A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA

Isidoro Martínezb,1 and José A. Melero1

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


   Abstract
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Abstract
Introduction
Methods
Results and Discussion 
References
 
Human respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues and 98 or 106 of them – for A/Mon/3/88 or Long HRSV strains, respectively – are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100–200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion 
References
 
Human respiratory syncytial virus (HRSV) is an enveloped, nonsegmented, negative-stranded RNA virus that belongs to the genus Pneumovirus, family Paramyxoviridae. HRSV is the most important cause of severe lower respiratory tract infections in neonates and infants. This virus is also a serious agent of disease in immunosuppressed adults and the elderly and it probably infects the general population (Simoes, 1999 ). Transcription and replication of the HRSV genome conforms the general model derived from other paramyxoviruses; i.e. the viral genes are transcribed sequentially from a single 3' end promoter and replication involves the synthesis of a positive-sense antigenome that is an exact complement of the genome (reviewed by Collins et al., 2001 ).

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 80–90 kDa, as estimated by SDS–PAGE (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 164–176) 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.


   Methods
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Abstract
Introduction
Methods
Results and Discussion 
References
 
{blacksquare} Virus nucleotide sequences.
The complete G gene RNA sequence of the group A HRSV strains used in this study have been described (García et al., 1994 ) or were obtained from the databases (specific details can be obtained from the authors upon request).

{blacksquare} 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 ).

{blacksquare} 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).


   Results and Discussion 
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Abstract
Introduction
Methods
Results and Discussion 
References
 
Antibody escape mutants with multiple A to G transitions (hypermutations)
A total of 51 antibody escape mutants of two HRSV group A strains has been selected in our laboratory with 19 different MAbs directed against the G glycoprotein (Table 1) (García-Barreno et al., 1990 ; Rueda et al., 1991 , 1994 , 1995 ; Martínez et al., 1997 ). Viruses with multiple nucleotide substitutions that involved almost exclusively A to G transitions (hypermutations) were selected only with antibodies whose epitopes were mapped in the conserved central region (aa 163–189, nt 502–582) of the G protein ectodomain (Fig. 1) (Rueda et al., 1994 ; Martínez et al., 1997 ). Five of 14 (35·7%) viruses selected with four of those antibodies were hypermutated. In contrast, none of 37 escape mutants selected with 15 other anti-G MAbs, whose epitopes lie outside the central conserved region, contained hypermutations (Table 1). In each hypermutated virus, all or most of the A to G changes were clustered in the conserved region (Fig. 1). Up to 12 A to G transitions were observed in the short gene segment spanning residues 495 to 581 of the 19G/8B/1 mutant (Fig. 1 and Table 2).


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Table 1. MAb escape mutants selected with antibodies against different regions of the HRSV glycoprotein

 


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Fig. 1. A to G transitions selected in the G protein of hypermutated escape mutants of strains A/Mon/3/88 and Long. The primary structure of the G protein is represented, indicating the transmembrane region (TM) and the cysteine residues ({blacksquare}). The gene segment of the identical sequences in all HRSV isolates of antigenic group A (aa 163–189) is indicated by the bar shown below the protein diagram. A to G changes in each mutant virus are indicated by vertical arrows. The mutant name and the wild-type virus from which the mutants were derived are indicated on the right. The bottom line is a scale of the G polypeptide in amino acids. Total length for the G protein of strains A/Mon/3/88 and Long is 297 and 298 aa, respectively (García et al., 1994 ).

 

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Table 2. A to G changes in the central region of the G gene in escape mutants of HRSV

 
The central conserved region of the G gene fulfils substrate requirements for dsRNA adenosine deaminase activity
The fact that hypermutated viruses were only selected with antibodies that mapped within the central conserved region of the G protein gene and that most of the A to G changes were clustered in this segment suggested to us that this region could be a ‘hot spot’ for A to G hypermutations.

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 495–582) 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 480–630 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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Fig. 2. Predicted RNA secondary structures for segments of 150 nt between residues 480 and 630 (upper part) and 720 and 870 (lower part) of the G genes of strains A/Mon/3/88 (A) and Long (B) are shown. The stability of the predicted structures is indicated as -kcal/mol. Dots denote A residues within the 480–630 segment that are changed in the hypermutated viruses of Fig. 1.

 
The percentage of changed adenosines in the central region of the G gene varies between 17·9% for the 19G/9A/2 mutant and 42·9% for the 19G/8B/1 mutant. In spite of this extensive modification, the pattern of changed adenosines is different between mutants (Fig. 1 and Table 2), according with the promiscuity of ADARs. However, some of the adenosines are modified in more than one mutant, indicating a preference for certain sites. Considering all of the mutants from Fig. 1 together, the 5' preferences for the A to G changes represented in Fig. 2 are 14A=14U>8C>>1G, which mirror the 5' preferences of ADARs (Bass, 1997 ). Some of the mutants illustrated in Fig. 1 also have certain A to G changes outside the central segment of the G protein gene, where secondary structures of lower stability are predicted (see Final remarks).

