NS Segment Research Articles

The Influenza A Virus PB2, PA, NP, and M Segments Play a Pivotal Role during Genome Packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.

Highly pathogenic avian influenza virus H5N1 controls type I IFN induction in chicken macrophage HD-11 cells: a polygenic trait that involves NS1 and the polymerase complex

BackgroundInfluenza A viruses are well characterized to antagonize type I IFN induction in infected mammalian cells. However, limited information is available for avian cells. It was hypothesised that avian influenza viruses (AIV) with distinct virulence may interact differently with the avian innate immune system. Therefore, the type I IFN responses induced by highly virulent and low virulent H5N1 AIV and reassortants thereof were analysed in chicken cells.ResultsThe highly pathogenic (HP) AIV A/chicken/Yamaguchi/7/04 (H5N1) (Yama) did not induce type I IFN in infected chicken HD-11 macrophage-like cells. This contrasted with an NS1 mutant Yama virus (Yama-NS1A144V) and with the attenuated H5N1 AIV A/duck/Hokkaido/Vac-1/04 (Vac) carrying the haemagglutinin (HA) of the Yama virus (Vac-Yama/HA), that both induced type I IFN in these cells. The substitution of the NS segment from Yama with that from Vac in the Yama backbone resulted in induction of type I IFN secretion in HD-11 cells. However, vice versa, the Yama NS segment did not prevent type I IFN induction by the Vac-Yama/HA virus. This was different with the PB1/PB2/PA segment reassortant Yama and Vac-Yama/HA viruses. Whereas the Yama virus with the Vac PB1/PB2/PA segments induced type I IFN in HD-11 cells, the Vac-Yama/HA virus with the Yama PB1/PB2/PA segments did not. As reported for mammalian cells, the expression of H5N1 PB2 inhibited the activation of the IFN-β promoter in chicken DF-1 fibroblast cells. Importantly, the Yama PB2 was more potent at inhibiting the IFN-β promoter than the Vac PB2.ConclusionsThe present study demonstrates that the NS1 protein and the polymerase complex of the HPAIV Yama act in concert to antagonize chicken type I IFN secretion in HD-11 cells. PB2 alone can also exert a partial inhibitory effect on type I IFN induction. In conclusion, the control of type I IFN induction by H5N1 HPAIV represents a complex phenotype that involves a particular viral gene constellation rather than a single viral protein. Collectively, these findings contribute to understand the high virulence of HPAIV H5N1 viruses observed in the chicken host.

Identification of novel conserved functional motifs across most Influenza A viral strains

BackgroundInfluenza A virus poses a continuous threat to global public health. Design of novel universal drugs and vaccine requires a careful analysis of different strains of Influenza A viral genome from diverse hosts and subtypes. We performed a systematic in silico analysis of Influenza A viral segments of all available Influenza A viral strains and subtypes and grouped them based on host, subtype, and years isolated, and through multiple sequence alignments we extrapolated conserved regions, motifs, and accessible regions for functional mapping and annotation.ResultsAcross all species and strains 87 highly conserved regions (conservation percentage > = 90%) and 19 functional motifs (conservation percentage = 100%) were found in PB2, PB1, PA, NP, M, and NS segments. The conservation percentage of these segments ranged between 94 - 98% in human strains (the most conserved), 85 - 93% in swine strains (the most variable), and 91 - 94% in avian strains. The most conserved segment was different in each host (PB1 for human strains, NS for avian strains, and M for swine strains). Target accessibility prediction yielded 324 accessible regions, with a single stranded probability > 0.5, of which 78 coincided with conserved regions. Some of the interesting annotations in these regions included sites for protein-protein interactions, the RNA binding groove, and the proton ion channel.ConclusionsThe influenza virus has evolved to adapt to its host through variations in the GC content and conservation percentage of the conserved regions. Nineteen universal conserved functional motifs were discovered, of which some were accessible regions with interesting biological functions. These regions will serve as a foundation for universal drug targets as well as universal vaccine design.

