Department of Virology I, National Institute of Infectious Diseases, 23-1, Toyama 1-chome, Shinjuku-ku, 162-8640 Tokyo, Japan1
Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Kokubunji-machi, Shimotsuga, 329-04 Tochigi, Japan2
Author for correspondence: Kuniaki Nerome. Fax +81 3 5285 1155. e-mail knerome{at}nih.go.jp
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Abstract |
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Introduction |
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The functional ribonucleoprotein (RNP) complex of influenza viruses responsible for transcription and replication of viral RNA exists as a heterotrimer polymerase complex consisting of the PB2, PB1 and PA proteins (Detjen et al., 1987 ; Digard et al., 1989
; Honda et al., 1990
; Kato et al., 1985
) bound to viral RNA and the nucleoprotein (NP) (Jambrina et al., 1997
; Krug et al., 1989
). Extensive analysis of protein subunits of the RNP complex of type A viruses has resulted in a fairly detailed understanding of the functional role of each subunit. Briefly, the PB1 protein has been shown to possess RNA polymerase catalytic activity (Biswas & Nayak, 1994
; Braam et al., 1983
; Kobayashi et al., 1996
; Nakagawa et al., 1996
; Toyoda et al., 1996b
), whereas the PB2 protein is responsible for the binding, cleavage and recruitment of cellular mRNA cap-1 structures essential for viral mRNA synthesis (Braam et al., 1983
; Nakagawa et al., 1995
; Ulmanen et al., 1983
). Although the PA protein has been shown to be essential in influenza virus gene expression, its function has not been fully elucidated. However, experiments have provided evidence indicating that the PA protein is required for switching from mRNA synthesis to cRNA synthesis during virus replication (Mahy, 1983
; Nakagawa et al., 1996
) and the ability to induce generalized proteolysis has been demonstrated (Sanz-Ezquerro et al., 1995
, 1996
).
It is generally believed that the functional roles of the PB2, PB1 and PA proteins of type B viruses are essentially similar to those of type A viruses based on structural and functional analysis (Akoto-Amanfu et al., 1987 ; Jambrina et al., 1997
; Kemdirim et al., 1986
). Also, Jambrina et al. (1997)
recently reported that all polymerase proteins and the NP protein of influenza B viruses are essential for replication of a model RNA template. Moreover, although the RNP complex of type A and type B viruses demonstrates type-specific subunit interactions, both RNA polymerase complexes were capable of binding and synthesizing heterotypic templates, albeit at reduced frequencies (Jambrina et al., 1997
; Stevens & Barclay, 1998
).
Although the complete nucleotide sequence of influenza B virus polymerase genes has been determined (DeBorde et al., 1988 ; Jambrina et al., 1997
; Kemdirim et al., 1986
), little is known of the evolutionary characteristics of these genes. In the present study, we investigated the phylogenetic patterns of the PB2, PB1 and PA polymerase genes of influenza B viruses isolated from 1940 to 1998 and analysed deduced protein variability among predicted proteins of these genes. Additionally, in order to understand the evolutionary relationships among the six internal genes and the HA gene, phylogenetic patterns of the three polymerase genes were compared in a parallel fashion with those previously determined for the NP, M, NS and HA genes of influenza B virus (Lindstrom et al., 1999
). Evolutionary characteristics of the polymerase proteins of influenza type A and B viruses are also compared.
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Methods |
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Phylogenetic analysis and evolutionary rates.
Phylogenetic trees were constructed using the neighbour-joining method (Gojobori et al., 1982 ; Nei & Gojobori, 1986
; Saitou & Nei, 1987
) and bootstrap analysis (n=500) (Felsenstein, 1985
) to determine the best fitting tree for each gene. Nucleotide distance matrices were estimated by the three-parameter method based on the total number of nucleotide substitutions (Gojobori et al., 1982
). Dendrograms were also constructed by maximum-parsimony and maximum-likelihood methods using PHYLIP (Phylogeny Inference Package) software version 3.57c (Felsenstein, 1995
) to evaluate the consistency of tree topologies. Evolutionary rates were calculated as described previously (Lindstrom et al., 1998a
).
