Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
A. J. Easton
a.j.easton{at}warwick.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY743909 and AY743910.
Present address: Intervet UK Ltd, Walton Manor, Walton, Milton Keynes, Bucks MK7 7AJ, UK.
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INTRODUCTION |
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PVM was originally isolated from apparently healthy laboratory mice (Horsfall & Hahn, 1939). Passage of lung tissue from these mice into healthy recipients resulted in fatal pneumonia (Horsfall & Hahn, 1939
, 1940
). Passage of PVM in tissue culture results in a significant reduction in LD50 levels (Harter & Choppin, 1967
). Serological evidence has since revealed that PVM is prevalent among many species of laboratory rodents, in which it causes a latent or inapparent infection (Horsfall & Curnen, 1946
; Gannon & Carthew, 1980
). Several wild rodent species also test seropositive for PVM, and wood mice population studies in the UK in 1978 suggested that an epizootic outbreak of PVM coincided with a decline in the population (Kaplan et al., 1980
). However, the impact of PVM infection in wild animals is not known. Horsfall & Hahn (1939
, 1940)
demonstrated that a significant proportion of humans were seropositive for PVM or an antigenically related virus, but no increase in neutralizing antibodies was observed on convalescence. Pringle & Eglin (1986)
showed that up to 80 % of the UK population was seropositive for PVM and the age distribution of seroconversion in humans was similar to that for RSV, suggesting a very early exposure. In addition, seroconversion to PVM was observed in 3·7 % of patients with respiratory symptoms of unknown aetiology and PVM may therefore represent a proportion of the unidentified agents causing respiratory disease in humans (Pringle & Eglin, 1986
).
Sequence analyses of PVM reported to date have mostly been conducted on PVM strain 15. Strain 15 was one of the original isolates of PVM reported by
Horsfall & Hahn (1940), which, since its original description, has been grown extensively in tissue culture and is no longer capable of causing disease in mice, consistent with early observations (Harter & Choppin, 1967
; Cook et al., 1998
; Domachowske et al., 2002
). A second strain of PVM, J3666, has been described, which has been maintained by animal-to-animal passage and is fully pathogenic for mice (Cook et al., 1998
; Domachowske et al., 2000a
, b
, 2002
; Bonville et al., 2003
). Strains 15 and J3666 were both isolated at the Rockefeller Institute, New York, and are believed to be descended from the same original virus isolate. Immediate responses to PVM strain J3666 infection of mice include pulmonary eosinophilia and production of the chemokine macrophage inflammatory protein 1
(Domachowske et al., 2000a
). Mortality occurs as early as 56 days post-infection and viral clearance is observed by day 10 (Cook et al., 1998
). The fast progression of infection suggests that a neutralizing-antibody response is unlikely to be involved in viral clearance. This is supported by the study of PVM infection in athymic mice: virus persisted in the alveolar wall of the mouse lung for the 20 day duration of the experiment, despite the presence of humoral antibody from day 11 (Carthew & Sparrow, 1980
). Recently, the comparative replicative abilities of PVM strains 15 and J3666 have been studied in the respiratory tract of C57BL/6 mice (Domachowske et al., 2002
). It was shown that both strains of PVM replicated to similar titres by 5 days post-infection, indicating a similar replicative ability in vivo.
The nucleotide sequences for nine of the PVM strain 15 genes and two of the PVM strain J3666 genes have been published (Barr et al., 1991, 1994
; Chambers et al., 1991
, 1992
; Randhawa et al., 1995
; Easton & Chambers, 1997
; Ahmadian et al., 1999
) and the sequences of the ORFs of six additional genes of PVM strain J3666 have been deposited in GenBank (accession numbers AY573811AY573816). The source of RNA, whether genomic or mRNA, for these latter sequences is not clear. The sequences of the 3'-leader region, the M2L intergenic region, the large (L) polymerase gene and the 5'-trailer region have yet to be described for either strain of PVM. Here, we report the completion of the genome sequence of PVM strain 15 and the determination of the complete genome sequence of strain J3666. Genetic differences between these viruses were determined, enabling potential genetic determinants of PVM pathogenicity to be identified.
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METHODS |
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Amplification and sequencing of the PVM leader region.
