Sequence polymorphism of the predicted human metapneumovirus G glycoprotein

Teresa C. T. Peret1,2, Yacine Abed3, Larry J. Anderson1, Dean D. Erdman1 and Guy Boivin3

1 Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
2 Task Force for Child Survival and Development, Atlanta, GA, USA
3 Research Center in Infectious Diseases of the Québec University Hospital Center, Department of Microbiology, Laval University, Québec City, Canada GIV 4G2

Correspondence
Dean D. Erdman
dde1{at}cdc.gov


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The putative G glycoprotein genes of 25 human metapneumovirus (hMPV) field isolates obtained during five consecutive epidemic seasons (1997 to 2002) were sequenced. Sequence alignments identified two major genetic groups, designated groups 1 and 2, and two minor genetic clusters within each major group, designated subgroups A and B. Extensive nucleotide and deduced amino acid sequence variability was observed, consisting of high rates of nucleotide substitutions, use of alternative transcription-termination codons and insertions that retained the reading frame. Deduced amino acid sequences showed the greatest variability, with most differences located in the extracellular domain of the protein: nucleotide and amino acid sequence identities for the entire open reading frame ranged from 52 to 58 % and 31 to 35 %, respectively, between the two major groups. Like the closely related avian pneumovirus and human and bovine respiratory syncytial viruses, the predicted G protein of hMPV shared the basic features of a type II mucin-like glycosylated protein. However, differences from these related viruses were also observed, e.g. lack of conserved cysteine clusters as seen in human respiratory syncytial virus and avian pneumovirus. The displacement of genetic groups of hMPV observed during the study period suggests that potential antigenic differences in the G glycoprotein, which have evolved in response to immune-mediated pressure, may influence the circulation patterns of hMPV strains.

GenBank accession numbers for the reported sequence data: AY485232AY485256.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human metapneumovirus (hMPV), a recently recognized paramyxovirus (van den Hoogen et al., 2001), has been linked to acute respiratory illness in children, the elderly and the immunocompromised (Boivin et al., 2002; Jartii et al., 2002; Pelletier et al., 2002; Peret et al., 2002; Stockton et al., 2002; Falsey et al., 2003; Boivin et al., 2003). Like human respiratory syncytial virus (HRSV), hMPV appears to be ubiquitous, infecting most children at an early age, and exhibits a distinct annual epidemic peak in the winter months (Falsey et al., 2003).

hMPV has been tentatively assigned to the genus Metapneumovirus (van den Hoogen et al., 2001, 2002) based upon sequence identity and similar genomic organization to avian pneumovirus (APV). Analyses of hMPV and APV sequences have shown that hMPV shares the closest relationship with APV type C (van den Hoogen et al., 2002; Bastien et al., 2003). hMPV, along with APV, HRSV and bovine respiratory syncytial virus (BRSV), belongs to the family Paramyxoviridae, subfamily Pneumovirinae, whose members encode the G protein, a type II mucin-like glycoprotein. The G glycoprotein of HRSV induces a group-specific protective immune response and is associated with enhanced HRSV disease (Sparer et al., 1998; Tripp et al., 2001). The G glycoproteins of HRSV and BRSV also participate but do not seem to be essential in virus attachment (Teng et al., 2002; Schlender et al., 2003).

The genetic variability of the hMPV G gene has not been fully examined, although limited studies have identified two major lineages of hMPV (Boivin et al., 2002; van den Hoogen et al., 2002; Peret et al., 2002; Bastien et al., 2003). To better assess the genetic variability of the hMPV G gene, we sequenced 25 full-length genes representing both major hMPV lineages from Canadian field isolates obtained over five consecutive epidemic seasons (1997 to 2002).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses.
hMPV G gene sequences were obtained from clinical specimens or culture isolates from 25 patients admitted to the Research Center in Infectious Diseases of the Québec University Hospital Center, Québec, Canada, between 1997 and 2002 (Table 1). Viruses were isolated in LLC-MK2 cells as previously described (Boivin et al., 2002).


