Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

Abstract

Success of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown.
we have constructed four mutant FMDVS that encode only one VPG: either VPg(1), VPg(3), or two chimeric versions containing part of VPg(1) and VPg(3). All mutants, except that encoding only VPg(1), were replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21 cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and detach BHK-21 cells from the monolayer, the first stage of cell killing.
The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses pathogenesis are discussed.

Full text

Deletion Mutants of VPg Reveal New Cytopathology
Determinants in a Picornavirus
Armando Arias
1¤
, Celia Perales
1,2
, Cristina Escarmı
´s
1
, Esteban Domingo
1,2
*
1Departamento de Virologı
´a y Microbiologı
´a, Centro de Biologı
´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Consejo Superior de Investigaciones Cientı
´ficas (CSIC), Campus
de Cantoblanco, Madrid, Spain, 2Centro de Investigacio
´n Biome
´dica en Red de Enfermedades Hepa
´ticas y Digestivas (CIBERehd), Barcelona, Spain
Abstract
Background:
Success of a viral infection requires that each infected cell delivers a sufficient number of infectious particles to
allow new rounds of infection. In picornaviruses, viral replication is initiated by the viral polymerase and a viral-coded
protein, termed VPg, that primes RNA synthesis. Foot-and-mouth disease virus (FMDV) is exceptional among picornaviruses
in that its genome encodes 3 copies of VPg. Why FMDV encodes three VPgs is unknown.
Methodology and Principal Findings:
We have constructed four mutant FMDVs that encode only one VPg: either VPg
1
,
VPg
3
, or two chimeric versions containing part of VPg
1
and VPg
3
. All mutants, except that encoding only VPg
1
, were
replication-competent. Unexpectedly, despite being replication-competent, the mutants did not form plaques on BHK-21
cell monolayers. The one-VPg mutant FMDVs released lower amounts of encapsidated viral RNA to the extracellular
environment than wild type FMDV, suggesting that deficient plaque formation was associated with insufficient release of
infectious progeny. Mutant FMDVs subjected to serial passages in BHK-21 cells regained plaque-forming capacity without
modification of the number of copies of VPg. Substitutions in non-structural proteins 2C, 3A and VPg were associated with
restoration of plaque formation. Specifically, replacement R55W in 2C was repeatedly found in several mutant viruses that
had regained competence in plaque development. The effect of R55W in 2C was to mediate an increase in the extracellular
viral RNA release without a detectable increase of total viral RNA that correlated with an enhanced capacity to alter and
detach BHK-21 cells from the monolayer, the first stage of cell killing.
Conclusions:
The results link the VPg copies in the FMDV genome with the cytopathology capacity of the virus, and have
unveiled yet another function of 2C: modulation of picornavirus cell-to-cell transmission. Implications for picornaviruses
pathogenesis are discussed.
Citation: Arias A, Perales C, Escarmı
´s C, Domingo E (2010) Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus. PLoS ONE 5(5):
e10735. doi:10.1371/journal.pone.0010735
Editor: Maciej Lesniak, The University of Chicago, United States of America
Received December 16, 2009; Accepted April 30, 2010; Published May 20, 2010
Copyright: ß2010 Arias et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Work supported by grants BFU2008-02816/BMC from Ministerio de Ciencia e Innovacion (MICINN), and Fundacion R. Areces. CIBERehd (Centro de
Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas) is funded by Instituto de Salud Carlos III. AA was supported by an I3P contract from
Centro Superior de Investigaciones Cientificas (CSIC) and CP is the recipient of a contract from CIBERehd. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: edomingo@cbm.uam.es
¤ Current address: Department of Virology, Imperial College London, London, United Kingdom
Introduction
Contrary to initiation of cellular DNA replication which is primed
by RNA molecules synthesised by cellular primases [1], viruses use a
wide variety of molecular mechanisms to initiate genome replication,
that include de novo initiation, priming by proteins or by self generated
39–ends of templates, and ‘cap–snatching’, among other mechanisms
[2]. Protein–primed initiation of genome replication is used by several
DNA and RNA viruses and some linear plasmids [3–5]. Picornaviridae
is a family of positive strand RNA viruses that use as protein–primer a
small peptide of about 20 residues in length, termed VPg or 3B
[3,6,7]. After replication, the protein–primer VPg remains bound to
the genomic RNA encapsidated into viral particles. Picornaviruses
encode only one copy of VPg, except foot–and–mouth disease virus
(FMDV) that expresses three similar but non–identical copies of VPg
(VPg
1–3
or 3B
1–3
) [8] (Figure 1). Each of the three VPgs are found
covalently bound to genomic viral RNA [9] and they can be
uridylylated in vitro by the viral polymerase, with VPg
3
.VPg
2
.VPg
1
as the order of substrate efficiency [10]. The biological meaning of
this unique in–tandem repetition in an RNA virus is not well
understood [11,12]. Molecular poliovirus clones constructed to
express two VPgs delete one of the two copies, and the polyprotein
harboring two VPgs underwent aberrant processing [13,14]. FMDV
encoding only VPg
3
is infectious in cell culture, showing that one copy
of VPg may be sufficient to complete the virus replication cycle [12].
The virus expressing only VPg
3
was infectious for hamster and bovine
fibroblasts (BHK and FBK cells), but not swine fibroblasts (FPK cells),
and was attenuated for swine [12]. FMDVs encoding VPg
1
and
VPg
2
, but lacking VPg
3
were not viable, suggesting that the presence
of VPg
3
was essential for FMDV viability [11]. The authors proposed
that this loss of viability could be due to a defect in the proteolytic
processing of the viral polyprotein precursor lacking VPg
3
[11].
Picornaviral proteins are generated by proteolytic processing of
a single viral polyprotein which is translated from a single ORF.
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During and after translation, different cleavages of the viral
polyprotein take place, most of them catalysed by the viral
protease 3C, resulting in the release of different processing
intermediates and mature proteins (reviewed in [15]). Specifically,
the capsid precursor (P1) is processed into VP0 (VP4–VP2), VP3
and VP1 which are assembled to form the mature virions. P2 and
P3 precursors render non-structural proteins 2A, 2B, 2C, 3A, 3B
(VPg), 3C, 3D and several processing intermediates which are
required for viral replication. 3D is the viral RNA-dependant
RNA polymerase (RdRp) that catalyses genomic RNA synthesis
and the critical VPg–uridylylation step at the initiation of
replication. 3C and its precursor 3CD stimulate the initial VPg–
uridylylation step, an activity additional to their role in polyprotein
processing [16–18]. It has been recently proposed that a precursor
form of VPg (either 3AB or 3BC) could act as the authentic
protein–primer molecule, while processing and release of 3B (VPg)
would be a step subsequent to initiation of replication, although
these hypotheses are still under discussion [16,17,19]. 2C and 3A
play also central roles in picornavirus replication. 2C includes
NTPase and RNA–binding activities [20–23], acts as an RNA
chaperone during picornaviral replication [24], and is involved in
viral RNA encapsidation [25], uncoating [26], and in host cell
membrane rearrangements required for replication [27–31]. 3A is
a membrane protein [32] that can establish interactions with 2C
[33], suggesting that 3A and 2C may constitute part of the same
protein complexes for some biological processes. In addition, 3AB
precursor (3A bound to VPg) may be involved in the recruitment
of 3D polymerase to membranes to form replication complexes in
which the viral genomes are synthesised [34,35].
