Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain1
Author for correspondence: José A. Melero. Fax +34 91 509 79 19. e-mail jmelero{at}isciii.es
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Abstract |
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Introduction |
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The virus envelope has two major glycoproteins, G and F, which are the main targets of neutralizing and protective antibodies (Hall et al., 1991 ). The F glycoprotein mediates the fusion of the viral and cell membranes (Walsh et al., 1985
) and the G glycoprotein is responsible for virus binding to the cell surface receptor (Levine et al., 1987
). HRSV isolates have been classified into two antigenic groups (A and B) based on antigenic diversity detected with panels of monoclonal antibodies (MAbs), preferentially recognizing the G glycoprotein (Anderson et al., 1985
; Mufson et al., 1985
).
The G protein shares neither sequence nor structural features with the attachment protein (HN or H) of other viruses of the same family. It is a type II glycoprotein with a single hydrophobic domain between residues 38 and 66, which serves as both a membrane anchor and a signal sequence. The protein precursor is synthesized as a 32 kDa polypeptide which is glycosylated by the addition of N- and O-linked carbohydrates to yield the mature protein of 8090 kDa, as estimated by SDSPAGE (Gruber & Levine, 1985 ; Wertz et al., 1989
; Collins & Mottet, 1992
). In the centre of the G protein ectodomain is a cluster of four cysteines (residues 173, 176, 182 and 186) and a short amino acid segment (residues 164176) of identical sequence among all HRSV isolates that extends from aa 163 to 189, when considering group A isolates only (Johnson et al., 1987
; Cane et al., 1991
; Sullender et al., 1991
; García et al., 1994
). This region was proposed as a putative receptor-binding site (Johnson et al., 1987
) and overlaps partially with a recently described heparin-binding site between residues 184 and 198 in group A viruses and 183 and 197 in group B viruses (Feldman et al., 1999
). Flanking the central region are two variable segments in the G protein ectodomain whose amino acid compositions resemble those of mucins, a class of O-glycosylated proteins secreted by epithelial cells (Apostolopoulos & McKenzie, 1994
).
The G glycoprotein is the viral gene product with the highest degree of antigenic and genetic diversity among virus isolates (Anderson et al., 1985 ; Mufson et al., 1985
; García-Barreno et al., 1989
; Cristina et al., 1990
; Cane et al., 1991
; Sullender et al., 1991
; García et al., 1994
; Cane & Pringle, 1995
; Martínez et al., 1999
). The plasticity of the G molecule in accommodating sequence changes is illustrated by previous analysis of escape mutants selected with MAbs (reviewed by Melero et al., 1997
). In addition to amino acid substitutions, some escape mutants had more drastic changes, including frame shift mutations that altered the sequence of the C-terminal third of the G protein (García-Barreno et al., 1990
), premature stop codons that shorten the G protein length between 1 and 42 aa (Rueda et al., 1991
, 1995
; Martínez et al., 1997
) and multiple A to G transitions (A to G hypermutations) that changed several amino acids, including one or two of the conserved cysteines (Rueda et al., 1994
; Martínez et al., 1997
; Walsh et al., 1998
).
Reiterative A to G transitions have been reported in other negative-stranded RNA viruses (Cattaneo, 1994 ), retroviruses and transcripts of the DNA virus, polyomavirus (reviewed by Bass, 1997
). Bass et al. (1989)
proposed that cellular adenosine deaminases that act on RNA (ADARs) could be involved in the generation of A to G hypermutations in viral RNA genomes by converting adenosines to inosines in double-stranded RNA molecules if collapsed transcription (mRNA transcripts hybridized to vRNA) occurs. In addition to intermolecular dsRNA, ADARs also modify intramolecular double-stranded regions of synthetic RNAs (Nishikura et al., 1991
) and intramolecular dsRNA structures are substrates for ADARs in editing of cellular and viral RNAs (Felder et al., 1994
; Netter et al., 1995
; Melcher et al., 1995
; Yang et al., 1995
). Here we present evidence that supports an alternative model to collapsed transcription for explaining the multiple A to G changes observed in antibody escape mutants of HRSV. This model predicts the formation of short intramolecular dsRNA segments during replication and/or transcription that may serve as substrates for ADARs.