Poly(A) polymerase was used to add a homopolymer tail to the 3' end of PVM genomic RNA, and the tailed template was reverse-transcribed with Moloney murine leukaemia virus reverse transcriptase (Promega) using the primer oli-T (5'-GGCCCGGGAAGCTTTTTTTTTTTTTTT-3'). PCR amplification comprised 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s using Pwo DNA polymerase (Roche Diagnostics) with primer oli-T and a PVM NS1 gene-specific primer, NS1B (5'-CTTGCCCTGTAGAACTAAACACG-3'). A semi-nested PCR was then performed using the initial PCR product as template with the primers oli-T and NS1A (5'-AATTGCAATCCCTTCCCACAAGG-3'), a second PVM NS1 gene-specific primer that anneals to the gene upstream of NS1B. The resulting PCR product of approximately 280 bp was purified and sequenced directly by using primer NS1A.
Amplification and sequencing of the PVM L gene.
A PVM M2 gene-specific primer, 22K4 (5'-GCCAGGCCAGATGATGTGG-3'), was used to reverse-transcribe genomic RNA and the cDNA was amplified with Pwo DNA polymerase (30 cycles of 94 °C for 30 s, 45 °C for 30 s, 72 °C for 30 s) by using the degenerate primers PL7 (5'-TGGATAAACACNATACTNGATGA) and PL8 (5'-GCTTGAGGRTCTCTCAT-3'), which were designed to anneal within a region of the L gene that was previously shown to be highly conserved among pneumoviruses (Randhawa et al., 1996) and which were expected to amplify a short central section of the PVM L gene. The resulting PCR product of approximately 500 bp was cloned and sequenced. Based on the novel sequence data, two PVM L gene-specific primers, the antisense PL20 (5'-TGCATCGGGACGGTAAGAAACAGTTC-3') and the sense PL21 (5'-GAACTGTTTCTTACCGTCCCGATGCA-3'), were designed. The 5' half of the PVM L gene was amplified with Elongase (Gibco-BRL Life Technologies) (30 cycles of 94 °C for 30 s, 55 °C for 30 s, 68 °C for 4 min) using primers 22K4 and PL20. Amplification of the 3' half of the PVM L gene (five cycles of 94 °C for 30 s, 30 °C for 30 s, 68 °C for 4 min, followed by 25 cycles at the raised annealing temperature of 55 °C) was achieved with primer PL21 and the degenerate primer AL4 (5'-GGGCTCGAGGATCCACGAGAAAAAAANN-3'), which contained the conserved 5'-terminal 12 nt of the RSV/APV trailer region (see Fig. 1c
). The two PVM L gene PCR products were cloned and sequenced. In order to obtain a consensus sequence, the PVM L gene was further amplified as six overlapping sections, which were sequenced directly by using a panel of L gene-specific primers.
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Amplification and sequencing of the PVM strain J3666 genome.
The leader and trailer regions of PVM strain J3666 were amplified and sequenced as described above. The remainder of the genome was amplified as ten overlapping fragments. Briefly, genomic RNA was either reverse-transcribed from within the leader region or the F gene, and the viral cDNAs were amplified with appropriate primer combinations. The ten fragments were sequenced directly with a panel of PVM sequence-specific primers. Alignments to compare the genome sequences of PVM strains 15 and J3666 were performed by using the MEGALIGN program (DNASTAR) with an alignment derived from the program CLUSTAL (Higgins & Sharp, 1988). All nucleotide differences between the two genomes were verified by sequencing additional RT-PCR-generated genomic fragments. Where appropriate, the sequence of the PVM strain 15 genome was also confirmed.
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RESULTS AND DISCUSSION |
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The PVM trailer region, in the negative genome sense, consisted of a short, 91 nt, A-rich sequence (Fig. 1b). The amplification strategy involved the addition of a G tail, which prevented the unambiguous identification of the 5'-terminal nucleotide of the PVM genome, but it is postulated to be an A residue to retain complementarity with the 3'-terminal U residue. In contrast to the pneumovirus leader regions, which were relatively conserved in length, the trailer regions were markedly variable. The PVM strain 15 trailer region was 91 nt and was thus intermediate in size compared with those of RSV strain A2 (155 nt) and APV strain CVL14/1 (40 nt) (Mink et al., 1991
; Randhawa et al., 1997
). The trailer regions of the Paramyxovirinae are also quite variable in size, with tupaia virus displaying an extremely long trailer region of 590 nt (Wang et al., 2000
). An alignment of the trailer regions of PVM, RSV and APV is shown in Fig. 1(c)
. The 5'-terminal 12 bases were conserved with the exception of position 4 and, whilst the degree of conservation among the pneumovirus trailer regions was high, with a mean nucleotide identity of 54 % over the 5'-terminal 40 nt, little conservation was observed elsewhere. In RSV, the trailer regions are also conserved poorly among subgroup A strains (Tolley et al., 1996
).