In the present report, we provide evidence of a functional link
between VPg and 2C in the release of FMDV from cells. We have
constructed FMDVs that encode a single VPg. Some mutants were
replication–competent but did not produce plaques on BHK–21 cell
monolayers. Passage of these viruses in BHK–21 cells selected for
mutants in 2C, 3A and 3B (VPg) that regained the ability to develop
plaques. These mutants displayed increased cytopathology and virus
shedding into the extracellular medium. The results provide evidence
for a function of the triplicated VPg in the detachment of cells from
the monolayers, which is the event that precedes cell killing by
FMDV [36,37]. These observations establish functional connections
between VPg and non–structural FMDV proteins.
Results
FMDVs with only one VPg copy are replication–
competent viruses
Previous studies showed that FMDV encoding VPg
3
as the only
copy of VPg (but not FMDV encoding only VPg
1
and VPg
2
)is
infectious in cell culture [11,12]. Itwasnotclearwhetherlethalitywas
due to absence of VPg
3
or to absence of the proteolytic cleavage site
between VPg
3
and 3C [11]. To address whether VPg
3
is essential for
FMDV replication, and to investigate the function of repeated VPg
genes in Aphthoviruses, we have designed FMDVs with only VPg
1
(termed V1), only VPg
3
(termed V3), and chimeric viruses containing
most of VPg
1
but a short C-terminal portion of VPg
3
that restores the
cleavage site between VPg
1
and 3C. The chimeric–VPg viruses have
been termed V19–4 (first 19 residues from VPg
1
and last 4 residues
from VPg
3
)andV15–9(first15residuesofVPg
1
and last 9 residues
from VPg
3
) (Figure 1). All mutant viruses, except V1, were competent
in replication when the corresponding RNAs were transfected into
BHK–21 cells, as evidenced by quantification of virus–specific RNA
and proteins (Figure 2). The results show that a complete sequence of
VPg
3
is not essential for viral replication, but VPg
1
–containing
mutants require additional residues from VPg
3
in their C–terminus
for replication.
Viral proteins, measured by metabolic labelling and Western
blot analysis, were detected with all mutants except V1 (Figure 2A–
C). As expected, all mutant constructs that expressed replication-
competent RNAs gave rise to a P3 polyprotein precursor that
displayed higher mobility than wild type (WT) P3, due to the
deletion of two VPg copies (Figure 2B and 2C). The pattern of
processed P3 proteins in the expression products of V19–4 and
V15–9 supports an efficient processing of the precursor 3BC into
3B and 3C, in agreement with the introduction of a functional 3C
cleavage site in the mutants (Figure 2C). Quantification of total
(intracellular and extracellular) viral RNA collected from cell
cultures transfected with mutant FMDVs yielded 10
4
–10
5
viral
RNA molecules/cell (vRNA/cell) for mutants V3, V15–9 and
V19–4, a value which is 10– to 100–fold lower than that obtained
for FMDV WT (Figure 2D). Mutant V1 showed a viral RNA level
that was 10,000-fold lower than WT (,10
2
viral RNA molecules/
cell), suggesting that replication of V1 is either drastically reduced
or abrogated (Figure 2). It cannot be excluded that the low viral
RNA level detected after transfection with V1 RNA could be a
remnant of the RNA used in the transfection (10
4
viral RNA
molecules/cell), and it confirms the positive replication of the
Figure 1. Schematic representation of the FMDV genome and of
the constructions with one copy of VPg.A, FMDV genome (VPg is the
protein covalently linked to the 59–end of the RNA; PolyC is the internal
polycytidylate tract; IRES is the internal ribosome entry site; A(n) is the
PolyA at the 39–end). The genomic region encoding the viral polyprotein is
boxed. Theviral polyprotein is processed into the different mature proteins
indicated in each corresponding box (based in [15]). Gene 3B (highlighted)
encodes 3 different but related copies of FMDV protein–primer VPg. B,
Amino acid sequence of VPg
1
,VPg
2
and VPg
3
of FMDV C–S8c1. Infectious
FMDV clones were constructed either to express VPg
1
(V1), VPg
3
(V3), a
chimeric VPg consisting of the first 19 residues from VPg
1
(N-terminus) and
the last 4 residues from VPg
3
(C-terminus) (V19–4), or a chimeric VPg
containing the first 15 residues from VPg
1
and the last 9 residues from VPg
3
(V15–9). The starting pMT28 plasmid and procedures for the construction
are detailed in Materials and Methods.
doi:10.1371/journal.pone.0010735.g001
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other mutants. In summary, there is no requirement of a specific
VPg sequence for FMDV viability since the three different VPg
versions confer replication–competence to a similar extent.
However, the presence of a 3C protease cleavage site between
VPg and 3C seems to be needed for viral RNA synthesis.
FMDVs expressing a single VPg lack plaque–forming
capacity
Samples from the cell culture supernatants of BHK–21 cells
transfected with V1, V3 or V19–4 RNAs did not include
detectable viral infectivity in a standard FMDV plaque assay
(,5 PFU/ml), with plaques visualised at 48 hours post–plating
(FMDV WT plaques are readily detectable at 24 hours post–
plating) (Figure 2E). Samples from transfection with V15–9
presented detectable infectivity [8(63)610
2
PFU/ml], albeit
1,000- to 10,000-fold lower than WT [3(62)610
6
PFU/ml].
