Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Sylvie van der Werf. Fax +33 1 4061 3241. e-mail svdwerf{at}pasteur.fr
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Abstract |
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Introduction |
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For all viral proteins, distinct evolutionary lineages of viral genes are associated with human and avian influenza A viruses (Webster & Bean, 1998 ). A number of reassortant viruses which contain human influenza HA and NA genes and various combinations of avian or human influenza internal genes have been generated by coinfection of cultured cells and evaluated for their ability to replicate in vitro and/or in vivo (Tian et al., 1985
; Buckler-White et al., 1986
; Clements et al., 1986
, 1992
; Snyder et al., 1987
). This has lead to the idea that specific constellations of gene segments encoding the internal proteins could be a determinant for the ability of reassortant viruses to replicate efficiently in mammalian cells and/or in the respiratory epithelium of primates. As far as the core proteins (PB1, PB2, PA and NP) are concerned, studies on reassortant viruses have shown that efficient virus replication is observed for some, but not all, constellations of human and avian virus-derived proteins, suggesting that specific interactions between the core proteins are required. On the other hand, co-immunoprecipitation and two-hybrid experiments have indicated that molecular assembly of the polymerase complex relies on PB1PB2 and PB1PA interactions, whereas no direct PB2PA interaction has been detected (Digard et al., 1989
; Gonzalez et al., 1996
; Toyoda et al., 1996
; Zürcher et al., 1996
). Biochemical and genetic data have established that the PB1 subunit displays RNA-dependent RNA polymerase activity (Braam et al., 1983
; Biswas & Nayak, 1994
) and that the PB2 subunit is involved in the cap-snatching mechanism by which viral mRNA synthesis is initiated (Blaas et al., 1982
; Braam et al., 1983
; Blok et al., 1996
; Shi et al., 1996
), while the only biochemical activity found to be associated with PA is the induction of proteolysis (Sanz-Ezquerro et al., 1995
). Studies on reassortant viruses have clearly shown that the origin of PB2 is a determinant for their efficiency of replication in mammalian cells (Almond, 1977
; Clements et al., 1992
). A reassortant virus that derived its PB2 gene from the AIV A/Mallard/NY/-6750/78 and its remaining genes from a human virus failed to produce plaques in mammalian cells and its replication was restricted in squirrel monkeys and humans (Clements et al., 1992
). Analysis of phenotypic revertants of the reassortant virus indicated that amino acid 627 of PB2 was a major determinant of the phenotype (Subbarao et al., 1993
). However, a major limitation from studies on reassortant viruses stems from the fact that all possible constellations among internal genes could not be examined as viruses with certain reassortant genotypes were not isolated, possibly because of a very low replication efficiency.
To address the question as to how efficiently the polymerase proteins derived from a human and an avian virus can interact with each other in the context of a mammalian cell, we made use of the genetic system described by Pleschka et al. (1996) for the in vivo reconstitution of functional ribonucleoproteins (RNPs). This was achieved by transfecting into COS-1 cells a plasmid that directed the expression of a viral-like reporter RNA together with plasmids from which the four core proteins were expressed. The synthetic reporter RNA contained the CAT reporter gene sequence in antisense orientation flanked by the non-coding sequences that serve as promoters for the viral polymerase, which are highly conserved among influenza A viruses (Desselberger et al., 1980
). Homospecific as well as heterospecific RNPs were reconstituted by expressing proteins derived (i) from two strains of human viruses, A/Puerto Rico/8/34 (which is anterior to the 1957 reassortment event) and A/Victoria/3/75 (which derives from the 1968 reassortant virus); (ii) from two strains of AIVs, the A/Mallard/NY/6750/78 strain, which was used in the reassortment experiments mentioned above (Clements et al., 1992
; Subbarao et al., 1993
), and A/FPV/Rostock/34, which is known to be highly pathogenic in chickens; (iii) from A/Hong Kong/156/97, the H5N1 strain of avian origin that was isolated from the index case in Hong Kong (Claas et al., 1998
; Subbarao et al., 1998
). This experimental system allowed us to examine how efficiently all possible heterospecific mixtures of the four core proteins permitted transcription/replication of the viral-like reporter RNA.
