Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N51
Author for correspondence: David Levin. Fax +1 250 472 4075. e-mail dlevin{at}uvic.ca
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Abstract |
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Introduction |
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These analyses suggested that lepidopteran NPVs evolved from a common ancestral virus and that the GVs diverged prior to the divergence of the Group I and Group II NPVs. Subsequent phylogenetic analyses based on the polyhedrin gene (Cowan et al., 1994 ) and the ecdysteroid UDP-glycosyltransferase (egt) gene (Hu et al., 1997
) generally supported the phylogenetic relationships suggested by Zanotto et al. (1993)
, but also resulted in conflicting placement of some branches, particularly in the Group II NPVs. More recent phylogenetic analyses of baculoviruses, based on both polyhedrin and DNA polymerase genes (Bulach et al., 1999
), revealed three Group II subclades, designated A, B and C.
Variations in amino acid frequencies determine the extent of dissimilarity for divergent but structurally and functionally conserved genes and thus significantly influence the outcome of phylogenetic analyses (Halpern & Bruno, 1998 ). Hence, it is important to consider variation in amino acid codon usage. The genetic code is degenerate. Most of the 20 amino acids are encoded by more than one codon. Synonymous codon use is distinctly non-random in both prokaryotic and eukaryotic genes (Li, 1997
). There is a range of minimal to extreme codon use bias in unicellular organisms (E. coli and yeast; Sharp & Li, 1987
) and in Drosophila species (Shields et al., 1988
; Powell & Moriyama, 1997
).
The Genome Hypothesis postulates that genes in any given genome use the same coding pattern with respect to synonymous codons. Genes in an organism or in related species generally show the same pattern of codon usage (Li, 1997 ). In the case of unicellular organisms, the degree of codon use bias is highly correlated with its level of expression in the cell (Gouy & Gautier, 1982
; Ikemura, 1985
). Highly expressed E. coli and yeast genes show a tendency to use a subset of 25 and 22 preferred codons, respectively, whereas genes with low levels of expression use codons more randomly (Bennetzen & Hall, 1982
).
Codon bias in Drosophila genes is consistent over long periods of evolution of Drosophila species. With the exception of aspartic acid, all amino acids contribute equally to the codon use bias of a gene. Some Drosophila genes, however, do display variant codon bias across species. G and C bases are favoured at synonymous sites in biased genes. Smaller genes tend to have more codon bias than longer genes. Highly and/or rapidly expressed genes tend to be highly biased in their codon usage. The preferred codons in highly biased genes optimally bind the most abundant isoaccepting tRNAs. Thus, codon bias in Drosophila genes appears to be driven by translational efficiency, as it is in E. coli and yeast (Powell & Moriyama, 1997 ).
We have examined codon usage in six genes (egt, fgf, pk-1, p10, p74 and polh) from six multinucleocapsid nucleopolyhedroviruses [AcMNPV, Bombyx mori (Bm)NPV, Orgyia pseudotsugata (Op)MNPV, Spodoptera littoralis (Spli)MNPV, Spodoptera litura (Splt)MNPV and Lymantria dispar (Ld)MNPV] to determine if the trends observed in unicellular organisms and in Drosophila are conserved within this group of viruses. We have found that: (1) there is significant variation in codon use by genes within the same virus genome; (2) there is significant variation in the codon usage of homologous genes encoded by different NPVs; (3) there is no correlation between the level of gene expression and codon bias in NPVs; (4) there is no correlation between gene length and codon bias in NPVs; and (5) that while codon use bias appears to be conserved between viruses that are closely related phylogenetically, the patterns of codon usage also appear to be a direct function of the GC-content of the virus-encoded genes.
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Methods |
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The genes examined, number of amino acids in each gene, GenBank accession numbers and relevant references are listed in Table 1. The GC-content of each gene within each NPV and the average GC-content of the NPV calculated across the six genes analysed are presented in Table 2
. The GC-content of NPV genes was determined from GenBank sequences using the DNAStar program.
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Analyses of codon usage frequency.
The frequency of occurrence of each codon was summed for each of the six NPV genes (egt, fgf, p10, p74, pk-1 and polh), from each of the six NPVs, yielding 61 values. The AcMNPV codons were arranged for lowest to highest frequency of occurrence. The percent frequency of each AcMNPV codon was calculated using the total number of codons from the six AcMNPV genes. Using AcMNPV as a base line, the percent differences in codon frequencies observed in the five other viruses were calculated [(% codon frequency NPV n/% codon frequency AcMNPV-1)x100] for each codon. These values were compared in a pair-wise manner, in the same codon order, with the AcMNPV base-line. We then compared the percent GC-rich and AT-rich codon frequency usage above and below the AcMNPV codon usage base-line. The frequencies of codon occurrence (in percent), arranged from most AT-rich (i.e. codons with 3 A and/or T nucleotides) to most GC-rich (i.e. codons with 3 G and/or C nucleotides) were also graphed in pair-wise comparisons for viruses with similar GC-content.
