Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK1
Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory, London NW9 5HT, UK2
Author for correspondence: Christopher Woelk. Fax +44 1865 310447. e-mail Christopher.Woelk{at}zoo.ox.ac.uk
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Abstract |
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Introduction |
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The L protein is thought to be the viral polymerase due to its low abundance, large size and localization to transcriptionally active viral cores. The centre of this protein comprises five regions of high homology, which form an ancestral polymerase fold that is conserved in the RNA-dependent RNA polymerases of other virus families (Lamb & Kolakofsky, 1996 ). The H protein is thought to interact with two different receptors, CD46 and SLAM (Manchester et al., 2000
; Tatsuo et al., 2000
), and together with the F glycoprotein facilitates MV entry into host cells (Lamb, 1993
). The H protein can be divided into three domains; a cytoplasmic domain, a transmembrane domain and a large ectodomain (Muller et al., 1993
). The ectodomain consists of a
-propeller structure projected from the cell surface by two helix-rich stem regions. Six antiparallel
-sheets of four strands each form the propeller such that the fourth strand of each sheet is connected by a loop to the first strand of the next sheet. Two such loops connecting sheets 4 to 5 and 5 to 6 are thought to delineate the CD46 receptor-binding domain (Langedijk et al., 1997
). Cysteine interactions allow pairing of amino acids 386 to 394 and 381 to 494, and the mature 78 kDa form of the H protein is produced by N-glycosylation of Asn residues 168, 187, 200 and 215, in the second stem region (Hu et al., 1994
; Langedijk et al., 1997
).
It was our aim to determine whether the H gene of MV might be subject to positive selection, such as that mediated by the immune response. In the context of comparing DNA sequences, this is most commonly done by counting the number of synonymous and nonsynonymous substitutions per site, referred to as dS and dN respectively. Omega () is the ratio of dN to dS (dN/dS) and an
<1 is indicative of purifying selection, an
=1 suggests complete neutrality, while an
>1 is an unambiguous signal of positive selection since it means that the rate of fixation is higher than the rate of mutation, which cannot be the case under neutral evolution (Yang & Bielawski, 2000
). Pairwise methods are commonly used for estimating
ratios but they suffer from the over-representation of distances associated with deeper branches in the phylogeny, averaging over large regions of sequence, and the movement of sites between the synonymous and nonsynonymous categories (Muse, 1996
; Nei & Gojobori, 1986
). A recently developed maximum likelihood (ML) method (Yang et al., 2000
) for calculating
ratios that accounts for phylogenetic structure as well as biases in both codon usage and transition/transversion ratio has been found to be superior to early pairwise methods and is able to detect localized positive selection (Zanotto et al., 1999
). This ML method provides more realistic models of sequence evolution, and allows the classification of individual amino acid sites into conserved, neutral or positively selected classes.
Although the current measles vaccine protects against all known genotypes of MV, antigenic differences have been detected between strains and it has been suggested that mutations in critical protective epitopes could lead to the generation of vaccine escape mutants (Jin et al., 1998 ; Tamin et al., 1994
). Since the H protein is the major target of the immune response we decided to determine if there were any sites, or phylogenetic lineages, under positive selection in this protein and, if so, whether they correspond to positions of T-cell or B-cell epitopes, suggesting that immune selection is in operation. For comparison purposes, the conserved L gene of MV, which is not thought to be under immune selection, was also analysed (Komase et al., 1995
). The effect of using passaged strains in selection analysis may introduce a false signal of positive selection (Woelk & Holmes, 2001
). Since the propagation of MV isolates in Vero cells has been well documented (Komase et al., 1990
; McChesney et al., 1997
; Rima et al., 1997
; Rota et al., 1992
, 1994
, 1996
) the effect of such passaging on selection analysis was also investigated.
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Methods |
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Multiple alignments.
All of the complete gene sequences available in GenBank for the H and L genes of MV were collected and aligned using CLUSTALW (Thompson et al., 1994 ) after the addition of the SSPE sequences determined in this study. One further H gene sequence from an SSPE patient was obtained from Michiko Watanabe (personal communication). A full list of the sequences analysed in this paper is available at http://evolve.zoo.ox.ac.uk. After identical sequences and vaccine strains had been removed, the L gene alignment contained 12 sequences, but the H gene alignment contained a much larger number of sequences such that its size would make the maximum likelihood analysis of selection pressures computationally unfeasible. Yang (1998)
proposed that the removal of sequences with a high level of similarity has insignificant effects on the results of selection analysis and thus H gene sequences with greater than 99% similarity were removed to produce a data set of 50 sequences. This H gene data set is referred to as the Global data set because it contains isolates from all of the genotypes of MV found worldwide. A further two data sets were created from this Global data set. The -Vero data set contains 25 H gene sequences and was created by removing MV isolates that had been passaged in Vero cells or had unknown passaging histories. The Vero data set contains 16 H gene sequences and consists of isolates that had only been Vero passaged.
Phylogenetic analyses.