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Methods |
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Molecular cloning and sequencing of cDNAs.
Virus isolates A/FPV/Rostock/34 (FPV), A/Mallard/NY/6750/78 (MAL) and A/-Hong Kong/156/97 (HK) were grown in 11-day-old embryonated chicken eggs (FPV, MAL) or on MDCK cells (HK). Viral genomic RNA was extracted using 5 M guanidium isothiocyanate and phenol. Full-length cDNAs were prepared by reverse-transcription of the viral RNA using a primer complementary to the 12 bp conserved at the 3' end of each viral segment and 20 U avian myeloblastosis virus reverse transcriptase with incubation at 42 °C for 1 h followed by incubation at 55 °C for 15 min. Amplification was performed next using primers specific for the coding sequences of PB1, PB2, PA or NP, with additional nucleotides providing an EcoRV site at their 5' end. The exact sequence of the primers can be obtained from the authors upon request. The PCR conditions were optimized for each sequence to be amplified: either Pwo or Expand High Fidelity polymerase (Boehringer Mannheim) were used, for 30 cycles, each consisting of 1 min at 94 °C, 1 min at 5060 °C (depending on the sequence), 29 min (depending on the sequence) at 72 °C. The PCR products were digested with EcoRV and cloned into the EcoRV site of plasmid pGEM5z(f+). At least three positive clones were sequenced using a dRhod or a Big Dye terminator sequencing kit (Perkin Elmer) and run on a 377 automated sequencer (Perkin Elmer). Clones without PCR-induced mutations were selected, and the EcoRV fragments derived from these were sub-cloned into the EcoRV site of plasmid pHMG.
Construction of mutants.
The PCR-based protocol from Tomic et al. (1990) was used to introduce point mutations at residue 627 (Gln
Lys) of MAL- and FPV-PB2. Primers were designed to provide two intermediate PCR products with a region of common sequence containing the mutation at codon 627 (GAA or GAG
AAA) and a site for BsaI. The exact sequence of the primers can be obtained from the authors upon request. Plasmids pGEM5z-MAL-PB2 or pGEM5z-FPV-PB2 were used as the starting template. Proof-reading Pwo polymerase (Boehringer Mannheim) was used for PCR amplification, for 20 cycles, each consisting of 1 min at 94 °C, 1 min at 50 °C, 90 s at 72 °C. The PCR products were digested with BsaI and ligated to each other. The ligation products were sub-cloned either in pGEM5z-MAL-PB2 using MfeI and EcoRI restriction sites or in pGEM5z-FPV-PB2 using two EcoRI sites. Positive clones were sequenced as previously indicated. Clones bearing the site-directed mutation but no unanticipated nucleotide change were selected, and the EcoRV fragments derived from these were sub-cloned into the EcoRV site of plasmid pHMG.
Transfections and CAT assays.
COS-1 cells were grown in Dulbeccos modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS). Subconfluent monolayers of COS-1 cells (3x105 cells in 35 mm dishes) were transfected using the Fugene 6-mediated method (Boehringer Mannheim). For the transient replication assay, a mixture of plasmids pHMG-PB1, -PB2, -PA, -NP (1; 1; 1; 2 µg) and pPolI-CAT-RT (1 µg) was transfected. Cells were incubated at 37 °C in DMEM containing 10% FCS until harvest. At 48 h post-transfection, cell extracts were prepared in 500 µl of lysis buffer and tested for CAT levels using the CAT ELISA kit (Boehringer Mannheim), which allowed detection of 0·05 ng/ml CAT. All experiments were done in duplicate.
Western blotting.
