Journal of General Virology (2015), 96, 1180–1189
DOI 10.1099/vir.0.000063
New enveloped dsRNA phage from freshwater
habitat
Sari Mäntynen,1 Elina Laanto,1 Annika Kohvakka,13 Minna M. Poranen,2
Jaana K. H. Bamford1 and Janne J. Ravantti1,2,3
Correspondence
Janne J. Ravantti
[email protected]
1
Department of Biological and Environmental Science and Nanoscience Center,
University of Jyväskylä, Jyväskylä, Finland
2
Department of Biosciences, University of Helsinki, Helsinki, Finland
3
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
Received 26 September 2014
Accepted 15 January 2015
Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a trilayered virion structure. Here, we present a new putative member of the Cystoviridae family,
bacteriophage wNN. wNN was isolated from a Finnish lake in contrast to the previously identified
cystoviruses, which originate from various legume samples collected in the USA. The nucleotide
sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for
the M-segments and ~84 % for the S-segments) to Pseudomonas phage w6, the type member of
the virus family. However, the relationship between wNN and other cystoviruses is more distant. In
general, proteins located in the internal parts of the virion were more conserved than those
exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA
viruses. Structural models of several putative wNN proteins propose that cystoviral structures are
highly conserved.
INTRODUCTION
RNA viruses evolve rapidly owing to their fast replication, large populations and extremely high mutation rates
(Domingo & Holland, 1997). Genetic recombination, occurring when two virus strains simultaneously infect the same
host cell, is also a major driving force for RNA virus diversity
(Domingo & Holland, 1997). The recombination occurs via
exchange of genetic material between homologous or nonhomologous regions by crossing-over or through reassortment of entire genome segments (Domingo & Holland,
1997). The genetic plasticity allows RNA viruses to rapidly
adapt to challenges posed by the environment, manifested
e.g. by evasion of host immune response (Malim &
Emerman, 2001), evolution of resistance to antiviral drugs
(Nora et al., 2007), increase in virulence (Khatchikian et al.,
1989) or host-range expansion (Brown, 1997; Duffy et al.,
2006; Ferris et al., 2007; Gibbs & Weiller, 1999).
The currently classified dsRNA viruses belong to eight
families, members of which infect a variety of hosts, including
bacteria, protozoa, fungi, plants, invertebrates and vertebrates
(Mertens, 2004). Despite the variability in host organisms and
3Present address: University of Tampere BioMediTech, Tampere, Finland.
The GenBank/EMBL/DDBJ accession numbers for the S-, M- and Lgenome segments of bacteriophage wNN are KJ957166, KJ957165
and KJ957164, respectively.
Five supplementary figures are available with the online Supplementary
Material.
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habitats, most dsRNA viruses have striking structural and
functional similarities. Typically, the virion of a dsRNA virus
is formed by concentric structural layers. The innermost
capsid layers, containing RNA polymerization activity, are
conserved among dsRNA viruses (Bamford et al., 2002; Luque
et al., 2010), whereas the outermost layer shows greater
genetic and structural diversity. The flexible composition of
the outer layer allows the virus to adapt for transmission and
initiation of infection in different host cells (Bamford et al.,
2002; Poranen & Bamford, 2012b).
Bacteriophages of the family Cystoviridae have genomes
composed of three dsRNA segments enclosed in polyhedral
protein capsids which in turn are surrounded by protein–
lipid envelope (Poranen & Bamford, 2012a). Owing to the
dsRNA genome and the outer membrane, cystoviruses
represent a unique group among known bacteriophages.
The inner core of the virion is composed of 120 copies of the
major capsid protein (Ktistakis & Lang, 1987; Olkkonen &
Bamford, 1987) arranged in a T51 icosahedral lattice
(Butcher et al., 1997; Huiskonen et al., 2006). This rare
architecture of the innermost protein layer is also seen in
eukaryotic dsRNA viruses, for example in the members of
the family Reoviridae (Poranen & Bamford, 2012b).
In addition to Pseudomonas phage w6 (Vidaver et al., 1973),
the type virus of the Cystoviridae, several putative
cystoviruses have been isolated (Mindich et al., 1999;
O’Keefe et al., 2010; Silander et al., 2005; Qiao et al., 2010),
but have not been formally classified by the International
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New enveloped dsRNA phage
Committee on Taxonomy of Viruses. All isolates were
obtained from plant debris in the USA, and they infect
Gram-negative bacteria, mainly phytopathogenic Pseudomonas
syringae strains. Complete genome sequences are available
for four cystoviral isolates in addition to w6 (Gottlieb et al.,
1988, 2002; Hoogstraten et al., 2000; McGraw et al., 1986;
Mindich et al., 1988; Qiao et al., 2000, 2010). Some cystoviruses are genetically remarkably similar to w6, whereas
others are only distantly related. Phage w6 and its distant
relative w2954 (Mindich et al., 1999; Qiao et al., 2010) attach
to a specific type IV pilus on the host bacterium, whereas the
other distant relatives of w6 (w8, w12 and w13) bind directly
to rough lipopolysaccharide on the host outer membrane
(Mindich et al., 1999).
