Journal of General Virology (2005), 86, 159–169
DOI 10.1099/vir.0.80315-0
Genome sequence of the non-pathogenic strain
15 of pneumonia virus of mice and comparison
with the genome of the pathogenic strain J3666
L. C. Thorpe3 and A. J. Easton
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
A. J. Easton
a.j.easton@warwick.ac.uk
Received 25 May 2004
Accepted 6 September 2004
Pneumonia virus of mice (PVM) is a member of the subfamily Pneumovirinae and is the closest
known relative of respiratory syncytial virus. Both viruses cause pneumonia in their respective
hosts. Here, the genome sequences of two strains of PVM, non-pathogenic strain 15 and
pathogenic strain J3666, are reported. Comparison of the genome sequences revealed
59 nucleotide differences between the two strains, 37 of which were coding. The nucleotide
differences were spread throughout the genome, affecting cis-acting regulatory regions and seven
of the ten genes. 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.
INTRODUCTION
Pneumonia virus of mice (PVM) is a member of the genus
Pneumovirus of the subfamily Pneumovirinae within the
Paramyxoviridae family of viruses (van Regenmortel et al.,
2000). The classification of PVM is based on virion
morphology, sizes of the viral proteins, the number of
mRNA species and the presence of the unique M2 gene
(Berthiaume et al., 1974; Cash et al., 1977; Chambers et al.,
1990a; Ahmadian et al., 1999). The genomic organization
of PVM is similar to that of respiratory syncytial virus
(RSV) and several PVM genes have been sequenced, with
each showing the greatest similarity to those of other
pneumoviruses such as RSV and avian pneumovirus
(APV) (Chambers et al., 1990a, b, 1991, 1992; Barr et al.,
1991, 1994; Randhawa et al., 1995; Easton & Chambers,
1997; Ahmadian et al., 1999).
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
3Present address: Intervet UK Ltd, Walton Manor, Walton, Milton
Keynes, Bucks MK7 7AJ, UK.
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are AY743909 and AY743910.
0008-0315 G 2005 SGM
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 1a (Domachowske et al., 2000a).
Mortality occurs as early as 5–6 days post-infection and
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159
L. C. Thorpe and A. J. Easton
viral clearance is observed by day 10 (Cook et al., 1998). The
fast progression of infection suggests that a neutralizingantibody 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 AY573811–AY573816). The
source of RNA, whether genomic or mRNA, for these latter
sequences is not clear. The sequences of the 39-leader region,
the M2–L intergenic region, the large (L) polymerase gene
and the 59-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.
METHODS
Preparation of viral RNA. Stocks of PVM strains 15 and J3666
were propagated in BS-C-1 monkey kidney cells at 33 uC. BS-C-1
cells were infected with PVM at a low m.o.i. and incubated at 33 uC
until extensive cytopathic effect was seen. Cells were harvested into
the medium and pelleted by low-speed centrifugation. The cell pellet
was resuspended in water and virus was precipitated with polyethylene glycol from the cleared supernatant as described by Randhawa
et al. (1996). Precipitated virus was pelleted by centrifugation at
6000 g for 30 min and the virus pellet was resuspended in water.
RNA was extracted from the cell pellet (total infected cellular RNA)
or from the virus pellet (genomic RNA) by using TRIzol LS (GibcoBRL Life Technologies).
Amplification and sequencing of the PVM leader region.
Poly(A) polymerase was used to add a homopolymer tail to the 39
end of PVM genomic RNA, and the tailed template was reversetranscribed with Moloney murine leukaemia virus reverse transcriptase
(Promega) using the primer oli-T (59-GGCCCGGGAAGCTTTTTTTTTTTTTTT-39). PCR amplification comprised 30 cycles of denaturation at 94 uC for 30 s, annealing at 55 uC for 30 s and extension at
72 uC for 30 s using Pwo DNA polymerase (Roche Diagnostics) with
primer oli-T and a PVM NS1 gene-specific primer, NS1B (59-CTTGCCCTGTAGAACTAAACACG-39). A semi-nested PCR was then
performed using the initial PCR product as template with the
primers oli-T and NS1A (59-AATTGCAATCCCTTCCCACAAGG-39),
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.