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 720–870 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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Fig. 3. Stability of predicted RNA secondary structures for 150 nt segments of the G gene of strains A/Mon/3/88 (A) and Long (B). The stability of predicted secondary structures (-kcal/mol) of 150 nt segments overlapping 120 nt was plotted against the central nucleotide of the corresponding segment. The central region where most of the A to G changes in escape mutants are clustered (nt 495–582) is indicated by a thick line. Positions where the epitopes of different MAbs map are also indicated by vertical lines. The epitopes of antibodies c793 and 74G have not been mapped to the single amino acid level but segments containing these epitopes are indicated by horizontal lines. The segments including nt 480–630 and nt 720–870, used for generating the structures shown in Fig. 2, are indicated by continuous lines. Segments including nt 180–330 and nt 615–765, used for the analysis in Fig. 4, are shown by dashed lines.

 


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Fig. 4. Frequency of A to G+G to A and U to C+C to U changes in the nt 180–330 and nt 615–765 regions of HRSV G protein from group A isolates. Circles represent the percentage of A to G+G to A (A–C) or U to C+C to U (D–F) changes observed when comparing, pairwise, different natural isolates in the nt 180–330 ({circ}) or nt 615–765 ({bullet}) regions. These percentages are plotted against the total number of nucleotides changed for each virus pair. Changes involving the same nucleotides (e.g. A to G+G to A) were considered together since ancestor sequences for each pair of viruses were unknown. Only comparisons between strains differing in more than 60 nt in the complete G gene sequence are shown because results from closely related viruses are dispersed in nature by statistical considerations. (C, F) Merged images of (A, B) and (D, E), respectively.

 
A to G+G to A substitutions between natural group A isolates predominate at G gene regions where stable secondary structures are predicted
We have reported previously that A to G+G to A substitutions predominate when comparing, pairwise, G gene sequences of strains from either group A (Martínez et al., 1997 ) or group B (Martínez et al., 1999 ) viruses. Changes involving the same nucleotides (e.g. A to G+G to A) were considered together since ancestor sequences for each pair of viruses were unknown. Since A to G transitions introduced by the hypermutation mechanism may be at the basis of that bias, we decided to explore whether A to G+G to A substitutions accumulate in regions where stable dsRNA structures are predicted. Since the central segment of the G protein ectodomain is highly conserved among virus isolates, we could not use this segment for comparing nucleotide changes between virus strains. Conservation of the central segment in virus strains, but not in escape mutants, may be related to functions that are essential for replication of HRSV in its natural host but not in tissue culture cells.

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 180–330 (where stable dsRNA structures are predicted) or nt 615–765 (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 180–330 nt region (Fig. 4A) than in the 615–765 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. 4D–F). The above observations could not be ascribed to differences in base composition because sequences with very similar A+G nucleotide content in the 180–330 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 180–330 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 (100–200 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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Fig. 5. Models for the generation of A to G hypermutations in HRSV. Details of the alternative models are explained in the text. (A) Replication or transcription of the viral genome (although RNA transcripts would not be encapsidated). (B) Synthesis of vRNA from cRNA as template. Black ovals and open circles represent viral RNA-dependent polymerase and nucleoprotein, respectively.

 
Final remarks
Walsh et al. (1998 ) have reported the isolation of five escape mutants of the Long strain of HRSV using a group cross-reactive MAb (obtained in their laboratory) that presumably recognized sequences of the conserved G protein region. Each of these viruses contained multiple A to G changes in their genomes. Four viruses had between six and eight A to G changes in the central segment of the G protein gene, resembling the mutants selected in our laboratory. One of the viruses accumulated seven A to G changes that led to four amino acid changes in the cytoplasmic and transmembrane region of the G molecule. This virus had reduced levels of the G protein incorporated into the virus particle and showed restricted growth in tissue culture cells. Most of the A to G changes selected in the mutants described by Walsh et al. (1998 ) were represented in the mutants described here, stressing the site specificity of the hypermutations selected in HRSV escape mutants.

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, O’Hara 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 521–922 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 (68–898) 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.


   Acknowledgments
 
We thank Joaquin Dopazo for helping in statistical analysis of the G gene sequences from natural HRSV isolates. This work was supported in part by grants PM99-0014 from Ministerio de Ciencia y Tecnología and 01/24 from Instituto de Salud Carlos III.


   Footnotes
 
b Present address: Department of Microbiology, University of Alabama School of Medicine, Birmingham, Alabama 35294, USA.


   References
Top
Abstract
Introduction
Methods
Results and Discussion 
References
 
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Received 14 January 2002; accepted 1 March 2002.