The absence of plaque development with V3 or V19–4
contrasted with the large amounts of total viral genomic RNA
detected in the same cell cultures (Fig 2D), and with the observed
intracellular levels of viral proteins (Figure 2A–C). The specific
infectivity (SI, ratio between viral titre and the number of viral
RNA molecules) of viruses expressing only one VPg is lower than
1:20,000,000, which is at least 500–fold lower than that of
Figure 2. Replication of mutant FMDVs in BHK–21 cells. A–C, Western blot assays for the specific detection of FMDV proteins using monoclonal
antibody (MAb) 1C8 specific for 2C (panel A), a polyclonal antibody (PAb) specific for 3D (panel B), and MAb 2D2 specific for 3C (panel C). A total of 10
6
BHK–
21 cells were mock–electroporated (BHK lane) or electroporated with either 2 mg of WT RNA or 8 mg of the mutant transcript indicated in each lane. A 4–fold
excess of transcript from mutant plasmids was required to reach a comparable level of viral proteins for WT and mutant RNAs. At 4 hours post–
electroporationcells were collected in lysis buffer, subjected to SDS–PAGE, and then transferred to a nitrocellulose membrane. The MAbs and the PAb have
been previously described[62]. Molecular size markers were run in parallel and their position is indicated on the left. The positions of viral proteins 2BC, 2C,
P3, 3CD, 3BC and 3C, determined with specific MAbs, are indicated [62]. Note that precursor P3 (3ABCD, highlighted with an asterisk) displayed a higher
mobility than WT in all the mutant transcripts, consistent with a decrease of the number of VPg copies. The right panel in C, (lanes V3, WT and V19–4)
correspond to the analysis of cells collected at 5 h instead of 4 h post–transfection, and is included here because of easier band identification. D,Average
number of viral RNA molecules per cell (quantitated both inside the cells and in the culture medium) at 72 hours post–transfection of 2610
6
BHK–21 cells
with 100 ng of the indicated RNA transcripts. The cells and supernatants were harvested at 72 hours post–transfection, and the number of genomic RNA
molecules was calculated by quantitative real–time RT–PCR, as described in Materials and Methods (limit of detection 8 viral RNA molecules/cell, indicated
as a dashed line). E, Viral titre (PFU/ml) in the transfected cultures (both the supernatant and cells subjected to freeze–thawing) described in D. Plaque
assays were performed as described in Materials and Methods. Plaque development was permitted for 48h (limit of detection 5 PFU/ml, indicated as a
dashed line). Measurements in D, E were carried out in triplicate and standard deviations are given.
doi:10.1371/journal.pone.0010735.g002
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FMDV WT (1:40,000). For standard FMDV C–S8c1, the ratio of
infectious particles to total number of physical particles was
previously estimated in 1:7,000 to 1:10,000 [38], and the ratio
decreases when FMDV is subjected to mutagenesis [39]. Thus,
viruses expressing a single VPg are competent for viral protein
synthesis and RNA synthesis, but defective in plaque develop-
ment (Figure 2).
The specific infectivity of mutant FMDVs is gradually
regained upon passage in BHK–21 cells
To investigate whether the capacity of FMDV mutants V1,
V3, V19–4 and V15–9 to form plaques could be restored upon
further virus replication, the virus present in supernatants
collected from cell cultures initially transfected with 100 ng
(series A) or 500 ng (series B) of V1, V3, V19–4 and V15–9 RNA
was serially passaged in BHK–21 cells. At the first passage (p1),
cytopathology was complete in cells infected with V3 and V15–9,
but affected only 10% of the cells infected with V19–4, at 70–
90 hours post–infection (hpi), and no cytopathology was observed
with the supernatants that should contain the progeny of V1
RNA. For FMDV WT, complete cytopathology was observed at
30 hpi, in agreement with previous observations [40]. Infectivity
was detected for all viruses (except for V1) at p1, although mutant
viruses yielded lower titres and SI values than WT. Successive
passages led gradually to a more extensive cytopathology at
shorter times pi, and to increases in viral titre and SI for all
mutants tested, except for V1 (Figure 3). Plaque development by
FMDV on BHK–21 cell monolayers is a reflection of cell
detachment as a manifestation of virus infection. Therefore, in
the assays described here, cytopathology refers as cell rounding
and detachment as a result of the cellular modifications
previously described for BHK–21 cells lytically infected by
FMDV [36,37].
Multiple genomic sites are involved in recovery of the
plaque–forming phenotype
To investigate whether plaque–forming capacity was regained
via adaptive mutations in the viral genome, the entire genomic
consensus sequence [except the genomic 59and 39termini, and
the nucleotides upstream and downstream from of the polyC
tract] of six mutant populations at passage 2 (series A and B for
mutants V3, V19–4 and V15–9) was determined. Although no
mutation was repeated in all populations, amino acid substitution
R55W in non–structural protein 2C was present in populations
V3B, V19–4B and V15–9B (Table 1). This result suggested that
substitution R55W in 2C could be involved in restoring the
plaque–forming phenotype in these three populations. The only
substitution that affected VPg was E8K found in population
V19–4A (Table 1).
Recovery of the plaque–forming phenotype was not associated
with any dominant mutation in two out of six populations at
passage 2 (V3A and V15–9A). To determine whether the mutant
spectrum of V3A included genomes with the same mutations
found in the other lineages, the P2–P3–coding region of virus
isolated from 5 individual plaques derived from population V3A at
passage 1 was sequenced. Each clone included non–synonymous
mutations in 2C and/or 3A, and two clones presented replace-
ment R55W in 2C (Table 1). The results suggest that plaque–
development in the VPg mutants can be attained by any of a
number of substitutions in non–structural proteins 2C, 3A and/or
VPg. Since R55W in 2C was found in several clones and
populations, its effect on FMDV progeny production and
cytopathology was further investigated.
2C substitution R55W increases the specific infectivity of
FMDV expressing a single VPg
To determine whether substitution R55W in 2C could restore
the plaque–forming capacity of FMDV mutants with alterations in
3B (FMDV mutants V3, V19–4 and V15–9, Figure 1), the
corresponding mutation was introduced in each of the mutant
constructs that express one VPg. All mutants regained the plaque–
forming phenotype (Figure 4A) that resulted in 17– to 190–fold
increase of specific infectivity of each viral RNA transcript
(Figure 4B). To minimize the emergence of adaptive mutations
during plaque development, serially diluted viral RNA transcripts
were transfected into cells, and plaque development was permitted
under semisolid agar medium. Plaques were formed by all mutants
expressing only one VPg and 2C with R55W, and they were
visible at 3 days post–transfection. No plaques were formed by
their RNA counterparts expressing wild type 2C, under the same
conditions. By 2 days post–transfection, plaques were already
detected for V15–9 and V3 with R55W in 2C, but not for V19–4
with R55W in 2C (Figure 4). Thus, 2C substitution R55W is
sufficient to restore the plaque–forming capacity of the mutants
tested that express one VPg.
R55W in 2C increases cytopathic effect and results in
enhanced viral release from cells
To investigate the effect of R55W 2C in FMDV replication,
viral RNA and viral protein levels of FMDVs expressing 2C with
either R55 (wild type) or W55 (adaptive substitution) were
quantified at different times post–electroporation. Positive RNA
replication was observed for all mutants (V3, V19–4 and V15–9
with or without R55W in 2C) with RNA levels reaching an
amount at least 10-fold higher than levels quantified at the time of
electroporation (Figure 5A). To ascertain the presence of newly
synthesised viral RNA molecules, the background RNA value
scored at the time of the electroporation was subtracted from each
of the values obtained at different times after electroporation. The
results (Figure 5A) indicate that the presence of R55W in 2C of the
FMDV mutants increased the amount of extracellular viral RNA
without any significant increase in the total viral RNA levels
(intracellular plus extracellular). The ratio of extracellular to total
viral RNA molecules was estimated to be in the range of 0.1 to 0.4
for mutants encoding R55W in 2C, and 0.03 to 0.04 for mutants
encoding wild type 2C (Figure 5B).
