1 Unidad de Biología Viral, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
2 Centro de Educación Médica e Investigaciones Clínicas, CEMIC, Hospital Universitario, Av. Galván 4102, Buenos Aires C1431FWO, Argentina
Correspondence
José A. Melero
jmelero{at}isciii.es
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ABSTRACT |
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MAIN TEXT |
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The G protein is a type II glycoprotein that shares neither sequence nor structural features with the attachment proteins (HN or H) of other paramyxoviruses (Wertz et al., 1985). Spontaneous mutants with deletions of the SH and G genes (Karron et al., 1997
) and genetically engineered viruses with deletions of the entire G gene have been isolated in tissue culture (Techaarpornkul et al., 2001
). These viruses can replicate efficiently in certain cell types (e.g. Vero cells) but replicate inefficiently in others (e.g. HEp-2 cells) and they are attenuated in BALB/c mice (Teng et al., 2001
). Therefore, it seems that the G protein, although not necessary for infection of certain cell types, is required for efficient infectivity, and this may be the reason for its presence in all virus isolates analysed to date. Nevertheless, the G protein shows extensive sequence and antigenic variation between viruses. The G protein is also one of the targets of neutralizing antibodies (reviewed by Melero et al., 1997
).
The capacity of the G protein to accommodate drastic sequence changes is illustrated by a series of escape mutants selected with certain monoclonal antibodies. Besides single amino acid substitutions, some escape mutants had: (i) frame-shift mutations that altered the C-terminal one-third of the G protein (García-Barreno et al., 1990); (ii) premature stop codons that shortened the length of the G polypeptide by between 1 and 42 amino acids (Rueda et al., 1991
, 1995
); and (iii) A
G hypermutations that were translated into several amino acid changes, some of them involving a conserved cluster of cysteines found in the middle of the G protein ectodomain (Rueda et al., 1994
; Martínez et al., 1997
; Walsh et al., 1998
).
There is some evidence that the changes mentioned above can also arise in the G protein during propagation of HRSV in its natural host. For instance, Sullender et al. (1991) described two viruses isolated from the same child 2 years apart that differed in 17 nucleotides of the G protein gene. These changes were translated into 11 amino acid differences, seven of them resulting from frame-shift mutations. Viruses with G proteins of different length (between 295 and 299 amino acids) due to mutations that determined termination codon usage have been isolated from clinical specimens (Sullender et al., 1991
; Martínez et al., 1999
). Finally, evidence for A
G hypermutations was provided by comparison of G gene sequences from certain natural isolates (Martínez & Melero, 2002
).
We now describe three clinical isolates of HRSV (BA3833/99B, BA3859/99B and BA4128/99B; named BA viruses), classified within antigenic group B, that contain a duplication of 60 nucleotides in the C-terminal one-third of the G protein gene. These viruses were isolated during an active surveillance study of respiratory infections in Buenos Aires, Argentina, from 1995 to 2001. Firstly, viral antigens were detected in clinical specimens by indirect immunofluorescence. Subsequently, viruses were isolated by inoculation of clinical samples in susceptible cells. A total of 38 RSVs were isolated in 1999; these viruses were classified in either antigenic group A (47·4 %) or antigenic group B (52·6 %) by reactivity with group-specific monoclonal antibodies. To gain further information about the phylogenetic relationship of the virus isolates, partial sequences of the C-terminal one-third of the G protein gene were obtained.
Initially, total RNA extracted from infected cells was used to obtain a cDNA segment of the G gene by hemi-nested RT-PCR. Reverse transcription was carried out with a negative sense primer that contained an oligo(dT) tail (LG3-, 5'-GGCCCGGGAAGCTTTTTTTTTTTTTTT-3'). Subsequently PCR amplification was done with Taq polymerase using LG3- and the primer LG5+ (5'-GGATCCCGGGGCAAATGCAAACATGTCC-3'), which included the start sequence of the G protein gene (in bold). For group B viruses, a second amplification was performed using primers LG3- and GB496+ (5'-GATGATTACCATTTTGAAGTGTTCA-3'), which started at nucleotide 496 of the G gene sequence of strain CH18547 (a prototype strain of antigenic group B) (Johnson et al., 1987). The DNA product of the hemi-nested RT-PCR from BA viruses migrated significantly more slowly than the equivalent DNA amplified from other viruses, suggestive of a larger size. When this DNA was sequenced using the Big-Dye Terminator Sequencing kit (Applied Biosystems), a duplication of 60 nucleotides was observed, as illustrated in Fig. 1
(A).
