Evolution and Structural Organization of the C Proteins of Paramyxovirinae

Abstract

The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus. We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, α-helical C-terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.

Full text

Evolution and Structural Organization of the C Proteins
of
Paramyxovirinae
Michael K. Lo
1.
, Teit Max Søgaard
2.
, David G. Karlin
2,3
*
1Centers for Disease Control and Prevention, Viral Special Pathogens Branch, Atlanta, Georgia, United States of America, 2Division of Structural Biology, Oxford
University, Oxford, United Kingdom, 3Department of Zoology, University of Oxford, Oxford, United Kingdom
Abstract
The phosphoprotein (P) gene of most Paramyxovirinae encodes several proteins in overlapping frames: P and V, which share
a common N-terminus (PNT), and C, which overlaps PNT. Overlapping genes are of particular interest because they encode
proteins originated de novo, some of which have unknown structural folds, challenging the notion that nature utilizes only a
limited, well-mapped area of fold space. The C proteins cluster in three groups, comprising measles, Nipah, and Sendai virus.
We predicted that all C proteins have a similar organization: a variable, disordered N-terminus and a conserved, a-helical C-
terminus. We confirmed this predicted organization by biophysically characterizing recombinant C proteins from Tupaia
paramyxovirus (measles group) and human parainfluenza virus 1 (Sendai group). We also found that the C of the measles
and Nipah groups have statistically significant sequence similarity, indicating a common origin. Although the C of the
Sendai group lack sequence similarity with them, we speculate that they also have a common origin, given their similar
genomic location and structural organization. Since C is dispensable for viral replication, unlike PNT, we hypothesize that C
may have originated de novo by overprinting PNT in the ancestor of Paramyxovirinae. Intriguingly, in measles virus and
Nipah virus, PNT encodes STAT1-binding sites that overlap different regions of the C-terminus of C, indicating they have
probably originated independently. This arrangement, in which the same genetic region encodes simultaneously a crucial
functional motif (a STAT1-binding site) and a highly constrained region (the C-terminus of C), seems paradoxical, since it
should severely reduce the ability of the virus to adapt. The fact that it originated twice suggests that it must be balanced
by an evolutionary advantage, perhaps from reducing the size of the genetic region vulnerable to mutations.
Citation: Lo MK, Søgaard TM, Karlin DG (2014) Evolution and Structural Organization of the C Proteins of Paramyxovirinae. PLoS ONE 9(2): e90003. doi:10.1371/
journal.pone.0090003
Editor: Darren P. Martin, Institute of Infectious Disease and Molecular Medicine, South Africa
Received December 19, 2013; Accepted January 24, 2014; Published February 25, 2014
Funding: This work was supported by the Wellcome Trust grant number 090005 to DK. 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: dkarlin@strubi.ox.ac.uk
.These authors contributed equally to this work.
Introduction
Paramyxovirinae is a large virus subfamily that contains 9 known
human pathogens: measles virus, mumps virus, human parainflu-
enza viruses type 1 (hPIV1), 2, 3 and 4, Menangle virus, and the
recently emerged, highly pathogenic Nipah and Hendra viruses
[1]. Paramyxovirinae encode multiple proteins from the phospho-
protein (P) gene transcription unit, including P, V, and C. In
almost all Paramyxovirinae, the P gene mRNA is edited, resulting in
the expression of at least two proteins, P and V, which share an
identical N-terminus (PNT), but have a unique C-terminus
(Figure 1A) (for a review, see [2]). In addition, several genera,
including Morbilliviruses,Henipaviruses, and Respiroviruses, encode a
third protein, C, within their P gene, from an overlapping reading
frame [2]. The C proteins are expressed by a variety of
mechanisms including: leaky scanning [3–5], non-AUG start
codons [6,7], ribosomal shunting [8], and proteolytic processing
[9]. The region of P that overlaps C, corresponding approximately
to PNT (Figure 1A), is disordered [10–13], and contains conserved
sequence motifs, such as soyuz1, found in all Paramyxovirinae, which
binds the viral nucleoprotein, and soyuz2, of unknown function
[14].
The two primary functions of the C proteins are their abilities to
regulate viral transcription/replication and to antagonize the
antiviral responses of the host. These functions are thought to be
interconnected, since a decrease in viral transcription/replication
often correlates with a decrease in the innate antiviral responses of
the host [15–18] (for a review, see [19]). Most paramyxoviral C
proteins inhibit viral RNA synthesis, and thereby presumably
regulate viral gene expression [20–24]. However, they differ in the
degree to which they block host antiviral responses [25]. These
responses are composed of two crucial signaling cascades: A)
Induction of type I interferon (IFN), following recognition of virus-
derived elements by pattern recognition receptors (PRRs) and B)
IFN signaling through the JAK/STAT pathway, leading to
transcription of antiviral effector genes [26,27].
Most paramyxoviral C proteins can inhibit IFN induction, but
only respiroviruses are known to inhibit IFN signaling. Morbillivirus C
proteins have two mechanisms to counteract IFN induction: 1) by
reducing levels of viral replication, which limits the production of
viral patterns recognized by PRRs and prevents them from
inducing IFN [17,21,28]; and 2) by inhibiting IFN transcription in
the nucleus [29,30]. An initial study reported that measles virus C
protein blocks IFN signaling [31], but subsequent studies indicated
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that this effect is not significant [17,32,33]. Similarly, although the
mechanistic details are less clear, Henipavirus C proteins block IFN
induction by decreasing viral RNA synthesis, which indirectly
inhibits type I IFN induction; but they have minimal effects on
IFN signaling [15,34–37]. Like the morbilliviruses,Respirovirus C
proteins also counteract IFN induction through two mechanisms:
1) by minimizing production of double-stranded RNA (dsRNA),
thereby avoiding PRR activation [16,38]; and 2) by inhibiting
IRF3-dependent induction of type I IFN [39]. However, the C
proteins of respiroviruses differ from those of Morbilliviruses and
Henipaviruses in being also able to inhibit IFN signaling [16,26,38–
53]. Finally, a new role has been reported recently for the C
proteins of respiroviruses: they regulate the levels of viral genomes
and antigenomes produced during infection [54].
Interestingly, henipaviruses and morbilliviruses can also block IFN
signaling, but do so by proteins encoded by the P frame rather
than the C frame (i.e. P, V, or a third protein called W), which
interfere with the localization or phosphorylation of STAT1
(Signal Transduction Activator of Transcription 1), among other
mechanisms [55–62].