The selective increase of extracellular RNA suggests increased
exit of viral particles. This implies that higher extracellular
concentrations of capsid proteins should be found for mutants
expressing 2C with R55W than for the same mutants expressing
wild type 2C. This was confirmed by determining extracellular
levels of capsid proteins VP1 and VP3. First, the synthesis of
capsid proteins VP1 and VP3 was ascertained in the electropo-
rated cells by pulse-labeling with [
35
S] Met-Cys at 3 h to 4 h post-
electroporation. The result (Figure 5C) revealed the presence of
VP1 and VP3 in the cells infected with mutants V15-9 and V3,
with or without R55W in 2C (excluding the cell culture
supernatant for the analysis). In contrast, when the extracellular
levels of VP1 and VP3 were examined at early times post-
electroporation, they were barely detectable for the mutants that
expressed 2C with R55 (Figure 5D). The difference tended to
diminish when the pulse-labeling was extended for several hours
post-electroporation (Figure 5D).
A standard cell killing assay previously developed for FMDV in
BHK-21 cells [40,41] could not be directly applied to measure the
effect of substitution R55W in 2C to mutants V19-4, V15-9 and
V3 because infectious (cell-detaching) particles could not be
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produced in sufficient amounts. However, the cell killing assay was
applied to wild type FMDV expressing either wild type 2C or 2C
with R55W and the results showed 8- to 20-fold increases of
cytopathology associated with substitution R55W in 2C
(Figure 6A).
In other assays, the number of detached cells versus the total
number of cells was measured following electroporation with V3,
V19–4 and V15–9 RNAs, expressing either 2C wild type or 2C
with R55W, as a means of circumventing the requirement of
infectious virus particles. The results (Figure 6B) show that the
proportion of cells detached was 6263%, 7966% and 72610%
in transfections with mutants V19–4, V15–9 and V3 encoding
R55W in 2C, respectively, while these values were 26611%,
45610% and 5863% in transfections with the same mutants
encoding the wild type 2C, and this difference was statistically
significant (p = 0.0002; Student’s t–Test). To prevent multiple–step
growth conditions that may have affected the interpretation of
results, the experiment was carried out by using 5 mg of viral RNA
for 10
6
BHK-21 cells, a sufficient amount to ensure that more than
90% of the cells were efficiently transfected Therefore, substitution
R55W in 2C favors both release of viral particles and
cytopathology.
Figure 3. Recovery of infectivity of FMDV mutants expressing one VPg, upon serial passages in BHK–21 cells. 2610
6
BHK-21 cells were
transfected with 100 ng (series A) (panels A, B, C) or 500 ng (series B) (panels D, E, F) of the FMDV transcripts indicated in the boxes. Cells and cell
culture supernatants were collected at 72 h post-transfection (passage 0). Successive passages were carried out by infecting BHK–21 cells with the
viruses from the cell culture supernatant of the previous passage, and samples were collected when cytopathology was complete (generally 20 to
30 hours pi). A,D, Viral infectivity as a function of passage number; plaque development was for 48 hours. B,E, Number of genomic RNA molecules
in the cell culture supernatants. C,F, Specific infectivity calculated as the number of PFU per 10
10
viral RNA genomes, from the data given in A, B and
D, E. Measurements were carried out in triplicate and standard deviations are given. Procedures for titration of infectivity and determination of viral
RNA levels are described in Materials and Methods.
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Further analyses with FMDV mutants expressing one VPg
The major defect of FMDV mutants V19-4, V15-9 and V3 that
express a single VPg is the absence of cytopathology associated
with limited release from cells with no impairment of intracellular
viral RNA levels. To test whether other viral functions might also
be affected in these mutants, cell entry and intracellular viral
protein synthesis were also examined.
To determine whether mutants expressing a single VPg could
also manifest a defect in cell entry, the capacity of mutants V19-4
and V3 expressing either wild type 2C or 2C with R55W to enter
cells was determined. For this purpose, viral samples were
collected from cell cultures at 4 hours post–transfection by
freeze-thawing the cells to liberate virus, and then applied to a
BHK–21 cell monolayer. The input FMDV RNA applied to the
monolayer and the FMDV RNA that entered BHK-21 cells were
quantitated by real time RT-PCR. The proportion of viral RNA
released from transfected cells that entered new cells was
3.060.3% for V19-4 and 3.562.0% for V3 that expressed wild
type 2C; the corresponding values were 2.661.3% and 3.061.2%
for the same mutants that expressed 2C with R55W. These data
suggest that defects in plaque-forming capacity observed are not
due to a defect in viral entry once the mutant FMDvs have been
released from the cells.
To compare intracellular protein synthesis directed by the mutant
FMDVs, proteins were metabolically labeled between 3 and 4 hours
post–electroporation. BHK–21 cells infected with mutants V3, V19–
4 and V15–9 encoding R55W in 2C presented modestly higher levels
of viral proteins than the same mutants expressing a wild type 2C
(Figure 7). Therefore, a limitation in protein synthesis cannot be
related to a defect in viral release from cells.
Substitution E8K in VPg contributes also to increased
viral release in mutant viruses expressing one VPg
Mutant V19–4 (R55W) showed a delayed plaque formation
relative to other mutants encoding R55W in 2C (Figure 4). To
investigate whether additional replacements could accelerate
plaque formation in this virus, populations V19–4A and V19–4B
at passage 2 (depicted in Table 1 and Figure 3) were subjected to
25 additional passages in BHK–21 cells. E8K in VPg
1
was the only
replacement repeatedly found in the two V19–4 lineages at
passage 27 (Table 2). Interestingly, R55W in 2C became dominant
during the 25 additional passages in lineage V19–4A in which
R55W was not present at passage 2 (compare Tables 1 and 2).
However, in lineage V19–4B W55 was dominant at passage 2 but
it had reverted to R55 by passage 27. This reversion was
accompanied by dominance of substitution I85V in 2C (Table 2).
Other amino acid replacements were identified in other viral
proteins of passage series A and B (Table 2).
Since R55W in 2C and E8K in VPg
1
were found together in
V19–4A at passage 27, the effect of both substitutions together in
the same genome in the sequence context of V19–4 was
investigated. The double mutation restored plaque–forming
capacity, with minute plaques detected at 48 h post–transfection
(Figure 8A). The specific infectivity of V19-4 increased 10- to
100-fold as a result of expressing 2C with R55W alone or
together with VPg with E8K (Figure 8B). The extracellular viral
RNA was 5– to 11–fold higher for V19–4 (R55W,E8K) than for
either V19–4 or V19–4 (R55W) (Figure 8C), and this difference
was statistically significant (P,0.0001; unpaired t–Test). Meta-
bolic labelling of cells transfected with V19–4 (R55W,E8K) RNA
revealed that viral protein synthesis was increased 2.2– to 4.4–
fold relative to V19–4 or V19–4 (R55W). Protein levels were 1.6–
fold higher for V19–4 (R55W,E8K) than for wild type FMDV
(Figure 8C). These measurements suggest that viral release into
the extracellular medium was enhanced by substitution E8K in
VPg.