For Western blot assays, total cell extracts were used. Transfected cells were resuspended directly in 300 µl of sample loading buffer, the proteins were resolved by electrophoresis in SDSpolyacrylamide gels and transferred onto a nitrocellulose membrane (Biorad). The filters were incubated for 1 h at room temperature or overnight at 4 °C with primary antibodies diluted in PBS containing 0·1% BSA (Sigma) and 0·25% Tween 20. Rabbit antisera directed against the PB1, PB2, and PA proteins of VIC virus were kindly provided by J. Ortin (Universidad Autonoma de Madrid, Spain) and were diluted 1/1000 (PB1, PA) or 1/5000 (PB2). For NP, a mouse antiserum was prepared by repeated intramuscular injection of plasmid pHMG-PR8-NP in mice, and used at a 1/500 dilution. After washing, the filters were incubated for 1 h at room temperature with peroxidase-conjugated secondary antibodies (donkey anti-rabbitperoxidase or goat anti-mouseperoxidase, Amersham) diluted 1/10000 in PBS containing 0·1% BSA and 0·25% Tween 20. After washing, the filters were incubated with peroxidase substrate (ECL Plus System, Amersham), scanned using a STORM820 optical scanner (Molecular Dynamics) and analysed using the Image Quant program (Molecular Dynamics).
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Results |
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Functional analysis of heterospecific replication complexes
In order to determine how efficiently heterospecific mixtures of PB1, PB2, PA and NP allowed transcription/replication of the viral-like reporter RNA, heterospecific RNPs were reconstituted with proteins derived from either human (PR8, VIC), avian (FPV, MAL) or HK viruses. For a given pair of viruses, all 16 possible combinations between the two sets of pHMG-PB1, -PB2, -PA and -NP plasmids were transfected together with plasmid pPolI-CAT-RT into COS-1 cells. The efficiency with which the various reconstituted heterospecific complexes permitted transcription/replication of the viral-like reporter RNA was determined by measuring the CAT levels in extracts prepared from transfected cells. In order to analyse separately the effects of the origin of PB2, PB1, PA or NP, we studied successively the variations of CAT levels that resulted from the permutation of one of the four proteins from one strain to another.
The human or avian origin of PB2 appeared to be a determinant for the efficiency with which heterospecific complexes allowed transcription/replication of the viral-like reporter RNA. Indeed, whatever the combination of PB1, PA and NP between PR8 and FPV viruses, high levels of CAT expression (>600 ng/ml) were measured in cell extracts when PB2 was derived from the human virus (PR8). Furthermore, except when PB1, PA and NP were of avian origin, CAT expression was systematically higher (5100-fold) when PB2 was derived from the human (PR8) rather than from the avian (FPV) virus (Fig. 2). Very similar results were obtained when PR8/MAL or VIC/FPV protein combinations were tested (not shown). In contrast, PB2 permutations had little effect among the MAL/FPV combinations (not shown). As amino acid 627 of PB2 (Glu in PB2 proteins of avian origin, Lys in PB2 proteins of human origin) has been identified as an important determinant of the host range of human/avian reassortant viruses (Subbarao et al., 1993
), we generated cDNAs encoding PB2 from MAL or FPV viruses with a Glu
Lys substitution at residue 627 (E627K), by site-directed mutagenesis. Western blot analysis showed that the wild-type and mutant proteins were expressed at similar levels (Table 1
). When the mutant MAL- or FPV-PB2 proteins were used in the replication assay, the levels of CAT expression were drastically increased as compared to the levels observed with the corresponding avian PB2 proteins (Table 1
). In the case of the combination where avian PB1, human PA and avian NP were used, the levels of CAT expression reached with the E627K mutant PB2 proteins were similar to those observed with PR8-PB2 (Table 1
). This confirmed that PB2 amino acid 627 is an important determinant of the ability of avian/human heterospecific RNPs to undergo transcription/replication in mammalian cells. When PR8 and HK proteins were mixed, substitutions of PR8-PB2 by HK-PB2 generally had the same effects as substitution of PR8-PB2 by MAL- or FPV-PB2 (not shown). However, in a number of combinations, such as the avian- or HK-PB1, PR8-PA, avian- or HK-NP combination shown in Table 1
, we repeatedly observed that CAT expression was reduced to a lesser extent when PR8-PB2 was replaced by HK-PB2 rather than by MAL- or FPV-PB2 (50% reduction compared to 85 and 66% reduction respectively, P<0·1).