Overlapping genes, such as those encoding P and C, are of
particular interest because they encode proteins originated de novo
(in contrast to origination by well-characterized processes such as
gene duplication or horizontal gene transfer [63,64]). Indeed,
overlapping genes are thought to arise by overprinting, a process
in which mutations within an existing (‘‘ancestral’’) protein-coding
reading frame allow the expression of a second reading frame (the
de novo frame), while preserving the expression of the first frame
[65–67]. De novo proteins have been little studied but are known to
play an important role in viral pathogenicity [68,69], for instance
by neutralizing the host interferon response [70] or the RNA
interference pathway [71]. In addition, de novo proteins char-
acterised so far have previously unknown 3D structural folds
[68,71,72] and novel mechanisms of action [71]. Thus, this class of
proteins may challenge the notion that nature only utilizes a
limited number of different protein folds and that this fold space is
well mapped [73,74]. Another particularly interesting feature of
overlapping genes is the evolutionary paradox they present, since
the overlap imposes sequence constraints which should restrict the
ability of the virus to adapt [75–81].
Our study was divided in three strands. First, we predicted the
structural organization of the C proteins, and determined whether
they had detectable sequence similarity, which could indicate a
common origin, guide experimental studies, and facilitate 3D
structure determination [82]. Second, we verified our predictions
experimentally, by expressing, purifying and characterizing several
C proteins in bacteria. Third, we investigated the evolutionary
history of the P/C gene overlap, and tried to determine which, of
P and C, is the novel frame.
Methods
Sequence Alignment
The accession numbers of the sequences of Paramyxovirinae P
used in this study, as well as the abbreviations of species names, are
in Table 1. The sequence of the C protein of Pacific salmon
paramyxovirus [83,84] was generously made available by Bill Batts
and Jim Winton. We used Psi-Coffee [85,86] for multiple sequence
alignments (MSAs). All alignments are presented using Jalview
[87] with the ClustalX colouring scheme (see Figure 2b and 2d in
[88]). The aligned sequences of the C proteins in text format are in
File S1.
Figure 1. Organization of the P/V/C gene of
Paramyxovirinae
and phylogeny of the C proteins. A. Organization of the P/V/C gene
transcription unit of Paramyxovirinae. PNT: N-terminal moiety of P; PCT: C-terminal moiety of P. The V protein is composed of PNT fused, by co-
transcriptional editing (arrow) of the P mRNA, to a zinc finger domain encoded in a different frame. For clarity, only the C-terminal zinc finger of V is
shown. B. Clustering of Paramyxovirinae C proteins by sequence similarity. The cladograms represent the measles, Nipah and Sendai groups.
doi:10.1371/journal.pone.0090003.g001
Evolution and Structure of Paramyxovirus C and PNT
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We used two criteria to estimate the reliability of alignments of
the C proteins: 1) the CORE reliability index, which is based on
the agreement between the different alignment programs used by
Psi-Coffee, and is part of the standard output of Psi-coffee [86]; 2)
in the case of the measles and Nipah groups, we also considered
the coherence between the alignments of either group separately
and the alignment of both groups. We considered as not reliably
aligned the positions that either have a low Psi-coffee CORE
index, or are not aligned in the same way in these alignments.
Finally, we used TranslatorX [89] to generate a nucleotide
alignment of the P/C gene corresponding to an amino acid
alignment of the C protein. The alignment of the C proteins (not
shown) was created using the MUSCLE program [90] built in
TranslatorX, and is thus slightly different from that generated by
Psi-coffee, mainly in the region between E
a2
and S/T
a4
. This has
no impact on the results presented.
Sequence Analyses
The secondary structure of individual sequences was predicted
using Jpred [91], and was verified in the context of multiple
alignments using PROMALS [92]. We predicted disordered
regions with MetaPrDOS [93], according to the principles
described in [94]. We used HHalign [95] to compare the MSAs
of the C proteins of various groups, with a cutoff E-value of 10
25
.
To identify and cluster homologous C proteins, we performed
iterative sequence searches [96] on the C proteins of each taxon,
using csi-blast [97] and HHblits [98] with a cutoff E-value of 10
23
,
as described in [99]. We identified 5 subgroups of homologs
(Figure 1), formed by the following taxons: 1) the genus morbillivirus
and Salem virus;2)Tupaia Paramyxovirus, Mossman virus, and Nariva
virus; 3) the genus henipavirus; 4) the newly proposed genus
jeilongvirus; and 5) the two genera respirovirus and aquaparamyxovirus
(called ‘‘Sendai group’’). Several proteins of subgroups 1 and 3 had
a subsignificant (E.10
23
) similarity with proteins of subgroups 2
and 4, respectively, indicating that these subgroups may be
homologous [99]. We confirmed their homology by using
HHalign [95] (E = 5.10
211
for the comparison between subgroups
1 and 2, and E = 2.10
29
for the comparison between subgroups 3
and 4). We called the combination of subgroups 1 and 2 ‘‘measles
group’’ and the combination of subgroups 3 and 4 ‘‘Nipah group’’.
Cloning of the C Genes
To maximize our chances of successfully expressing C proteins,
we adopted a high-throughput approach. We cloned full-length
synthetic cDNAs (obtained from Genscript) of the C proteins of all
24 species in the measles, Nipah and Sendai groups into the vector
pOPIN-F [100] using the InFusion procedure, as described in
[100,101]. The resulting fusion proteins have an N-terminal
hexahistidine tag followed by a 3C cleavage site immediately
upstream of the coding sequence of the C proteins.
Expression of the C Proteins
Proteins were expressed in the bacteria Escherichia coli (E. coli)
using the BL21(DE3) Rosetta pLysS strain (Novagen), following
the ZYM-5052 auto-induction protocol [102]. Briefly, large scale
cultures were inoculated to OD600 of 0.02 and grown for 16 h at
25uC. Cells were harvested and the pellet resuspended 1:3 (w/vol)
in lysis buffer (50 mM TrisHCl, 500 mM NaCl, 30 mM Imidazole
pH 8.0, 1% vol/vol Protease inhibitor mix (Sigma P8849)) and
frozen in liquid nitrogen before storage at 280uC.