The PAUP* package was used to construct maximum likelihood (ML) trees from sequences of L and H gene (Global, -Vero and Vero) data sets using the HKY85+ model of nucleotide substitution (Swofford, 2000
). Maximum likelihood trees for the L gene and Global data sets are presented in Fig. 1(a)
and Fig. 1(b)
, respectively. Values for both the transition/transversion (TS/TV) ratio and the shape parameter (
) of a gamma distribution of rate variation among sites (with eight categories) were estimated from the data (Table 1
).
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Results |
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When Vero-passaged isolates were analysed separately the parameter estimates differed extensively from results of the Global and -Vero data sets (Table 2). Although the significance of positive selection under M3 and M8 was still confirmed by LRTs (Table 3
), both these models estimated a smaller proportion of positively selected sites (0·6%) with a relatively high
ratio (>5 in both cases). M3 again had the best likelihood score and divided the remainder of sites such that 53·2% were moderately conserved (
0=0·387) and 46·2 % were strongly conserved (
1=0·001). For both M3 and M8, posterior probabilities could only assign two sites at positions 546 and 562 to the positively selected class above the 90% level (Table 4
).
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Discussion |
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Extensive work has been done on the cellular immune response to MV (Jacobson et al., 1984 , 1989
; Kreth et al., 1979
; Lucas et al., 1982
; Richert et al., 1985
; Sethi et al., 1982
; van Binnendijk et al., 1990
), the most recent of which showed specific cytotoxic T-lymphocyte (CTL) responses in Caucasian and West African adults to MV fusion (F) and H proteins and to a lesser extent to the nucleoprotein (NP) (Jaye et al., 1998
). They concluded that different MHC class I and class II molecules bind to different sets of peptides scattered along the entire sequence of F, H and NP, but that the main CTL response was mediated through CD8 HLA class I-restricted cells that were largely directed to the F and H proteins. Synthetic peptides comprising amino acids 2937, 3145 and 4155 of the H protein (Fig. 2
) have been shown to induce T-cell proliferation when exposed to either peripheral blood mononuclear cells (PBMC) or peripheral blood leukocytes (PBL) (Muller et al., 1993
; Nanan et al., 1995
). These peptide regions did not correlate to any putative sites of positive selection identified in our study. Obeid et al. (1993)
did distinguish peptides bearing TCEs that corresponded to positively selected sites, namely peptides 423437 (423), 443457 (451), 473487 (476 and 481) and 543557 (546) (Table 4
). However, these peptides were identified by testing responses of spleen cells from peptide-primed mice to MV in vitro and this is unlikely to be relevant to the immune response in humans.
MV infection is facilitated through the binding of the H protein to two different receptors. Vero-passaged isolates are thought to have an affinity for binding the CD46 receptor (Manchester et al., 2000 ) whereas isolates passaged in B-cells appear to prefer the SLAM receptor (Tatsuo et al., 2000
). Binding of CD46 by the H protein leads to receptor down-regulation, syncytium formation, haemagglutination and haemadsorption (Lecouturier et al., 1996
). The relevance of the CD46 receptor in natural measles infection has recently been confirmed (Manchester et al., 2000
) and its down-regulation appears to be modular (Sakata et al., 1998
). Low-affinity CD46-binding isolates can be converted to the high-affinity CD46-binding phenotype by a substitution of Asn for Tyr at position 481 (Hsu et al., 1998
). Likewise, a substitution of Ser for Gly at site 546 has been shown to confer both the properties of CD46 binding and haemadsorption to isolates that were previously defective for these traits (Li et al., 1999
). These are clearly selectable traits because they facilitate efficient spread of MV to other cells.
For positive selection at a particular site to be attributed to Vero passaging, the signal for positive selection should be lost at this particular site when Vero-passaged isolates are removed from the Global data set, and then this site should be positively selected when only Vero-passaged isolates are analysed. This pattern was only seen for the positive selection at site 546. This site was found to be under strong selection pressure (>5) when only Vero-passaged isolates were analysed, and this selection is therefore most likely the result of Vero passaging. Although positive selection at site 481 was lost in the -Vero data set, it could not be assigned to the positively selected class when only Vero isolates were analysed and cannot be confirmed to result from Vero passaging. Indeed, analysis of a larger number of MV strains than were used in this study suggests that isolates of several different passaging types contain a Tyr at position 481. Hence, it seems probable that the positive selection at site 481 results from natural selection for the CD46 receptor as opposed to artificial selection attributable to Vero passaging. This is further supported by the fact that site 481 was not found to correlate with any antigenic features discussed earlier. Site 562 was also deemed to be under strong positive selection when only Vero-passaged isolates were analysed. However, because positive selection at site 562 was not lost when Vero-passaged isolates were removed from the Global data set, it is also unlikely to result from Vero passaging.