In cystoviruses, the general mechanism of recombination is
template switching (Qiao et al., 1997). Exogenous genetic
material, such as cellular transcripts or RNA strands from
other viruses, may also be incorporated into cystovirus
procapsids and recombine with endogenous viral RNA
(Onodera et al., 2001). However, the probability of the
packaging of two cystoviral RNA molecules of the same
segment class into the same procapsid is very low owing to
the strictly controlled packaging mechanism (Mindich,
1999), making homologous recombination an extremely
rare event, both in vitro (Onodera et al., 2001) and in
natural populations (Silander et al., 2005).
Reassortment of genome segments occurs frequently among
cystoviruses in laboratory conditions as well as in nature
(Onodera et al., 2001; Silander et al., 2005). Surprisingly, the
rate of reassortment may vary among populations sampled
from different geographical locations (O’Keefe et al., 2010).
Additional genetic diversity is acquired via mutations
occurring at the rate of y1026 per locus per generation
(Burch et al., 2007; Chao et al., 2002; Ferris et al., 2007),
although higher values have also been suggested, especially
in terms of host-range mutations (Ferris et al., 2007).
Here, we present a putative new member of the Cystoviridae,
which we name Pseudomonas phage wNN. wNN has a unique
freshwater habitat among cystoviruses, demonstrating that
this group is more diverse and widely distributed than
previously known. Phylogenetic relationships in light of the
putative new member are also discussed. wNN shares
striking genetic similarity to w6, whereas the other previously identified cystoviruses are more distantly related.
There are several X-ray and electron microscopic structures
available for various cystoviral proteins. Thus, it was feasible
to build atomic-resolution models of the wNN protein in
order to investigate its relationship with the known cystoviruses at the structural level also.
RESULTS
wNN virion
Bacteriophage wNN was isolated with its host Pseudomonas
sp. B314 from Lake Vehkalampi in Central Finland (N 62u
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149 42", E 25u 429 54"). Virus production was most efficient
when the host cells were infected in the middle of the
exponential growth phase (~1.56109 c.f.u. ml21) with
m.o.i 2–5. The duration of the latent period (~100 min;
Fig. 1a) was similar to that described for bacteriophage w6
(80–160 min, depending on the culture conditions; Vidaver
et al., 1973), while the measured mean burst size (~80) was
somewhat lower than in w6 (125–400, depending on the
culture conditions; Vidaver et al., 1973). However, the
duration and extent of the bacterial lysis as well as the overall
phage production in wNN infection varied somewhat
between different cultures.
The chloroform sensitivity of wNN indicated a possible
lipid component. Transmission electron microscope (TEM)
imaging revealed that the size and shape of wNN resemble
those of w6 and other putative cystoviruses. The mature
virion is tail-less and round; however, empty icosahedrally
symmetrical structures analogous to the core particles
of w6 were also observed from purified wNN samples
(Fig. 1b).
Based on SDS-PAGE, the protein profile of wNN was
similar to that of w6 (Fig. 1c). The proteins of wNN were
named according to the corresponding proteins of w6 (Fig.
1c, d, Table 1). Bands for the internally located RNAdependent RNA polymerase, P2, and the external spike
protein, P3, were assigned based on their stoichiometry. To
confirm this interpretation, the electrophoretic analysis was
repeated after treating the virions with butylated hydroxytoluene, which specifically removes the P3 spikes from
the virion surface (Bamford et al., 1995; and data not
shown). Furthermore, Triton X-100 solubilized/removed
proteins P3, P6 and P9, which indicates their presence on
the outer membrane (data not shown). The sensitivity of
P3 and the resistance of P2 to these treatments support the
interpretation made by stoichiometry.
Agarose gel electrophoresis (AGE) of dsRNA extracted
from purified wNN virions revealed the presence of three
dsRNA fragments, which were named according to their
size as small (S), medium (M) and large (L). Mobility in
agarose gel indicated that the sizes of the S- and L-segments
resemble those of w6, whereas the M-segment is smaller
than that of w6 (Fig. 1e).
Host interaction of wNN
Cross-infection studies of bacteriophages wNN and w6 were
conducted with their isolated hosts Pseudomonas sp. B314
and P. syringae pv. phaseolicola strain HB10Y (Vidaver et al.,
1973), respectively. HB10Y has type IV pili and its outer
membrane is covered with smooth lipopolysaccharide
(Mindich et al., 1999). In spot assay, wNN and w6 were
incapable of infecting each other’s host strains (data not
shown). TEM imaging of wNN infecting its host showed
virions attached to long, thin protrusions (Fig. 1f), possibly
indicating type IV pilus-mediated infection, previously shown
for w6 and w2954 (Mindich et al., 1999; Qiao et al., 2010).