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Table 1. Distribution of hMPV isolates

 
RT-PCR amplification and sequencing of the hMPV G gene.
Primers complementary to conserved regions of the hMPV SH, G and L genes were designed for amplification and sequencing of the full hMPV G gene (Table 2). Total RNA was extracted from clinical specimens or infected culture lysates using TRIZOL LS Reagent (Invitrogen) or QIAamp Viral RNA Mini kit (QIAGEN). Viral RNA was reverse transcribed using 20 mM of primer MPVGLRV2 or MPVGLRV1 at 55 °C for 1 h with ThermoScript RNase H reverse transcriptase following the manufacturer's instructions (Invitrogen). After cDNA synthesis, PCR was performed with 2 µl template, 15 mM dNTP mixture, 10 mM forward and reverse primers, 2·5 U Platinum Pfx DNA Polymerase (Invitrogen), 5 µl 10x Pfx Amplification Buffer and 1 µl 50 mM MgSO4, brought to 50 µl with distilled water. Amplification conditions consisted of 2 min at 94 °C, followed by 35 cycles of PCR (94 °C/15 s; 55 °C/30 s; 68 °C/1 min per kb). PCR amplification products were purified with either the QIAquick Gel Extraction Kit or QIAquick PCR Purification Kit (QIAGEN). PCR amplification products were sequenced at least twice from independent RNA extractions. Both strands were sequenced on ABI 377 and ABI 3100 DNA sequencers using a fluorescent dye-terminator kit (Applied Biosystems). Each isolate was sequenced in both laboratories, in most instances from the same passage material. Insertions or deletions were confirmed by repeated sequencing from the earliest available passage. Where differences in sequence between the two laboratories were observed, additional sequencing was performed and the consensus sequence was chosen for analysis. The nucleotide sequences were edited with Sequencher version 3.1.1 (Power Macintosh) (Gene Codes Corp.) or ABI Prism 3100 Genetic Analyser data collection software (version 1.1, Applied Biosystems).


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Table 2. Primers used in RT-PCR and nucleotide sequencing

 
Sequence analysis.
Nucleotide and deduced amino acid sequences constituting the predicted open reading frame (ORF) of the hMPV G genes were compiled using CLUSTALW, version 1.7, for Unix. Phylogenetic trees were computed by distance (not shown), maximum-likelihood (not shown) and maximum-parsimony criteria analysis with PAUP* version 4.0.d10 (Swofford, 1999). For the bootstrapping analysis, sequences were added randomly and one tree was held at each step (100 bootstrap replicates). Pairwise nucleotide and amino acid identities within and between hMPV groups were calculated using the ‘Multiple Comparison Programs' in the Wisconsin Package Version 10.2 (Genetics Computer Group). Amino acid alignments were used to calculate mean hydrophobicity profiles using the method of Kyte & Doolittle (1982).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
hMPV G gene nucleotide sequence analysis
Nucleotide sequences of the 25 hMPV strains and a previously published hMPV G gene (van den Hoogen et al., 2001) were compared. The G gene transcription initiation codon (ATG) was located four bases downstream of the gene start sequence (GGGACAAGT), which was conserved among all isolates and was identical to those described for the G gene of APV (Ling et al., 1992; Bäyon-Auboyer et al., 2000; Alvarez et al., 2003). A single potential ORF of variable length (654–711 nt) was identified. All other reading frames were blocked by numerous termination codons as previously described (van den Hoogen et al., 2002). With the exception of two hMPV isolates (hMPV80-1999 and hMPV83-1997), which possessed TAG as transcription termination codon, all G gene ORFs were terminated by a TAA codon.

Phylogenetic analysis of the G gene ORFs identified two major clusters, designated groups 1 and 2 (Fig. 1). Minor clusters that segregated within each major group were designated subgroups A and B, respectively. Bootstrapping analysis showed strong support for both groups and subgroups. Subgroup 1A ORFs varied in length from 660 (hMPV80-1999 and hMPV83-1997) to 654 nt (hMPV16-2000-16 and hMPV17-2000) as the result of early termination due to a single base substitution at position 654 (CAA->TAA). Subgroups 1B and 2A ORFs were all 711 nt in length. Subgroup 2B sequences were distinguished by a 15 nt in-frame insertion located in a polypurine stretch at nucleotide position 480 and a stop codon at nucleotide positions 694–696 which resulted in a 696 nt ORF.