Figure 2. Alignment of the C proteins of the measles group. Numbering corresponds to measles virus. The N-terminus of C is highly variable
and shown for information only. Only the C-terminal moiety of C (helices a2toa4) is reliably aligned; positions that appear conserved but are outside
this region are thus not indicated. Residues that have been experimentally substituted (Table 2) are in bold. N-terminal sequences of fragments of
Tupaia PMV C obtained after limited proteolysis are underlined. Overlapping motifs of the PNT frame overlapping C are indicated above the
alignment.
doi:10.1371/journal.pone.0090003.g002
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Purification of the C Proteins
We purified both C proteins in two steps: Nickel Immobilized
Affinity Chromatography (IMAC) followed by size-exclusion
chromatography (SEC). Pellets were thawed and homogenized
(Constant Systems homogenizer) at 25 kpi at 4uC. The lysate was
cleared at 50,000 g for 30 minutes before batch incubation of the
supernatant (i.e. the soluble fraction of bacteria) on Ni-NTA
sepharose FF resin (Qiagen) for 2 hrs at 4uC. The material was
collected in an Econo-Pac column (Biorad) and washed in 100
Column Volumes (CV) of lysis buffer. Elution was done in 0.5 CV
fractions with lysis buffer containing 500 mM Imidazole. Fractions
containing protein were pooled and loaded onto a preparative
Superdex 75 (GE Healthcare Life Sciences) size exclusion column
pre-equilibrated in 20 mM Tris 150 mM NaCl, 1 mM EDTA,
pH 7.5. Peak fractions were pooled and concentrated using 15 ml
spin concentrators (Millipore).
Circular Dichroism (CD)
Protein samples were extensively dialyzed into 20 mM NaPho-
sphate, 20 mM NaCl pH7.5 and then concentrated to 0.2 mg/ml
in spin concentrators (0.5 ml, 3KDa MWCO, Millipore). The
Circular dichroism (CD) analysis was done on a JASCO 815 CD
spectropolarimeter. Data are averages of 5 independent scans in
the 190 nm –250 nm range, and were normalized to the baseline
of the dialysis buffer. The data were smoothed using the
manufacturer’s software (Jasco SpectraManager) before interpre-
tation. The percentage of a-helix was calculated according to the
formula: percentage of a-helix = (h
208–
4000)/(-33000-4000)6100,
where h
208
is the ellipticity at 208 nm [103].
Limited Proteolysis
From 1 mg/ml protease stocks, we made 10-fold serial dilutions
in 20 mM Hepes, 50 mM NaCl, 10 mM MgSO4, pH 7.5.
Proteins were concentrated to 0.6 mg/ml by spin concentrators
(0.5 ml, 3 MWCO, Millipore). For limited proteolysis, 10 mlof
protein was mixed with 3 ml of protease and incubated on ice for
30 min, 60 min or 2 hrs. Reactions were stopped by adding 2 ml
protease inhibitor mix (Sigma P8849). To each reaction, 5 mlof4x
SDS PAGE sample buffer was added and samples were heated to
95uC for 2 min before loading on a 1 mm 15% SDS-PAGE gel. A
subtilisin digest of hPIV1 C and an a-chymotrypsin digest of
Tupaia PMV C gave rise to stable fragments which were blotted to
PVDF before submitting the samples for N-terminal sequencing
(ALTA bioscience, UK).
Analytical Size Exclusion Chromatography (SEC)
Analytical size exclusion chromatography (SEC) was performed
at a flow-rate of 0.5 ml/min using a Superdex 75 10/300 column
(GE Healthcare Life Sciences) pre-equilibrated in 20 mM TrisCl,
150 mM NaCl, 1 mM EDTA pH = 7.9. The column was
calibrated with a separate run of appropriate globular marker
Table 1. Accession numbers of Paramyxovirinae C proteins.
Virus species Abbreviation Genus Genbank accession number
Atlantic salmon paramyxovirus Atlantic PMV Unclassified ABW38050.1
Beilong virus Beilong Jeilongvirus* YP_512248.1
Bovine parainfluenza virus 3 bPIV3 Respirovirus P06164
Canine distemper virus Canine DV Morbillivirus NP_047203.1
Dolphin Morbillivirus Dolphin MV Morbillivirus NP_945026.1
Bat paramyxovirus/Eid_hel/GH-M74a/GHA/2009 Bat PMV Henipavirus AFH96008.1
Cedar virus Cedar Henipavirus AFP87276.1
Feline morbillivirus Feline MV Morbillivirus AFH55514.1
Hendra virus Hendra Henipavirus O55779
Human parainfluenza virus 1 hPIV1 Respirovirus NP_604434.1
Human parainfluenza virus 3 hPIV3 Respirovirus NP_599251.1
J virus J virus Jeilongvirus* YP_338079.1
Measles virus Measles Morbillivirus NP_056920.1
Mossman virus Mossman Unclassified NP_958051.1
Nariva virus Nariva Unclassified YP_006347585.1
Nipah virus Nipah Henipavirus AAY43913.1
Pacific salmon paramyxovirus Pacific PMV Unclassified AFF60402.1
Peste des petits ruminants virus PPRV Morbillivirus YP_133824.1
Phocine distemper virus Phocine DV Morbillivirus P35940.1
Porcine parainfluenza virus 1 pPIV1 Respirovirus AGR39559.1
Rinderpest virus Rinderpest Morbillivirus ADF32062.1
Salem virus Salem Morbillivirus-like Q9IZB9
Sendai virus Sendai Respirovirus NP_056872.1
Tailam virus Tailam Jeilongvirus* AEU08859.1
Tupaia paramyxovirus Tupaia PMV Unclassified Q9WS38
(*) Proposed genus.
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proteins (Gel Filtration LMW Calibration Kit, GE Healthcare Life
Sciences).
Results
The C Proteins of Paramyxovirinae Cluster in three
Groups: the Measles, Nipah and Sendai Groups
On the basis of sequence analyses (see Methods), the C proteins
of Paramyxovirinae can be divided into three groups: the measles,
Nipah and Sendai groups (Figure 1B). The measles group is
composed of morbilliviruses, of the unclassified Salem virus, and of a
subgroup comprising the unclassified Tupaia paramyxovirus, Mossman
virus and Nariva virus. The Nipah group comprises henipaviruses and
jeilongviruses. Finally, the Sendai group is composed of respiroviruses
and of the recently described genus aquaparamyxovirus, composed of
fish viruses [83,84,104] related to respiroviruses [105,106]. The
classification of C into measles and Nipah groups is supported by
an examination of the PNT domain of P, which is encoded by the
same region as C but in a different frame (Figure 1A). Indeed, the
PNT of all species in the Nipah group differ from the PNT of the
measles group in having a soyuz2 motif (see Introduction) [14].