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S. Mäntynen and others
Turbidity (OD550)
(a)
(c)
2.5
P1
P2
P3
2.0
Φ6 ΦNN
(d)
Host attachment protein P3
Fusogenic membrane protein P6
Major capsid protein P9
Membrane proteins P10 and P13
Phospholipids
Major capsid protein P1
RNA dependent RNA polymerase P2
Packaging NTPase P4
Assembly factor P7
Major outer capsid protein P8
Muralytic enzyme P5
P1
P2
P3
1.5
L
M
1.0
P4
P4
P5
P5
P7
P6
P7
0.5
S
0
0
100 200 300 400
Time (min)
(b)
(f)
(e)
Φ6
ΦNN
L
P8
M
P8
P9
S
P9
Fig. 1. wNN life cycle and the virion. (a) Turbidity (OD550) of the infected (dashed line) and uninfected (solid line) Pseudomonas
sp. B314 culture. The culture was infected with wNN virions at m.o.i. 2. Time 0 indicates the time of infection. (b) Transmission
electron micrograph of bacteriophage wNN. Purified virions were negatively stained in ammonium molybdate (pH 7.4) and
imaged by a TEM at 80 kV. In addition to mature virions, empty core particles were also seen (box in upper right corner). Bar,
80 nm. (c) Protein profiles of w6 and wNN by SDS-PAGE analysis. Purified virions were disrupted by boiling and loaded onto a
15 % polyacrylamide gel. The mobility of the w6 proteins is indicated on the left and that of corresponding proteins of wNN on
the right. Protein P6 of w6 is not visible in the image but was seen on the gel. (d) Schematic representation of w6 virion. Three
dsRNA genome segments (S, M and L) are enclosed in an inner core (proteins P1, P2, P4 and P7), which is surrounded by an
outer protein shell (protein P8). The outermost surface consists of a lipid envelope with embedded phage-encoded proteins and
P3 spikes. (e) AGE analysis of dsRNA genomes of bacteriophages w6 and wNN. Extracted dsRNA was subjected to
electrophoresis in 1 % agarose gel. The mobility of the w6 segments L (6374 bp), M (4063 bp) and S (2948 bp) is indicated on
the left and that of corresponding wNN segments on the right. (f) Transmission electron micrograph of wNN virions attaching to a
pilus on Pseudomonas sp. B314 at 2 min post-infection. Bar, 400 nm.
Table 1. Amino acid sequence similarities between ORFs of wNN and those of previously identified cystoviruses
wNN
L
M
S
P14
P7
P2
P4
P1
P10
P6
P3
P13
P8
P12
P9
P5
MW (kDa)
7
17
75
35
85
6
19
71
8
16
20
10
24
Function based on w6 annotation
Non-structural, non-essential
Assembly factor
RNA-dependent RNA polymerase
Packaging NTPase
Major capsid protein, forms the T51 shell
Membrane protein, needed in cell lysis
Fusogenic membrane protein, binds to P3
Spike protein, binds to the host receptor
Minor membrane protein
Forms the T513 nucleocapsid shell
Assembly factor in viral membrane morphogenesis
Major membrane protein
Muralytic enzyme, needed in host entry and lysis
Amino acid similarity (%)*
w6
w13
w12
w2954
w8
94
93
98
89
98
65
64
40
69
95
91
91
92e
2
58
60
45
52
38
37
17a
2
41
36
26
29
10
26
34
33
30
56
36
19b
2
17
22
31
32
6
34
33
32
31
31
21
28
2
26
21
35
31
9
30
31
29
29
16
36
17c
28d
14
17
30
11
ORFs are listed in their genomic order.
*Corresponding cystoviral ORF was not detected. The most similar peptide/protein based on genome location and annotation for wNN P3 from
w13 is P3a (a), for wNN P3 from w12, it is P3c (b), for wNN P3 from w8, it is P3b (c), for wNN P13 from w8, it is PF (d) and for wNN P5 from w6, it is
P5a (e), respectively.
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Journal of General Virology 96
New enveloped dsRNA phage
The host bacterium of wNN was characterized by 16S rRNA
sequence analysis and selected biophysical tests (conducted
by DSMZ). According to these assays, the strain belongs to
the genus Pseudomonas, and is presumably P. syringae or a
closely related species.
Sequencing and genome analysis of wNN
The dsRNA genome of wNN was converted into complementary DNA (cDNA) fragments which were sequenced.
Overlapping sites of the sequences were aligned to produce
complete nucleotide sequences for the three genome
segments (Fig. 2). The sizes of the segments are 2945 (S),
3814 (M) and 6503 (L) bp, comparable with the genome
segments of w6, which are 2948, 4063 and 6374 bp,
respectively (Fig. 1e). The extreme 59 end of the wNN Lsegment could not be confirmed, and it may contain a few
additional nucleotides. The GC-content is 53, 55 and 55 %
for wNN S, M and L, respectively, compared with about
56 % GC-content of the w6 genome (Mindich, 1988).