In summary, FMDV expressing only one VPg is defective in
cell–to–cell propagation and this defect is reverted by an increase
in viral release, concomitantly with restoration of cytopathology,
mediated by substitutions in 2C, VPg or other non-structural
proteins.
Table 1. Substitutions found in FMDV mutants that encode one VPg, upon passage in BHK–21 cells
a
.
Genomic region
b
Mutation
c
Amino acid
substitution
c
Presence in biological
clones from V3A at passage 1
d
Presence in uncloned
population at passage 2
d
LA1244G D69G — V19–4A
A1292U Q85L — V3B
2C C4507U
e
R55W
e
c1, c4
e
V19–4B; V15–9B; V3B
e
C4778U T135I c2 —
A4988G K215R — V15–9B
3A A5422C I42L c3, c5 —
A5609C Q104P c4 —
VPg
1
-coding region G5779A E8K — V19–4A
3C C6060U — c1 —
3D
pol
A7487G E293G c1 —
a
The consensus sequence of the entire viral genome was determined for FMDV populations V3, V19–4 and V15–9 (series A and B) after 2 passages in cell culture
(experiment described in Figure 3). The sequence of viral genomic regions 2C, 3A, 3B (VPg), 3C and 3D (residues from genomic position 4200 to 8115) was determined
for 5 independent biological clones isolated from population V3A at passage 1. The origin of each population is described in the text and in Figure 3.
b
FMDV genomic region analysed [15].
c
Mutations and deduced amino acid substitutions are relative to the sequence of the parental clone FMDV WT (pMT28, described in Materials and Methods); residue
numbering is according to [49]. Amino acid residues (single–letter code) are numbered individually for each protein from the N– to the C–terminus. Procedures for
nucleotide sequencing and identification of FMDV genomic regions are described in Materials and Methods.
d
Populations and biological clones in which the substitutions indicated in
c
were found.
e
Mutation C4507U (that corresponds to amino acid substitution R55W in 2C) was repeatedly found in 3 out of 6 mutant FMDV populations at passage 2, and 2 outof5
biological clones from population V3A at passage 1 (underlined).
doi:10.1371/journal.pone.0010735.t001
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Discussion
Two features of RNA virus evolution are relevant to the
interpretation of the results with VPg mutants of FMDV reported
here. One is the multifunctional nature of many (probably most)
proteins encoded in the compact RNA genomes, particularly in
the case of picornaviruses in which several intermediates obtained
in the processing of the polyprotein play essential roles in the virus
life cycle. In the present study, multifunctionality was manifested
by the participation of 2C in the compensation of a defect in the
release from cells of FMDVs that do not express the 3 VPg
versions encoded by wild type FMDV [42]. Such compensation
suggests a role of 2C in functions additional to those described for
this non–structural protein [20,21,24-27,43]. The second relevant
feature of RNA virus evolution is the functional connection among
different genomic regions, in this case between VPg and other
non–structural proteins, regarding expression of phenotypic traits
[44].
The results reported here indicate that the replication-
competent FMDV mutants that expressed a single VPg were
deficient in plaque formation, a defect related to the inability to
cause cytopathology. Cell rounding and detachment from the
monolayer precede cell death. The mechanism (apoptosis,
necrosis, autophagy) by which FMDV kills BHK-21 cells in cell
culture has not been elucidated [45], and our attempts to confirm
or exclude apoptosis of BHK-21 cells following infection by
FMDV have not been conclusive (C. Perales, unpublished
observations). We have observed that the first step towards cell
death –as measured either by trypan blue staining or by
fluorescence-activated cell sorting (FACS) using propidium iodide
that gave equivalent quantifications [46]– is cell rounding and
detachment of the monolayer. The capacity to produce cytopa-
thology was rapidly restored upon passage of the mutant viruses in
BHK-21 cells, that acquired amino acid substitutions in non-
structural proteins, including R55W in 2C. The results suggest
that substitution R55W in 2C permitted FMDVs that expressed a
single VPg to attain a critical number of viral particles released
from cells, that are associated with cytopathology. An approximate
value of 5 infectious particles released per BHK–21 cell was
estimated as the minimum number needed to develop a plaque
under our experimental conditions (Table 3). This is the number
of particles released per cell estimated for mutant V19–4 (R55W)
Figure 4. Restoration of the plaque–forming phenotype in FMDV mutants by replacement R55W in 2C. RNA transcripts (10 ng for
48 hours of plaque development and 1 ng for 72 hours plaque development) were transfected into 2610
6
BHK–21 cells. At 2 hours post–transfection
cell culture supernatants were removed and the cells were overlaid with semisolid agar medium. A, Plaque assay of directly transfected FMDV RNA
transcripts encoding either wild type 2C (left) or 2C with substitution R55W (right). For mutants V15–9, V19–4 and V3, plaques were detected when
2C included substitution R55W. WT(0.2x) represents transfections with a fifth of the amount used for the rest of the assays. Plaques for mutant V19–4
(R55W) were not detected at 48 hours post–transfection. V3 formed some plaques at 72 hours post–transfection but not at 48 hours post–
transfection. The number of plaques formed by V3 was 18–fold lower than the number formed by V3 encoding R55W in 2C. B, Specific infectivity
measured as the ratio of the number of PFU at 72 hours post–transfection to the amount of viral transcript used in the electroporation. Assays were
carried out in triplicate, and standard deviations are given. Procedures for plaque assays of transfected cells are described in Materials and Methods.
doi:10.1371/journal.pone.0010735.g004
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Figure 5. Effect of replacement R55W in 2C in the release of FMDV RNA from cells. 10
6
BHK–21 cells were electroporated with 8 mgof
either V19–4, V3, V15–9 or WT RNAs. Then the cells were transferred to M24–wells. A, Total viral RNA (intracellular and extracellular) and extracellular
RNA were quantified by real–time RT–PCR, at the indicated hours post-electroporation (hpe). The amount of FMDV RNA was normalised to the
number of cells seeded in the corresponding wells. The viral RNA (total and extracellular) measured just after electroporation was subtracted from
each corresponding value. Note that RNAs encoding R55W in 2C are significantly more efficient in promoting release of viral RNA from cells at
6 hours post–transfection than those expressing wild type 2C (p,0.05; paired t–Test). B, Ratio of FMDV RNA released from the cells (ratio of
extracellular viral RNA to total RNA) at 6 hours post–electroporation, determined from the data shown in A. Assays in A and B were carried out in
triplicate and standard deviations are given. C, Detection of capsid proteins VP3 and VP1 in mutant FMDVs expressing 2C with or without
replacement R55W. 10
6
BHK–21 cells were electroporated with 8 mg of viral RNA. At 3 hours post–electroporation, supernatants were removed and
DMEM without Met and Cys, but supplemented with [
35
S] Met–Cys, was added. At 4 hours post–electroporation, supernatants were removed and
cells were collected to analyse the synthesis of the viral capsid proteins VP3 and VP1. D, Extracellular samples of cell cultures electroporated with the
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which is near the threshold of not being competent in plaque
development; the plaques formed by this virus were visible only at
late times post–transfection. The functional basis for this critical
number is not known.
