, particularly catechins, are known to have strong anti-influenza activity. The goal of this study was to determine whether green tea by-products could function as an alternative to common antivirals in animals compared to original green tea. Inhibition of viral cytopathic effects ascertained by neutral red dye uptake was examined with 50% effective (virus-inhibitory) concentrations (EC50)determined. Against the H1N1 virus A/NWS/33, we found the anti-influenza activity of green tea by-products (EC50 = 6.36 µg/mL) to be equivalent to that of original green tea (EC50= 6.72 µg/mL). The anti-influenza activity of green tea by-products was further examined in mouse and chicken influenza infection models. In mice, oral administration of green tea by-products reduced viral titers in the lungs in the early phase of infection, but they could not protect these animals from disease and death. In contrast, therapeutic administration of green tea by-products via feed or water supplement resulted in a dose-dependent significant antiviral effect in chickens, with a dose of 10 g/kg of feed being the most effective (P < 0.001). We also demonstrated that unidentified hexane-soluble fractions of green tea by-products possessed strong anti-influenza activity, in addition to ethyl acetate-soluble fractions, including catechins. This study revealed green tea by-product extracts to be a promising novel antiviral resource for animals.

The humoral immune response to the inactivated influenza A (H1N1) 2009 monovalent vaccine in patients with Type 2 diabetes mellitus in Korea.

AIMS: We evaluated the antibody response to a single-dose adjuvanted, inactivated, pandemic H1N1 influenza vaccination in patients with diabetes and assessed factors associated with the failure to induce antibody responses. METHODS: Eighty-two patients with Type 2 diabetes were vaccinated and antibody responses were determined with haemagglutination inhibition assay and anti-haemagglutinin antibody ELISA. RESULTS: Among 70 antibody-negative patients at baseline, 34 (48.6%) achieved seroconversion; 28 (60.9%) in the young adults group and six (25%) in the elderly group acquired H1N1-specific antibodies. Patients in the older age range or with longer duration of diabetes had a lower seroconversion rate. CONCLUSIONS: Our data show low cross-reactive antibody carrying rate and low seroconversion rate in patients with diabetes. Until larger-scale, case-controlled trials become available, older patients and patients with a longer duration of diabetes should be considered for the two-dose vaccination or have antibody titres measured after the first vaccination.

Rifamycin B oxidase from Monocillium spp., a new type of diphenol oxidase.

It was found that enzyme from a microbial strain, Monocillium spp. ATCC 20621, catalyzed the oxidative reaction of rifamycin B to form rifamycin O. The identification of the reaction products suggested that the reaction proceeded by the oxidative cyclization of rifamycin B to give rifamycin O, which spontaneously hydrolyzed to rifamycin S in neutral aqueous milieu. The characteristic of the enzyme was different as compared with that of other polyphenol oxidases such as laccase. It is proposed that this new type of enzyme be classified into a subgroup EC 1.10.3.6 with a trivial name rifamycin B oxidase.

Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.

The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.

Influencing the influenza virus: genetic analysis and engineering of the negative-sense RNA genome.

The genome of influenza virus has become amenable to genetic analysis as a result of newly developed methods for reconstitution of the ribonucleoprotein (RNP) complex. This allows a dissection of the regulatory signals and promoters required for transcription and replication of the viral genome. The ability to rescue chimeric viruses in vivo after transfection opens up a way to engineer viruses suitable for human immunization and carrying desirable antigenic determinants.

Mutational analysis of influenza B virus RNA transcription in vitro.

The roles of the 3'- and 5'-terminal nucleotides and the panhandle structure of influenza B virus virion RNA were analyzed in vitro by transcription of model RNA templates with influenza B virus RNA polymerase. The results suggest that the stability of the panhandle and breathing of the extreme ends of the panhandle are important factors for efficient transcription. Influenza B virus polymerase appears to be more tolerant of mutations in the panhandle structure than influenza A virus polymerase. This is consistent with the greater degree of heterogeneity observed naturally in the 3'-terminal nucleotides of the virion RNA of influenza B virus than in influenza A virus.

Escherichia coli formylmethionine tRNA: mutations in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation of protein synthesis and conformation of anticodon loop.