Thirteen potential ORFs were identified from the genome
of wNN. The genetic organization of wNN resembles that of
w6 and other identified cystoviruses (Gottlieb et al., 1988,
2002; Hoogstraten et al., 2000; McGraw et al., 1986;
Mindich et al., 1988; Qiao et al., 2000, 2010), and putative
functions could be assigned to the ORFs based on chromosomal position, size and sequence homology with w6 (Fig.
2, Table 1).
Similarly to other cystoviruses, all the ORFs of the wNN
genome are oriented in the same direction (Gottlieb et al.,
1988, 2002; Hoogstraten et al., 2000; McGraw et al., 1986;
Mindich et al., 1988; Qiao et al., 2000, 2010). The ORFs
contain a translational initiation codon AUG, except for
ORFs 5 and 10, which begin with the codon GUG. ORFs 2,
5 and 12 did not seem to have a well-defined ribosomebinding site (Shine & Dalgarno, 1975) and probably
depend on upstream genes (genes 7, 9 and 8, respectively;
Fig. 2) for ribosome loading and subsequent translation.
Similarly, the ribosome-binding sites of ORFs 6 and 13
were non-optimal. Polar relationships between genes have
ORF
8
12
9
also been demonstrated for other cystoviruses (Gottlieb
et al., 2002; Hoogstraten et al., 2000; McGraw et al., 1986;
Mindich et al., 1988; Qiao et al., 2000, 2010).
The nucleotide sequences of wNN genome segments were
compared with previously identified cystoviruses for which
complete genome sequences are available (w6, w8, w12, w13
and w2954), and phylogenetic trees were reconstructed
from these comparisons (Fig. 3). Each segment of wNN is
closely related to the corresponding segment of w6, the Msegments being the most divergent. Furthermore, the Lsegments of wNN and w13 are moderately related.
The high amino acid sequence relatedness between wNN
and w6 was evident when visualizing the relationships
between wNN and the previously identified cystoviruses
(Fig. S1, available in the online Supplementary Material).
The highest similarities (89–98 %; Table 1) between translated
wNN ORFs and their putative w6 homologues were observed
in ORFs/genes located in the S- and L-segments (highest in
ORF 1/gene 1 and ORF 2/gene 2), whereas similarity in the
ORFs/genes of the M-segment was significantly lower (40–
69 %; Table 1), being lowest in the ORF/gene encoding the
host attachment protein P3 (40 % similarity; Table 1). The
sequence relatedness in ORFs predicted to encode nucleocapsid proteins (P1, P2, P4, P7, P8; 41–60 % similarity;
Table 1) was also prominent between wNN and w13. In
general, putative ORFs of essential enzymes and proteins in
the internal parts of the virion (Fig. 1d) shared greater
amino acid sequence similarity between wNN and the more
distant relatives, w8, w12, w13 and w2954 (11–60 %; Table 1),
whereas the ORFs of the host recognition protein P3 (17–
28 % similarity; Table 1) and the other membraneassociated proteins differed more.
Structural modelling of wNN putative proteins
Structures of all predicted wNN proteins were modelled
and the models were quality controlled. The models for
putative proteins of the capsid protein, P1, polymerase
complex, P2, packaging NTPase, P4, and the nucleocapsidassociated lytic enzyme, P5 (Fig. 1d, Table 1), were
5
Small segment (S)
ORF 10
ORF 14
6
Medium segment (M)
7
2
3
13
4
1
Large segment (L)
Fig. 2. Genetic maps of the wNN genome segments. ORFs, indicated by numbered boxes, were assigned based on similarity
with w6. The sizes of the S-, M- and L-segments are 2945, 3814 and 6503 bp, respectively. Proteins that form the
nucleocapsid or are associated with it are indicated with dark grey and membrane-associated proteins with light grey.
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S. Mäntynen and others
0.1
Φ13
Φ6
ΦNN
Φ8
Φ2954
Φ12
Small (S) segments
0.1
Φ13
Φ6
Φ12
ΦNN
Φ8
Φ2954
Medium (M) segments
0.1
Φ13
Φ6
ΦNN
Φ8
The model of the putative P1 major capsid protein of wNN
is based mainly on P1 of w6 (PDBid 4BTG; Nemecek et al.,
2013; 98 % sequence similarity). The Homology Structure
Finder (HSF)-program (Ravantti et al., 2013) found 600
residues structurally equivalent between wNN and w8 P1
(4BTP; El Omari et al., 2013a; 78 % coverage of wNN P1),
although sequence-level similarity is only 29 %. The wNN
P2 polymerase model (Fig. 4) is based on the w6 structure
(PDBid 1HI8; Butcher et al., 2001), and shares 574
equivalent residues (86 % coverage of wNN P2) with the
w6 and w12 P2 structures (Butcher et al., 2001; Ren et al.,
2013). This is expected, as viral RNA polymerases are
structurally highly conserved (Bruenn, 2003; Mönttinen
et al., 2014; Poch et al., 1989). There are several cystoviral
packaging NTPase P4 structures available in the Protein
Data Bank (PDB) (PDBid 4BLO, 4BLP, 4BLQ and 1W44;
El Omari et al., 2013b; Mancini et al., 2004; Fig. 4). The
wNN P4 model is most similar to the w6 P4 structure (Fig.