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Results |
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Through analysis of the PA gene of B virus isolates (Fig. 3), it became apparent that the PA genes of viruses isolated before 1994 were evolving in a similar manner to the PB2 gene. Like the PB2 gene, the PA genes of SIN79 and NOR84 were the earliest divergent genes of lineages I and II, respectively, and evidently diverged from a putative gene similar to that of AA66, with branch confidence probabilities of 100%. Additionally, the phylogenetic locations of the PA genes of viruses subsequently isolated until 1994 were similar to those of the PB2 and PB1 genes. In contrast to the PB2 and PB1 genes, however, the PA genes of viruses isolated in China and Japan in 1997 and 1998 were all located in lineage I, forming two minor clades, I-i (BEI97, HEN97, CHI98, BEI97) and I-ii (SHI3098, YAN98). Thus, unlike the PB2 and PB1 genes, distinct PA genes of both lineages were shown not to have co-circulated in 1998 in Japan.
Comparison of predicted amino acid sequences
PB2 genes of all strains investigated here contained a conserved open reading frame (ORF) of 2313 nt encoding a predicted polypeptide of 769 aa, which was consistent with that previously described (DeBorde et al., 1988 ; Jambrina et al., 1997
). Comparison of deduced amino acids of PB2 proteins (Table 2
) revealed lineage-specific amino acid variability among PB2 proteins of lineages I and II, although proteins of lineage I demonstrated lower variability than those of lineage II. PB2 proteins of lineage I were found to contain two amino acid changes at positions 301 (A to T) and 641 (I to V), whereas those of lineage II contained six differences at positions 115 (K to R), 383 (M to L), 397 (K to R), 468 (L to S), 639 (R to K) and 641 (M to V). Curiously, similar amino acid substitutions occurred independently in both lineages resulting in a valine residue at position 641. Regardless of this, phylogenetically distinct viruses isolated in Japan in 1998, were observed to differ by six conserved amino acids (0·8%).
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Deduced nucleotide sequences of RNA segment 3 determined in this study all contained a conserved ORF encoding a PA protein of 726 aa. Interestingly, a three nucleotide deletion in the non-coding region of the 5' end of the genomic RNA was observed in isolates of lineage I (19931998), which was similar to that found in B/Panama/45/90 (Jambrina et al., 1997 ). As shown in Table 2
, PA proteins of influenza B viruses showed higher amino acid variability than the PB2 or PB1 proteins. A total of nine lineage-specific amino acid changes was observed in the PA proteins of lineage I, whereas those of lineage II contained four amino acid differences. As a result, the PA protein of the most recent strain of lineage II (GUA94) differed from 1998 isolates of lineage I by 17 aa (2·3%) or 18 aa (2·5%). Since recent viruses from 1998 were observed to form two branch clusters within lineage I, variability among these viruses was more limited, with PA proteins of SHI5198 (I-i) and SHI3098 (I-ii) differing by only one conserved amino acid (0·1%). It should be noted that a deletion of three nucleotides resulting in an amino acid deletion at position 486 (V) of a previously sequenced PA gene of SIN79 (Akoto-Amanfu et al., 1987
) was not observed in any other viruses examined in this study.
Evolutionary rates
Evolutionary rates of the polymerase PB2, PB1 and PA genes were calculated based on the total number of nucleotide and amino acid changes. As shown in Table 3, respective rates of change for lineages I and II of each gene were calculated separately. For comparison, the rates of change of the polymerase genes of human influenza A H3N2 viruses were also calculated based on previously published sequences of viruses isolated in 19681997. Both lineages of the PB2 gene of B viruses were estimated to be evolving at a rate of approximately 0·76x10-3 nucleotide substitutions per site per year (ns/site/year) which was slower than that of human influenza A H3N2 viruses (1·27x10-3 ns/site/year). However, the corresponding rates of change of the PB2 protein of each lineage of influenza B virus varied considerably, from no detectable change in lineage I to 0·14x10-3 amino acid changes per site per year (aas/site/year) in lineage II. The rate of substitution in the PB2 protein of type A viruses was considerably higher (0·40x10-3 aas/site/year).