We have generated mutants of Escherichia coli formylmethionine initiator tRNA in which one, two, and all three G X C base pairs in the GGGCCC sequence in the anticodon stem are changed to those found in E. coli elongator methionine tRNA. Overproduction of the mutant tRNAs using M13 recombinants as an expression vector and development of a one-step purification scheme allowed us to purify, characterize, and analyze the function of the mutant tRNAs. After aminoacylation and formylation, the function of mutant formylmethionyl tRNAs was analyzed in an MS2 RNA-directed in vitro protein-synthesizing system, in AUG-dependent ribosomal P site binding, and in initiation factor binding. The mutant tRNAs show progressive loss of activity in initiation, the mutant with all three G X C base pairs substituted being the least active. The mutations affect binding to the ribosomal P site. None of the mutations affects binding to initiation factor 2. We also show that there is a progressive increase in accessibility of phosphodiester bonds in the anticodon loop of the three mutants to S1 nuclease, such that the cleavage pattern of the mutant with all three G X C base-pair changes resembles that of elongator tRNAs. These results are consistent with the notion that the contiguous G X C base pairs in the anticodon stem of initiator tRNAs impart on the anticodon loop a unique conformation, which may be important in targeting the initiator tRNA to the ribosomal P site during initiation of protein synthesis.

Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro.

The 12 nucleotide conserved sequence at the 3' end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.

A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo.

The influenza RNA polymerase is known to catalyse three distinct copying activities: (i) transcription of minus-sense virion RNA (vRNA) into mRNA, (ii) transcription of vRNA into full-length complementary RNA (cRNA), and (iii) transcription of cRNA to vRNA. Ever since the discovery of the conserved 13 and 12 long sequences at each end of all the influenza RNA segments, these have been good candidates for promoters of transcription. By devising a new, simple method for preparing influenza polymerase complex capable of transcribing in vitro added short model RNA templates without interference from endogenous viral RNA, we have now tested the promoter hypothesis. We conclude that the 13 long and the 12 long 3' conserved sequences of cRNA and vRNA of influenza A virus are by themselves sufficient to promote vRNA and cRNA synthesis in vitro. Using our new method, we also show that chloramphenicol acetyl transferase (CAT) activity can be detected in MDBK (bovine kidney) cells, after transfection of influenza polymerase assembled with a negatively stranded CAT RNA, even in the absence of helper virus. As in a previously described method (Luytjes et al., 1989), CAT activity is amplified by helper virus and can be rescued in infectious recombinant virus.

Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro.

We show that the absence of a Watson-Crick base pair at the end of the amino acid acceptor stem, which is a hallmark of all prokaryotic initiator tRNAs, is one of the key features that prevents them from acting as an elongator in protein synthesis. We generated mutants of Escherichia coli formylmethionine tRNA that have a base pair at the end of the acceptor stem. The mutants generated were C1----T1, which had a U.A base pair, A72----G72, which had a C.G base pair, and the C1A72----T1G72 double mutant, which lacked the base pair. After aminoacylation, the activity of these and other mutant initiator methionyl-tRNAs (Met-tRNAs) in elongation were assayed in a MS2 RNA-directed E. coli protein-synthesizing system and in binding to the elongation factor Tu (EF-Tu). Unlike wild-type initiator tRNA or the T1G72 double mutant, the T1 and G72 mutant Met-tRNAs were active in elongation, the G72 mutant being more active than the T1 mutant. The T1 and G72 mutant Met-tRNAs also formed a ternary complex with elongation factor EF-Tu.GTP, and their relative affinities for EF-Tu.GTP paralleled their activities in elongation. Combination of the T1 or G72 mutation with mutations in the GGG.CCC sequence conserved in the anticodon stem of initiator tRNAs led to a further increase in the activities of these mutant tRNAs in elongation such that one of these mutants was now almost as good an elongator as E. coli elongator methionine tRNA.

Nucleotides in the panhandle structure of the influenza B virus virion RNA are involved in the specificity between influenza A and B viruses.

Influenza A and B viruses share common sequences and potentially similar panhandle structures in the terminal noncoding regions of virion RNA (vRNA). Interesting differences exist, however, in the number of conserved nucleotides at the 5' and 3' ends of the vRNAs, in base pairs constituting the panhandle duplex, and the length of uridine stretch (U stretch) juxtaposed to the RNA duplex. To analyse the contribution of these signals to the specificity between the two viruses, a transient ribonucleoprotein transfection method was used for the expression of the chloramphenicol acetyltransferase (CAT) reporter gene flanked by the noncoding nucleotides derived from influenza B vRNA. While the base pairing in the RNA duplex was primarily important for template activity, mismatch mutations G11 x G12' and C12 x A13' in the terminal RNA duplex region were utilized by influenza B virus, whereas these mutations were detrimental for influenza A virus. Different activity profiles were observed in the length preference of the RNA duplexes: maximum template activity was observed with 11 base pairs for influenza B virus, and 8 base pairs for influenza A virus. When the mutants with various lengths of U stretch were tested, highest CAT activities were observed with 5 to 7 uridine residues in influenza A virus, whereas in influenza B virus the activity was drastically decreased with 7 uridine residues. We suggest that the specific interaction of influenza virus RNA polymerase with these noncoding cis-acting signals in transcription of the RNA genome, along with unique coding strategies adopted by influenza B virus, has contributed to the divergence of these two closely related viruses.