4), but interestingly the structural similarity to the w8 P4 is
higher than to the w12 P4, although the w12 P4 has higher
sequence similarity to the putative wNN P4. These cystoviral NTPases, including the wNN model presented here,
share a common catalytic core (172 equivalent residues by
HSF-program), but they have different specificities and
control mechanisms, which can be mapped onto the
divergent N- and C-terminal domains (El Omari et al.,
2013b).
Since the overall sequence similarity between w6 and wNN
is high (Table 1), it is not surprising that most structural
models for wNN proteins are based on structures derived
from w6, the muralytic enzyme, P5, being no exception.
The wNN P5 model (Fig. 4) covers all 160 residues solved
by X-ray crystallography for w6 P5 (PDBid 4DQ5).
DISCUSSION
Fig. 3. Phylogenetic trees showing relationships between cystoviruses based on nucleotide sequence comparisons of complete
genome segments constructed by CLUSTAL Omega (Sievers et al.,
2011). Dendroscope3 (Huson & Scornavacca, 2012) was used
for visualization. Bars, 0.1 units.
A freshwater bacteriophage, wNN, was isolated and shown
to have a tripartite dsRNA genome and a lipid-containing
virion. Further characterization revealed that the wNN
virion is tail-less and contains an icosahedrally symmetrical
core particle (Fig. 1b). Furthermore, the genome content
and protein profile of wNN resemble those of w6, the type
member of the family Cystoviridae (Fig. 1c–e). Owing to
these distinctive similarities, we propose that wNN is a new
member of the Cystoviridae.
considered to have a correct fold. The models of P1, P2, P4
and P5 all had ProSA-web Z-scores (29.84, 210.57, 27.69
and 27.12, respectively) that are well within acceptable
range and characteristic for native proteins (Figs S2–S5).
These models were also mainly based on templates from
homologous proteins of cystoviruses. The best models are
presented, along with structural alignments to other known
cystoviral structures, in Fig. 4, and additional details are
provided in Figs S2–S5.
Bacteriophage w6, isolated over 40 years ago, is still the
only classified member of the Cystoviridae (Poranen &
Bamford, 2012a). However, it has been shown that bacteriophages with a tripartite dsRNA genome can be readily
isolated from agricultural plant species (Mindich et al.,
1999; O’Keefe et al., 2010; Silander et al., 2005; Qiao et al.,
2010). Although these additional viral isolates have not
been specifically identified or classified, their high frequency in agricultural samples indicates that dsRNA
bacteriophages are more common in terrestrial habitats
than previously estimated. All the previous cystoviruses are
Φ2954
Φ12
Large (L) segments
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New enveloped dsRNA phage
P1
ΦNN model
Φ6
Φ8
ΦNN model
Φ6
Φ12
P2
P4
ΦNN model
Φ6
Φ13
Φ8
Φ12
P5
ΦNN model
Φ6
Fig. 4. Predicted structural models for putative wNN proteins. Four models were considered reliable by I-TASSER (P1, P2, P4 and
P5). The corresponding known cystoviral protein structures are presented on the right in descending order based on their
similarity to each wNN model and coloured according to their secondary structures (a-helices, red; b-sheets, yellow). Equivalent
residues between the model and the known cystoviral structure were detected using the HSF-program (Ravantti et al., 2013).
Cyan indicates non-equivalent residues.
from legume samples from certain parts of the USA
(Nebraska, New England, California, Connecticut). In contrast, wNN was isolated in Central Finland from a freshwater
sample, demonstrating that cystoviruses are more widely
distributed than previously known.
Despite the clear similarities between wNN and w6, they did
not infect each other’s natural host strains. According to
16S rRNA sequence analysis and several biophysical tests,
the host strain of wNN belongs to the genus Pseudomonas and is presumably P. syringae or a closely related
species. Electron microscope imaging (Fig. 1f) indicated
that wNN likely uses type IV pilus as its primary receptor,
similarly to w6 and w2954 (Mindich et al., 1999; Qiao et al.,
2010). However, the sites recognized by wNN and w6 are
apparently non-conserved in their Pseudomonas hosts.
The sizes and gene arrangements of wNN genome segments resemble closely those of w6 (Figs 1e and 2). All in
all, the genetic organization is relatively conserved within
the identified cystoviruses, and the ORFs/genes cluster into
functional groups (Gottlieb et al., 1988, 2002; Hoogstraten
et al., 2000; McGraw et al., 1986; Mindich et al., 1988; Qiao
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et al., 2000, 2010). For instance, the highly conserved Lsegment includes ORFs/genes encoding the components of
the virion core containing the polymerase activity (Figs 1d
and 2) whereas the M-segment, which varies the most
among cystoviruses, contains ORFs/genes encoding envelope proteins (Figs 1d and 2).