The PVM L gene was 6332 nt and was particularly rich in A and U residues (63 % of the L gene sequence). The gene-start and -end sequences were confirmed by rapid amplification of cDNA ends (RACE) (data not shown) and these conformed to the pattern described by Chambers et al. (1990b) for the other nine genes. The first translation initiation codon in the PVM L gene was located at nt 1012 in the mRNA and was in a favourable context for the initiation of eukaryotic translation (Kozak, 1986
), having a G residue at the +4 position and an A residue at 3. This ORF terminated at the stop codon located at nt 61306132 and was followed by a 3' non-translated region of 199 nt. The predicted PVM L protein was 2040 aa, which is comparable to the L proteins of RSV strain A2 (2165 aa) and APV strain CVL14/1 (2004 aa) (Stec et al., 1991
; Randhawa et al., 1996
). The degree of conservation among the pneumovirus L proteins was high, with a mean amino acid identity of 45 %. Alignment of the L proteins of PVM strain 15, RSV strain RSS-2 and APV strain CVL14/1 (not shown) revealed that the RSV L protein had an additional sequence at the N terminus when compared with the polymerase proteins of both PVM and APV. In addition, most of the six conserved domains that are proposed to constitute functional motifs in the non-segmented negative-strand RNA virus polymerases (Poch et al., 1990
) were identified in the PVM L protein, as shown in Fig. 2
. In particular, the invariant GHP tripeptide in domain I, the putative RNA-binding KERE motif in domain II, motifs AD of the core polymerase module in domain III and the putative GEGAG ATP-binding motif in domain VI, all of which are conserved in other pneumovirus L proteins (Stec et al., 1991
; Randhawa et al., 1996
), were present in the PVM L protein.
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The data presented above confirmed the genomic organization of PVM as 3'-LeNS1NS2NPMSHGFM2LTr-5', which is the same as for RSV (Collins et al., 1996). The PVM strain 15 genome sequence was 14 887 nt and, in common with other pneumoviruses, the genome of PVM does not obey the rule of six.
Determination of the PVM strain J3666 genome sequence
The only sequence data available for PVM pathogenic strain J3666 prior to this study were for the G and F genes, both of which were derived from cDNA clones (Randhawa et al., 1995). By using the data obtained above and the previously published sequences for the PVM strain 15 genes, primers were designed to establish the complete nucleotide sequence of J3666. The sequence was determined from multiple, independent RT-PCRs that were performed directly on genomic RNA. The pathogenicity of the stock of PVM strain J3666 used in this study was confirmed prior to sequencing. As anticipated for an RNA virus, direct sequencing of genomic RNA generated sequences that contained some alternative bases at specific locations. These alternatives reflect the natural sequence variation that is seen in quasispecies populations of RNA viruses. The variable sequences were confirmed in a number of repeated sequencing reactions. Whilst it is possible to plaque-purify viruses, amplification of stocks produces a similar sequence distribution. An additional factor for PVM is the known attenuation by passage in tissue culture. For these reasons, the sequences presented here contain the spectrum of variation observed in the virus population. In the discussion below, reference is made to the sequences deposited in GenBank by K. D. Dyer and H. F. Rosenberg, where they suggest that a sequence difference between the two strains identified in this study may reflect variation in the virus population.
The data showed that the strain J3666 genome was 14 885 nt, 2 nt shorter than that of strain 15. Single nucleotide deletions in both the leader region and the G gene were responsible for the length difference. In common with the genome of PVM strain 15, that of strain J3666 does not obey the rule of six.
As shown in Table 1, a comparison of the genomes of PVM strains 15 and J3666 revealed 59 nucleotide differences, 37 of which were coding. In establishing the sequence differences between the two strains, the PVM strain 15 genome was also sequenced. This showed ten nucleotide differences compared with the published sequences. In all cases, the corrected sequences were identical to those seen in strain J3666. The complete sequences of both strains of PVM have been deposited in GenBank. Table 1
shows all the differences between strains 15 and J3666, including those identified previously by others. Four sequence differences identified in this work, in the N gene (aa 149), P gene ORF2 (aa 62) and SH gene (aa 8 and 87), and which are not seen in the strain J3666 sequences deposited in GenBank by K. D. Dyer and H. F. Rosenberg, are indicated. Sequence differences between the G and F protein genes of PVM strains 15 and J3666 have been reported (Randhawa et al., 1995
) and were confirmed by the sequence analysis repeated in this study. Only those identified in this analysis are discussed below. The nucleotide differences between the two strains were distributed throughout the genome and the only genes that were entirely conserved between the two strains of PVM were those encoding the NS1, NS2 and M2 proteins. The non-structural NS1 and NS2 proteins of PVM act co-operatively to counteract the antiviral interferon response (Bossert et al., 2001
), as described for bovine RSV (Schlender et al., 2000
; Bossert et al., 2003
), and the two proteins encoded by the M2 gene have been shown for RSV to be intimately associated with the control of virus replication and transcription (Bermingham & Collins, 1999
; Fearns & Collins, 1999
).