Pair-wise differences in codon usage were analysed for statistical significance by Chi-square cumulative analysis using the Syntax1 Statistical Package for Social Sciences (SPSS) for Windows Viewers software. In this analysis, Chi-square values for 61 degrees of freedom <80·23 were not statistically significant.
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Results |
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In unicellular organisms and in Drosophila, codon use bias is highly correlated with the level of expression in the cell and/or with the length of the gene (Gouy & Gautier, 1982 ; Bennetzen & Hall, 1982
; Ikemura, 1985
; Powell & Moriyama, 1997
). The polh and p10 genes are the most highly expressed genes in NPVs. Codon usage in polh appears biased in OpMNPV and LdMNPV (ENC values of 36·3 and 36·2, respectively; Table 3
), and more random in AcMNPV and BmNPV (ENC values of 50·1 and 54·6, respectively; Table 3
). Codon usage by p10 displays the same variability and appears highly biased in LdMNPV, AcMNPV and BmNPV, and more random in SpliMNPV (ENC values of 27·2, 33·9, 35·0 and 56·6, respectively; Table 3
). Thus, highly expressed genes such as polh and p10 do not display consistent codon use bias. Comparison of the ENC values displayed by a very short gene (p10; Table 1
) with those of long genes (egt and p74; Table 1
) also failed to reveal a consistent pattern of codon bias (Table 3
).
To evaluate the degree of variation in codon usage between the different NPV genomes, we plotted the percent frequency of all codons from the six genes studied, from the lowest to highest occurrence within the AcMNPV genome (Fig. 2). We then superimposed the percent frequencies of each codon for the six genes, in the same order as they occur in the AcMNPV genome, from BmNPV (Fig. 2A
), OpMNPV (Fig. 2B
), SpliMNPV (Fig. 2C
), SpltMNPV (Fig. 2D
) and LdMNPV (Fig. 2E
). These codon percent frequency comparison plots revealed some very interesting patterns of codon usage in the different NPVs. Fig. 2(A)
reveals a relatively low degree of variation in codon usage between AcMNPV (base line) and BmNPV genes. The observed variation, however, was not statistically significant by Chi-square cumulative analysis (Chi-square value=33·58). Fig. 2(B)
reveals a large degree of variation in codon usage between AcMNPV (base-line) and OpMNPV genes, and this variation was statistically significant (Chi-square value=336·49). Fig. 2
(C
, D
) reveals a moderate degree of variation in codon usage between AcMNPV (base-line) genes and those of SpliMNPV and SpltMNPV genes, respectively. Variations in codon usage between AcMNPV and SpliMNPV, and between AcMNPV and SpltMNPV, were statistically significant (Chi-square value=159·46 and 107·64, respectively). Fig. 2(E)
reveals the variation in codon usage between AcMNPV (base line) and LdMNPV genes. LdMNPV displays the largest variance of codon usage compared with AcMNPV. Chi-square analysis revealed that this variation was also statistically significant (Chi-square value=659·89).
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Discussion |
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Analysis of a partial sequence of the SpltMNPV DNA polymerase gene placed this virus in the Group II-B subclade (Bulach et al., 1999 ) along with Helicoverpa armigera mutlinucleocapsid NPV (HearMNPV) and H. zea single nucleocapsid NPV (HzSNPV). Comparison of the SpliMNPV DNA polymerase gene amino acid sequence with the partial amino acid sequence of SpltNPV DNA polymerase gene (our unpublished data) revealed a very high level of sequence identity (94% over 603 amino acids). Phylogenetic analyses, based on both the nucleotide and amino acid sequences, suggest that SpliMNPV and SpltMNPV form a separate subclade, while the other relationships within the Group II NPV delineated by Bulach et al. (1999)
remain unchanged (our unpublished data). As with AcMNPV and BmNPV, the close similarities between the average GC-content of the six genes examined from SpliMNPV and SpltMNPV (48·2and 48·3%, respectively) most probably reflects the close evolutionary relationship between these NPVs.