Phylogenetic trees of the genome segments indicate a close
relationship between wNN and w6 (Fig. 3). The sequence
alignments reveal that the predominant amino acid sequence variation between wNN and w6 is in the spike
protein P3 (Fig. S1). This putative host attachment protein
of wNN differs greatly from the corresponding proteins of
other identified cystoviruses, the most similar one being
the P3 of w2954 (Table 1). Cystoviruses w6, wNN and w2954
use pilus-mediated infection (Mindich et al., 1999; Qiao et
al., 2010; Fig. 1f) and have attachment complexes
consisting of a single P3 polypeptide (Gottlieb et al.,
1988; Qiao et al., 2010; Fig. 1c, Table 1), whereas other
previously identified cystoviruses (w8, w12 and w13)
interact directly with the layer of rough lipopolysaccharide
and have a more complex heteromeric attachment
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S. Mäntynen and others
apparatus with at least two or three polypeptides (Gottlieb
et al., 2002; Hoogstraten et al., 2000; Mindich et al., 1999;
Qiao et al., 2000). The similarity in infection mechanisms
could explain the moderate sequence similarity between the
host attachment proteins of w6, wNN and w2954.
The frequent genome segment reassortment of cystoviruses
has been demonstrated in laboratory conditions and in
nature (Onodera et al., 2001; Silander et al., 2005). Such
a phenomenon could also explain the higher sequence
variability between the M-segments of wNN and w6 compared with the other two segments. Furthermore, strong
selection pressure is presumably exerted on the M-segmentencoded proteins during co-evolution with the host, as
demonstrated by the susceptibility of the host attachment
protein P3 of w6 to spontaneous mutations, resulting in
phages with varying host range (Duffy et al., 2006; Ferris
et al., 2007). Recombination by crossing-over with genetic
material of the host cell or other viruses is an unlikely
explanation for the observed sequence variation (Onodera
et al., 2001) as homologous sequences for wNN were not
found outside previously identified cystoviruses.
In contrast to protein components of the outer membrane,
the internal major structural proteins and the key viral
enzymes, encoded by the S- and L-segments (Table 1), are
relatively conserved, indicating that host–virus interactions
are not significantly driving their evolution. The similarity
of these proteins appears to be even higher at structural
level (Fig. 4), underlining how RNA viruses undergo rapid
evolution affecting the primary sequence yet conserving the
structures needed to maintain a viable virus.
Structural variation in the outermost layer of the virion is
commonly observed in animal viruses. For instance, the
outermost layer of different reoviruses consists of a variety
of unrelated proteins (Poranen & Bamford, 2012b). Significant variation is also seen in the surface glycoproteins
(haemagglutinin and neuraminase) of influenza viruses.
Since these proteins are the major target of neutralizing
antibodies of the host immune system, their rapid
evolution is presumed to be at least partly driven by the
selection pressure caused by the host’s antibody-mediated
immunity system (Webster et al., 1992). However, antibodymediated selection pressure cannot be the driving force for
the variation detected here in the external layers of bacterial
viruses. Apparently, changes in the outer layer accumulate to
broaden the host range of the virus, whereas protein–protein
associations inside the virion are highly coupled, so that
even minor structural changes in any of the internal proteins
may disturb virion assembly. Furthermore, essential viral
enzymes, which likely are more sensitive to variations, are
typically located in the interior of the virion.
Even though wNN and w6 were isolated from highly diverse
ecological habitats on different continents at a .40-year
interval, these two phages are significantly similar. Their
resemblance reflects the conservation of key viral elements,
while simultaneously the inherent viral ability to broaden
the host range leads to ecological plasticity.
1186
METHODS
Bacteriophages, bacterial strains and plasmids. wNN and its
host bacterium Pseudomonas sp. B314 were isolated in this study.
Pseudomonas phage w6 and its natural host P. syringae pv. phaseolicola strain HB10Y (Vidaver et al., 1973) were obtained from the
laboratory of D. H. Bamford (University of Helsinki). A high-copynumber plasmid, pUC18 (Yanisch-Perron et al., 1985), was used for
the cloning of cDNA copies of the wNN genome. Recombinant
plasmids were propagated in Escherichia coli DH5a (Sambrook et al.,
1989).
Bacteria were grown in Luria–Bertani (LB) medium (Sinclair et al.,
1976). Selective plates contained 200 mg ampicillin ml21 in LB agar
and were supplemented with 0.1 mM IPTG and 50 mg X-Gal ml21 for
blue–white screening (Maniatis et al., 1982).