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Methods |
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MAbs and HRSV escape mutants.
MAbs directed against the G glycoprotein of group A strains A/Mon/3/88 (prefixed 021) (Martínez et al., 1997 ) and Long (Mufson et al., 1985
; García-Barreno et al., 1989
) were described previously. Escape mutants of either A/Mon/3/88 or Long viruses have also been described (García-Barreno et al., 1990
; Rueda et al., 1991
, 1994
, 1995
; Martínez et al., 1997
). Briefly, each virus was passed from five to ten times in the presence of individual antibodies until cell extracts from infected cells did not react with the antibody used in the selection process. Multiple passages in the presence of MAbs were needed to select escape mutants because the neutralizing capacity of individual MAbs is low (Martínez & Melero, 1998
).
Statistical analysis.
The ANDIST program (Joaquín Dopazo, unpublished) was utilized to find out the frequency of different nucleotide changes between pairs of virus. RNA secondary structure analysis was carried out with the RNAFOLD program based on the method of M. Zuker (http://bioinfo.math.rpi.edu/mfold/rna/form1.cgi).
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Results and Discussion |
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It has been proposed that cellular ADARs may be implicated in the generation of reiterative A to G substitutions (Bass et al., 1989 ). This activity modifies adenosines to inosines in either inter- or intramolecular dsRNAs using a hydrolytic deamination mechanism (Polson et al., 1991
; Nishikura et al., 1991
; Polson & Bass, 1994
). The enzyme was first characterized using as substrates completely base-paired dsRNAs, although it can also deaminate adenosines within RNAs that are largely base-paired but interrupted by mismatches, bulges and loops. AU-rich duplexes are preferred as substrates and deaminated adenosines show a 5' nearest-neighbour preference (A=U>C>G). Multiple adenosines (up to 50%), whether base-paired, mismatched or contained in a loop, can be modified in a single dsRNA, especially in those of
50 bp (Nishikura et al., 1991
; Polson & Bass, 1994
; Herb et al., 1996
; Maas et al., 1996
).
Computer prediction of dsRNA structures of the complete G gene showed that most residues could participate in predicted large intramolecular stems but did not reveal special characteristics for the region (nt 495582) where most of the A to G transitions are localized in the escape mutants (data not shown). It is hard to envisage a scenario in the replicative cycle of HRSV, where large segments of either vRNA or cRNA could be intramolecularly base-paired because both strands are encapsidated with the nucleoprotein and their synthesis is coupled to encapsidation (Huang & Wertz, 1982 ). However, it is conceivable that nonencapsidated short segments (between 100 and 200 residues) of either the genome or the antigenome are transiently formed during replication/transcription of HRSV (see below). Therefore, we decided to explore the capacity of 150 nt segments around the conserved region of the G protein gene to form RNA secondary structures. Interestingly, computer predictions showed a highly stable base-paired segment (-37·7 kcal/mol) in the 480630 nt region of the G gene of strain A/Mon/3/88 (Fig. 2A
). This is an AU-rich region (61·6%) containing 49 bp within 137 nt. Both base composition and length characteristics fulfil substrate requirements for ADARs (Kimelman & Kirschner, 1989
; Nishikura et al., 1991
; Polson & Bass, 1994
). A very similar structure is predicted for the same segment of the Long strain (Fig. 2B
): the percentage of AU being 60·9% with 53 bp in a 137 nt segment conferring a stability of -42·6 kcal/mol. The adenosines changed to guanidines in the hypermutated escape mutants of strains A/Mon/3/88 and Long (denoted by dots in Figs 2A
, B
) were found predominantly in paired bases of the predicted secondary structures.