While LdMNPV may be considered a Group II NPV, based on phylogenetic analyses of the DNA polymerase gene and polypeptide sequences (Bulach et al., 1999 ), it displays some significant differences in terms of genome size (161 Kb) and genetic organization compared with other NPVs studies to date (Kuzio et al., 1999
). The much higher average percentage of GC-nucleotides of the six genes examined (61·4%) reflects the overall higher GC-content of LdMNPV.
ENC is a very simple, but effective way of measuring codon use bias (Wright, 1990 ). ENC is analogous to the effective number of alleles. It is related to the homouzygosity of codons: the probability that two randomly chosen synonymous codons are identical. ENC ranges from 20, if only one codon is used for each amino acid, to 61, if all codons are used equally. ENC values of 35 or less are considered biased and genes with low ENC values are restricted in the use of synonymous codons compared with genes with high ENC values, which have significantly greater flexibility in the use of synonymous codons (Powell & Moriyama, 1997
).
We observed that there is significant variation in codon use of genes within the same virus genome and that there is significant variation in the codon usage of homologous genes encoded by different NPVs. Despite the relatively similar average percent GC-content between AcMNPV, BmNPV, SpliMNPV and SpltMNPV, the p10 genes of AcMNPV and BmNPV have ENC values indicative of codon use bias (33·9 and 35·0, respectively), while the p10 genes of SpliMNPV and SpltMNPV have ENC values that are indicative of more random codon usage (56·6 and 41·6%, respectively). Moreover, while the average percent GC-contents of OpMNPV and LdMNPV are more similar to each other, and much higher than those of AcMNPV, BmNPV, SpliMNPV and SpltMNPV, the p10 gene of OpMNPV has an ENC value indicative of random codon usage, while LdMNPV p10 has an ENC value indicative of extreme bias (42·1 vs 27·2, respectively). Similar patterns were observed for each of the other genes observed in each of the six NPVs. Thus, based on ENC values, codon usage patterns do not appear to be consistent, neither within a particular NPV genome, nor for a particular gene between viral genomes. Nor did we find a correlation between codon use bias and the level of gene expression and/or gene length. Highly expressed genes such as polh and p10 do not display consistent codon use bias. Comparisons of ENC values displayed by a very short gene (p10) with those of long genes (egt and p74) also failed to reveal a consistent pattern of codon bias. These observations are consistent with a previous analysis of codon usage of AcMNPV genes (Ranjan & Hasnain, 1995 ), in which it was found that certain genes displayed significant variation from the overall AcMNPV codon use pattern. While the p10 and polh genes displayed the greatest variation from the overall codon usage profile, genes from both the early (ie-2) and late (39k, sod) gene classes also displayed variations in codon usage.
Taking the GC-content of each gene within each virus into account, however, the pair-wise comparisons of codon usage in AcMNPV with codon usage for each of the other NPVs revealed that the greater the GC-content difference between AcMNPV and the other NPVs examined, the greater the variance in codon usage. The variance in codon usage between AcMNPV and BmNPV, which have very similar GC-contents (44·8 and 44·5%, respectively) averaged across the six genes used in this analysis (a difference in average GC-content of only 0·3%), was not statistically different (Chi-square value=33·58). In contrast, variance in codon usage between AcMNPV and LdMNPV, which differ greatly in GC-content (44·8 vs 61·4%, a difference of 16·6%), was highly significant statistically (Chi-square value=659·89). LdMNPV codons that were used more frequently tended to be GC-rich codons, while LdMNPV codons that were used less frequently tend to be AT-rich codons, a phenomenon also observed in unicellular organisms and Drosophila, where GC-nucleotides are favoured at synonymous sites (Powell & Moriyama, 1997 ).
Pair-wise comparisons of NPVs of similar GC-content (AcMNPV vs BmNPV; SpliMNPV vs SpltMNPV; OpMNPV vs LdMNPV) also revealed that NPVs with very similar GC-content had highly similar patterns of codon usage. Two pairs of closely related NPVs (AcMNPV and BmNPV; SpliMNPV and SpltMNPV) displayed extremely high concordance in codon use patterns, and the variation in codon usage between each pair was not significantly different by Chi-square cumulative analysis (Chi-square values of 33·58 for AcMNPV vs BmNPV and of 36·55 for SpliMNPV vs SpltMNPV).