We found that other Paramyxovirinae that do not not express a C
frame [2,107] can be classified in two groups based on the
phylogeny of their P gene: the mumps group (comprised of the
sister genera rubulavirus and avulavirus) and the Fer de lance group
(formed by the genus ferlavirus [108]). This classification corre-
sponds to that of previous analyses [105].
The C Proteins of the Measles and Nipah Groups are
Homologous
We separately aligned the C proteins of the measles, Nipah and
Sendai groups (Figures 2, 3, and 4 respectively; the aligned
sequences in text format are in file S1). In these three groups, we
observed a similar organization of the C proteins, composed of a
variable N-terminus predicted to be disordered, and of a C-
terminus predicted to be ordered and a-helical. We compared
these alignments to each other using the profile-profile comparison
software HHalign [95] (see Methods). Briefly, a sequence profile is
a representation of a multiple alignment that contains information
about which amino acids (aas) are ‘‘tolerated’’ at each position of
the alignment, and with what probability. Comparing profiles is
much more sensitive than comparing single sequences, because the
profiles contain information about how the sequences can diverge
and thus can identify weak similarities which remain after both
sequences have diverged [99,109,110].
HHalign reported that the C proteins of the measles and Nipah
groups have statistically significant similarity (E = 4610
26
) over a
region of about 50aa in their C-terminus (shown in Figure 5). This
high similarity could in theory result either from convergent
evolution or from homologous descent. The fact that the measles
and Nipah groups are phylogenetically related [105], and that
their C proteins are encoded in the same genomic location makes
homologous descent a much more likely explanation. On the other
hand, HHalign did not detect any similarity between the C
proteins of the Sendai group and those of the measles and Nipah
Table 2. Selected experimental substitutions in C and their effect.
Group Virus name C ORF mutation Functional effect(s) of mutation References
MEASLES GROUP Measles virus R44G Ablates nuclear localization [29,156]
S134Y Associated with temperature-sensitive vaccine virus [114]
D127–138 (deletion) Ablated interaction with SHCBP1, reduced ability to
inhibit minigenome replication
[115]
NIPAH GROUP Nipah virus No fine mutational data –
SENDAI GROUP Sendai virus C D10–15 (deletion) Inability to interact with and modulate levels of
phosphorylated STAT1
[48]
Series of deletions in
aa149–157
Loss of nuclear translocation of Y1 by Ran-GTPase pathway [120]
K151A/E153L/R157L (Cm*) Increased IFN-binduction and dsRNA production, induction of
antiviral state, increased CPE, apathogenic in vivo
[39,127]
K77A/D80A (Cm2), M139A/
D142A (Cm4), R173A/
E175A/E176A (Cm8)
Inability to bind STAT1, ablated ability to inhibit RNA
synthesis, decreased binding to viral polymerase (L protein)
[46,118]
K77R/D80A (Cm2’), D80A Increased cytopathic effect, increased nuclear translocation
of IRF3, increased IFN-binduction and production of dsRNA
[39]
K151A/E153A/R154A (Cm5) Attenuated virulence in vivo, inability to block IFN signaling,
inability to inhibit replication, inability to skew STAT1/2
phosphorylation and to bind STAT1, decreased binding
to L protein
[39,46,118,127]
Human parainfluenza
virus 1 (hPIV1)
R84G Increased IFN-bproduction, increased IRF3 nuclear
translocation, reduced plaque sizes, non-temperature
sensitive mutation contributing to attenuation in vivo
[43,157]
Human parainfluenza
virus 3 (hPIV3)
CND25, CND50 (deletions) Increased inhibition of viral RNA synthesis, suppression of viral
replication, decreased ability to block Type 1 IFN signaling
[121,122]
K3A, K6A, K12A,
E16A, R24A
Increased inhibition of viral RNA synthesis, decreased
ability to block Type 1 IFN signaling
[121,122]
aa 90–195 of C Region required for STAT1 binding [119]
These studies used either recombinant viruses, minigenome systems, or eukaryotic expression systems. Substituted residues that are conserved in a group are in bold.
For a more comprehensive list of studies on Paramyxovirinae C, please see Table S1.
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groups. Thus, either they are not homologous, despite their similar
organization, or they are homologous but have diverged in
sequence beyond recognition. The latter scenario is possible, in
theory, since the relative frame of C compared to P (+1) is the
same in the Sendai group and in the measles/Nipah groups
(Figure 1A).
Sequence Analysis of the C Proteins of the Measles and
Nipah Groups
Figures 2 and 3 present alignments of the C proteins of the
measles and Nipah groups, respectively. Above the alignments, we
indicated regions of C that overlap conserved motifs of the P
frame. The C proteins of the measles and Nipah groups are all
composed of a 30–60 amino acid (aa) N-terminus predicted to be
at least partially disordered, and of a 90–120 aa C-terminus
comprising a predicted a-helix (a1), a loop of 10–20aa (‘‘loop
1–2
’’),
and three further a-helices (a2toa4), followed in some species by
C-terminal extensions of at most 20aa (forming helix a5 in some
species of the Nipah group).
In the C proteins of the measles group, only the region from a2
to a4 is well conserved in sequence; it contains many conserved
positions (Figure 2), of which six (boxed) are also conserved in the
C proteins of the Nipah group (see below). In contrast, the C
proteins of the Nipah group contains two additional, conserved
regions (Figure 3): 1) a short N-terminus with a-helical potential
(a0, aa 2–19 in Nipah virus), containing a hydrophobic region
followed by a basic region (boxed in Figure 3); and 2) a short
region at the C-terminus of a1 (aa 74–83 in Nipah virus) that
Figure 3. Alignment of the C proteins of the Nipah group. Conventions are the same as in Figure 2. Numbering corresponds to Nipah virus.
Several residues that appear conserved have not been indicated, because their alignment is not reliable, or their conservation is probably imposed by
the P frame (see text).
doi:10.1371/journal.pone.0090003.g003
Figure 4. Alignment of the C proteins of the Sendai group. Conventions are the same as in Figure 2. Numbering corresponds to the C protein
of Sendai virus. Arrows indicate the start of the different isoforms of C. For information, the arrowhead indicates the well-characterized F residue of
respiroviruses (F170 in Sendai virus), whose substitution by S reduces innate immune antagonism and attenuation of in vivo pathogenesis by C
[39,53,153–155] (see Table S1). The N-terminal sequence of the fragment of hPIV1 C obtained after limited proteolysis is underlined. The variable
region between basic region 1 and residue G89 is not reliably aligned and is presented for information only.