160
Amplification and sequencing of the PVM L gene. A PVM
M2 gene-specific primer, 22K4 (59-GCCAGGCCAGATGATGTGG39), was used to reverse-transcribe genomic RNA and the cDNA was
amplified with Pwo DNA polymerase (30 cycles of 94 uC for 30 s,
45 uC for 30 s, 72 uC for 30 s) by using the degenerate primers PL7
(59-TGGATAAACACNATACTNGATGA) and PL8 (59-GCTTGAGGRTCTCTCAT-39), 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 genespecific primers, the antisense PL20 (59-TGCATCGGGACGGTAAGAAACAGTTC-39) and the sense PL21 (59-GAACTGTTTCTTACCGTCCCGATGCA-39), were designed. The 59 half of the PVM L
gene was amplified with Elongase (Gibco-BRL Life Technologies)
(30 cycles of 94 uC for 30 s, 55 uC for 30 s, 68 uC for 4 min) using
primers 22K4 and PL20. Amplification of the 39 half of the PVM L
gene (five cycles of 94 uC for 30 s, 30 uC for 30 s, 68 uC for 4 min,
followed by 25 cycles at the raised annealing temperature of 55 uC)
was achieved with primer PL21 and the degenerate primer AL4 (59GGGCTCGAGGATCCACGAGAAAAAAANN-39), which contained
the conserved 59-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.
Amplification and sequencing of the PVM trailer region.
PVM genomic RNA was reverse-transcribed by using the L genespecific primer PL43 (59-GCTAACATGTCATCCCCACTAC-39). A
G homopolymer tail was added to the 39 end of the viral cDNA by
using terminal deoxynucleotidyl transferase and the tailed template
was amplified (30 cycles of 94 uC for 30 s, 50 uC for 30 s, 72 uC
for 30 s) with Pwo DNA polymerase using the primers PL43
and oli-C (59-AGATCTAGAAGCTTGGATCCCCCCCCCCCC-39). A
semi-nested PCR was then performed using primers oli-C and PL48
(59-CTAGACATGTGAGAAGGTCCCA-39); PL48 is a PVM L genespecific primer that anneals downstream of PL43. The resulting PCR
product of approximately 260 bp was purified and sequenced
directly by using the primer PL48.
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.
RESULTS AND DISCUSSION
Completion of the PVM strain 15 genome
sequence
A similar strategy to that used to determine the leader and
trailer region sequences of RSV and APV was employed for
PVM (Mink et al., 1991; Randhawa et al., 1997). The PVM
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Journal of General Virology 86
Pathogenic and non-pathogenic PVM strain sequences
Fig. 1. PVM strain 15 leader and trailer region sequences. All sequences are shown in the negative genome sense (39R59).
Alignments were generated by using the MEGALIGN program with an alignment derived from the program CLUSTAL.
(a) Alignment of the PVM strain 15 leader region with the leader regions of RSV strain A2 and APV strain CVL14/1 (Mink
et al., 1991; Randhawa et al., 1997). Conserved residues are shaded. (b) Sequence of the PVM strain 15 trailer region.
(c) Alignment of the PVM strain 15 trailer region with the trailer regions of RSV strain A2 and APV strain CVL14/1 (Mink et al.,
1991; Randhawa et al., 1997). Conserved residues are shaded. Due to the large variation in length of the pneumovirus trailer
regions, only the 59-terminal 40 nt were aligned.
leader region was shown to consist of a short, 43 nt, U-rich
sequence (negative genome sense) and is shown aligned with
other pneumovirus leader regions in Fig. 1(a). Although the
amplification strategy resulted in the addition of a stretch
of A residues to the 39 end of PVM genomic RNA, thus
preventing the 39-terminal nucleotide from being identified
unambiguously, the 39-terminal residue of the PVM leader
region is believed to be a U, in common with all members
of the Paramyxoviridae for which the leader region terminal
residue has been determined (Wang et al., 2000). The degree
of conservation between the pneumovirus leader regions
was high, with an overall nucleotide identity of 67 %. The
39-terminal 11 bases were conserved with the exception of
position 4 but, thereafter, little conservation was seen apart
from a short U tract at the 59 end.
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
59-terminal nucleotide of the PVM genome, but it is postulated to be an A residue to retain complementarity with
the 39-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 59-terminal 12 bases were conserved
with the exception of position 4 and, whilst the degree of
conservation among the pneumovirus trailer regions was
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high, with a mean nucleotide identity of 54 % over the 59terminal 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 10–12 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 23. This ORF
terminated at the stop codon located at nt 6130–6132 and
was followed by a 39 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 A–D of the core polymerase
module in domain III and the putative GEGAG ATPbinding motif in domain VI, all of which are conserved in
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L. C. Thorpe and A. J. Easton
Fig. 2. Predicted amino acid sequence of
the PVM strain 15 L protein. Conserved
motifs located in domains I, II, III and VI are
indicated and asterisks show the four amino
acid residues that are almost invariant
among the RNA-dependent polymerases
(Poch et al., 1989, 1990).
other pneumovirus L proteins (Stec et al., 1991; Randhawa
et al., 1996), were present in the PVM L protein.
et al., 1996). This indicates that overlapping mRNAs are
not essential for pneumovirus L gene expression.