The similarities in codon usage between these NPVs, however, may not be attributable to phylogenetic relatedness alone. OpMNPV is phylogenetically more similar to AcMNPV and BmNPV than to LdMNPV. While the variation in codon usage between OpMNPV and LdMNPV was statistically significant (Chi-square value=241·55), OpMNPV and LdMNPV have GC-contents and patterns of codon usage that are much more similar to each other than does OpMNPV compared with AcMNPV and BmNPV. Similarly, while the variation in codon usages between SpliMNPV (a Group II NPV) and AcMNPV, and between SpliMNPV and BmNPV, are significantly different (Chi-square values of 136·91 and 127·91, respectively), the pattern of codon usage displayed by SpliMNPV is more similar to AcMNPV and BmNPV than it is to LdMNPV. The same is true of SpltMNPV when compared with AcMNPV and BmNPV. Thus, while codon use bias appears to be conserved between viruses that are closely related phylogenetically, the patterns of codon usage also appear to be a direct function of the GC-content of the virus-encoded genes.
The overall conclusion from this study is that codon usage within NPV genes does not follow the patterns observed from E. coli, yeast and Drosophila. These organisms, however, are not viruses and codon usage has been analysed in only a few viruses. Human immunodeficiency virus type 1 (HIV-1) and other lentiviruses demonstrate an inverse correlation between the extent of codon bias and the rate of translation of the viral genes. The pol genes of lentiviruses display a shift toward AT-rich codons (Bronson & Anderson, 1994 ). The gag genes do not display this bias toward AT-rich codons, despite the fact that expression of the gag genes exceeds that of the pol genes by a factor of 20, due to infrequent frame-shifting during translation of the gagpol mRNA. It was hypothesized that the aminoacyl-tRNA availability within the host cell restricts the lentivirus preference for AT-rich codons (van Hemert & Berkhout, 1995
).
Analyses of bovine papillomavirus (BPV) late gene expression have demonstrated that BPV late genes (capsid proteins L1 and L2) mRNAs are efficiently translated in non-replicating, terminally differentiated keratinocytes, but not in actively dividing undifferentiated cells (Stoler et al., 1992 ). Zhou et al. (1999)
hypothesized that BPV late mRNAs may not be efficiently translated in dividing, undifferentiated cells due to a mis-match between codon usage of the viral genes and aminoacyl-tRNA availability in host cell. They suggest that translation of BPV capsid genes may be limited in actively dividing cells due to competition between viral and host mRNAs for rare tRNAs. In terminally differentiated cells, the number of competing host mRNAs would be greatly reduced, permitting increased levels of translation of the viral mRNAs (Zhou et al., 1999)
.
BPV capsid genes, however, are translated efficiently in insect cells using the AcMNPV baculovirus expression system (BEVS) (Volpers et al., 1994 ), which is used for high-level synthesis of heterologous proteins (OReilly et al., 1992
). Efficient translation of BPV capsid gene mRNAs in NPV-infected cells may be due to the lack of competition between host mRNAs and the BPV mRNAs because NPV infections result in a shut-down of host cell mRNA and protein synthesis (Ooi & Miller, 1988
; Carstens et al., 1979
). Thus, one could argue that the lack of consistency in codon bias displayed by NPV genes, as well as the lack of correlation between codon usage and expression levels of NPV genes, may be due to a lack of competition between host and viral mRNAs for aminoacyl-tRNAs. Selection for translational efficiency in NPV genes would be minimal in NPV-infected cells as aminoacyl-tRNAs would not be a limiting factor. The strong codon bias displayed by some NPV genes, p10 in LdMNPV for example, may simply reflect the strong GC-bias in these genomes.
Our data suggest that NPVs display a wide variation in codon usage which, when coupled with a lack of competition between host and viral mRNAs for aminoacyl-tRNAs, provides a working hypothesis to explain why the BEVS are so useful for high-level expression of heterologous proteins. Not all heterologous proteins, however, are successfully expressed at high levels. The beta subunit of human chorionic gonadotropin (hCG) is expressed at very low levels in the AcMNPV BEVS compared with other heterologous proteins. A comparison of the
hCG codon usage pattern with the codon use patterns of a heterologous protein that is expressed at high levels [the firefly luciferase (luc) gene product], and with the codon usage patterns of numerous AcMNPV genes, revealed that
hCG displayed a significantly distinct codon usage that could account for its low level of expression (Ranjan & Husnain, 1994
). Moreover, a comparison of the
hCG and AcMNPV consensus translation initiation codon sequences revealed major differences that could also influence the low level of
hCG expression (Ranjan & Husnain, 1994
). Thus, the basis for the differences in the levels of heterologous protein expression in BEVS may be a consequence of several factors, including extreme bias in codon usage and differences in translation initiation codon context.
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Acknowledgments |
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References |
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Received 8 February 2000;
accepted 10 May 2000.
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