The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens.

Within the sequence motif conserved at the extreme ends of the influenza virus vRNAs, a unique natural variation, U or C, is observed at position 4 of the 3' end. To test the role of this nucleotide, two isogenic A/WSN/33 viruses, carrying either C4 or U4 nucleotide at the 3' end of the neuraminidase (NA) gene, were generated. Compared with the C4 virus, the U4 virus exhibited delayed synthesis of vRNA and stimulation of mRNA synthesis with prolonged accumulation in influenza virus-infected cells. The mRNA/ vRNA ratio was increased up to 20-fold by the C4 --> U4 substitution suggesting that the U4 nucleotide greatly stimulated transcription of the vRNA template. In isolated virion, the U4 virus had higher NA activity than the C4 virus. In MDBK cells, the U4 virus grew to lower haemagglutination (HA) titres but with higher infectivity than the C4 virus, with a corresponding increase in the ratio of p.f.u./HA units of about 10- to 40-fold. Western blot analysis of isolated virion showed that the ratio of two surface proteins, HA/NA, was greatly decreased in the U4 virus. This suggests that the position 4 nucleotide is a genetic determinant for the repertoire of surface antigens and their ratio could be changed without detrimental effects on virus growth. Results could be used to design genetically engineered influenza virus for vaccination. The observed down-regulation of transcription by C4 nucleotide is consistent with its potential role in segment-specific regulation of influenza virus gene expression, especially PB1, PB2 and PA proteins, during virus infection.

Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem.

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A. "mismatch" in E. coli initiator tRNA. Mutation of this C.A. to U:A, a weak base pair, or U.G., a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kappm by 495-fold. Mutation of the second basepair G2:C71 to U2:A71 lowers Vmax/Kappm by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kappm by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation.

Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter.

A previous study of the 12 nucleotide-long influenza A virion RNA promoter has shown that three nucleotides, residues 9 to 11, were crucial for transcription in vitro, although other nucleotides play a significant but less important role. A model for polymerase-promoter recognition was proposed, according to which there were two sites: a binding site at residues 9 to 11 and a regulatory site at or near the site of initiation at residue 1. By studying the effect of point mutations in the promoter on the binding efficiency of the polymerase using a photochemical cross-linking assay, we now show that residues 9 to 12 are crucial for binding. In addition residues 4 to 8, though not as important, are involved in binding, possibly by stabilizing the polymerase-promoter complex. Both PB1 and PB2 apparently play an important role during virion RNA promoter recognition and binding.

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis.

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.

Mutants of initiator tRNA that function both as initiators and elongators.

We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU----CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C.P., Seong, B. L., and RajBhandary, U.L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli beta-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such "dual" functions in tightly regulated expression of genes for proteins in E. coli.

In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle.

An in vitro transcription assay was used to study transcription from synthetic RNA corresponding to the 3' terminus of influenza A virus cRNA. Micrococcal nuclease-treated influenza virus ribonucleoprotein was used as a source of active polymerase complex. Mutations at two regions of the 13 nucleotide-long conserved cRNA 3' terminus were shown to reduce transcription templated by the short added model RNAs. The first region, at positions 1 and 2 from the 3' terminus, was shown to be affected by the exact nature of the dinucleotide primer used in the in vitro transcription reactions and may not be relevant in vivo. The second region, centred on positions 11 and 12, may be involved in base pairing with conserved nucleotides at the 5' terminus of the cRNA. Evidence for this comes from the finding that RNA corresponding to 5' conserved sequences, but mutated to restore the postulated base pairing with the mutated 3' ends, could partly restore transcription. Binding of the influenza virus polymerase complex to a set of 5'-mutated RNAs was investigated using a photochemical cross-linking assay. Specific binding to two regions of the cRNA 5' terminus was demonstrated, at positions 1 to 3 and positions 8 to 10. Together, these observations suggest that a panhandle forms from the termini of the cRNA molecule and that this structure may play a role in transcription to produce virion RNA.