NS Reassortment of an H7-Type Highly Pathogenic Avian Influenza Virus Affects Its Propagation by Altering the Regulation of Viral RNA Production and Antiviral Host Response

Highly pathogenic avian influenza viruses (HPAIV) with reassorted NS segments from H5- and H7-type avian virus strains placed in the genetic background of the A/FPV/Rostock/34 HPAIV (FPV; H7N1) were generated by reverse genetics. Virological characterizations demonstrated that the growth kinetics of the reassortant viruses differed from that of wild-type (wt) FPV and depended on whether cells were of mammalian or avian origin. Surprisingly, molecular analysis revealed that the different reassortant NS segments were not only responsible for alterations in the antiviral host response but also affected viral genome replication and transcription as well as nuclear ribonucleoprotein (RNP) export. RNP reconstitution experiments demonstrated that the effects on accumulation levels of viral RNA species were dependent on the specific NS segment as well as on the genetic background of the RNA-dependent RNA polymerase (RdRp). Beta interferon (IFN-β) expression and the induction of apoptosis were found to be inversely correlated with the magnitude of viral growth, while the NS allele, virus subtype, and nonstructural protein NS1 expression levels showed no correlation. Thus, these results demonstrate that the origin of the NS segment can have a dramatic effect on the replication efficiency and host range of HPAIV. Overall, our data suggest that the propagation of NS reassortant influenza viruses is affected at multiple steps of the viral life cycle as a result of the different effects of the NS1 protein on multiple viral and host functions.

Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus

Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b(+)CD11c(-), CD11b(-)CD11c(+), and CD11b(+)CD11c(+), as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.

Limited Compatibility of Polymerase Subunit Interactions in Influenza A and B Viruses

Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.

A seven plasmid-based system for the rescue of influenza C virus

We report the establishment of a reverse-genetics system for the rescue of recombinant influenza C/JJ/50 virus from seven plasmids. The nucleotide sequence of the whole C/JJ/50 genome was determined and full-length cDNAs were cloned into an RNA pol I/pol II-based bidirectional vector. Transfection of Vero cells and subsequent amplification on MDCK cells yielded viral HA titres of 128. The utility of this bidirectional approach was proved by generating a reassortant virus encoding the NS segment from strain C/JHB/1/66 and a virus with mutations in the noncoding ends of PB1. The latter virus, which has a base-pair mutation within the proposed double-stranded region of the PB1 termini, exhibited impaired replication. In conclusion, our efficient seven-plasmid system for the rescue of recombinant influenza C virus may be used to study the influenza C virus life cycle in more detail and for generation of influenza C virus-based vectors.

The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV

A reassortant avian influenza virus (designated FPV NS GD), carrying the NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able to replicate more efficiently in different human cell lines and primary mouse epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and death in experimentally infected mice and was detected in mouse lungs; in contrast, rFPV was not able to replicate in mice effectively. These results indicated an altered host range and increased virulence. Furthermore FPV NS GD showed pronounced pathogenicity in chicken embryos. In an attempt to define the molecular basis for the apparent differences, we determined that NS1 proteins of the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable efficiencies in vitro. However, FPV NS GD infection resulted in (i) increased expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger beta interferon promoter inhibition than rFPV. Taken together, the results shed further light on the importance of the NS segment of an H5N1 strain for viral replication, molecular pathogenicity, and host range of HPAIVs and the possible consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.

Cis -Acting Packaging Signals in the Influenza Virus PB1, PB2, and PA Genomic RNA Segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.

Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.

Mapping of Genomic Segments of Influenza B Virus Strains by an Oligonucleotide Microarray Method

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.

Genetic diversity of influenza B virus: the frequent reassortment and cocirculation of the genetically distinct reassortant viruses in a community.

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439-447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses.

Intranasal inoculation of a recombinant influenza virus containing exogenous nucleotides in the NS segment induces mucosal immune response against the exogenous gene product in mice

To establish a mucosal vaccine system, we evaluated the immunogenicity of a recombinant influenza virus, designated NS2Acat, in which the chloramphenicol acetyltransferase ( CAT) reporter gene is fused in frame to the NS1 gene in the NS gene segment. The NS2Acat replicated slightly within the lungs in BALB/c mice after intranasal administration, accompanying expression of the CAT and the viral HA mRNA. NS2Acat induced delayed-type hypersensitivity (DTH) and sensitized the CAT-specific T lymphocytes in the regional lymph nodes, which proliferated and synthesized several cytokines in vitro upon antigenic challenge. These results suggest that NS2Acat is capable of eliciting a respiratory immune response against the fused gene product.

