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 fusion (F) and haemagglutinin (H) glycoproteins of MV are type I and type II integral membrane proteins, respectively, and reside on the viral envelope. The F protein is primarily synthesized as an inactive precursor (F0), which is cleaved by host cell proteolytic enzymes to form the biologically active disulphide-linked F1 and F2 subunits (Scheid & Choppin, 1974 ). N-Glycosylation of the F2 subunit at Asn residues 29, 61 and 67 produces the fusion active conformation of the F protein (Alkhatib et al., 1994
; Hu et al., 1995
). The mature form of the H protein also results from disulphide bridge formation and N-glycosylation of Asn residues 168, 187, 200 and 215 (Hu et al., 1994
; Langedijk et al., 1997
). The fusion process is thought to result from aggregation of the envelope proteins such that H contacts the receptor of a neighbouring cell and F controls the actual membrane fusion (Billeter et al., 1994
; Lamb, 1993
). The CD46 and SLAM host cell receptors have been identified as the targets for H protein interaction (Manchester et al., 2000
; Tatsuo et al., 2000
). The matrix (M) protein is not an integral membrane protein but it does associate with membranes and is found in dense concentrations underlying the lipid bilayer after host cell infection (Lamb & Kolakofsky, 1996
). The M protein is thought to play a pivotal role in the formation of budding viral particles because it interacts with the cytoplasmic tails of H and F, the lipid bilayer and the nucleocapsids (N) (Lamb & Kolakofsky, 1996
).
The development of SSPE is thought to be determined by a combination of host cell factors, immune system activity and mutations in the MV genome. For example, mutations in the M gene of SSPE cases result in sites of cryptic initiation and premature termination, as well as adenosine to inosine (A/I) hypermutations, which lead to instability and limited interaction with the N protein. Truncations in the cytoplasmic domain of the F protein impede efficient virus assembly and budding (Cattaneo & Rose, 1993 ), and loss of glycosylation in the H protein leads to inefficient membrane transport (Schneider-Schaulies et al., 1995
). These features often lead to reduced amounts of envelope protein on the cell surface as well as dysfunctional M protein, which maintain persistence by hampering the assembly and budding of mature infectious MV particles (Liebert, 1997
).
The body overcomes acute MV infection through activation of T-cell immunity and protection against reinfection results from induction of humoral responses. High titres of antibodies against the F, H, N and P proteins are found in the serum and cerebrospinal fluid (CSF) of SSPE patients (Liebert, 1997 ; Schneider-Schaulies et al., 1995
). Therefore, it appears that virus spread through the CNS in SSPE cases occurs in the presence of a considerable immune response but without detectable virion formation or virus fusion (Billeter et al., 1994
). It is thought that, despite downregulation of the envelope glycoproteins, the fusion function is still maintained in the CNS, possibly by axonal transport through synaptic clefts. Establishment of virus persistence probably results from interactions between MV and cells of the CNS, whereas sequence alterations are thought to lead to the maintenance of persistent infections over long incubation periods (Schneider-Schaulies & Liebert, 1991
).
This study aims to investigate the differences between acute and persistent MV infection in terms of sequence evolution and specifically to determine if any mutations (beyond those currently identified) that occur in viral genes facilitate persistence either through aberrant protein function or avoidance of the immune system. Sites under positive selection (adaptive evolution) in genes encoding envelope proteins of SSPE patients are good candidates for such mutations because they may represent substitutions in epitopes that abolish immune recognition, or changes in structural domains that result in abnormal proteins. Sites under putative positive selection can be identified by calculating the number of synonymous (dS) and nonsynonymous (dN) substitutions per site, whose ratio (dN/dS) is termed . An
<1 suggests purifying (negative) selection, whereas an
>1 is indicative of positive selection because the rate of fixation is higher than the background rate of mutation, which defies neutrality (
=1) (Yang & Bielawski, 2000
). A maximum likelihood (ML) method using models of sequence evolution can be employed to calculate
ratios and to identify amino acid sites as conserved, neutral or positively selected (Yang et al., 2000
). This method accounts for phylogenetic structure, biases in codon usage and the transition/transversion (TS/TV) ratio, and has already been applied to the H and L genes of MV (Woelk et al., 2001
). This previous research investigated the difference in sites under positive selection between acute and persistent infections but lack of sequence data led to inconclusive results. In this work, new SSPE sequence data for the H gene have been obtained and F gene sequences from both acute and persistent patients have been analysed. In addition, phylogenetic methods are used to estimate the rate of evolution and the extent of hypermutation in genes from SSPE cases.