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Discussion |
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The origin of PB2, and especially the nature of PB2 amino acid 627, appeared to be an important determinant of the functional efficiency of heterospecific complexes in COS-1 cells. Our observations being in accordance with previous observations on human/avian reassortant viruses (Clements et al., 1992 ; Subbarao et al., 1993
), we believe that they constitute a validation of our experimental approach for the identification of other molecular determinants of the compatibility between core proteins from human and avian strains of influenza A viruses. Moreover, they demonstrate that in order to manifest, the PB2-mediated restriction of transcription/replication in mammalian cells does not need the expression of viral genes other than PB1, PA and NP. Detailed analysis of the RNA species will be needed to determine precisely which transcription and/or replication process is affected by the origin of PB2. The poor efficiency of heterospecific complexes comprising a PB2 subunit of avian origin could result from an incompatibility with other subunits of human origin and/or an altered interaction with a host factor. If protein incompatibility only was involved, poor efficiency would probably characterize both an avian/human complex and its human/avian counterpart, which does not correspond to our observations. Indeed our analysis of all possible constellations among the polymerase genes (which was not provided by studies on reassortant viruses) shows that in the context of a mammalian cell, PB2 of human origin cooperates efficiently with any combination of PB1, PA or NP proteins. If interaction of PB2 with a host factor was the only mechanism involved, avian homospecific complexes and any heterospecific complex comprising a PB2 protein of avian origin would function with an equally poor efficiency, which does not correspond to our observations either. Indeed avian-derived homospecific complexes achieved similar levels of transcription/replication of the viral-like reporter RNA as compared to human-derived complexes. On the whole, our observations suggest that the mechanism by which differences in the PB2 protein, and especially the nature of amino acid 627, alter the efficiency of transcription/replication is complex, relying on interactions of PB2 with both viral and cellular proteins. Interaction of PB2 residue 627 with a host factor is likely to be the major determinant, as the efficiencies of the MAL and FPV complexes were increased when MAL-E627K-PB2 and FPV-E627K-PB2 were combined with MAL-PB1, -PA, -NP and FPV-PB1, -PA, -NP, respectively (results not shown). Further experiments involving both mammalian and avian cell lines will probably help clarify this point.
We observed that homospecificity between PB2 and NP appeared to enhance the functional efficiency of chimeric complexes. The NP, in addition to its structural role for the formation of the RNPs, is required for efficient elongation of RNA chains and is thought to be involved in the switch from transcription to replication (Beaton & Krug, 1986 ; Honda et al., 1988
). In accordance with previously published results based on genetic (Mandler et al., 1991
) or biochemical (Biswas et al., 1998
) studies, our observations suggest that PB2NP interactions could be involved in the transcription/replication process, and could therefore impose some constraints on reassortment events that involve the corresponding segments. Replication assays using human/avian chimeric proteins may help identify the domains of interaction between PB2 and NP. In this study, the human or avian origin of NP per se was not found to be a determinant for the efficiency of transcription/replication of the viral-like reporter RNA in COS-1 cells. In the context of a reassortant virus which derived all remaining genes from a human virus, the NP gene from the MAL avian virus was shown to specify restriction of virus replication in squirrel monkeys (Tian et al., 1985
; Clements et al., 1992
), but not in humans or in MDCK cells (Clements et al., 1992
). Our observations suggest that the attenuation which has been observed in squirrel monkeys does not result from an impairment of the transcription/replication process.