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To search for analogous structures along the complete G gene, the stability of predicted secondary structures of 150 nt segments overlapping 120 nt was plotted against the representative central nucleotide of each segment (Fig. 3). The results obtained with the A/Mon/3/88 and Long strains were very similar and confirmed that the conserved central region constitutes a peak of stability surrounded by two deep valleys. Most of the MAbs that did not select hypermutated viruses have their epitopes mapped within these valleys or within regions of low energy for dsRNA structures. More importantly, when the actual predicted structures were represented, they differed considerably from the structure predicted for the central segment. As an example, the structures predicted for a 150 nt segment between nt 720 and 870 of strains A/Mon/3/88 and Long are shown in Fig. 2(A
, B
). This segment contains variable epitopes and their antibodies selected escape mutants without hypermutations (Table 1
). In both cases, the structures of the 720870 segment contain several loops with bulges in short stems of dsRNA. It is worth stressing that ADARs are highly dependent on the length of the dsRNAs and that internal loops in dsRNA structures reduce the efficiency of ADARs (Lehmann & Bass, 1999
).
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Two other segments of the G protein gene were, therefore, selected to test whether A to G+G to A changes in virus isolates accumulated preferentially in regions where more stable dsRNA structures are predicted. Fig. 4 shows the frequency of A to G+G to A and U to C+C to U nucleotide changes when 45 sequences of the G protein gene from natural group A isolates were compared, pairwise, in the regions spanning nt 180330 (where stable dsRNA structures are predicted) or nt 615765 (where the stability of predicted dsRNA structures is very low) [note that the similarity of the energy profiles from two distantly related strains as A/Mon/3/88 and Long (García et al., 1994
) validate this approach] (Fig. 3
). The percentages of A to G+G to A transitions seen in Fig. 4
are higher in the 180330 nt region (Fig. 4A
) than in the 615765 nt region (Fig. 4B
, see panel C for comparison). In contrast, the percentages of U to C+C to U transitions are similar in both gene segments (Fig. 4DF
). The above observations could not be ascribed to differences in base composition because sequences with very similar A+G nucleotide content in the 180330 nt region show, nevertheless, significant differences in the percentage of A to G+G to A changes when compared with another strain. As an example, the base composition of strains A/Mad/2/88 (31A, 48G, 54U and 17C) and A/Mad/9/93 (29A, 46G, 56U and 19C) is very similar in the 180330 nt region of the G gene. However, when compared with the Long strain, A/Mad/9/93 shows ten nucleotides changes, four (40·0%) are A to G+G to A and four are U to C+C to U. In contrast, A/Mad/2/88 and Long strains differ in 17 nt changes, ten (58·8%) of them being A to G+G to A changes and only five (29·4%) being U to C+C to U changes.
A model for A to G hypermutations
To explain the observations presented in the previous sections, we propose two alternative models to that of collapsed transcription (Bass et al., 1989 ), where ADARs could introduce A to I mutations on intramolecular dsRNA segments during replication/transcription of viral RNA (Fig. 5
). Neither collapsed transcription nor intramolecular base pairing are compatible with the idea that both vRNA and cRNA are encapsidated (Huang & Wertz, 1982
). However, it is conceivable that short RNA segments (100200 nt) devoid of nucleoprotein are transiently formed during replication/transcription and that transient dsRNA structures could be formed. In model A (Fig. 5A
), a transient dissociation between template RNA and nucleoprotein is depicted during replication/transcription at the sites where the polymerase complex is operating (Fig. 5
, black oval). In model B (Fig. 5B
), the incorporation of nucleoprotein to nascent vRNA is delayed during replication. The following steps are common to both models and are essentially identical to those of collapsed transcription (Bass et al., 1989
). ADAR activity would modify adenosines to inosines in the duplexes destabilizing the secondary structures and facilitating the association with the nucleoprotein. Because inosines are equivalent to guanosines in nucleotide pairing, inosine residues would direct the incorporation of cytosine residues during replication, leading to U to C mutations in the positive strand that become A to G mutations in the negative strand (viral genome). Considering that the nucleotide composition is approximately the same over the entire G gene, the model of collapsed transcription would not explain the accumulation of A to G changes in certain segments of the G gene, since ADARs would be acting on a dsRNA formed over the entire gene. In contrast, the models depicted in Fig. 5
would favour the accumulation of A to G changes in segments with a propensity to form stable intramolecular dsRNAs.