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contains two conserved acidic positions (E/D). The apparent
conservation of other regions of C, which overlap the soyuz1 and
soyuz2 motifs of the P frame (Figure 3), should not be over-
interpreted, since it may be due to constraints imposed by selection
pressures acting in fact on the P frame, which is much more
conserved than the C frame in these regions (not shown).
An alignment of the C proteins of both groups (Figure 5)
revealed four remarkable positions conserved in nearly all viruses
(boxed in Figure 5): a Tyrosine (Y) upstream of helix a2(Y
a2
); a
Glutamate (E
a2
) at the C-terminus of the same helix; a residue
with an alcohol group (Serine/Threonine, S/T
a4
) at the N-
terminus of helix a4; and a Glutamate (E
a4
) two residues
downstream. Two other positions of hydrophobic nature (indicat-
ed by ‘‘h’’) are conserved in both groups. These conserved residues
are also boxed in Figures 2 and 3, in the separate alignments of the
measles and Nipah groups. Other positions that appear conserved
in Figure 5 or in Figures 2 and 3 may in fact not be reliably aligned
(see Methods) and are therefore not boxed.
Sequence Analysis of the C Proteins of the Sendai Group
Figure 4 shows the alignment of the C proteins of the Sendai
group. In Sendai virus and human parainfluenza virus 1 (hPIV1), as
many as four products (C’, C, Y1, Y2) are expressed from the C
reading frame by a combination of alternative initiation codons
[6–8] and proteolytic processing [9]. Their respective N-termini
are indicated by arrows. The C proteins of the Sendai group have
a similar organization to that of the measles and Nipah groups.
They are composed of a variable, disordered N-terminus of about
80aa, rich in Prolines (P), Serines (S) and Threonines (T), followed
by a conserved C-terminus composed of four a-helices (aAtoaD).
The N-terminus contains a basic region (boxed in Figure 4) within
a predicted a-helix (aZ), like the C protein of the Nipah group
(Figure 3). In the C protein of Sendai virus, the first half of aZ was
reported to act as a membrane-targeting signal, perhaps by
forming an amphipathic a-helix [111]. There are 11 residues
strictly conserved in C across the Sendai group, clustered
predominantly in the C-terminus of aC and in aD. aCis
particularly rich in K and R (‘‘basic region 2’’ in Figure 4),
suggesting it might bind a negatively charged partner.
Obtaining a Reliable Alignment of the Region of PNT
Containing STAT1-binding Sites in measles virus and
Nipah virus
We present in Figure 6 a summary of the structural and
functional organization of PNT and C in the different taxa of
Paramyxovirinae, to scale, with their functional motifs vis-a`-vis of
each other. PNT contains sequences that bind the protein STAT1
in several morbilliviruses (measles virus [55,56], canine distemper virus
[57], Rinderpest virus [60]) and henipaviruses (Nipah virus [58] and
Hendra virus [59]). The region of PNT that contains these sites is
highly variable in sequence (Figure 7), and thus its alignment is not
reliable. In contrast, the overlapping region of C is well conserved,
and its alignment reliable (Figure 5). Therefore, we used the C
frame to construct a reliable alignment of PNT. We proceeded in
two steps (see Methods). First, we used the amino acid alignment of
the C proteins (Figure 8, top panel) to generate an alignment of the
nucleotide sequences of the P/C gene (Figure 8, middle panel and
File S2), using TranslatorX [89]. Second, we translated this
nucleotide alignment into an amino acid alignment in the P frame
(Figure 8, bottom panel). The resulting alignment of PNT of the
measles and Nipah groups is presented in Figure 9.
The STAT1-binding Sites of PNT of Nipah Virus and
Measles Virus Overlap Different Regions of C and thus
Probably Evolved Independently
From the reliable alignment of PNT corrected by using the C
frame (Figure 9), we made three observations:
i) The STAT1-binding sites of measles virus and Nipah virus PNT
are conserved in sequence only in very closely related species
Figure 5. Alignment of the C proteins of the measles and Nipah groups. Conventions are the same as in Figure 2. Several positions appear
conserved but have not been indicated, because their alignment is not reliable (see text).
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(thick boxes in Figure 9). For instance, in PNT of Feline
morbillivirus, which is more distantly related to measles virus
than other morbilliviruses, only 2 aa out of 11 (E110 and I116)
correspond to conservative substitutions with respect to the
STAT1-binding motif of measles virus (Figure 9). Such a high
number of non-conservative substitutions within a short
peptide suggests that it may not bind STAT1.
ii) The STAT1-binding sites of measles virus and Nipah virus PNT
are not aligned together (Figure 9) (although they overlap
Figure 6. Summary of the organization of
Paramyxovirinae
PNT and C. The figure is to scale, with PNT and C vis-a
`-vis of each other. The PNTs
are all positioned so that their soyuz1 motifs match. Regions whose homology is proven (by statistically significant similarity) have the same color.
Homology of soyuz1 motifs is suspected but not proven [14], thus they have a same color, but different patterns. STAT1b: STAT1-binding site. Ust1:
‘‘upstream of STAT1’’ motif.
doi:10.1371/journal.pone.0090003.g006
Figure 7. Alignment of the central region of PNT of the measles and Nipah groups (not corrected by using the C frame). Note the
high variability of the alignment. The [Y/H]DH[S/G]GE motifs common to the STAT1-binding sites of PNT of measles virus and Nipah virus are
underlined. In bold are the residues Y110 of measles virus PNT and Y116 of Nipah virus PNT, which were suggested to be analogous (see text).
doi:10.1371/journal.pone.0090003.g007
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slightly, by 4aa), which indicates that they are encoded in
different locations of the P/C gene. It is thus highly likely that
they have originated independently (see Discussion).
iii) The STAT1-binding sites of measles virus and Nipah virus PNT
have some limited sequence similarity, as reported earlier
[58]: they share a [Y/H]DH[S/G]GE motif, underlined in
Figure 8. Procedure to reliably align the central region of PNT of the measles and Nipah groups by using a C frame alignment.