In the context of a mammalian cell, PB1 of avian origin appeared to cooperate with PB2, PA and NP of human origin more efficiently than PB1 of human origin appeared to cooperate with PB2, PA and NP of avian origin. In particular, in our experiments the PB2 humanPB1 avianPA human constellation (HAH), associated either with a human or avian NP, permitted efficient replication of the viral-like RNA reporter. This suggests that the phenotype of influenza A/Pintail/119/79xA/Washington/897/80 reassortant viruses, which possessed the HAH constellation and were restricted in MDCK cells and in monkeys (Snyder et al., 1987 ), was not determined by the HAH constellation only, but probably by the origin of the NS and M segments also. Our results may also be related to the fact that segments found to be of avian origin in the 1957 and 1968 pandemic viruses encoded not only the surface glycoproteins (NA and/or HA) but also PB1 (Kawaoka et al., 1989
). Taken together, these observations suggest that PB1 proteins of avian origin might have a higher specific activity than PB1 proteins of human origin, and that the acquisition of the PB1 segment from an avian virus may have conferred a selective advantage to the 1957 and 1968 reassortant viruses. In an alignment of PB1 sequences of human or avian origin (given the fact that only four sequences, including PR8-PB1, are available for human viruses isolated before 1957), five amino acids appeared as characteristic of the origin of the sequences (Fig. 1
). Remarkably, in all PB1 sequences derived from human viruses isolated later than 1957, including six virus strains isolated in Japan in 19931996 (Lindstrom et al., 1998
), avian-type residues were found at all but one of these specific positions (Fig. 1
). Conservation over a period of 28 years (running from the 1968 reassortment event up to 1996) may reflect some selective advantage conferred by these avian-type residues. In addition to the four conserved motifs found among all viral RNA-dependent polymerases (Poch et al., 1989
), putative nucleotide-binding domains (Asano & Ishihama, 1997
) and RNA-binding sites (Li et al., 1998
; Gonzalez & Ortin, 1999a
, b
) have recently been identified on PB1. A number of amino acids that appear to be characteristic of the human or avian origin of the sequences are located within these domains and may therefore affect the specific activity of the PB1 proteins.
Interestingly, PB2 and PB1 proteins from the HK virus were found to behave in an intermediate fashion with respect to the corresponding proteins from human and avian viruses. Indeed, in the replication assay, both the disadvantage of HK-PB2 over PR8-PB2 and the advantage of HK-PB1 over PR8-PB1 were reduced in comparison to FPV or MAL proteins in a number of combinations. As far as PB2 is concerned, the HK strain used here shows a Glu at residue 627 (typical of avian viruses). Our data suggest that some molecular characteristics other than the nature of residue 627 could render HK-PB2 closer to human virus-derived PB2 than FPV or MAL-PB2. A potential candidate is residue 199 that is of the human type (Ser) in HK-PB2 (Fig. 1), while it is of the avian type (Ala) in the sequence of PB2 from an H5N1 virus (A/Chicken/Hong Kong/220/97) circulating in chickens in Hong Kong during the same period (Suarez et al., 1998
). Mutagenesis experiments will be needed to confirm this hypothesis. The fact that not only one but all three PB1, PB2 and PA proteins from HK show some characteristics of human viruses at the level of their amino acid sequence (Fig. 1
) is probably related to the fact that proteins associated physically and functionally undergo co-evolution. Our observations suggest that PB1 and PB2 proteins from HK may also have acquired some functional characteristics from human viruses, which could partially account for the ability of this virus strain to replicate in humans.
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Acknowledgments |
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This work was supported in part by the Ministère de lEducation Nationale, de la Recherche et de la Technologie (EA 302).
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Footnotes |
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References |
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Asano, Y. & Ishihama, A. (1997). Identification of two nucleotide-binding domains on the PB1 subunit of influenza virus RNA polymerase.Journal of Biochemistry 122, 627-634.[Abstract]
Beare, A. S. & Webster, R. G. (1991). Replication of avian influenza viruses in humans.Archives of Virology 119, 37-42.[Medline]
Beaton, A. R. & Krug, R. M. (1986). Transcription antitermination during influenza viral template RNA synthesis requires the nucleocapsid protein and the absence of a 5' capped end.Proceedings of the National Academy of Sciences, USA 83, 6282-6286.[Abstract]
Biswas, S. K. & Nayak, D. P. (1994). Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.Journal of Virology 68, 1819-1826.[Abstract]
Biswas, S. K., Boutz, P. L. & Nayak, D. P. (1998). Influenza virus nucleoprotein interacts with influenza virus polymerase proteins.Journal of Virology 72, 5493-5501.