Residue 55 of 2C [and also residue 85, substituted in the R55W
revertant of V19–4B (see Results)] is located in the region
comprised between the expected membrane-binding domain and
the predicted NTPase/helicase domain A, and it is a variable
residue when 2Cs of different picornaviruses are aligned. Positions
55 and 85 have not been identified as related to resistance to
guanidine in FMDV [47,48] and therefore, from the data
presently available no connection between 2C positions 55 and
85 and FMDV replication is apparent. It cannot be excluded that
the established role of 2C in cellular membrane rearrangements
during picornaviral infection [27–31] might be connected with its
effect in virus release and cytopathology.
With the assumption that cytopathology by FMDV precedes
cell death, several determinants of BHK–21 cell killing were
previously mapped in the IRES, the viral capsid, and non–
structural proteins of the virus [40,49,50]. A lack of correlation
between replicative fitness and BHK–21 cell killing capacity was
unveiled by analysis of the behavior of chimeric FMDVs. While
fitness determinants were scattered along the genome, cell killing
determinants were concentrated in several specific genomic
regions, including 2C [40]. Three substitutions in 2C (S80N,
T256A and Q263H) enhanced BHK–21 cell killing. Since these
substitutions are distant from substitution R55W that multiple sites
in 2C are related to FMDV cytopathology.
The molecular basis of the increased cytopathology observed for
mutants that encode 2C with substitution R55W remains unknown.
Since a modest increase in viral protein synthesis in these mutants is
observed despite no increase in viral RNA levels, an appealing
hypothesis is that 2C has some involved in translation, and that
R55W might enhance protein synthesis modestly but to a level
sufficient to contribute to enhanced cytopathology. The effect of
substitution R55W could be exerted during polyprotein processing,
by rendering 2C more sensitive to polyprotein cleavage mediated by
3C. Distance effects of amino acid substitutions on FMDV
poplyprotein processing have been previously documented [51].
Other interpretations are possible. For example, since 2C is a
multifunctional protein [20–31], substitution R55W may alter virus-
host interactions that lead to enhanced cytopathology, being the slight
increase of protein synthesis an indirect consequence of alterations in
other viral functions.
Substitution E8K was also found associated with the one-VPg
mutants that regained capacity to cause cytopathology. The crystal
structure of the complex between 3D and VPg predicts that E8
participates directly in the interaction with the viral polymerase.
Although E8 is far from the UMP residue bound to VPg (covalent
bond Tyr3-UMP), substitution E8A has been shown to affect
drastically the uridylylation of VPg by 3D [52]. The molecular
basis of the phenotypic effect of VPg replacement E8K remains
unknown.
Despite the fact that the behavior of the one-VPg FMDV
mutants studied here suggests a functional connection between 2C
and VPg in FMDV, to our knowledge a direct interaction between
the two proteins has not been reported in picornaviruses.
Nevertheless, PV 2C interacts with the VPg precursor 3AB (3B)
[33]. In an FMDV–infected cell, a possible 3AB–2C interaction
could be mediated by more than one VPg copy, and the alteration
indicated mutant FMDV RNAs, labelled as described in C, were collected at 2 (0-2 hours) and 20 (2-24 hours) hours post–labelling (6 and 24 hours
post–electroporation). Samples were analysed by SDS–PAGE (15% acrylamide). VP3 and VP1 were detected at early time (4 to 6 hours post–
transfection) in the supernatant of cells transfected with 8 mg of V15–9 or V3 encoding 2C with R55W, but not with the same amount of RNA from
constructs encoding wild type 2C. At later times (6 to 24 hours post–transfection), VP3 and VP1 were detected in the supernatant of cells transfected
with any of the constructs tested. Procedures are further detailed in Materials and Methods.
doi:10.1371/journal.pone.0010735.g005
Figure 6. Effect of replacement R55W in 2C on FMDV cytopathology. A, BHK-21 cell-killing assay [40] carried out with either FMDV WT
(expressing three VPgs) with the standard 2C (abbreviated as WT) or FMDV WT with substitution R55W in 2C (abbreviated as WT R55W). Serial
dilutions of either FMDV WT or FMDV R55W were applied to wells containing 10
4
BHK-21 cells each. The plot depicts the minimum MOI (PFU/cell)
required to cause complete cytopathology (detachment of all cells from the monolayer) at the indicated hours post-infection, as previously described
[40]. Results are the average of four independent determinations. Note that the presence of R55W in 2C decreases the MOI needed to cause complete
cytopathology for a given time. B, Quantification of cytopathology (percentage of cells detached from the monolayer) in cells with RNA from wild
type (WT) or mutants V19-4, V15-9 or V3 expressing either wild type 2C or 2C with substitution R55W. At 8 h post-electroporation the total number of
cells and the number of detached cells were counted; a correction was introduced to account for the number of cells that were detached due to the
electroporation by parallel measurements with mock-electroporated cells (around 50% of cell detachment). Results are the average of three
determinations.
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Figure 7. Viral proteins expressed by mutant FMDVs encoding either wild type 2C or 2C with R55W. A, Electrophoretic analysis of
proteins labelled with [
35
S] Met–Cys. 10
6
BHK–21 cells were electroporated with 2 or 8 mg of the indicated viral RNAs; At 3 hours post–
electroporation, supernatants were removed and DMEM without Met and Cys but supplemented with [
35
S] Met–Cys was added. One hour later, cell
culture supernatants were removed and cells were collected in loading buffer, proteins were resolved in SDS–PAGE (15% acrylamide), and the gel was
dried and exposed. Viral proteins, identified by their reactivity with specific MAbs [62], are indicated with an asterisk. B, Close-up view of the bands in
A corresponding to 3CD and 3BCD precursors. Wild type FMDVs expressing 3 copies of VPg show three precursors of lower mobility than 3CD that
may correspond to 3BCD, 3BBCD and 3BBBCD, respectively. Mutant FMDVs expressing one VPg show only one precursor above 3CD that may
correspond to 3BCD. C, Western blot analysis of samples described in A. Proteins were visualised with a specific polyclonal antibody against FMDV 3D
[62]. Molecular size markers were run in parallel and their position is indicated on the left. The positions of viral proteins P3, 3CD, and 3D are indicated
[62]. Note that precursor P3 (3ABCD) from the mutant transcripts expressing one VPg displayed a higher mobility than WT P3. A band above 3CD was
detected in some lanes (indicated with an asterisk) that may correspond to precursor 3BCD in V19–4 R55W and to precursors 3BCD, 3BBCD and/or
3BBBCD in FMDV WT, with or without R55W in 2C. D, Proportion of viral polymerase 3D (as percentage of the value obtained in the electroporation
with 8 mg of FMDV WT transcript), measured by densitometry of electropherograms as that shown in A. Values are the average of triplicate protein
analyses of independent labelling experiments, and standard deviations are given. WT (0.25x) indicates transfections with 2 mg of WT. The assignment
of viral proteins was based on reactivity with specific monoclonal antibodies [39,62,66]. Procedures are described in Materials and Methods.
doi:10.1371/journal.pone.0010735.g007
Table 2. Mutations in populations V19–4A and V19–4AB after 27 passages in BHK–21 cells
a
.
