Download Domain organization of the N-terminal portion of hordeivirus

Journal of General Virology (2009), 90, 3022–3032
DOI 10.1099/vir.0.013862-0
Domain organization of the N-terminal portion of
hordeivirus movement protein TGBp1
Valentin V. Makarov,13 Ekaterina N. Rybakova,13 Alexander V. Efimov,2
Eugene N. Dobrov,1 Marina V. Serebryakova,3 Andrey G. Solovyev,1,4
Igor V. Yaminsky,5 Michael E. Taliansky,6 Sergey Yu. Morozov1,7
and Natalia O. Kalinina1
Correspondence
Natalia Kalinina
1
[email protected]
2
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow
119992, Russia
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290,
Russia
3
Institute of Physico-Chemical Medicine, Moscow 119828, Russia
4
Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow
127550, Russia
5
Physical Faculty, Moscow State University, Moscow 119992, Russia
6
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK
7
Department of Virology, Biological Faculty, Moscow State University, Moscow 119992, Russia
Received 28 May 2009
Accepted 6 August 2009
Three ‘triple gene block’ proteins known as TGBp1, TGBp2 and TGBp3 are required for cell-tocell movement of plant viruses belonging to a number of genera including Hordeivirus. Hordeiviral
TGBp1 interacts with viral genomic RNAs to form ribonucleoprotein (RNP) complexes competent
for translocation between cells through plasmodesmata and over long distances via the phloem.
Binding of hordeivirus TGBp1 to RNA involves two protein regions, the C-terminal NTPase/
helicase domain and the N-terminal extension region. This study demonstrated that the extension
region of hordeivirus TGBp1 consists of two structurally and functionally distinct domains called
the N-terminal domain (NTD) and the internal domain (ID). In agreement with secondary structure
predictions, analysis of circular dichroism spectra of the isolated NTD and ID demonstrated that
the NTD represents a natively unfolded protein domain, whereas the ID has a pronounced
secondary structure. Both the NTD and ID were able to bind ssRNA non-specifically. However,
whilst the NTD interacted with ssRNA non-cooperatively, the ID bound ssRNA in a cooperative
manner. Additionally, both domains bound dsRNA. The NTD and ID formed low-molecular-mass
oligomers, whereas the ID also gave rise to high-molecular-mass complexes. The isolated ID was
able to interact with both the NTD and the C-terminal NTPase/helicase domain in solution. These
data demonstrate that the hordeivirus TGBp1 has three RNA-binding domains and that
interaction between these structural units can provide a basis for remodelling of viral RNP
complexes at different steps of cell-to-cell and long-distance transport of virus infection.
INTRODUCTION
Transport of plant viruses from cell to cell occurs through
plasmodesmata and requires virus-encoded movement
proteins (Boevink & Oparka, 2005; Lucas, 2006; Epel,
2009). In the genus Hordeivirus, viral movement proteins
are represented by three proteins encoded by a ‘triple gene
block’ (TGB) and referred to as TGBp1, TGBp2 and
TGBp3 (Solovyev et al., 1996). The TGB is a conserved
3These authors contributed equally to this work.
3022
module of overlapping genes found in a number of virus
groups (Morozov & Solovyev, 2003). Hordeivirus TGBp1
binds viral genomic RNA forming a cell-to-cell transportcompetent complex (Brakke et al., 1988; Kalinina et al.,
2001; Lim et al., 2008; Jackson et al., 2009). TGBp2 and
TGBp3 are small membrane proteins necessary for
intracellular transport of complexes containing TGBp1
and viral RNA to plasmodesmata (Morozov & Solovyev,
2003; Zamyatnin et al., 2004; Haupt et al., 2005; Jackson
et al., 2009).