Blaas, D., Patzelt, E. & Kuechler, E. (1982). Cap-recognizing protein of influenza virus.Virology 116, 339-348.[Medline]
Blok, V., Cianci, C., Tibbles, K. W., Inglis, S. C., Krystal, M. & Digard, P. (1996). Inhibition of the influenza virus RNA-dependent RNA polymerase by antisera directed against the carboxy-terminal region of the PB2 subunit.Journal of General Virology 77, 1025-1033.[Abstract]
Braam, J., Ulmanen, I. & Krug, R. M. (1983). Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription.Cell 34, 609-618.[Medline]
Buckler-White, A. J., Naeve, C. W. & Murphy, B. R. (1986). Characterization of a gene coding for M proteins which is involved in host range restriction of an avian influenza A virus in monkeys.Journal of Virology 57, 697-700.[Medline]
Centers for Disease Control & Prevention (1998). Update: isolation of avian influenza A (H5N1) viruses from humans Hong Kong, 19971998. Morbidity & Mortality Weekly Report 46, 12451247.
Claas, E. C., Osterhaus, A. D., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus [published erratum appears in Lancet (1998) 351, 1292].Lancet 351, 472-477.[Medline]
Clements, M. L., Snyder, M. H., Buckler-White, A. J., Tierney, E. L., London, W. T. & Murphy, B. R. (1986). Evaluation of avianhuman reassortant influenza A/Washington/897/80xA/Pintail/119/79 virus in monkeys and adult volunteers.Journal of Clinical Microbiology 24, 47-51.[Medline]
Clements, M. L., Subbarao, E. K., Fries, L. F., Karron, R. A., London, W. T. & Murphy, B. R. (1992). Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers.Journal of Clinical Microbiology 30, 655-662.[Abstract]
Desselberger, U., Racaniello, V. R., Zazra, J. J. & Palese, P. (1980). The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity.Gene 8, 315-328.[Medline]
Digard, P., Block, V. C. & Inglis, S. C. (1989). Complex formation between influenza virus polymerase proteins expressed in Xenopus oocytes.Virology 171, 162-169.[Medline]
Gonzalez, S. & Ortin, J. (1999a). Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates.EMBO Journal 18, 3767-3775.
Gonzalez, S. & Ortin, J. (1999b). Characterization of influenza virus PB1 protein binding to viral RNA: two separate regions of the protein contribute to the interaction domain.Journal of Virology 73, 631-637.
Gonzalez, S., Zurcher, T. & Ortin, J. (1996). Identification of two separate domains in the influenza virus PB1 protein involved in the interaction with the PB2 and PA subunits: a model for the viral RNA polymerase structure.Nucleic Acids Research 24, 4456-4463.
Guan, Y., Shortridge, K. F., Krauss, S. & Webster, R. G. (1999). Molecular characterization of H9N2 influenza viruses: were they the donors of the internal genes of H5N1 viruses in Hong-Kong?Proceedings of the National Academy of Sciences, USA 96, 9363-9367.
Honda, A., Ueda, K., Nagata, K. & Ishihama, A. (1988). RNA polymerase of influenza virus: role of NP in RNA chain elongation.Journal of Biochemistry 104, 1021-1026.[Abstract]
Kawaoka, Y., Krauss, S. & Webster, R. G. (1989). Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.Journal of Virology 63, 4603-4608.[Medline]
Li, M. L., Ramirez, B. C. & Krug, R. M. (1998). 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.EMBO Journal 17, 5844-5852.