Genomic region
b
Mutation
c
Amino acid substitution
c
V19–4 population
d
LU1103C L22P V19–4B
A1244G D69G V19–4A
VP1 A3649G T148A V19–4B
C3650C/U T148M V19–4A
C3653A/C T149K V19–4A
2C C4507U R55W V19–4A
U4507C
e
reversion to R55
e
V19–4B
e
A4597G I85V V19–4B
3A A5573G E92G V19–4B
VPg
1
-coding region G5779A
f
E8K
f
V19–4A; V19–4B
f
3C U6534A — V19–4B
3D U7341C/U — V19–4A
a
The consensus sequence of the entire viral genome was determined for FMDV populations V19–4A and V19–4B after 27 passages in cell culture.
b
FMDV genomic region analysed [15].
c
Mutations and deduced amino acid substitutions are relative to the sequence of the parental clone FMDV WT (pMT28, described in Materials and Methods); residue
numbering is according to [49]. Amino acid residues (single–letter code) are numbered individually for each protein from the N– to the C–terminus. Two residues
separated by a bar indicate a mixture of two nucleotides in the population, according to the sequence chromatogram pattern. Procedures for nucleotide sequencing
and identification of FMDV genomic regions are described in Materials and Methods.
d
Populations in which the substitutions indicated in
c
were found.
e
Mutation C4507U (that corresponds to amino acid substitution R55W in 2C) was dominant in V19–4B at passage 2 (Table 1) and had reverted by passage 27
(highlighted in italics).
f
Substitution E8K (G5779A) was found repeated in population V19–4 at passage 27 in series A and B (underlined).
doi:10.1371/journal.pone.0010735.t002
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in the number of VPg copies or their amino acid sequence could
perturb these 2C–3AB interactions. In addition to the replace-
ments in 2C and 3B, substitutions in 3A were also found in
populations and biological clones that regained the plaque–
forming phenotype upon passage in BHK–21 cells, supporting
that recovery of this phenotype can be mediated by any of a
number of readjustments in the interaction among several non–
structural proteins (Tables 1 and 2).
The present investigation has established non–structural
proteins 2C, 3A and VPg as key determinants for modulating
cytopathology in cell culture. For several picornaviruses it has
been shown that replacements either in 2C or 3A can be
associated with modifications of virulence, cell tropism or host
range [40,53–57]. Altogether, these data highlight the role of
non–structural proteins in the adaptability to changing environ-
ments during picornavirus infections, with clear implications for
viral pathogenesis.
Materials and Methods
Plasmids, cells and viruses
pMT28 is a pGEM–1 plasmid (Promega) that contains the
complete cDNA genomic sequence of FMDV serotype C (FMDV
WT: C-S8c1 as described in [41,58,59]). To produce infectious
transcripts, pMT28 was linearised with NdeI (NEB), and
transcribed with SP6 polymerase (Promega), following described
procedures [60,61]. Mutant plasmids encoding only one VPg copy
were constructed by mutagenic PCR with primers harboring the
desired deletion (overlapping upstream and downstream sequences
of the region to be deleted). Two amplifications were made and
then the DNA products were shuffled to introduce the deletion.
The first amplification was performed with a forward primer
spanning residues 3988 to 4009 and a mutagenic reverse primer
spanning either residues 5980–5971 and 5826–5804 (to obtain the
deletion of 5827 to 5970, mutant V1), or residues 5910–5899 and
Figure 8. Effect of replacement E8K in VPg on the specific infectivity of V19–4 (R55W). A, Plaque assay with cells transfected with RNA
transcripts of WT or the indicated mutant constructs. At 2 hours post–transfection, the cell culture supernatants were removed and the cells were
covered with semisolid agar. Plaques were visualized either at 48 or 72 hours post–transfection. Note that substitutions R55W in 2C and E8K in VPg
enhanced the plaque-forming capacity of V19–4. B, Specific infectivity (calculated as the ratio of the number of PFUs obtained to the ng of RNA used
in the electroporation) for WT and the indicated V19–4 mutants. C, Total and extracellular FMDV RNAs measured at 6 hours after electroporation of
BHK–21 cells with WT and the indicated V19–4 mutant RNAs. D, Amount of viral polymerase (3D) determined by metabolic labelling after
electroporation of BHK–21 cells with the indicated RNAs, and densitometry of the relevant ptotein band. The results are expressed as percentage of
the amount measured following electroporation with WT RNA, which is taken as 100%. Values in B, C, D are the average of 3 determinations; standard
deviations are given. Procedures are described in Materials and Methods.
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5757–5735 (deletion of 5758–5898, mutant V3), or residues 5977–
5959 and 5814–5792 (deletion of 5815–5958, mutant V19–4), or
residues 5955–5944 and 5802–5781 (deletion of 5803–5943,
mutant V15–9). The second amplification was performed with an
antisense primer spanning either residues 7160 to 7141 and a
mutagenic forward primer spanning residues 5816–5826 and
5971–5992 (to obtain the deletion of 5827 to 5970, mutant V1), or
residues 5746–5757 and 5899–5912 (deletion of 5758–5898,
mutant V3), or residues 5804–5814 and 5959–5992 (deletion of
5815–5958, mutant V19–4), or residues 5782–5802 and 5944–
5958 (deletion of 5803–5943, mutant V15–9). Each pair of
amplification products was shuffled by PCR amplification with
internal primers spanning residues 4189 to 4211 (sense) and 7019
to 6997 (antisense). The mutagenised PCR products were inserted
into pMT28, previously digested with BglII and ClaI (located at
positions 4199 and 7003, respectively), by recombination using the
In–Fusion Dry and Down Mix kit (Clontech).
For the construction of mutants encoding R55W in 2C (that
correspond to mutation C4507U) two additional cloning sites, NotI
(position 5412) and NsiI (position 6393), were introduced into
pMT28, leading to plasmid pMT30. We designed mutagenic
primers that introduced substitutions C5412G and C5418G (NotI
site) and G6393A (NsiI site), without affecting the coding sequence.
To obtain plasmid FMDV WT (R55W), the resulting pMT30
plasmid was digested with BglII (position 4199) and NotI (position
5412). RT–PCR amplification of RNA from population V19–4A
at passage 27, that encodes 2C with R55W, was performed with
primers spanning residues 4189 to 4211 (sense) and 5432 to 5392
(antisense). The amplification product was introduced into plasmid
pMT30 by recombination using the In–Fusion Dry and Down Mix kit
(Clontech).
Mutant FMDVs encoding one VPg and 2C with R55W were
constructed by amplification of the mutant plasmids described above
(V3, V19–4, V15–9) with primers spanning residues 5403 to 5442
(sense) and 6412 to 6374 (antisense). PCR amplification products
were inserted into plasmid FMDV WT (R55W) previously digested
with NotIandNsiI (positions 5412 and 6393, respectively) by
recombination with In–Fusion Dry and Down Mix kit (Clontech).