Characterization of the host bacterium. The 16S rRNA gene of
Pseudomonas sp. B314 was amplified by PCR using primers fD1 and
rD1 (Weisburg et al., 1991) and DreamTaq DNA polymerase
(Thermo Scientific). Bacterial genome extracted with the GeneJET
Genomic DNA Purification kit (Thermo Scientific) was used as a
template. The 16S rRNA-specific DNA was purified with the
QIAquick PCR Purification kit (Qiagen), and its nucleotide sequence
was determined using the BigDye Terminator v3.1 kit and ABI Prism
Genetic Analyzer 3100 (Life Technologies). The microbiological
characterization of Pseudomonas sp. B314 strain was commercially
conducted by DSMZ.
Isolation, growth and purification of viruses. Pseudomonas sp.
B314-specific viruses were isolated from a filtered water sample
(Micropore, 0.45 mm filter) using enrichment culture containing the
host bacteria in 20 % (v/v) LB. After incubation at room temperature
(RT) for 2 days with agitation (110 r.p.m.), samples of the enrichment culture were mixed with 0.7 % (w/v) soft agar in 20 % (v/v) LB
and plated on the same medium supplemented with 1 % (w/v) agar.
The plates were incubated at RT for 2–3 days, then independent
plaques were picked, suspended in 20 % LB medium and stored at
4 uC. Virus stocks were prepared by collecting the top agar from
plates showing semi-confluent lysis of the bacterial lawn by the phage
as previously described for phage w6 (Bamford et al., 1995).
For large-scale liquid culture, host cells grown (26 uC, 220 r.p.m.)
to exponential growth phase (~1.56109 c.f.u. ml21) were infected
by wNN or w6 at m.o.i. 2–5. Culturing was continued (26 uC,
110 r.p.m.) in the presence of 5 mg DNase I ml21 (Sigma-Aldrich)
until lysis. Cell debris was removed by centrifugation (Sorvall
SLA3000 rotor, 10 800 g, 15 min, 4 uC) and virions were concentrated by precipitation with 10 % (w/v) polyethylene glycol 6000 and
0.5 M NaCl (Bamford et al., 1995). Phage precipitate was collected by
centrifugation (Sorvall SLA3000, 10 800 g, 20 min) and suspended in
20 mM potassium phosphate buffer (PPB, pH 7.2). Aggregates were
removed from the concentrated phage preparation by centrifugation
(Sorvall SS-34, 3000 g, 5 min, 4 uC) and the cleared supernatant was
layered on top of a 5–20 % (w/v) sucrose gradient (in 20 mM PPB,
pH 7.2) for subsequent centrifugation (Beckman Optima L-90K
ultracentrifuge, SW28 rotor, 76 000 g, 40 min, 15 uC). The lightscattering zone, containing the phage, was collected and subjected to
equilibrium centrifugation in a 20–70 % (w/v) sucrose gradient (in
20 mM PPB, pH 7.2; Beckman SW41 rotor, 126 000 g, 18–19 h,
5 uC). Phage particles were finally collected by centrifugation
(Beckman 70 Ti rotor, 80 000 g, 3 h, 5 uC), suspended in 20 mM
PPB (pH 7.2) and stored at 280 uC. The non-ionic detergent Triton
X-100 was used to remove the viral membrane and membraneassociated proteins from the virions (Bamford & Palva, 1980). The
protein profiles of the purified virions were analysed by SDS-PAGE
(Laemmli, 1970) in 15 % polyacrylamide gel and subsequently
scanned using a Bio-Rad imaging device.
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Journal of General Virology 96
New enveloped dsRNA phage
The analysis of the burst size was carried out as described by Dennehy
& Turner (2004). The unbound viruses were removed by centrifugation of the culture and resuspension of the cells into fresh medium
at 30 min post-infection.
Host-range investigation and chloroform-sensitivity assay.
Pseudomonas strains were grown in LB overnight, after which 200 ml
each culture was mixed with 3 ml molten soft agar and laid onto an LB
plate. Ten microlitres of viral dilutions was spotted onto the solidified
plate. After overnight incubation at RT, the plates were examined for
bacterial lysis.
For the chloroform-sensitivity assay, a drop of chloroform (~50 ml) was
added to 100 ml virus stock and the solution was mixed with vigorous
shaking. After 15 min, 5–10 ml of the aqueous phase or an untreated
sample of the phage stock was spotted onto a lawn of the host bacterium.
Electron microscopy. The morphology of virions and their attachment to host cells were examined by negative-stain transmission
electron microscopy. Purified viral particles or samples of infected
host culture were deposited onto coated copper grids for 1–3 min.
The grids were subsequently stained with ammonium molybdate
(pH 7.4) for 30 s–1 min and examined by means of a JEOL JEM1200EX TEM at an accelerating voltage of 80 kV.
Isolation of viral dsRNA and cDNA synthesis. Genomic dsRNA
was isolated from purified virions using Tri Reagent extraction (Life
Technologies). The integrity and size distribution of the extracted
dsRNA was assessed on a 1 % (w/v) agarose gel (in Tris/acetate EDTA
buffer) stained with ethidium bromide.
