The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation

A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.     

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.     

Nuclear import and export of influenza virus nucleoprotein

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Compound heterozygosity for a nonsense mutation and a splice site mutation in the type VII collagen gene (COL7A1) in recessive dystrophic epidermolysis bullosa

The genes encoding the nucleoprotein, PB1, PB2, and PA proteins of the influenza virus strain B/Panamá/45/90 have been cloned under control of the T7 RNA polymerase promoter of plasmid pGEM-3. Transfection of the recombinant plasmids obtained into mammalian cells, which had been infected with a vaccinia virus encoding the T7 RNA polymerase, resulted in expression of the expected influenza B virus polypeptides. Moreover, it is shown that coexpression of the four recombinant core proteins in COS-1 cells reconstituted a functional polymerase capable of expressing a synthetic influenza B virus-like CAT RNA. By using the influenza B virus recombinant plasmids and a set of pGEM-derived plasmids encoding the homologous core proteins of the influenza A virus A/Victoria/3/75 (I. Mena et al. (1994). J. Gen. Virol. 75, 2109-2114), the capabilities of homo- and heterotypic mixtures of the four core proteins to express synthetic type A and B CAT RNAs were analyzed. Both the influenza A and B virus polymerases were active in expressing, albeit with reduced efficiencies, the heterotypic model CAT RNAs. However, none of all possible heterotypic mixtures of the core proteins reconstituted a functional polymerase. In order to fully characterize the recombinant plasmids obtained, the nucleotide sequences of the cloned genes were determined and compared to sequences of other type B virus isolates. The results obtained from these latter analyses are discussed in terms of the conservation and evolution of the influenza B virus core genes.     

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 degreesC 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.     

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.     

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.     

Inhibition of influenza virus hemagglutinin-mediated membrane fusion by a compound related to podocarpic acid

Entry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformation that is fusogenic. A compound related to podocarpic acid (180299) was identified that inhibits multicycle replication of influenza A/Kawasaki/86 (H1N1) virus in culture. Treatment of Madin-Darby canine kidney (MDCK) cells with 180299 at 1 h before infection resulted in the inhibition of viral protein synthesis. Addition of 20 microgram of 180299/ml at 1 h p.i. had no effect, indicating that 180299 affects an early step of the influenza viral replication cycle. Genetic analysis of reassortants between sensitive and resistant viruses demonstrated that hemagglutinin (HA) conferred the 180299-resistant (180299(r)) phenotype. Twelve independent isolates of influenza A/Kawasaki/86 were selected for resistance to 180299, and sequence analysis revealed that each of these viruses contained amino acid substitutions in the HA. These mutations are dispersed throughout the HA primary amino acid sequence and cluster in one of two regions: the interface between HA1 and HA2 and in a region near the fusion domain of HA2. When compared with the parent virus, the pH-of-inactivation of the resistant mutants was increased by 0.3 to 0.6 pH unit, suggesting that the mutant HAs undergo the conformational change at an elevated pH. Fusion of human erythrocytes to MDCK cells infected with parent influenza A/Kawasaki/86 was inhibited by 180299 (0.1-10 microgram/ml) in a concentration-dependent manner, whereas fusion of erythrocytes to MDCK cells infected with 180299(r) mutants was not affected. These results suggest that 180299 interacts with the neutral pH conformation of influenza A HA and prevents the low-pH-induced change of HA to its fusogenic conformation.     

Memories of a WHO expert in midwifery

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the nicked (NDRDON) and circular (CDRDON) dumbbell RNA/ DNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA, which gave RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligonucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON with four target sites (influenza virus A RNA polymerases (PB1, PB2, PA) and nucleoprotein (NP)) was synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The circular dumbbell DNA/ RNA chimeric oligonucleotide (CDRDON-PB2-as) containing an AUG initiation codon sequence as the target of PB2 showed highly inhibitory effects.     

Replication of influenza virus in organ cultures of human and simian urogenital tissues and human foetal tissues

A survey of human adult tissues in organ cultures showed that influenza viruses (A/Moscow/1019/65 (h2n2) or a recombinant virus virulent for man (PR/8-A/England/939/69 Clone 7a(H3N2)) could infect uterus, bladder and conjunctiva but not oesophagus under the conditions employed; simian bladder and uterus were also susceptible. These results were similar to those already described for corresponding ferret tissues. Organ cultures of human foetal nasal mucosa, trachea, oesophagus, small and large intestine, and bladder consistently supported replication over 4 days or more with high virus yields. Lung, conjunctiva and umbilical cord were less consistently susceptible and gave lower yields. Placenta and kidney cultures allowed replication of virus in one of 8 and one of 4 experiments respectively, the yields being low and of short duration. Organ cultures of neural tissue (meninges and brain), lymphopoietic tissue (spleen, liver and thymus) and amnion did not support significant viral replication. The results are discussed in relation to possible infection of the foetus in utero with influenza virus.