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Methods |
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Multiple alignments and phylogenetic analysis.
Complete sequences of H, F and M genes were downloaded from GenBank and aligned after the removal of identical, vaccine and highly passaged strains. The M gene alignment of 35 sequences includes five SSPE variants of isolate S(B) (Baczko et al., 1993 ). Within this alignment, sequences were divided into Acute-M (n=20) data sets and SSPE-M [n=10; S(B) variants and the S(C) MIBE isolate removed]. The F gene alignment (Total-F) contains 22 sequences and was further divided into Acute-F (n=12) and SSPE-F [n=9; S(C) MIBE isolate removed]. Amino acids after site 523 were stripped from the Total-F and SSPE-F alignment due to the presence of frameshifts and premature stop codons. Three H gene alignments of SSPE isolates were created; SSPE-H (n=20), SSPE-H(NP) (n=12) and SSPE-H(CP) (n=7). The latter two data sets contain SSPE isolates that were non-passaged (NP) and cell passaged (CP), respectively. For comparison to SSPE, we incorporated an alignment of 37 H genes from acute strains of MV (Acute-H) that was not analysed separately in our previous paper (Woelk et al., 2001
). A full list of the sequences used in this study is presented in Table 1
.
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Selection analyses.
Yang et al. (2000) have developed an ML method for determining selection pressures in protein coding DNA sequences. This method originally employed 14 models that use statistical distributions to account for variable
(dN/dS) ratios among codon sites. Subsequent research has shown that models M0, M1, M2, M3, M7 and M8 are sufficient for accurate selection analysis (Woelk & Holmes, 2001
; Woelk et al., 2001
). Models M0, M1 and M7 do not allow for the existence of positively selected sites. M0 calculates a single
ratio (between the bounds 0 and 1) averaged over all sites, whereas M7 uses a discrete beta distribution (between the same bounds) to model different
ratios among sites. The shape of the beta distribution is governed by the parameters p and q. M1 accounts for neutral evolution by estimating the proportion of conserved (
=0) and neutral (
=1) sites. Alternatively, models M2, M3 and M8 account for positive selection using parameters that estimate
>1. Models M2 and M8 extend M1 and M7, respectively, through the addition of two parameters (p2 and
2 for M2 and p1 and
1 for M8) that have the potential to estimate
>1 for an extra class of sites. M3 provides the most sensitive test for positive selection by estimating an
ratio for a predetermined number of classes. Three classes were used in this analysis (p0, p1 and p2) such that three corresponding
ratios (
0,
1 and
2) were estimated. The first step in the identification of amino acid sites under positive selection is to test whether sites exist with
>1 by comparing nested models using likelihood ratio tests (LRTs). M0 and M1 are both special cases of M2 and M3, while M7 is a special case of M8, and such nested models can be compared with LRTs. Once positively selected sites have been shown to exist, the second step uses Bayesian methods to locate their position. Sites having high posterior probabilities (>90%) of belonging to a site class with
>1 are good candidates for positively selected sites. Posterior probabilities are conditional on the observed data such that they refer to the probability that a site, given the data at that site, is from a particular site class. The methods and models described here were implemented using the CODEML program of the PAML package, version 3.0c (Yang, 1997
).