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Fig. 1. Estimated maximum-parsimony (MP) tree showing the genetic clusters obtained in the analysis of the hMPV G sequences. Bootstrap proportions, obtained from a 50 % majority rule consensus tree, were plotted at the main internal branches of the phylogram to show the support values. For the bootstrap analysis under the MP assumption, sequences were added randomly and one tree was held at each step (100 replicates, 100 bootstrap replicates) applying the TBR branch-swapping algorithm. Trees were midpoint-rooted using MINF optimization. Trees were drawn to scale. NLD, The Netherlands. GenBank accession no. AF371337. Bar represents 5 nucleotide changes.

 
Genetic distances between the G gene ORFs of the two major hMPV groups were substantial; sequence identities ranged from 52 to 58 % at the nucleotide and 31 to 35 % at the amino acid level (Table 3). In contrast, sequence identities within the two groups ranged from 74 to 100 % at the nucleotide and 61 to 100 % at the amino acid levels for group 1 viruses, and 77 to 99 % at the nucleotide and 63 to 99 % at the amino acid level for group 2.


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Table 3. hMPV G gene and protein sequence identity (%)

 
Predicted hMPV G proteins
hMPV G group 1 ORFs predicted polypeptides of 217, 219 or 236 residues, with molecular masses ranging from 23·6 to 25·8 kDa (Fig. 2, upper panel). Group 2 polypeptides were 231 or 236 residues in length, with a deduced molecular mass ranging from 25·5 to 25·8 kDa (Fig. 2, lower panel). Sequences of isolates hMPV23-2001, 28, 29 and 31-2001 and hMPV193-2002 and 228 were identical and therefore only one sequence per cluster was included in the alignments. Whereas the predicted intracellular and transmembrane domains of hMPV groups 1 and 2 were conserved within each group (Table 3), the extracellular domains of both groups were highly variable. Cysteine residues were rare overall; one cysteine residue was located in the intracellular domain of most group 1 isolates and two cysteine residues were located in the intracellular and extracellular domains of all group 2 isolates.



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Fig. 2. Deduced amino acid alignments of hMPV G proteins of group 1 and group 2. Proposed intracellular, transmembrane and extracellular domains are indicated by the arrows above the alignment. Conserved amino acid residues along the alignment are indicated by shaded boxes. Potential O-glycosylation sites in an hMPV group and subgroup are indicated by {bullet} and {circ}, respectively. Potential N-glycosylation sites are underlined.

 
Despite a high degree of divergence in the predicted amino acid sequences between the two major groups, the structural features of the proteins were remarkably similar (Fig. 3). Hydropathy analysis showed patterns consistent with an anchored type II transmembrane protein as previously described (van den Hoogen et al., 2002): an amino-terminal hydrophilic cytoplasmic domain; a strongly hydrophobic region spanning amino acids 40 to 51; and a mostly hydrophilic extracellular carboxyl-terminal domain of variable length. Extracellular domain profiles were similar for all G protein sequences within each group, although it was necessary to remove the 5 amino acid insertion in hMPV82-1997 and hMPV33-2001 (subgroup 2B) to obtain overlapping profiles. The similarity in profiles of the extracellular domain between the two major groups suggests a conservation of amino acids with similar biochemical properties.



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Fig. 3. Hydropathy profiles for the hMPV groups 1 and 2 G proteins. The distribution of hydrophobic and hydrophilic regions along the predicted amino acid sequence of the G protein, as displayed above and below the central axes, respectively, was obtained using the Kyte and Doolittle algorithm. The hydropathy value of each amino acid was calculated over a window of 13 residues. The bottom scale indicates the amino acid residue beginning with the amino-terminal methionine.