Transfection of viral RNA transcripts into BHK-21 cells
Viral replication of each mutant FMDV was analysed by
transfection of the corresponding transcript into BHK-21 cells. For
this purpose we used both lipofection and electroporation
methods. Lipofection was used to obtain mutant viral samples
that were then passaged on BHK–21 cell monolayers, and also in
plaque assays of viral transcripts. Electroporation was used to
detect intracellular FMDV protein synthesis, and in the quanti-
fication of intracellular FMDV RNA synthesis relative to viral
RNA release. Lipofection and electroporation were carried out as
previously described [41,62].
Extraction of RNA, cDNA synthesis, PCR amplification,
and nucleotide sequencing
RNA was extracted from the supernatants of infected cells by
treatment with Trizol (Invitrogen) as previously described [63].
Reverse transcription (RT) was carried out using avian myelo-
blastosis virus reverse transcriptase (Promega) or Transcriptor
reverse transcriptase (Roche), and PCR amplification was
performed using EHF DNA polymerase (Roche) as specified by
the manufacturer. PCR amplifications to obtain mutant infectious
clones were carried out using Pfu Turbo DNA polymerase
(Stratagene) because of its high copying fidelity [64]. Nucleotide
sequencing was carried out as described [61].
Plaque assays of viral samples
Plaque assays were performed as previously described [65].
Serial dilutions of viral samples were applied to confluent BHK–21
cell monolayers and incubated for 1 h at 37uC. Then, superna-
tants were removed and semisolid agar medium was added.
Plaque development was permitted for 48 h post-infection.
Plaque assay of directly transfected infectious RNA
transcripts
Serial dilutions of in vitro transcribed infectious RNA (10, 1 and
0.1 ng) were mixed with lipofectin (Invitrogen) as indicated by the
manufacturer. The mixture was added to a monolayer of 2610
6
BHK–21 cells and incubated for 2 h at 37uC. Then, the medium
was removed, the cell monolayer was washed with DMEM and
semisolid agar medium was added. Plaque development was
permitted for either 48 or 72 h.
Quantification of viral RNA in cell culture samples
Quantitative real time RT–PCR was carried out using the Light
Cycler RNA Master SYBR Green I kit (Roche), according to the
instructions of the manufacturer. Oligonucleotides spanning
residues 3175 to 3194 (sense orientation) and 3518 to 3496
(antisense orientation), or 4924 to 4944 (sense) and 5026 to 5047
(antisense) were used in the amplification. Quantification was
Table 3. Relative amounts of mutant FMDV RNA released
from cells
a
.
Electroporated
FMDV
transcript
b
Viral RNA
released (%)
c
Calculated number of
infectious particles
released per cell
d
Plaque-
forming
capacity
e
WT 100648 34630 Yes
WT R55W 356665 121 Yes
V19–4 7612 No
V19–4 R55W 14610 5 At 72 hpt
V15–9 5612 No
V15–9 R55W 32615 11 Yes
V3 9653 No
V3 R55W 79630 27 Yes
a
10
6
BHK–21 cells were electroporated with 8 mg of FMDV RNA, and the amount
of extracellular FMDV RNA at 6 hours post–electroporation was quantitated as
detailed in Materials and Methods.
b
Mutant FMDVs analysed. Their construction is described in Materials and
Methods, and depicted in Figure 1; R55W indicates the presence of
replacement R55W in 2C.
c
Viral RNA released per cell for the indicated transcripts. Values are expressed as
the percentage of the amount of RNA released per cell in the transfections
with FMDV WT. Each value is the average of at least three determinations;
standard deviations are given. Relative viral RNA released per cell in mutant
FMDVs defective in plaque–forming capacity is below 20% of FMDV WT
(calculated from data shown in Figure 5).
d
Calculated number of infectious particles released per cell for wild type and
mutant FMDVs. An average number of 34 PFU are released per cell in
infections with FMDV WT (data in Figure 3). The calculation assumes that one
FMDV WT infectious particle is equivalent to one PFU, and that the number of
infectious particles released by each mutant virus correlates with the viral RNA
released.
e
Plaque–forming capacity of mutant FMDV transcripts (data in Figure 4). Yes
indicates the ability of mutant transcripts to develop plaques in 48 h. No
indicates that no plaques were observed at 72 hours post–electroporation.
Plaque development with V19–4 R55W was only observed at 72 hours post–
electroporation. Procedures for plaque assays of viral transcripts are detailed in
Materials and Methods.
doi:10.1371/journal.pone.0010735.t003
Cell-Killing Determinants
PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10735

relative to a standard curve obtained with known amounts of
FMDV RNA, synthesised by in vitro transcription of the infectious
plasmid pMT28 [41]. For the quantification of extracellular
FMDV RNA, the supernatants were collected, freed from
detached cells by centrifugation, and extracted with Trizol
(Invitrogen).
Metabolic labelling of viral proteins
To detect viral protein synthesis in transfected cells, 2 to 8 mgof
viral transcript were electroporated into 2610
6
BHK–21 cells.
Electroporated cells were incubated for 3–4 hours in culture
medium (DMEM, 1% FCS). Then, the medium was removed and
cells were incubated for 1 h at 37uC in DMEM without Met and
Cys, but supplemented with [
35
S] Met–Cys (Perkin–Elmer)
(400 mCi/mmol). After metabolic labeling, cells were collected
in 0.1 ml of sample buffer [160 mM Tris–HCl pH 6.8; 2% SDS;
11% glycerol; 0.1 M DTT, 0.033% bromophenol–blue]. The
samples were heated at 90uC for 5 min and aliquots were
subjected to SDS–PAGE (15% acrylamide).
Western blot assays
Proteins were transferred to a 0.45 mm pore size nitrocellulose
membrane (BioRad). Western blots were developed with the
following antibodies at a dilution of 1:2,000: mouse monoclonal
anti–2C (1C8) and anti–3C (2D2) (a gift from E. Brocchi, Istituto
Zooprofilattico Sperimentale della Lombardia e dell’Emilia
Romagna, Brescia, Italy) and rabbit polyclonal anti–3D. Goat
anti–rabbit IgG antibody coupled to peroxidase and goat anti–
mouse IgG antibody coupled to peroxidase (Pierce) were used at
1:10,000 dilutions. Each sample was analysed by Western blot to
identify virus–specific proteins, following previously described
procedures [62].
Acknowledgments
We are indebted to E. Brocchi for the generous supply of MAbs, and to A.
I. de A
´vila and E. Garcı
´a–Cueto for expert technical assistance.
Author Contributions
Conceived and designed the experiments: AA CE ED. Performed the
experiments: AA CP. Analyzed the data: AA CP CE ED. Contributed
reagents/materials/analysis tools: AA CP CE ED. Wrote the paper: AA
ED.
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