Conventions are the same as in Figure 2. An alignment of C (top panel) is converted in a nucleotide alignment (middle panel) by using TranslatorX
(see text), then translated into the P frame (bottom panel), yielding a reliable alignment of the PNT domain of P, which overlaps C. The nucleotide
alignment of the P/C genes corresponding to the middle panel is in File S2.
doi:10.1371/journal.pone.0090003.g008
Figure 9. Alignment of the region of PNT of the measles and Nipah groups containing STAT1-binding sites, corrected by using the
C frame. Conventions are the same as in Figure 2. This reliable alignment of PNT is based on an alignment of the C frame by the procedure
described in Figure 8. The [Y/H]DH[S/G]GE motifs common to the STAT1-binding sites of measles virus and Nipah virus PNT are underlined. Note that
contrary to the alignment of Figure 7, here they are not aligned together. Y110 of measles virus PNT and Y116 of Nipah virus PNT, which were
suggested to be analogous (see text) are in bold.
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Figures 7 and 9. However, this similarity is unlikely to be due
to homologous descent, since the motifs are not aligned
together in the reliable alignment of PNT (Figure 9).
Likewise, the tyrosine residues immediately upstream of this
motif (Y110 in measles virus PNT, critical for STAT1
inhibition [33,55,112,113], and Y116 in Nipah virus PNT),
which were perceived to occur in a similar sequence context
[58], are not aligned together either in the reliable alignment
of PNT (Figure 9), indicating that they are not homologous
either.
Finally, we also noticed an 8aa motif (aa 104–111 in Nipah virus)
conserved in the PNT of all henipaviruses (Figure 9, thin box). We
called this motif ust1 (for ‘‘upstream of STAT1’’). Its function is
unknown, though aa 81–113 of Nipah virus P, which include ust1,
are required for the synthesis of viral RNA [58]. We cannot
exclude, however, the possibility that the conservation of ust1 is
due to constraints imposed by the overlapping C frame.
Functional Organization of the C Proteins in Relation to
Their Sequence
We systematically examined mutational studies of Paramyxovir-
inae C and their phenotypic impact. The most relevant studies are
in Table 2 and a more extensive list of studies is in Table S1. We
found that very few conserved positions identified herein have
been subjected to targeted mutagenesis; notable substitutions are
indicated in bold in Figures 2 and 4.
In the measles group, experimental substitutions have been
performed mostly in the C-terminus of C. In a comparison of a
temperature-sensitive strain of measles vaccine, AIK-C, with its
parental strain, Edmonston [114], one of several substitutions
identified, S134Y, occurs in the S/T
a4
position conserved in the
measles and Nipah groups (Figures 2 and 5) (Table 2). Although
this particular substitution is not responsible for the temperature
sensitive phenotype [114], we note that it is located within a 12aa
peptide (aa 127–138) recently shown to inhibit the viral
polymerase by interacting with SHCBP1 (Shc Src homology 2
domain-binding protein 1) [115]. This peptide, underlined in
Figures 2 and 5, contains two other positions conserved in the
measles/Nipah groups (a hydrophobic residue and E
a4
). Such
conservation suggests that other viruses in the measles/Nipah
groups may also bind SHCBP1 to block the viral polymerase.
Finally, the role of the disordered N-terminus of measles virus Cis
poorly known, although it contributes to nuclear localization,
which correlates with its ability to block IFN induction [29]
(Table 2).
In the Nipah group, there are no fine mutational data
published, but it is known that both the N-terminus and the C-
terminus of Nipah virus C are required to inhibit minigenome
replication [116].
In the Sendai group, experimental substitutions have delineated
multiple residues in the C-terminus of C responsible for
antagonizing both IFN induction and IFN signaling, and for
regulating viral transcription and replication [46,49,117,118]
(Table 2 and Table S1). For both Sendai virus and hPIV3, the
minimal region required for STAT1-binding corresponds to the
structured, well-conserved C-terminus of C [117,119]. Within that
domain, aas 149–157 (corresponding roughly to basic region 2,
underlined in Figure 4) are critical for nuclear translocation of the
Y1 isoform of Sendai virus C, and may also play a role in the
inhibition of type-I IFN-stimulated gene expression [120]. This
region contains several conserved residues, suggesting that its
function may be conserved in the Sendai group. Studies of the N-
terminus of C in the Sendai group indicate that it also contributes
to antagonizing the innate immune response and to regulate viral
transcription and replication [121,122] (Table 2 and Table S1).
Taken together, these studies suggest that both the N- and C-
terminus of Sendai group C proteins may need to act in
Figure 10. Circular Dichroism (CD) spectra of the C proteins of hPIV1 and Tupaia PMV.
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coordinated fashion in order to perform their complete suite of
antagonistic and regulatory functions.
Experimental Characterization of one C Protein of the
Measles/Nipah Group and of One C Protein of the Sendai
Group
In order to check our predictions of structural organization, we
attempted to characterize biophysically at least one C protein of
the measles/Nipah groups and one of the Sendai group. We
systematically tested, in the bacteria E. coli, the expression and
solubility of the C proteins of all species in the measles, Nipah and
Sendai groups (see Methods). We found that the C proteins of
tupaia paramyxovirus (Tupaia PMV) and of hPIV1 were by far the
best candidates, for the measles/Nipah groups and Sendai group
respectively, in terms of yield and solubility (not shown). We
expressed both proteins as hexahistidine-tagged N-terminal fusion
proteins in Escherichia coli and purified them from the soluble
fraction by immobilized metal affinity chromatography (IMAC)
and size exclusion chromatography (SEC) (see Methods). Mass
spectrometry confirmed that the C proteins had the exact expected
mass. In SDS-PAGE analysis (Figure S1), hPIV1 C migrated at a
notably larger size (,31kD) than expected (25.9kD), while Tupaia
PMV C migrated at ,21kD, only slightly above the expected size
(19.7kD). This anomalous migration may be caused by regions
that are disordered or have a biased aa composition [123].
Accordingly, the N-terminus of both proteins is predicted
disordered, and has a biased composition in the case of hPIV1 C.