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Results |
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Hypermutation investigation and selection analysis of the M gene
To determine the extent of hypermutation in genes of SSPE isolates, the TS/TV ratio (Table 2) and substitution rate parameters for all possible transitions and transversions (not shown) were estimated for the acute and SSPE alignments of the F, H and M genes. The TS/TV ratio was comparable between acute and SSPE alignments for the F and H genes but was approximately double in SSPE-M when compared to Acute-M. Further analysis reveals that the increased ratio in SSPE-M isolates predominantly results from U to C changes. Selection analysis of isolates of the M protein from acute patients (results not shown) indicated that this protein is strongly conserved, which is not surprising considering its critical functional role during lytic infection. Selection analysis of the M gene from SSPE cases was not performed because sequence data contained too many frameshifts and premature stop codons in the N terminus.
Selection analysis of the F gene
Selection analysis of the Acute-F data set did not identify any positively selected sites. M0 estimated a similar likelihood compared to models able to account for positive selection (M2, M3 and M8) and suggested that sites are strongly conserved (=0·098) (results not shown). In contrast, positive selection was identified in the SSPE-F data set by M2, M3 and M8, which rejected models M0, M1 and M7 in likelihood ratio tests (LRTs) (Table 3
). M2 and M3 produced similar results and estimated a large proportion of conserved sites and a small proportion of positively selected sites (
>5). Using Bayesian methods, M2 only assigned site 62 to the positively selected class with a posterior probability of 0·9356. M3 and M8 both assigned sites 62, 167, 446 and 461 to the positively selected class with posterior probabilities >99% for M3 and >95% for M8. Although M3 could not reject M2 in an LRT, sites 167, 446 and 461 should still be considered under possible positive selection because they were also identified by M8. M3 further identified sites 462 and 465 as under potential positive selection but this was with posterior probabilities of
90% and, since they were not confirmed by M8, these two sites are not discussed further.
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Stronger evidence for positive selection was found in the SSPE-H alignment. M2 and M8 estimated similar parameters for positive selection such that a small proportion of sites is under a very strong force of positive selection (=10·704 and 9·350, respectively) (Table 3
). The M3 model suggested that two classes of positive selection with different
ratios exist but the significance of this could not be confirmed because M3 was unable to reject M2. Bayesian methods assigned sites 12, 62 and 618 with posterior probabilities >99% to the positively selected class estimated by M2 and M8.
When SSPE strains detected directly from brain material [SSPE-H(NP)] were analysed, M2 and M3 were able to reject M0 but not M1, suggesting that a neutral class of sites fits the data just as well as a class of positively selected of sites (Table 3). Interestingly, the M7 vs M8 comparison contradicted this, since M8 was able to reject M7. This lends significance to positive selection (
1=3·331) at sites 7, 8, 12, 62, 302, 423, 560 and 575 estimated by M8 with posterior probabilities >90%. Although the alignment of cell-passaged SSPE strains [SSPE-H(CP)] contained fewer sequences than SSPE-H(NP), models accounting for positive selection were consistently strongly supported (Table 3
). Similar to the results obtained for SSPE-H, M3 estimated two classes of positively selected sites, but since it could not reject M2 the existence of both classes is unconfirmed. M2 and M8 both estimated a very strong positive selection pressure for site 62 (
2=15·314 and
1=14·211, respectively), which was the only site that could be assigned to the positively selected class with a posterior probability >90% (Table 3
).
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Discussion |
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The estimated rate of molecular evolution for SSPE strains (3·4x10-4 substitutions/site/year) is very close to the acute rate of 4·0x10-4 substitutions/site/year estimated for epidemics of genotypes C2 and D3 in Spain (Rima et al., 1997 ) and by using TipDate on a diverse set of sequences (Jenkins et al., 2002
). This similarity in rates is probably because a large proportion of the evolutionary history linking SSPE strains to one another has occurred within acute infections, such that the transition from acute to persistent infection occurs quite recently in the overall phylogenetic tree.