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The N genes of the two strains of PVM differed by 1 nt. This changed arginine 149 to glutamine, but despite the high level (60 %) of amino acid identity between the N proteins of PVM and RSV, this position was not conserved (Barr et al., 1991). The N gene sequence for strain J3666 that was deposited by K. D. Dyer and H. F. Rosenberg (GenBank accession no. AY573813) does not show a difference between the two strains at this position, suggesting that the difference may be due to sequence variability in the virus population. A much greater degree of variation was observed among the P genes, with nine nucleotide differences between the two strains (Table 1
). The P gene of PVM directs the synthesis of multiple proteins from two overlapping reading frames (Barr et al., 1994
). The major ORF1 encodes a polypeptide of 295 aa and also directs the synthesis of several carboxyl co-terminal products by internal initiation, and the minor ORF2 encodes a polypeptide of 137 aa. The presence of a second ORF within the P gene is unique among the Pneumovirinae and is more reminiscent of Paramyxovirinae P gene expression.
Alignment of the P proteins of PVM and RSV has revealed a region of the PVM P ORF1 protein spanning aa 18180 that is poorly conserved (Barr et al., 1994). Within this region, there is also little conservation between the P proteins of RSV and bovine RSV. The apparent lack of need for strict conservation of amino acids in this region was proposed to have allowed the creation of the PVM P gene second ORF (Barr et al., 1994
). Nine nucleotide differences were found between the two strains of PVM, all of which resulted in coding changes in the ORF1 and/or ORF2 proteins. All of these nucleotides were located within this region of poor conservation. One of these differences, at aa 62 of ORF2, was not observed by K. D. Dyer and H. F. Rosenberg (GenBank accession no. AY573814). The function of the PVM P ORF2 protein is unknown; however, the C protein of Sendai virus, also expressed from an alternative reading frame in the P gene mRNA, has been implicated in counteraction of the antiviral interferon response (Garcin et al., 1999
). It is possible that the PVM P ORF2 protein also plays a role in the pathogenicity of the virus, although no similarity with the Sendai virus C protein was detected. Specifically, the strain J3666 P ORF2 protein, which displayed only 96 % amino acid identity to that of strain 15, may be adapted to act as a viral virulence factor in the mouse lung.
The SH gene is the smallest in the PVM genome (Easton & Chambers, 1997), but 21 of the nucleotide differences observed between strains J3666 and 15 were located within this gene, making it the most divergent. A gene encoding a small hydrophobic (SH) protein has been found in the pneumoviruses and certain members of the genera Rubulavirus and Avulavirus of the subfamily Paramyxovirinae (Chang et al., 2001
). There is little conservation in terms of size or sequence among the SH proteins, but they all possess a hydrophobic domain of approximately 30 aa at the N terminus, with most variation occurring in the C-terminal region (Chang et al., 2001
). The RSV SH protein has been shown to be a type II integral membrane protein (Collins & Mottet, 1993
) that accumulates in lipid rafts in infected cell membranes (Rixon et al., 2004
), but its function is unknown. However, several lines of evidence suggest that it is a virulence factor that is non-essential for virus replication. A recombinant RSV lacking the SH gene replicated normally in cell culture, but was attenuated in the mouse and chimpanzee (Bukreyev et al., 1997
; Whitehead et al., 1999
). Also, the RSV G and/or SH proteins have been shown to inhibit chemokine expression in the mouse model, impairing the Th1 T-cell response, which is vital for virus clearance (Tripp et al., 2000
). By analogy, the PVM SH protein may also play a role in the pathogenicity of the virus. Given that the SH proteins of RSV are reasonably well-conserved within a subgroup (Chen et al., 2000
), the lack of sequence conservation between the SH proteins of the two strains of PVM is striking. Two of the strain differences, at aa 8 and 87, were not seen in the sequence reported by K. D. Dyer and H. F. Rosenberg, although their sequence showed two additional changes and the protein was 92 aa in size, compared with the 94 aa seen for the SH protein of strain 15. These data also emphasize the variability and potential plasticity of the SH protein.