We analyzed the secondary structure of the C proteins by
Circular Dichroism (CD). The CD spectrum of both proteins
(Figure 10) is typical of a-helical content [124], with two dips in
ellipticity at around 208 and 222 nm. The estimated a-helical
content was 57% for hPIV1 C and 33% for Tupaia PMV C (see
Methods). We also examined the C proteins by analytical SEC
(Figure 11). Tupaia PMV C elutes at an apparent molecular mass
of 21.4 kDa, close to its theoretical mass of 19.7 KDa. In contrast,
hPIV1 C elutes at a much larger MW (38.7 kDa) than expected
(25.9 kDa). This discrepancy could correspond to an extended
shape, or to self-association in a fast equilibrium between a
monomeric and dimeric form (see below).
Limited Proteolysis of hPIV1 C and Tupaia PMV C
Confirms That they Have a Flexible N-terminus and a
Structured C-terminus
We used limited proteolysis combined with N-terminal
sequencing to probe the structural organization of the C proteins
of hPIV1 and Tupaia PMV. We tested a range of proteases with
different substrate requirements (see Methods), and identified
fragments resistant to proteolysis, indicative of folded domains.
Digestion of hPIV1 C by subtilisin yielded a stable degradation
product of around 14 kD (Figure 12, left panel), whose N-terminal
sequence, starting at aa 104, is underlined in Figure 4. The size of
this fragment indicates that it comprises the whole C-terminus of
C (expected size 14.16 kD), which corresponds well to our
sequence predictions (Figure 4). These results are also coherent
with cellular experiments that identified a proteolysis-sensitive N-
Figure 11. Size Exclusion Chromatography (SEC) on the C proteins of hPIV1 and Tupaia PMV. The curves represent two SEC purifications
runs, on the same column and in similar conditions (see text): one for Tupaia PMV C (blue), and one for hPIV1 C (green).
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terminus in the C’ proteins of Sendai virus [125]. We note that the
presence of a long, disordered region in hPIV1 C is compatible
with its high apparent molecular weight observed in SEC (see
above) [126].
Digestion of the C protein of Tupaia PMV by a-chymotrypsin
yielded a series of bands ranging from 14 kD to 6 kD (Figure 12,
right panel); further digestion (not shown) yielded a single 6kD
fragment. We obtained N-terminal sequences of the three most
abundant fragments, of ,14.4, 13, and 6 kD (arrows to the right of
Figure 12). They start respectively at aa 30, 43 and 84. This
pattern of proteolytic digestion indicates that Tupaia PMV C is
composed of a disordered N-terminus and of an ordered C-
terminus. This is compatible with our predictions, in which aa 1–
56 are devoid of secondary structure (Figure 2) and aa 1–42
disordered, and in which a predicted loop, a
1–2
(aa 81–92), could
be accessible to proteolysis. The observed fragments of 14.4 and
13kD correspond exactly to C proteins where aa 1–29 and 1–43,
respectively, have been digested, whereas the size of the smaller
fragment (6kD) corresponds to aa 81–135, indicating that the last
18 C-terminal aa are digested upon extended proteolysis.
In summary, our experiments confirm that in vitro, the C
proteins of hPIV1 and Tupaia PMV are predominantly a-helical
and contain a disordered N-terminus, whose boundaries are in
good agreement with our sequence-based predictions.
Discussion
Substituting the conserved, charged residues we have identified
herein should be a powerful way to dissect the function of C.
Indeed, charged residues are often on the surface of proteins and
thus their conservation is generally the result of functional
constraints, rather than constraints imposed by a mere structural
role. The power of this approach has been shown by studies on
several regions of respirovirus C [39,46,127], and our thorough
sequence analysis of the full-length C proteins of all Paramyxovirinae
should greatly extend its applicability. In addition, knowing the
structural organization of C will allow the design of deletions that
have less risk of disrupting its three-dimensional structure.
A Common Origin of the C Proteins?
The C proteins of the Sendai group have no detectable
sequence similarity with those of the measles/Nipah groups.
However, we consider it unlikely that they have an independent
origin, because they are located in the same region of the P gene,
in the same frame relative to P, and have a similar structural
organization and several similar functions [118,128,129]. Thus we
consider that all C proteins most probably have a common origin,
as proposed earlier [67,130]. The absence of a C protein in the
mumps group is probably due to a loss in the ancestor of that
group, since the Sendai group, which has a C protein, is basal in a
phylogeny of the P gene [105]. This common origin would imply
that in Sendai virus, it is the Y1 isoform of C that is the equivalent of
C of the measles/Nipah groups, because their start codon have the
same location immediately upstream of the soyuz1 motif of the P
frame (Figure 6; compare also Figure 4 and Figure 3). Therefore,
the C and C’ proteins of Sendai virus would have presumably
originated by mutations creating new, alternative start codons
upstream of Y1. A common origin of Paramyxovirinae C proteins
would also imply that the basic regions in the N-terminus of C
have originated independently in the Sendai and Nipah groups,
since they occupy different positions with respect to soyuz1
(Figure 6).
Figure 12. Limited proteolysis of the C proteins of hPIV1 and Tupaia PMV. Digestion profiles of hPIV1 C (left) and Tupaia PMV C (right),
visualized by SDS-PAGE and Coomassie blue staining. Several fragments (arrowheads) were N-terminally sequenced. Their N-terminal sequences are
underlined in Figures 2 and 4. a–chymotrypsin is not visible on the digestion profile of Tupaia PMV C.
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Which Frame Originated Earlier, PNT or C?
Overlapping genes typically encode an ancestral frame and a
novel frame originated by overprinting it (see Introduction). Our
analyses in this work and in an earlier study [14] suggest that the C
and PNT frames were probably both present in the ancestor of
Paramyxovirinae, making it impossible to conclude which frame is
ancestral on the basis of phylogeny. Analysis of codon usage [131]
cannot determine which frame is ancestral either, because the
codon usages of PNT and C are indistinguishable in Paramyxovirinae
(Angelo Pavesi, personal communication). However, functional
considerations suggest that the PNT frame originated earlier, since
it is indispensable to viral replication in vitro [2,132], unlike C
[20,133,134]. The ancestry of PNT is supported by a comparison
with families related to Paramyxovirinae (Mononegavirales). Most
Mononegavirales also encode P proteins with a disordered N-
terminus [14,135]; at least in Rhabdoviridae, this N-terminus has the
same function as Paramyxovirinae PNT, i.e. preventing the
nucleoprotein from self-assembling illegitimately [136–139]. Thus,
it is reasonable to speculate that the P of the ancestral
Mononegavirales already had a disordered N-terminus, which was
overprinted by C in the ancestor of Paramyxovirinae.