Selection in the F gene
Positive selection appears to have affected the evolution of the F gene in persistent (amino acid sites 62, 167, 446 and 461) but not acute MV cases (Table 4). The F1 portion of the F protein contains two heptad repeat regions termed A (140188) and B (463484) (Lamb & Kolakofsky, 1996
). Heptad repeat A is located directly after the fusion peptide (Chambers et al., 1990
) while heptad repeat B resides just prior to the transmembrane domain and contains a leucine zipper motif (Buckland et al., 1992
). In parallel to observations concerning other viral envelope glycoproteins (Gething et al., 1986
; Kreis & Lodish, 1986
; Spruce et al., 1991
), heptad repeat regions are thought to form long helices integral in tetramerization and higher oligomerization of the F protein which in turn govern the properties of fusion pore formation and cellular transport. Mutations in the leucine zipper motif of heptad repeat B have been shown to abolish fusion activity without disrupting the oligomeric form or surface expression of the protein (Chambers et al., 1990
). Putative positively selected site 167 lies within heptad repeat A and site 461 resides just prior to heptad repeat B. It is possible that substitutions at these sites have been selected to maintain persistence of MV within cells because they can lead to disruption of fusion pore formation and limit transport of the F protein to the cell surface. For instance, substitutions at site 167 appear to be from Ala to Thr or Val. Ala and Val are classified as hydrophobic but Thr contains a hydroxyl group, which increases hydrophilicity, and such a change could disrupt helix structure.
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Putative positive selection at sites 62 and 167 also correlates with synthetic peptides shown to react with antibodies in human sera from donors previously exposed to MV (Muller et al., 1993 ; Wiesmüller et al., 1992
). Whether the peptides identified by these studies correspond to bona fide BCEs is unclear because no immune function was shown for the antigenic regions identified and some reactions may represent non-specific binding of sera to peptide since experiments were performed in the absence of anti-MV-negative antisera (Atabani et al., 1997
). However, site 167 has already been discussed in association with structural features of the F protein and site 62 was found to correlate with a TCE.
Selection in the H gene
We have previously presented an analysis of a combined acute and SSPE data set of H gene sequences (Woelk et al., 2001 ). That study identified weak positive selection at sites 12, 62, 211, 303, 348, 423, 476, 481, 546, 562 and 575, and demonstrated that removal of SSPE isolates from the analysis led to the loss of positive selection at seven of them 12, 62, 211, 303, 348, 423, and 476. Here we show that positive selection in the remaining acute isolates could only be confirmed at sites 481, 546, 562 and 575 (Table 4
). Sites 481 and 546 are thought to result from selection for the CD46 receptor and sites 562 and 575 were shown to correlate with potential BCEs. Previously we were unable to confirm which sites were positively selected in SSPE isolates because of a lack of data. The larger data set of H gene sequences from SSPE cases (SSPE-H) presented here overcomes this limitation and it appears that sites 12, 62 and 618 are under extremely strong positive selection (Table 4
). Therefore, we suggest that different sites are under positive selection in SSPE and acute cases with selection at sites 12, 62 and 618 exclusive to SSPE isolates and selection at sites 481, 546, 562 and 575 exclusive to acute isolates. Positive selection at sites 211, 303, 348, 423 and 476 appears to have affected the evolution of both types of infection since a combined data set of SSPE and acute isolates is required for their identification.
The exact effect of the positive selection in the H gene at sites exclusive to SSPE isolates is unclear, but plausible explanations exist. Site 12 lies in the cytoplasmic domain and it is possible that substitutions at this site could affect M protein interaction during virus budding and hence lead to the maintenance of MV persistence. Substitutions at site 12 are from an ancestral Tyr (highly hydrophobic) to His, which can exist as uncharged or positively charged (hydrophilic) depending on its environment. This change could potentially hinder hydrophobic contacts between proteins. In our previous analysis (Woelk et al., 2001 ) we were unable to find an antigenic feature that correlated with site 62 and the reason for positive selection at this site remains unclear. Site 62 lies within the stem 1 domain of the H protein, which projects the neuraminidase head above the envelope (Langedijk et al., 1997
). Substitutions at site 62 are from an ancestral hydrophilic Arg to either the highly hydrophobic Trp, or the extremely small Gln. Whether such changes have structural implications for the H protein is not known but major distortions of the stem 1 region could affect efficient H protein function and help to maintain persistence. SSPE strains Osaka (1, 2 and 3), Yamagata-1, Sma79 and Sma94 have H proteins of abnormal length resulting from substitutions at site 618 which abolish the stop codon at this position. Whether this assists the maintenance of persistence in some way through irregular function or limited cellular transport is unclear.