Analysis of the sequencing data showed eight positions within the PVM strain J3666 SH gene that were variable in the virus population. In each instance, one of the two possible nucleotides that were present at each position was also present in the strain 15 sequence. As such sequence variation is common in all RNA virus populations and it is not known whether one or all versions of the SH gene are expressed during infection, for the purpose of this discussion, all possibilities are considered. The contribution of each of these potential differences in sequence to the pathogenicity of the virus is unclear. However, it is likely that more than one sequence is associated with, but may not contribute to, the pathogenic phenotype. Based on the sequence data, if all eight positions where there is dual usage of bases are utilized equally, there is potential for 256 possible sequence variations, but it has been shown that as few as 100 infectious units will result in severe disease, with 1020 % mortality on average (Cook et al., 1998), indicating that more than one genomic sequence must be capable of generating disease. The nucleotide sequence and coding potential of the PVM strain J3666 SH gene are shown in Fig. 3
. All versions of the PVM strain J3666 SH gene contained coding changes when compared with the strain 15 sequence, and these differences affected seven amino acid residues (aa 8, 24, 25, 27, 29, 32 and 54). Of particular note was a cluster of five amino acid differences that was located within a short stretch of the putative transmembrane domain. However, hydrophilicity profiles (Kyte & Doolittle, 1982
) did not reveal any significant differences in the overall hydropathy of the two SH proteins (data not shown). In addition, as a result of alternative nucleotide usage in the strain J3666 SH gene, aa 12 and 87 exhibited amino acid variability. Five of the nucleotide differences that were identified between the two strains of PVM were located in the 3' non-translated region of the prototype strain 15 SH gene and, in several independently generated sequences, the translation termination codon for the SH gene of strain J3666 was altered to allow the expression of proteins of 92, 96 or 114 aa. Studies with RSV have detected similar C-terminal heterogeneity within the G protein in populations of G antibody-escape mutant viruses (Garcia-Barreno et al., 1990
).
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In addition to the coding changes identified above, several non-coding differences in the M, SH and L genes were identified between the two PVM strains. Most of these differences were located within coding regions, but one non-coding difference in the SH gene and two in the L gene were found in the non-translated region of the mRNA. The potential importance of these differences in affecting pathogenicity is not clear.
The data presented here describe the comparison of the genome sequences of a pathogenic and a non-pathogenic strain of a second member of the Pneumovirinae and the closest known relative of RSV. As shown here for PVM, attenuated versions of RSV (cp-RSV and RSS-2 ts1C) have also been shown to possess mutations in a wide range of genes and in cis-acting regulatory regions (Connors et al., 1995; Crowe et al., 1996
; Tolley et al., 1996
). Also, a vaccine strain of the paramyxovirus rinderpest virus was shown to have 87 nucleotide changes relative to wild-type virus, with differences in the leader and trailer regions and coding mutations in five of the six genes (Baron et al., 1996
). Whilst most PVM genes showed only a limited number of differences between the two strains, several regions in the PVM genome were particularly variable, implying that they are quite tolerant to change. These included a section of the P gene encoding ORF2, the SH gene and the 5' region of the G gene.
Due to the similar replicative abilities of both strains of PVM in the respiratory tract of C57BL/6 mice (Domachowske et al., 2002), the non-pathogenic phenotype of strain 15 cannot be due to a reduced ability of this virus to grow in vivo. Although both strains of PVM grow to similar titres in C57BL/6 mice, only infection with PVM strain J3666 results in pulmonary eosinophilia and expression of proinflammatory cytokine genes (Domachowske et al., 2002
). Therefore, PVM replication alone is insufficient to stimulate the inflammatory antiviral response, and one or more of the six proteins that are divergent between the two strains is likely to be associated with the differential response to infection, with the most likely candidates being the SH, G and F proteins. The presence of a mutation in the L protein at the same position in pathogenic and non-pathogenic isolates of RSV and PVM is striking and may indicate a similarity between the two systems. Recent studies have demonstrated that attenuating mutations found in diverse murine and human paramyxoviruses may be imported into other paramyxoviruses for rapid attenuation (Newman et al., 2004
). PVM is the closest known relative of RSV and causes a similar disease in its natural host. A better understanding of PVM pathogenicity may therefore aid the rational design of live-attenuated RSV vaccine candidates and even those of more diverse paramyxoviruses. Research into the differential inflammatory responses caused by infection with PVM strains 15 and J3666 is beginning to unravel the intricacies of virus-specific pathogenicity (Domachowske et al., 2002
; Moreau et al., 2003
) and the development of a reverse-genetics system for PVM should allow further elucidation of the functional importance of the genetic differences between the two strains identified here.
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ACKNOWLEDGEMENTS |
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Received 25 May 2004;
accepted 6 September 2004.
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