The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA

The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA.

Course of impaired left ventricular function after acute myocardial infarction predicted with planar thallium-201 chloride and F18-fluorodeoxyglucose imaging

Planar rest myocardial thallium-201 chloride ( 201T1)/F18-fluorodeoxyglucose (FDG) imaging has been shown to distinguish between viable and non-viable tissue. Twenty-five patients (60 ± 9 years) with acute myocardial infarction were studied using this technique within 6 ± 2 days (T1) after infarction and again after 42 ± 4 days (T6). Serial assessment of wall motion with 2D-echocardiography was performed to determine the predictive value of radionuclide indices for the course of impaired regional left ventricular function. No revascularization procedure was performed. Segmental 201T1 and FDG uptake was evaluated using circumferential profiles. Echocardiographic wall motion was scored as normal, hypokinetic or akinetic. Myocardial segments were considered non-viable if a match between 201T1 and FDG uptake was present, which is a concordant reduction in 201T1 and FDG uptake (Group A). Myocardial segments were considered viable if: a mismatch was present between 201T1 and FDG uptake which was defined as a segmental FDG uptake exceeding 201T1 uptake by >- 20% in a segment with reduced 201T1 uptake (Group B); a normal FDG uptake (>- 75%) was present without a mismatch pattern in a segment with reduced 201T1 uptake ( 201T1 < 75% of peak activity) (Group C); a normal 201T1 uptake was present in the area of wall motion abnormality (Group D). Corresponding scintigraphic images obtained at T1 and T6 were compared. Results: 51 segments were normokinetic, 37 were hypokinetic and 26 were akinetic at T1. Of the 63 segments with wall motion abnormalities at T1, 18 regions showed a match (FDG- 201T1 < 20%) (Group A). Regional function improved in only one (6%) of these segments. In 19 regions a mismatch was present (FDG- 201T1 >- 20%) (Group B) of which three (16%) showed spontaneous improvement in function ( P = NS vs. matched segments), although recovery varied considerably among patients. Regional function in two segments deteriorated. In 14 regions with reduced 201T1 uptake, FDG uptake was normal (Group C) of which five (36%) were improved after 6 weeks ( p < 0.05 vs. Match; p = NS vs. mismatched segments). Of the 12 segments with normal 201T1 uptake (Group D), seven (58%) showed improvement in function, whereas five (42%) did not show improvement ( p < 0.05 vs. Match). In addition, all scintigraphically selected viable segments were grouped (Group B+C+D) and compared with the non-viable segments (Group A). The predictive value of a positive viability test for spontaneous functional improvement was 33%. The predictive value of a negative viability test for lack of functional improvement was 94%. Conclusions: absence of residual FDG uptake shortly after infarction is associated with irreversible injury, while preservation of metabolic activity identifies segments with variable outcome. Wall motion alone is not a good indicator for the presence of viable tissue. Planar 201T1/FDG imaging allows early identification of viable but jeopardized tissue and may help select patients who will benefit from aggressive therapy to salvage endangered myocardium.

Characterization of a unique protein produced by influenza a virus recovered from a long-term persistent infection

Virus isolated from a persistent infection initiated in BHK cells with influenza A/WSN/33 (H1 N1) produced an unusual pattern of protein synthesis in productive infections of BHK cells: The levels of NP and M1 proteins were slightly reduced compared to an infection with wild-type WSN, while the other proteins (Pb1, Pb2, Pa, HA, NS1, and NS2) were synthesized at very low or undetectable levels. In addition, a new viral protein with an approximate molecular weight of 11 kDa (Pi protein) is made ( Frielle et al., Virology 138,103–117,1984). When viral RNA was analyzed by the Northern blot technique, a deletion was found in the NS gene segment and in NS1 mRNA; all other RNAs were full-sized. Immunoprecipitation of in vitro translation products demonstrated that the Pi protein reacts specifically with anti-NS1 serum. In addition, the Pi protein, like the NS1 of the parental wild-type virus, accumulated in the nucleus of infected cells. These results indicate that the Pi protein is a mutated form of the NS1 protein encoded by a deleted NS segment and suggest that this mutation may be involved in the expression of the persistent virus phenotype.