The PVM strain 15 genome was shown to possess a 9 nt
M2–L intergenic region. In contrast, the RSV M2 gene-end
signal is 68 nt downstream of the start of the L gene and
thus there is no M2–L gene junction (Collins et al., 1996).
In this regard, PVM is similar to APV, which contains an
intergenic region between the G and L genes (Randhawa
The data presented above confirmed the genomic organization of PVM as 39-Le–NS1–NS2–N–P–M–SH–G–F–M2–L–
Tr-59, 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.
162
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Journal of General Virology 86
Pathogenic and non-pathogenic PVM strain sequences
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
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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).
All intergenic regions and gene-start and -end signals were
completely conserved between the two strains of PVM.
However, differences were identified in both the leader
and trailer regions. The genomic termini of members of
the order Mononegavirales are believed to contain the cisacting signals that are essential for encapsidation, transcription, replication and assembly of the virus. As strains 15
and J3666 of PVM are believed to be related, varying only
in their ability to cause disease, it was striking that differences occurred in these crucial control regions. Recent
studies with recombinant viruses have identified mutations
within the genomic termini as important determinants of
pathogenicity in Sendai virus (Fujii et al., 2002).
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 18–180
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.
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L. C. Thorpe and A. J. Easton
Table 1. Nucleotide differences between PVM strains 15 and J3666
Differences between the genome sequences of prototype PVM strain 15 and strain J3666 are shown in the positive mRNA sense. The
nucleotide change and amino acid substitution, where appropriate, are indicated. Sequence differences noted in this work, but not present
in the sequences deposited by K. D. Dyer and H. F. Rosenberg, for the N gene (aa 149), P gene ORF2 (aa 62) and SH gene (aa 8 and 87)
are indicated with an asterisk. Nucleotide differences between the G and F genes have been reported previously (Randhawa et al., 1995).
Sequence
Leader
N gene
P gene
Nucleotide length
(strain 15)
Nucleotide
position
Nucleotide
change
Coding/
non-coding
43
1219
907
40
477
211
220
303
A deletion
GRA
GRA
GRA
GRA
Non-coding
Coding
Coding
Coding
Coding
311
GRA
Coding
313
ARG
Coding
314
ARA/G
Coding
414
449
GRA
GRA
Coding
Coding
462
700
32
43
44
46
70
80
82
84
90
96
105
171
175
269
283
287
292
293
299
353
367
25
30
51
62
65
68
78
97
102
123
GRA
GRU
CRU
CRU
CRC/U
CRU
URU/C
CRU
CRU
CRU
CRU
CRU
CRU
CRU
CRU
URU/C
URU/C
URU/C
URU/C
URU/C
URU/C
CRU
CRU
CRU
CRU
CRU
CRU
ARU
CRU
CRU
ARU
ARU
CRU
Coding
Non-coding
Coding
Non-coding
Coding
Non-coding
Non-coding
Coding
Coding
Coding
Coding
Coding
Coding
Coding
Non-coding
Coding
Non-coding
Coding
Coding
Coding
Coding
Coding
Non-coding
Non-coding
Non-coding
Non-coding
Non-coding
Non-coding
Non-coding
Non-coding
Coding
Coding
Coding
M gene
SH gene
931
396
G gene
1334
164
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Amino acid
change
–
149ArgRGln*
ORF1 68GluRLys
ORF1 71AlaRThr
ORF1 98MetRIle
ORF2 58CysRTyr
ORF1 101CysRTyr
ORF2 61ValRMet
ORF1 102LysRGlu
ORF2 61ValRMet
ORF1 102LysRGly
ORF2 62ArgRGly*
ORF2 95ArgRLys
ORF1 147ArgRLys
ORF2 107GlyRArg
ORF2 111ArgRLys
–
8HisRTyr*
–
12LeuRPhe
–
–
24HisRTyr
24HisRTyr
25ThrRIle
27ProRLeu
29ProRLeu
32ProRLeu
54ThrRIle
–
87TyrRHis*
–
93StopRGln
Non-codingRHis
Non-codingRTyr or His
Non-codingRStop or Gln
Non-codingRStop
–
–
–
–
–
–
–
–
Non-codingRPhe
Non-codingRVal
Non-codingRLeu
Journal of General Virology 86
Pathogenic and non-pathogenic PVM strain sequences
Table 1. cont.