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As mentioned before, some hypermutated viruses selected with MAbs contain A to G changes outside the central region of the G protein gene (Fig. 1). We can only speculate at present how these extra A to G changes are generated but it is conceivable that once an RNA molecule is engaged in a hypermutation event, regions of the molecule outside the segment predicted to have the most stable dsRNA structure are also prone to changes introduced by ADARs.
Reiterative A to G transitions have been reported in the case of deficient genomes of paramyxoviruses (parainfluenza virus type 3, Murphy et al., 1991 ; measles virus, Cattaneo et al., 1988
) and related viruses (vesicular stomatitis virus, OHara et al., 1984
). However, it is only in HRSV that viable hypermutated viruses have been isolated. This is probably due to the extreme plasticity of the G protein to accumulate drastic sequence changes without losing its function (Melero et al., 1997
). Nevertheless, the viability of hypermutated virus described here suggests that the same mechanism operating to generate these mutants could also function during replication of HRSV and, perhaps, other negative-stranded RNA viruses. Extensive hypermutations would probably be selected against but limited hypermutations may be allowed in other regions of the genome and could be considered to be simple misincorporations. However, a bias in the frequency of nucleotide changes towards A to G transitions would be expected if changes introduced by limited hypermutations represent a significant percentage of total nucleotide substitutions.
In agreement with that hypothesis, A to G+G to A transitions are the most frequent changes observed in the G protein gene of HRSV isolates of antigenic groups A (Martínez et al., 1997 ) and B (Martínez et al., 1999
) and in other gene segments (unpublished results). This is true despite the fact that the models depicted in Fig. 5
would predict that A to G hypermutations occurring in the positive-sense RNA (cRNA) would lead to U to C transitions in the genome (vRNA). Interestingly, predictions for the positive strand of the G gene of strains A/Mon/3/88 and Long, as done in Fig. 3
for negative-sense sequences, showed a low tendency to form stable dsRNA structures.
Hypermutation events may be more easily observed when comparing sequences of closely related strains, before misincorporations blur the hypermutated changes. Supporting this assumption, A to G transitions are predominantly observed between some closely related sequences of the group A viruses analysed in Fig. 4. For instance, seven of 13 nucleotide differences between the G gene sequences of strains A/Mon/5/91 and A/Mon/9/92 were A to G changes (García et al., 1994
). A striking example was observed when comparing two group B strains (B/Mon/19/94 and B/Mon/3/94): 16 of 23 changes in the 521922 nt region of the G gene were U to C changes (Martínez et al., 1999
). All of these U to C changes were clustered within a region of 211 nt (68898) that is predicted to form a stable dsRNA structure in the positive polarity of strain B/Mon/10/94 but not in the group A strains (A/Mon/3/88 and Long) (data not shown). In the context of the ADAR hypothesis, hypermutations might influence drastically the evolution of HRSV by one step generation of viruses that originate new phylogenetic lineages (Martínez et al., 1999
).
In summary, to explain the multiple A to G changes observed in HRSV escape mutants, we propose a model in which ADARs convert adenosines to inosines on dsRNA secondary structures, transiently formed during HRSV replication and/or transcription. We extend this model to explain the bias accumulation of A to G changes (and in some cases U to C changes) during the natural evolution of this virus.
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Acknowledgments |
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Footnotes |
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References |
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Received 14 January 2002;
accepted 1 March 2002.