Lindstrom, S. E., Hiromoto, Y., Nerome, R., Omoe, K., Sugita, S., Yamasaki, Y., Takahashi, T. & Nerome, K. (1998). Phylogenetic analysis of the entire genome of influenza A (H3N2) viruses from Japan: evidence for genetic reassortment of the six internal genes.Journal of Virology 72, 8021-8031.
Mandler, J., Muller, K. & Scholtissek, C. (1991). Mutants and revertants of an avian influenza A virus with temperature-sensitive defects in the nucleoprotein and PB2.Virology 181, 512-519.[Medline]
Murphy, B. R., Hinshaw, V. S., Sly, D. L., London, W. T., Hosier, N. T., Wood, F. T., Webster, R. G. & Chanock, R. M. (1982). Virulence of avian influenza A viruses for squirrel monkeys.Infection and Immunity 37, 1119-1126.[Medline]
Pleschka, S., Jaskunas, R., Engelhardt, O. G., Zurcher, T., Palese, P. & Garcia-Sastre, A. (1996). A plasmid-based reverse genetics system for influenza A virus.Journal of Virology 70, 4188-4192.[Abstract]
Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.EMBO Journal 8, 3867-3874.[Abstract]
Sanz-Ezquerro, J. J., de la Luna, S., Ortin, J. & Nieto, A. (1995). Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.Journal of Virology 69, 2420-2426.[Abstract]
Scholtissek, C., Rohde, W., von Hoyningen, V. & Rott, R. (1978). On the origin of the human influenza subtype H2N2 and H3N2.Virology 87, 13-20.[Medline]
Scholtissek, C., Hinshaw, V. S. & Olsen, C. W. (1998). Influenza in pigs and their role as the intermediate host. In Textbook of Influenza, pp. 137-145. Edited by K. G. Nicholson, R. G. Webster & A. J. Hay. Oxford: Blackwell Science.
Shi, L., Galarza, J. M. & Summers, D. F. (1996). Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit.Virus Research 42, 1-9.[Medline]
Snyder, M. H., Buckler-White, A. J., London, W. T., Tierney, E. L. & Murphy, B. R. (1987). The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avianhuman influenza A/Pintail/79 reassortant viruses for monkeys.Journal of Virology 61, 2857-2863.[Medline]
Suarez, D. L., Perdue, M. L., Cox, N., Rowe, T., Bender, C., Huang, J. & Swayne, D. E. (1998). Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.Journal of Virology 72, 6678-6688.
Subbarao, E. K., London, W. & Murphy, B. R. (1993). A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.Journal of Virology 67, 1761-1764.[Abstract]
Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C., Huang, J., Hemphill, M., Rowe, T., Shaw, M., Xu, X., Fukuda, K. & Cox, N. (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness [see comments].Science 279, 393-396.
Tian, S. F., Buckler-White, A. J., London, W. T., Reck, L. J., Chanock, R. M. & Murphy, B. R. (1985). Nucleoprotein and membrane protein genes are associated with restriction of replication of influenza A/Mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract.Journal of Virology 53, 771-775.[Medline]
Tomic, M., Sunjevaric, I., Savtchenko, E. S. & Blumenberg, M. (1990). A rapid and simple method for introducing specific mutations into any position of DNA leaving all other positions unaltered.Nucleic Acids Research 18, 1656.[Medline]
Toyoda, T., Adyshev, D. M., Kobayashi, M., Iwata, A. & Ishihama, A. (1996). Molecular assembly of the influenza virus RNA polymerase: determination of the subunitsubunit contact sites.Journal of General Virology 77, 2149-2157.[Abstract]
Webster, R. G. & Bean, W. J.Jr (1998). Evolution and ecology of influenza viruses: interspecies transmission. In Textbook of Influenza, pp. 109-119. Edited by K. G. Nicholson, R. G. Webster & A. J. Hay. Oxford: Blackwell Science.
Zürcher, T., de la Luna, S., Sanz-Ezquerro, J. J., Nieto, A. & Ortín, J. (1996). Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.Journal of General Virology 77, 1745-1749.[Abstract]
Received 8 November 1999;
accepted 26 January 2000.