Convergent Evolution between the STAT1-binding Sites
of measles virus and Nipah virus?
The STAT1-binding sites of measles virus and Nipah virus do not
align together in the reliable alignment of PNT, generated using
the C frame (Figure 9). This strongly suggests that they have
originated independently. Alternatively, since they overlap by 4aa
(Figure 9), these STAT1-binding sites might, in theory, have
originated from a common, short peptide, providing some
STAT1-blocking capability, and later have extended respectively
upstream and downstream of PNT. However, this scenario is not
parsimonious because it would imply several losses in the lineages
separating measles virus and Nipah virus. Also, the common 4aa
stretch is chemically very different in both viruses (G
117
EAV in
measles virus and V
115
YHD in Nipah virus, Figure 9). We thus
consider it most likely that the STAT1-binding sites of measles virus
and Nipah virus have originated independently.
Their limited sequence similarity (they share an [Y/H]DH[S/
G]GE motif, underlined in Figure 9) would thus not be the result
of homologous descent, but could instead result either from
convergent evolution (owing to a common mechanism), or from
random chance. Convergent evolution seems a definite possibility,
since the mechanisms by which PNT acts are somewhat similar in
both viruses (PNT interferes with the phosphorylation of
cytoplasmic STAT1) [55,58,112,140], and since the PNT of both
viruses bind a similar part of STAT1 [141].
The P/C Gene Exemplifies Three Keys to the Evolutionary
Paradox of Overlapping Genes
Overlapping genes are an evolutionary paradox, because they
simultaneously encode two proteins whose freedom to mutate is
constrained by each other, which should severely reduce the ability
of the virus to adapt [75–81].
A first key to the paradox has been suggested earlier
[67,77,78,142–146]: overlapping genes frequently encode an
‘‘ancillary’’ frame that can tolerate a higher substitution rate than
the other, ‘‘dominant’’ frame; the ancillary frame is often
Figure 13. Three patterns of sequence constraints in the overlapping frames P and C. PNT and C are represented vis-a
`-vis of each other
with same conventions as in Figure 6. Sequence constraints of PNT and C were estimated by their sequence variability.
doi:10.1371/journal.pone.0090003.g013
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structurally disordered [68]. Accordingly, a previous sequence
analysis of Sendai virus indicated that PNT and C are generally not
both under strong constraint [142]; rather, the N-terminus of PNT
is markedly more conserved than that of C, whereas the C-
terminus of PNT is markedly more conserved than that of C [142].
This is also the case for most of the PNT and C of measles and
Nipah virus (Figure 13, evolutionary pattern 1 or 2), with the
exception of the region corresponding to the STAT1-binding sites
of PNT (see below).
A second key to the paradox of overlapping genes is that it may
be beneficial for a virus, under certain conditions, to encode
functional motifs simultaneously by using overlapping frames
[147]. Initially, we were very surprised to discover that a region of
the P/C gene encodes simultaneously, in different frames, two
well-conserved regions: the STAT1-binding motif of PNT, and the
a2–a4 region of C (Figure 13, evolutionary pattern 3). Intuitively,
this arrangement seems to dramatically restrict the capacity of the
virus to mutate and to escape host defenses. We were all the more
surprised that this arrangement originated twice independently, in
measles virus and in Nipah virus (see Figure 6). This seems beyond
coincidence, and strongly suggests that the loss of fitness of the
virus due to its reduced ability to mutate is compensated by an
evolutionary advantage. In fact, this phenomenon had been
predicted on the basis of mathematical modeling [147]. Given a
high mutation rate, it may be advantageous to encode crucial
functional motifs in overlapping frames (provided that they are
short), because the superposition of critical amino acids reduce the
number of vulnerable positions in the genome. The conditions of
application of the model are met here: RNA viruses have one of
the highest mutation rate of all organisms [148], and the STAT1-
binding sites are short (10–26aa). It will be interesting to
investigate whether this evolutionary pattern, in which two
reading frames are both under strong constraint, is common in
viruses, and whether it does entail a selective advantage. The
genome of Hepatitis B virus, for instance, also contains short regions
where both the overlapping Polymerase and Glycoprotein frames
are under strong constraint [149,150]. A recent innovative
methodology that combines experimental and computational
approaches [151] could help to tease out the different factors
(structural, functional and co-evolutionary) constraining overlap-
ping motifs.
Finally, a third key to the paradox of overlapping genes is that
they provide a regulatory advantage that may offset the increased
constraints they impose on the virus, by encoding two proteins that
are co-regulated and have complementary functions [131]. For
instance, the expression levels of the C and V proteins of Nipah or
measles viruses are co-regulated, since they are transcribed from
the same gene transcription unit; in addition, their roles are
complementary, since together they inhibit both viral RNA
synthesis and type I IFN induction, enabling an efficient block
of the first stage of the host antiviral response [15,17,20,24,152]. In
the same vein, the expression of C and P is also co-regulated and
they have complementary effects on viral transcription, mediated
by binding the same cellular protein, SHCBP1 [115].
Conclusion
In conclusion, we predict that the C proteins of the Sendai
group and of the measles/Nipah groups will have the same
structural fold, testifying to a common origin, and that this fold will
be a previously unobserved one, in keeping with their de novo origin
[68].
Supporting Information
Figure S1 Purification of the C proteins of hPIV1 and
Tupaia PMV. The purifications are visualized by Coomassie
blue-stained SDS-PAGE.
(TIF)
Table S1 Effect of experimental substitutions in Para-
myxovirinae C proteins.
(DOCX)
File S1 Multiple sequence alignment of the C proteins
of the measles, Nipah, and Sendai groups.
(DOC)
File S2 Multiple sequence alignment of the P/C genes
of the measles and Nipah groups, based on an alignment
of the C proteins
(DOC)
Acknowledgments
We thank B Bankamp, JM Bourhis, P Devaux, M Jamin, R Neme and A
Vianelli for comments on the manuscript. We thank the OPPF-UK for
help with expression of the C proteins, and the organizers of the EMBO
training ‘‘High-throughput methods for protein production and crystalli-
zation’’.
Disclaimer: The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the Centers for
Disease Control and Prevention.
Author Contributions
Conceived and designed the experiments: TMS DGK. Performed the
experiments: TMS DGK. Analyzed the data: MKL TMS DGK.
Contributed reagents/materials/analysis tools: MKL TMS DGK. Wrote
the paper: MKL TMS DGK.
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