 
The predicted hMPV G proteins had a relatively high content of serine and threonine residues, potential sites for O-glycosylation, and were rich in proline, a consistent feature of mucin-like glycoproteins (Fig. 2). The threonine/serine content of the predicted G proteins ranged from 28·6 to 34 %. Of interest, the ratio of threonine to serine residues was considerably higher for group 2 G proteins (2·4 : 1) than for group 1 (1·1 : 1). Most conserved O-glycosylation sites were located in the extracellular domain, where they were distributed without notable clustering. Of the 19 amino acids within the extracellular domain that were conserved between both groups, 12 (63 %) were potential O-glycosylation sites.

Potential N-glycosylation acceptor sites (Asn-X-Thr/Ser) were also identified among the hMPV G proteins. Among group 1 proteins, two potential N-glycosylation sites unique to subgroup 1A were identified at amino acid positions 101–103 and 153–155, and one site unique to subgroup 1B was identified at positions 233–235. Among group 2 proteins, one site unique to subgroup A was identified at positions 202–204 and four sites unique to subgroup 2B were identified at positions 101–103 (also present in subgroup 1A), 169–171, 188–190 and 215–217. One N-glycosylation site located at positions 181–183 in the extracellular domain was conserved among group 2 proteins, and one site was conserved among all G protein sequences; however, this site was located in the intracellular domain (positions 30–32).

Proline content also varied among the G protein groups and subgroups. Subgroups 1A and 2B had a similar proline content, ranging from 6·4 % to 7·8 %, whereas subgroups 2A and 1B had relatively lower (4·2 to 5·1 %) and higher (8·4 to 9·3 %) proline content, respectively. No N-myristylation sites were identified.

Molecular epidemiology
Cocirculation of hMPV groups and subgroups during a single epidemic season within the same community (Québec City) was observed as previously described (Boivin et al. 2002; Peret et al. 2002) (Table 1). Eight of 9 (89 %) hMPV field isolates obtained in years 1997 and 1998 were group 2 viruses and all isolates from the 1998 epidemic period were subgroup 2A. In contrast, 15 of 16 (94 %) isolates obtained after 1998 were group 1 viruses, with most isolates from the 2001 and 2002 epidemic periods subgroup 1B. There was no apparent association between the different virus groups/subgroups and age or clinical presentation of the patients, although too few isolates were examined to adequately address these issues.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence analysis of the G gene of multiple hMPV isolates obtained over five consecutive years from Québec City, Canada, revealed substantial genetic heterogeneity. Previous studies of the hMPV N, P, M and F genes identified two major genetic groups, with sequence identities between groups ranging from 78 to 86·1 % and 81·6 to 97·6 % at the nucleotide and amino acid levels, respectively (Bastien et al., 2003). In contrast, variability of the hMPV G genes was substantially greater, with identities ranging from 52 to 58 % at the nucleotide and 31 to 35 % at the amino acid levels. Moreover, the G gene of both major groups display well-defined genetic subgroups supported by high bootstrap values.

Sequence variation of the hMPV G gene occurred through nucleotide substitutions, use of alternative transcription termination codons, and insertions that retained the original reading frame. Similar types of change have been described for the G gene of other members of the subfamily Pneumovirinae; use of alternative transcription termination codons has been described for BRSV (Furze et al., 1997) and HRSV (Sullender et al., 1991; Peret et al., 1998; Martinez et al., 1999), and in-frame and frame-shift insertions have been identified with HRSV (Sullender et al., 1991; Peret et al., 1998). Like APV, HRSV and BRSV (Johnson et al., 1987; Juhasz et al., 1994; Bäyon-Auboyer et al., 2000; Valarcher et al., 2000), the nucleotide substitutions resulted in a higher rate of amino acid changes, especially in the ectodomain. This suggests amino acid changes are advantageous, possibly due to immunological pressure.

Comparisons of the hMPV G gene with APV, the virus most closely related to hMPV, showed no significant identity. In contrast, amino acid identities between hMPV and APV N, P, M and F genes ranged from 55 to 78 % for APV A; 51 to 77 % for APV B; and 66 to 88 % for APV C (Bastien et al., 2003).