Sites 211, 243, 451 and 476 have also been implicated in CD46 interaction (Bartz et al., 1996 ; Patterson et al., 1999
) and our analysis of the -Vero data set suggested that sites 211, 451 and 476 are potentially under positive selection. As discussed previously, the positive selection at sites 211 and 476 could be the result of immune selection from antibodies but a possible selective force from the CD46 receptor cannot be ruled out entirely. Early studies indicated that Val and Tyr at sites 451 and 481, respectively, are critical for inducing CD46 down-regulation (Bartz et al., 1996
; Lecouturier et al., 1996
). A more recent study suggests that site 451 may be the more influential of this pair since it describes a strain generated by site-directed mutagenesis with a Tyr at position 481 that did not induce significant down-regulation (Xie et al., 1999
). Down-regulation of CD46 is thought to confer an advantage to the virus because it disrupts antigen presentation of the H protein to T-cells (Rivailler et al., 1998
). Interestingly, the majority of isolates have a Val at position 451, indicating that this is probably the ancestral state. On a number of occasions after the mid 1970s, the Val at position 451 appears to have been replaced by Ala, Glu, Lys or Met. This is unexpected since such replacements would increase the efficiency of H protein presentation to the immune system. Hence, it is likely that the replacement of Val at 451 and disruption of down-regulation may confer some other selective advantage upon the virus. For instance, the presence of CD46 upon the cell surface protects the cell from complement lysis and may allow for greater efficiency of virus generation (Schnorr et al., 1995
).
A similar analysis to that performed with Vero-passaged isolates was attempted with B95a-passaged isolates (results not shown) in order to determine if any of the positively selected sites were the result of selection for the SLAM receptor. However, selection analysis of 15 isolates from the Global data set that had only undergone B95a passaging did not estimate a positively selected class. If the positive selection pressure exerted by the SLAM receptor is weaker than that from the CD46 receptor, or if the SLAM receptor selects for a single site, then a larger data set of B95a-passaged isolates is needed to detect this. This work is ongoing.
Finally, we attempted to determine if any of the potential positively selected sites resulted from the presence of the SSPE strains in the Global data set. Removing the 13 SSPE strains from the analysis (results not shown) led to a loss of positive selection at sites 12, 62, 211, 303, 348, 423 and 476 in comparison to results from the Global data set. Unfortunately, positive selection at these sites could not be confirmed when the 13 SSPE strains were analysed as a separate data set, because the models that identified a positively selected class did not perform significantly well. Loss of positive selection at site 12 presents an interesting result because it lies in the cytoplasmic domain of the H protein (Fig. 2). The matrix (M) protein is thought to interact with the nucleocapsid protein and the cytoplasmic tails of the F and H glycoproteins to enable the morphogenesis of viral particles by a budding process (Peeples, 1991
). Viral factors thought to abolish viral budding and, thus in part, facilitate the generation of SSPE cases, are defective M proteins and F proteins with truncated and/or distorted cytoplasmic domains (Schneider-Schaulies et al., 1995
). It is possible that changes at site 12 may block interaction with the M protein and are selected so that viral budding is further impeded in SSPE-associated viruses. The serum of SSPE patients has been shown to have a high antibody titre directed against the H protein (Liebert, 1997
). Consequently, even though positive selection at sites 211, 348, 423 and 476 was lost on removal of SSPE strains their discussion in terms of epitopes can still be considered valid. Selection analysis of a larger number of SSPE strains should confirm significant positive selection in these strains and further research will be concentrated in this area.
In summary, the conserved nature of the L protein was confirmed since we failed to predict any sites under positive selection. The putative positively selected sites identified in the H protein could have arisen for a variety of reasons (Table 4). Although positively selected sites were not shown to correlate with TCEs presently identified, sites 211, 348, 423, 476, 562 and 575 were shown to coincide with potential BCEs and may have been generated by immune selection. Further work is clearly needed to confirm whether these epitopes are protective. Some of the aforementioned sites are also associated with areas that are known to interact with the CD46 receptor and this may contribute to their selection. The selection at site 546 is probably generated by Vero passaging, which selects for more efficient binding of the CD46 receptor. Selection for limiting down-regulation of the CD46 receptor and for increased binding of this receptor probably led to the positive selection identified at sites 451 and 481, respectively. These two sites appear to have arisen naturally rather than by artificial selection from Vero cell passaging. Possible explanations for the positive selection at a number of sites could not be deduced (Table 4
). These may have resulted from selection for the SLAM receptor during B95a passaging or coincide with some, as yet undetermined, antigenic feature. Further work is needed to confirm this.
Previous analyses of viral glycoproteins have estimated the selection pressure at positively selected sites to range from 2·433 (attachment glycoprotein of respiratory syncytial virus) to 6·898 (haemagglutinin gene of influenza virus) (Woelk & Holmes, 2001 ; Yang et al., 2000
). In comparison, the positive selection pressure estimated for the H gene of MV is relatively weak (2·319 under M3 and 1·658 under M8). Although vaccines protect against all known strains of MV, substitutions at positively selected sites should still be monitored because they have the potential to generate escape mutants. Ideally, selection analysis in future studies should be performed using strains obtained directly from clinical material of acute cases, which does not suffer from passaging problems. This will provide the most accurate picture of the selection pressures affecting the H gene of MV during acute infections and determine the precise nature of adaptive evolution.
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Acknowledgments |
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References |
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Received 12 March 2001;
accepted 9 July 2001.