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Lineages of the PA gene of type B viruses were calculated to be evolving at similar rates of 1·24x10-3 ns/site/year (lineage I) and 1·16x10-3 ns/site/year (lineage II), which were comparable with that of influenza A H3N2 viruses (1·46x10-3 ns/site/year). At the amino acid level, the PA proteins also evolved at similar rates of 0·26x10-3 aas/site/year (lineage I) and 0·37x10-3 aas/site/year (lineage II), which were somewhat slower than that of type A human H3N2 viruses (0·49x10-3 aas/site/year).
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Discussion |
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Similarly to previous analysis of the HA, NP, M and NS genes (Lindstrom et al., 1999 ), nucleotide substitution rates estimated for the polymerase genes of influenza B virus appeared to be slightly slower than those of human influenza A H3N2 virus. Protein evolutionary rates of divergent lineages of the polymerase proteins, in particular the PB1 protein, tended to differ considerably, suggesting variable functional constraints or other evolutionary pressures on these proteins. Genetically variable PA proteins of type B virus demonstrated higher variability than the PB1 or PB2 proteins which was consistent with the observed variability among polymerase proteins of influenza A viruses (Gorman et al., 1990
; Kawaoka et al., 1989
; Okazaki et al., 1989
). Most lineage-specific amino acid substitutions were located in the N-terminal half of the PA protein which may have significance as most host-specific substitutions observed in the PA protein of influenza A viruses were also located in this region (Okazaki et al., 1989
). Additionally, the N-terminal 247 aa region of the PA protein of influenza A virus has demonstrated induction of proteolytic activity and contains the signal for nuclear localization (Sanz-Ezquerro et al., 1995
, 1996
). It was interesting to note that four of nine conserved amino acid substitutions in the PB1 protein of type B viruses were located in regions implicated in recognition of the 5' and 3' ends of vRNA and the cRNA panhandle of influenza A virus (Gonzalez & Ortin, 1999a
, b
).
In order to understand the phylogenetic relationships among gene segments of influenza B virus, the evolutionary patterns of the three polymerase genes were compared with those of the internal NP, M, NS and surface HA genes recently described by Lindstrom et al. (1999) (Table 4
). As shown in Table 4
, evolution of the HA and internal gene segments of influenza B virus was characterized by genetic reassortment of phylogenetically divergent genes. Indeed, comparison of 18 viruses isolated following phylogenetic divergence revealed eight distinct genome constellations, whereas only five isolates (YAM88, PAN90, BEI93, MIE93, HAR94) contained all gene segments of a single lineage corresponding with the Yamagata/16/88-like HA lineage. It was previously demonstrated that the NP, M and NS genes evolved independently of the HA gene (Lindstrom et al., 1999
). Therefore, it was noteworthy to observe that while the evolutionary pathway of the PA gene was similar to those of the NP and M genes (Lindstrom et al., 1999
), dendrogram topologies of the PB2 and PB1 genes were, in contrast, quite similar to that of the HA gene. Thus, despite reassortment among co-circulating viruses, it appeared that the six gene segments encoding the internal proteins of influenza B virus demonstrated three general evolutionary patterns including: (i) PB2 and PB1 genes; (ii) PA, NP and M genes; and (iii) the NS gene.
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Phylogenetic characterization of all six internal genes of influenza B viruses provided evidence suggesting that genetic reassortment among co-circulating divergent lineages of the internal genes may not be a random process but instead involves mechanisms which lead to selective reassortment of genes. Future investigation into specific protein interactions as well as terminal non-coding sequence variability of genetic RNA segments may provide evidence for understanding co-evolutionary patterns of influenza B virus genes. Genetic characterization of the internal genes of influenza B virus allows for subsequent analysis of the functional significance of lineage-specific amino acid changes in the internal proteins of influenza B viruses to be undertaken through construction of viruses with novel genome constellations by genetic reassortment or reverse genetics techniques.
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Acknowledgments |
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Received 12 August 1999;
accepted 9 December 1999.