Sequence
Nucleotide length
(strain 15)
F gene
1662
L gene
6332
Trailer
91
Nucleotide
position
Nucleotide
change
Coding/
non-coding
Amino acid
change
165
170
954
1122
737
966
992
318
1617
4591
5021
5159
5800
6156
6209
71
URG
A deletion
GRC
ARU
ARG
GRU
CRU
GRA
CRU
CRC/U
ARU
GRA
CRA
URU/C
GRA
CRU
Coding
Coding
Coding
Coding
Coding
Non-coding
Coding
Non-coding
Non-coding
Non-coding
Coding
Coding
Coding
Non-coding
Non-coding
Non-coding
Non-codingRGly
Non-codingRORF extension
258GluRGln
314ThrRSer
243LysRArg
–
328AlaRVal
–
–
–
1671GlnRLeu
1717ArgRGln
1931LeuRIle
–
–
–
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.
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.
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 Cterminal 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
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 10–20 % 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
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L. C. Thorpe and A. J. Easton
Fig. 3. Nucleotide sequence of the PVM
strain J3666 SH gene and predicted amino
acid sequence of the encoded protein. The
nucleotide sequence is shown in positive
mRNA sense and the sequence of the predicted protein uses the single-letter amino
acid code. Numbers on the left refer to the
nucleotide sequence and those on the right
to the amino acid sequence. The 21 nucleotide differences observed between the two
strains of PVM are shaded, as are coding
changes.
Where
nucleotide
variability
occurred, the nucleotide and amino acid
present in the strain 15 sequence are
shown on top. The putative hydrophobic
transmembrane domain is shown in bold and
potential N-linked glycosylation sites are
underlined (Easton & Chambers, 1997).
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 39 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).
The L genes of the two strains of PVM differed by a total
of eight nucleotide substitutions, three of which resulted
in coding changes. This represented a very low level of
differences, given that the L gene constitutes 43 % of the
PVM genome. It is possible that the polymerase gene is not
particularly susceptible to spontaneous mutation or, more
likely, that most mutations are deleterious or lethal. The
three amino acid differences that were identified between
the L proteins of PVM strains 15 and J3666 were located
in the C-terminal 20 % of the protein, at aa 1671, 1717 and
1931. Alignment of the L proteins of PVM strain 15, RSV
strain RSS-2 and APV strain CVL14/1 (not shown) revealed
that aa 1671 of the PVM L protein is a conserved glutamine residue. Comparison of the sequences of the L genes
of the attenuated RSS-2 ts1C mutant of RSV with that of
its parent identified an amino acid substitution at aa 1842
166
of the L protein (Tolley et al., 1996). Due to the presence
of an additional sequence at the N terminus of the RSV L
protein, comparison of RSV and PVM L proteins (not
shown) indicated that aa 1842 in the RSV protein aligned
with aa 1717 of the PVM L protein. This position is 11 aa
upstream of the conserved GEGAG motif in the putative
ATP-binding domain VI that was identified by Poch et al.
(1990). In light of this coincidence, it is tempting to
speculate that the residues at one or both of these locations
in the L protein may contribute to pathogenicity. Additional mutations in the RSV L gene that are responsible
for virus attenuation by restricting growth in vivo have
been identified. The cpts530/1009 vaccine candidate, which
was derived from the cold-passaged, attenuated cp-RSV
mutant of RSV strain A2, contained two mutations (affecting aa 521 and 1169 of the L protein) when compared with
the parental virus (Juhasz et al., 1999). These mutations were
introduced individually into recombinant RSV and each
was shown to confer an attenuated phenotype (Juhasz et al.,
1999). As the two strains of PVM, one pathogenic and one
non-pathogenic, are able to replicate to a similar level in vivo
(Domachowske et al., 2002), differences in the polymerase
protein are unlikely to result in a modification of enzymic
activity.
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
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Journal of General Virology 86
Pathogenic and non-pathogenic PVM strain sequences
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 59 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 virusspecific 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
We would like to thank Oliver Dibben for clarification of the PVM
strain 15 sequence. L. C. T. was supported by a University of Warwick
Graduate Scholarship and by grants from the British Federation of
Women Graduates, the General Charities of the City of Coventry and
the Newby Trust Ltd.
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