Several SSPE strains were sequenced after various degrees of cell passaging (CP) and we were concerned that such strains were not truly representative of natural SSPE infection. For instance, the essence of persistent viruses is that they cannot replicate lytically and SSPE strains that have been cell passaged must have overcome this barrier. We therefore compared SSPE isolates that had been passaged with those that had not. Since most CP strains were passaged through Vero cells, it was surprising that sites known to interact with the CD46 receptor (see Woelk et al., 2001 ) were not identified as being under positive selection. Some evidence for positive selection, although not significant, was observed at sites involved in CD46 interaction (481 and 546). Only positive selection at site 62 could be confirmed when cell passaged SSPE strains were analysed separately. This site has not previously been implicated in CD46 interaction (Langedijk et al., 1997
) and since it was also identified in the data set that was not cell passaged [SSPE-H(NP)] it is unlikely to be the result of Vero passaging. When this non-passaged data set was analysed, it was initially unclear as to whether a small class of sites was positively selected or neutral, but further testing (M7 vs M8) provided some evidence for positive selection at sites 7, 8, 12, 62, 302, 423, 560 and 575. Hence, this result suggests that selection at site 575 may not be exclusive to acute MV strains. Selection at sites 12 and 62 in the H gene have been discussed above, while the selection at sites 7 and 8 may be due to the same pressures hypothesized for site 12 (i.e. hindrance to M protein interaction). Sites 423 and 575 correspond to antigenic areas recognized by antibodies (Woelk et al., 2001
), but the relevance of selection at sites 302 and 560 is unknown (Table 4
).
In summary, different selection pressures appear to be affecting the evolution of the envelope glycoproteins from acute and persistent cases of MV. For the F protein, positive selection was not evident in acute isolates but strong selection was seen in persistent cases. The value estimated under the M3 model of codon substitution 5·589 is equivalent to that seen in the haemagglutinin gene of influenza virus (
=47) (Yang, 2000
; Yang et al., 2000
). Passaging was not a major problem for the F gene data set used here because nonsynonymous substitutions were seen at all of the positively selected sites in non-passaged strains S(A) and S(B). A more complicated scenario of positive selection exists in the H gene. Acute strains are undergoing moderate selection (
>2), whereas analysis of all the available SSPE isolates suggests the influence of an extremely high force of positive selection (with
ranging from 9·350 to 10·705), comparable to that seen in the V3 region of the HIV-1 envelope protein (
=630) (Nielsen & Yang, 1998
). However, SSPE-H contains passaged strains such that this high positive selection pressure may not be indicative of natural SSPE evolution. Larger alignments of non-passaged isolates should help to obtain a more accurate picture of positive selection in the H gene of SSPE isolates. In any case, it is clear that far stronger positive selection pressure exists in SSPE cases compared to acute strains. Cattaneo et al. (1993)
have proposed that single or multiple mutations in the envelope glycoproteins may be pivotal in the establishment as well as the maintenance of SSPE cases by impeding virus assembly and possibly reducing cell surface expression. This proposition correlates with amino acid sites under possible selection in the H protein from SSPE cases (sites 12, 62 and 618), which have the potential to reduce M protein interaction and cellular transport, as well as distort structure. Sites under potential selection in the F protein (62, 167, 446 and 461) show limited association with structural and antigenic features. Further investigation is needed to confirm the exact roles of the sites under potential positive selection in these envelope glycoproteins in establishing SSPE infection.
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Acknowledgments |
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Received 8 November 2001;
accepted 25 January 2002.