Regulation of the Replication of Influenza Virus RNA Segments: Partial Suppression of Protein Synthesis Restores the 'Early' Replication Pattern

The replication of influenza virus RNA was monitored by RNA-RNA hybridization with subsequent analysis of hybrid duplexes, as well as by immunosorption of viral nucleocapsids from extracts of [3H]uridine-labelled cells followed by the isolation and characterization of nucleocapsid-associated RNA. The nucleocapsid-associated RNA preparations contained mostly negative-strand genomic RNA. Electrophoresis of the hybrid RNA duplexes or single-stranded nucleocapsid-associated RNA in polyacrylamide gel revealed an 'early' replication pattern, with a predominance of the NP and NS gene segments, in cells labelled from 0 to 1 h post-infection. At later stages of infection the pattern changed to the 'late' one, with the M gene segment in excess of NS, and the NP gene no longer predominant. Cycloheximide added as late as 2 or 3 h post-infection suppressed RNA replication. Moderate concentrations of cycloheximide inhibited the replication of NS and NP gene segments to a lesser degree than the replication of the other RNA segments, thus restoring the 'early' replication pattern. Cycloheximide treatment resulted in a slight increase in the percentage of positive strands in nucleocapsid-associated RNA. The role of protein synthesis in the transition from the 'early' to the 'late' pattern of influenza virus RNA synthesis is discussed.

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.

Localization of the ts defects of ts mutants of influenza A virus using complementation analysis and gel analysis of the RNA segments of recombinants

Two sets of temperature-sensitive ( ts) mutants of influenza A virus (derived by mutagenesis of the Hong Kong (HK) and WSN strains, respectively) were analyzed for shared lesions using a complementation-recombination assay which was performed directly on tissue culture monolayers. Each of these sets had previously been shown to consist of seven complementation groups. By this assay three of the HK groups were shown to correspond to WSN complementation groups: HK group 1 and WSN I shared a common ts lesion as did HK group 5 and WSN group III; finally, HK group 6 and WSN group II were shown to be related. The relationship of the WSN groups IV, V, VI, and VII and HK groups 2, 3, 4, and 7 could not be interpreted from the complementation-recombination studies. We therefore sought to localize the lesions of the HK mutants by preparing ts + recombinants between the HK groups 6 and 7 mutants and PR8 wildtype or WSN ts mutant 51. In this way we have localized the lesions in these mutants to RNA segments 3 (P2) and 5 (NP), respectively. The findings with HK group 6 confirm the genetic analysis while those with HK group 7 suggest the occurrence of three independent groups of lesions on the NP segment of HK (groups 2, 3, and 7). The cotransfer of the NS segment with the NP and P2 genes is discussed, as are the factors which make genetic analysis by biological methods difficult, but perhaps useful in understanding the functional interactions of the influenza polypeptides.

Nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS RNA: Conservation of the NS1/NS2 overlapping gene structure in a divergent influenza virus RNA segment

The complete nucleotide sequence of the influenza A/duck/Alberta/60/76 virus NS gene was determined. A direct sequence comparison of this NS gene with that of influenza A/PR/8/34 virus showed a difference of 27.3%. In contrast, the differences among the previously sequenced NS genes of the A/PR/8/34, A/FPV/Rostock/34, and A/Udorn/72 viruses were much lower and ranged from 8–11%. The NS genes of the latter three strains contain open reading frames in their virion (−) RNAs which potentially code for polypeptides of 167 and 216 amino acids. Although the NS segment of the A/duck/Alberta/60/76 virus lacks such an agnogene in its negative strand sequence, it conserves the overlapping NS1 and NS2 gene arrangement identified in the other NS RNA segments. A comparison of the deduced amino acid sequences of the A/duck/Alberta/60/76 and A/ PR/8/34 virus NS genes showed that the NS2 polypeptides are more highly conserved (18.2% differences) than the NS1 polypeptides (33.0% differences). Surprisingly, the NS1 polypeptides of the two strains show this high degree of variation over their entire lengths, including the amino acids encoded by the conserved RNA domain of the NS1 gene and NS2 gene overlap.