Despite the genetic variability in the hMPV G protein, the overall structural features varied little. The overall serine/threonine content of the hMPV G protein (32 to 34 %), a predictor of degree of glycosylation, was similar to HRSV (29 to 31 %) (Johnson et al., 1987), and greater than that reported for APV (23·5 to 24·6 %) (Alvarez et al., 2003). Similar to HRSV groups A and B, the hydrophobicity plots of the predicted hMPV G proteins of both major groups were strikingly similar, despite limited amino acid identity.

Previous studies have identified conserved clusters of cysteine residues in the G glycoprotein of HRSV (reviewed by Collins et al. 2001), APV (Bäyon-Auboyer et al. 2000; Alvarez et al., 2003) and BRSV (Furze et al., 1997; Elvander et al., 1998; Valarcher et al. 2000), although BRSV G gene isolates lacking the central four cysteine residues have been described (Valarcher et al., 2000). Cystine nooses have been associated with protein conformation and biological signalling (Lapthorn et al., 1995). In addition, a CX3C motif has been identified in the HRSV G glycoprotein that mediates chemokine mimicry (Tripp et al., 2001) and an identical motif was recently identified in APV C (Alvarez et al., 2003). In contrast, there were no analogous cysteine clusters or other conserved amino acid motifs identified in the G ectodomain of hMPV. The lack of conserved sequences in the ectodomain of the G protein between the two major groups of hMPV may reflect differences in the functional properties of these proteins.

The extensive polymorphism of the hMPV G gene seen in this study raised concerns that sequence variability may have resulted from mutations occurring during virus propagation in cell culture. Although culture isolation and passage of hMPV was necessary in most cases to obtain sufficient viral RNA for sequencing, sequences of two virus strains from subgroup 1B (hMPV228-2002 and hMPV193-2002) were obtained directly from clinical specimens. These sequences were virtually identical to those of other hMPV strains from the same subgroup obtained after multiple passages in cell culture. Moreover, sequences from multiple passages of virus strain hMPV82-1997, which displayed a unique insertion sequence, were identical (data not shown). These observations suggest that isolation and limited passage of hMPV in cell culture does not contribute significantly to nucleotide changes in the G gene, which is consistent with findings for BRSV (Larsen et al., 1998) and HRSV (Cane et al., 1994).

In contrast to our findings, Biacchesi et al. (2003) recently reported nucleotide heterogeneity among cloned sequences of the SH and G genes of strain hMPV75-1998 (CAN98-75), a virus originally isolated from an immunocompromised child with acute lymphoplastic leukaemia (Peret et al., 2002; Boivin et al., 2002; Pelletier et al., 2002). One possible explanation is that a mixed virus population (or quasispecies), an intrinsic feature of RNA viruses (Elena et al., 2000), was present in the original clinical specimen or arose during in vitro propagation. Alternatively, nucleotide misincorporations during cloning and sequencing may have occurred. We obtained similar sequences (only two third-position mismatches) for the entire G gene from multiple independent RT-PCR amplifications of hMPV75-1998 and, unlike the sequence reported by Biacchesi et al. (2003), the hMPV75-1998 G gene sequences we obtained were nearly identical with sequences from independently isolated hMPV strains circulating during the same epidemic period (hMPV76-1998; hMPV77-1998; hMPV78-1998 and hMPV79-1998).

Community studies of HRSV circulation patterns have documented the temporal displacement of G gene variants of HRSV in successive years, attributing this to changes in the herd immunity of the population in response to antigenic differences between the predominant circulating strains (Cane & Pringle, 1995; Peret et al., 1998). Although our dataset is limited and geographically restricted, there was a clear indication of a shift between the two major groups of hMPV during the study period, which we speculate may be in response to immune-mediated pressure.

In conclusion, our study documents the extensive polymorphism of the hMPV G gene and confirms its basic features of a type II mucin-like glycoprotein. Long-term studies sampling wider geographical areas will be necessary to provide a more complete picture of the sequence diversity of hMPV. The importance of the G protein variation to the immunobiology of hMPV has yet to be determined and warrants further studies.


   ACKNOWLEDGEMENTS
 
The authors thank Brian Holloway and staff (CDC Biotechnology Core Facility, DNA Chemistry Section) for the oligonucleotide synthesis and the two anonymous referees for valuable suggestions.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 July 2003; accepted 13 November 2003.