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Eight other viruses of antigenic group B isolated in Buenos Aires during the same outbreak as the BA viruses were sequenced. None of these viruses had the 60 nucleotide duplication. Phylogenetic analysis of all group B isolates revealed that viruses from different genetic branches circulated in Buenos Aires during the 1999 epidemic (M. Galiano and others, unpublished data). One of these viruses (BA3737/99B) was closely related to the BA isolates, with only eight nucleotide differences in the last 400 nucleotides of the G gene (excluding the duplication) and none of them in the duplicated segment of the later viruses. Thus, a virus similar to BA3737/99B could have been the ancestor of the viruses with the 60 nucleotide duplication.
The extra sequence in the BA viruses starts with a motif of four nucleotides, CACA (nucleotides 732735, mRNA sense), which is repeated at the end of the duplicated segment (Fig. 1A). This introduces an uncertainty about the starting site of the duplication, which could equally start in any of these four nucleotides. This ambiguity, however, does not alter the amino acid sequence deduced for the G protein of the three BA viruses.
A relatively stable secondary structure of the vRNA sequence that is duplicated in BA viruses was predicted using the algorithms developed by Zuker et al. (1999) (Fig. 1C
). This structure suggests a possible mechanism for generating the duplicated segment if the viral polymerase switched to the original vRNA strand and copied again the 60 nucleotides represented in Fig. 1(C)
before continuing the synthesis of the cRNA intermediate. It is worth stressing that no stable structures were predicted in that region of the BA viruses antigenome. Consequently, the above mechanism is less likely to occur during synthesis of the vRNA strand from the cRNA intermediate. Although the CACA motif is found repeatedly throughout the HRSV genome, the generation of stable secondary RNA structures and, most importantly, the viability of mutations may restrict the incorporation of nucleotide duplications in natural isolates.
The nucleotide sequence of the G gene from BA viruses is translated in a polypeptide of 315 amino acids, the largest found so far among HRSV isolates (Fig. 2). This protein shares structural features with the G proteins of other HRSV strains, such as the cluster of cysteines and the presence of multiple potential sites for O- and N-glycosylation in the protein ectodomain. The duplicated sequence lies in the C-terminal one-third of the G polypeptide and includes some of the potential O-glycosylation sites.
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The C-terminal one-third of the G molecule has been shown to be immunologically relevant. Epitopes recognized by strain-specific monoclonal antibodies directed against the G protein of group A viruses have been mapped in that segment of the G polypeptide (Melero et al., 1997). In addition, human convalescent sera react with the G protein C-terminal one-third of certain HRSV strains (Palomo et al., 2000
) and with synthetic peptides derived from them (Cane, 1997
). Thus, it is possible that the duplicated amino acids in BA viruses change the antigenic structure of the G molecule, conferring to them an evolutionary advantage to re-infect individuals exposed previously to the ancestor virus. However, the antigenic properties of the BA virus G proteins cannot be assessed at present due to the lack of specific reagents.
Viruses with a three nucleotide duplication in the G protein gene have been reported (Sullender et al., 1991; García et al., 1994
). Another virus isolated in Buenos Aires in 2001 had a six nucleotide duplication (to be reported). Thus, it seems that the HRSV polymerase is prone to copy repeatedly limited sequences of the G protein gene. In fact, when analysed in detail, the G protein sequence of many virus strains contain multiple short sequence repeats. The 60 nucleotide duplication reported here represents an extreme example of repeated sequences in the G protein gene. Whether this duplication originated from a partial vRNA secondary structure, as illustrated in Fig. 1(C)
, is not known. This structure could not be formed if the vRNA is bound tightly to the nucleoprotein. However, it is possible that short segments of vRNA devoid of nucleoprotein are generated during the process of RNA replication. Then, transient RNA secondary structures could be formed. These structures could be also at the basis of other mechanisms to generate RSVs with multiple A
G changes (hypermutations) (Martínez & Melero, 2002
) or defective genomes, as described for other negative-stranded RNA viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cane, P. A. (1997). Analysis of linear epitopes recognized by the primary human antibody response to a variable region of the attachment (G) protein of respiratory syncytial virus. J Med Virol 51, 297304.[CrossRef][Medline]
Collins, P. L. & Mottet, G. (1993). Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus. J Gen Virol 74, 14451450.[Abstract]
Collins, P. L., Chanock, R. M. & Murphy, B. R. (2001). Respiratory syncytial virus. In Fields Virology, pp. 14431484. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
García, O., Martín, M., Dopazo, J. & 8 other authors (1994). Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein. J Virol 68, 54485459.[Abstract]
García-Barreno, B., Portela, A., Delgado, T., López, J. A. & Melero, J. A. (1990). Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus. EMBO J 9, 41814187.[Abstract]
Johnson, P. R., Spriggs, M. K., Olmsted, R. A. & Collins, P. L. (1987). The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A 84, <@?show=[to]>56255629.