Genomic dsRNA (~3 mg) was denatured by boiling for 5 min in 15 %
(v/v) DMSO and cooled in ice–water slurry. Poly(A) tails were added
to the 39 ends of the RNA strands using E. coli poly(A) polymerase (Life
Technologies). The RNA was then purified by phenol/chloroform
extraction, precipitated with ammonium acetate and ethanol, and
resuspended in 10 ml nuclease-free H2O.
The poly(A)-tailed RNA was combined with sequence-specific
primers (0.5 mg mg21 RNA) and heated to 96 uC for 3 min. The
sample was cooled on ice before the cDNA was synthesized with the
Universal RiboClone cDNA Synthesis kit (Promega) according to
manufacturer’s instructions. The resulting cDNA was again purified,
precipitated and resuspended in 10 ml nuclease-free H2O.
Plasmid pUC18 was digested with SmaI (Thermo Scientific),
dephosphorylated with FASTAP Thermosensitive Alkaline Phosphatase
(Thermo Scientific), extracted from agarose gel after electrophoresis,
and purified using the Qiagen Gel Extraction kit. The cDNA fragments
of the dsRNA genome (~100–400 ng) were blunt-end ligated into the
SmaI site of pUC18 (~50 ng) using T4 DNA ligase (400 U; New
England Biolabs) in 16 ligation buffer containing 20 % (w/v)
polyethylene glycol 6000. After overnight incubation at 16 uC, the
resulting DNA molecules were transformed into competent E. coli
DH5a cells using a heat-shock method. The transformants were plated
onto selective LB plates containing IPTG and X-Gal, and cultivated at
37 uC overnight. Plasmid-DNA was purified from selected clones by
using the Qiagen Miniprep kit and digested with EcoRI and HindIII
(Thermo Scientific) to verify the presence of an insert.
B314, using the Basic Local Alignment Search Tool (http://www.blast.
ncbi.nlm.nih.gov/Blast.cgi). The genome of wNN was screened using
the programs NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/
orfig.cgi), Genemark (Besemer et al., 2001) and VectorNTI (Life
Technologies) for possible protein-encoding ORFs. Each ORF was
visually inspected and compared with those of existing putative
cystoviruses to confirm its location within the genome segments. The
GenBank accession numbers for the nucleotide sequences of wNN
genome segments S, M and L are KJ957166, KJ957165 and KJ957164,
respectively. wNN was compared with those previously identified
cystoviruses for which complete genome sequences are available.
These are w6 (GenBank accession numbers for S, M and L, respectively: NC_003714.1, NC_003716.1, NC_003715.1), w8 (NC_003301.1,
NC_003300.1, NC_003299.1), w12 (NC_004174.1, NC_004175.1,
NC_004173.1), w13 (NC_004170, NC_004171.1, NC_004172.1) and
w2954 (NC_012093, NC_012092, NC_012091.2). The genome segments were aligned separately per segment type (S, M and L) using
CLUSTAL Omega (Sievers et al., 2011) and visualized using
Dendroscope3 (Huson & Scornavacca, 2012).
The ORFs were translated into amino acid sequences with the Emboss
Transeq program (Rice et al., 2000). Statistics for putative proteins
were calculated using Emboss Pepstat (Rice et al., 2000). Each putative protein was compared against all other putative cystoviral proteins using Emboss Needle (Rice et al., 2000). The most similar
proteins to wNN ORFs from each phage were selected and their
similarity was visualized (Fig. S1) using Circos (Krzywinski et al.,
2009).
An online version of the I-TASSER program (Roy et al., 2010; Roy &
Zhang, 2012) was used to model all predicted wNN proteins. Only
those putative proteins that had a TM-score of the model .0.5 or had
a known cystoviral structure available for comparison (i.e. putative
protein P5, TM-score 0.49) were used. The model quality was assessed
using ProSA-web (Sippl, 1993; Wiederstein & Sippl, 2007). The
models were aligned and their equivalent residues detected by the
HSF-program (Ravantti et al., 2013). The cystoviral structures were
downloaded from the PDB and visualized using PYMOL (www.pymol.
org).
ACKNOWLEDGEMENTS
This study was supported by the Academy of Finland Centre of
Excellence Program in Virus Research (1129684, 2006–2011 to
J. K. H. B.), the Center of Excellence Program in Biological Interactions (252411, 2012–2017 to J. K. H. B.), the Academy of Finland
grants 251106 (to J. K. H. B.), 250113, 256069, 283192 and 272507 (to
M. M. P.), and a grant from the Finnish Cultural Foundation (00110600
to S. M.). We thank the Academy of Finland (grants 271413 and
272853) and University of Helsinki for their support to the EU ESFRI
Instruct Centre for Virus Production. We thank Petri Papponen for
technical support in electron microscope experiments and Riitta
Tarkiainen for assistance with SDS-PAGE assays.
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