Karron, R. A., Buonagurio, D. A., Georgiu, A. F. & 8 other authors (1997). Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc Natl Acad Sci U S A 94, 1396113966.
Levine, S., Klaiber-Franco, R. & Paradiso, P. R. (1987). Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus. J Gen Virol 68, 25212524.[Abstract]
Martínez, I. & Melero, J. A. (2002). 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. J Gen Virol 83, 14451455.
Martínez, I., Dopazo, J. & Melero, J. A. (1997). Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants. J Gen Virol 78, 24192429.[Abstract]
Martínez, I., Valdés, O., Delfraro, A., Arbiza, J., Russi, J. & Melero, J. A. (1999). Evolutionary pattern of the G glycoprotein of human respiratory syncytial viruses from antigenic group B: the use of alternative termination codons and lineage diversification. J Gen Virol 80, 125130.[Abstract]
Melero, J. A., García-Barreno, B., Martínez, I., Pringle, C. R. & Cane, P. A. (1997). Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein. J Gen Virol 78, 24112418.
Mufson, M. A., Örvell, C., Rafnar, B. & Norrby, E. (1985). Two distinct subtypes of human respiratory syncytial virus. J Gen Virol 66, 21112124.[Abstract]
Palomo, C., García-Barreno, B., Peñas, C. & Melero, J. A. (1991). The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity. J Gen Virol 72, 669675.[Abstract]
Palomo, C., Cane, P. A. & Melero, J. A. (2000). Evaluation of the antibody specificities of human convalescent-phase sera against the attachment (G) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side chains. J Med Virol 60, 468474.[CrossRef][Medline]
Perez, M., García-Barreno, B., Melero, J. A., Carrasco, L. & Guinea, R. (1997). Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus. Virology 235, 342351.[CrossRef][Medline]
Roberts, S. R., Lichtenstein, D., Ball, L. A. & Wertz, G. W. (1994). The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons. J Virol 68, 45384546.[Abstract]
Rueda, P., Delgado, T., Portela, A., Melero, J. A. & García-Barreno, B. (1991). Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies. J Virol 65, 33743378.[Medline]
Rueda, P., García-Barreno, B. & Melero, J. A. (1994). Loss of conserved cysteine residues in the attachment (G) glycoprotein of two human respiratory syncytial virus escape mutants that contain multiple AG substitutions (hypermutations). Virology 198, 653662.[CrossRef][Medline]
Rueda, P., Palomo, C., García-Barreno, B. & Melero, J. A. (1995). The three C-terminal residues of human respiratory syncytial virus G glycoprotein (Long strain) are essential for integrity of multiple epitopes distinguishable by antiidiotypic antibodies. Viral Immunol 8, 3746.[Medline]
Sullender, W. M., Mufson, M. A., Anderson, L. J. & Wertz, G. W. (1991). Genetic diversity of the attachment protein of subgroup B respiratory syncytial viruses. J Virol 65, 54255434.[Medline]
Techaarpornkul, S., Barretto, N. & Peeples, M. E. (2001). Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75, 68256834.
Teng, M. N. & Collins, P. L. (2002). The central conserved cysteine noose of the attachment G protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo. J Virol 76, 61646171.
Teng, M. N., Whitehead, S. S. & Collins, P. L. (2001). Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. Virology 289, 283296.[CrossRef][Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 48764882.
Walsh, E. E. & Hruska, J. (1983). Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein. J Virol 47, 171177.[Medline]
Walsh, E. E., Falsey, A. R. & Sullender, W. M. (1998). Monoclonal antibody neutralization escape mutants of respiratory syncytial virus with unique alterations in the attachment (G) protein. J Gen Virol 79, 479487.[Abstract]
Wertz, G. W., Collins, P. L., Huang, Y., Gruber, C., Levine, S. & Ball, L. A. (1985). Nucleotide sequence of the G protein of human respiratory syncytial virus reveals an unusual type of viral membrane. Proc Natl Acad Sci U S A 82, 40754079.[Abstract]
Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology, pp. 1143. Edited by J. Barciszewski & B. F. C. Clark. Dordrecht: Kluwer.
Received 19 May 2003;
accepted 15 July 2003.