Downloaded from www.microbiologyresearch.org by
013862 G 2009 SGM
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Printed in Great Britain
Domain organization of hordeivirus TGBp1
Amino acid sequence analyses of TGB-encoded proteins,
together with functional studies, have revealed two distinct
TGB types termed ‘hordei-like’ and ‘potex-like’ TGB
(Morozov & Solovyev, 2003). Viruses with hordei-like TGB,
in contrast to viruses with potex-like TGB, require no coat
protein for viral cell-to-cell and long-distance transport (Petty
& Jackson, 1990; Morozov & Solovyev, 2003). Analysis of
RNA–protein complexes isolated from plants infected with
barley stripe mosaic virus (BSMV; the type member of the
genus Hordeivirus) demonstrated that TGBp1 is the major if
not the only protein that interacts with viral genomic and
subgenomic RNAs in infected cells to form ribonucleoprotein
(RNP) complexes (Brakke et al., 1988; Lim et al., 2008;
Jackson et al., 2009). Such TGBp1–RNA complexes are
considered to be a form of viral genome capable of cell-to-cell
and long-distance transport in plants (Morozov & Solovyev,
2003; Lim et al., 2008). In potex-like TGBs, the whole TGBp1
sequence is represented by the NTPase/helicase domain
(HELD) with seven conserved motifs of the superfamily 1
NTPases/helicases (Gorbalenya et al., 1989), whereas the
hordei-like TGBp1 has an additional N-terminal ‘extension
region’ (Morozov & Solovyev, 2003). Deletion analysis of
BSMV TGBp1 has revealed that the protein has multiple
RNA-binding sites, some of which map to the N-terminal
extension region (Donald et al., 1997). As shown for TGBp1
of poa semilatent virus (PSLV; genus Hordeivirus), the Nterminal extension region expressed as a separate polypeptide
binds RNA in a non-cooperative manner (Kalinina et al.,
2001). The N-terminal 24 aa of TGBp1 of beet necrotic yellow
vein virus (BNYVV; genus Benyvirus), another virus with a
hordei-like TGB, also binds RNA (Bleykasten et al., 1996).
Interestingly, BSMV and BNYVV TGBp1s are able to bind
both ssRNA and dsRNA (Bleykasten et al., 1996; Donald et al.,
1997). A double mutation in PSLV TGBp1 that disrupts two
clusters of positively charged amino acids responsible for RNA
binding blocks long-distance but not cell-to-cell transport of
viral infection (Kalinina et al., 2001), whereas deletion of the
24 aa N-terminal region containing an RNA-binding site
prevents BNYVV cell-to-cell transport (Bleykasten et al.,
1996). Thus, the RNA-binding ability of the TGBp1 Nterminal extension region is necessary for viral transport in
plants.
In this paper, we showed that the N-terminal ‘extension
region’ of the hordeivirus TGBp1 consists of two
structurally and functionally distinct domains.
METHODS
Amino acid sequence analysis. For computer-assisted secondary
structure prediction, the SCRATCH server (Cheng et al., 2005; available at
http://www.igb.uci.edu/servers/psss.html), the FoldIndex server (Prilusky
et al., 2005; available at http://bioportal.weizmann.ac.il/fldbin/findex) and
the GOR IV server implementing an improved prediction algorithm
(Garnier et al., 1978; available at http://cn.expasy.org/) were used.
constructed for expression of the N-terminal domain (NTD) (aa 1–
190 of the 63K protein) and the internal domain (ID) (aa 190–290).
The NTD-encoding DNA fragment was obtained by PCR using
primers 59-TTGTGAGCGGATAACAATTTC-39 and 59-CCTCTCTAGATTATTTATCTTTCGTTTGCTTCT-39, whilst the ID-encoding DNA fragment was obtained with primers 59-AAAGGATCCGCTGAAGACTTAAATGCA-39 and 59-AAGCTCTAGATTATGTCTTCTTTAAGTGCTCCA-39. PCR products were digested with BamHI and
XbaI, and ligated with similarly digested pQE30. E. coli strain M15 cells
were transformed with plasmids pQE30-N63K, pQE30-NTD and
pQE30-ID. Expression, purification and Western blot analysis were
performed according to the method described by Leshchiner et al. (2006).
Circular dichroism (CD) spectroscopy. CD spectra of the
recombinant proteins in the far-UV region (198–250 nm) were
measured in a modified Jobin–Jvon Mark V dichrograph interfaced
with a computer, using RDA and Wtest programs developed in our
laboratory. The spectra were measured at 25 uC in 1 mm cells at a
protein concentration of 200 mg ml21. The spectra were calculated as
[h] values per mole of amino acid residues. The concentration of the
recombinant ID protein was measured spectrophotometrically using
calculated absorption coefficients at 280 nm (E280, 1 cm, 0.1 %) of
0.24. In the case of the NTD, which does not contain Trp and Tyr, the
concentration was determined by SDS-PAGE with Coomassie blue
staining using BSA as a standard or by a Bradford assay.
RNA-binding assays. North-Western analysis was carried out as
described previously (Kalinina et al., 1996). For gel-shift assays,
increasing concentrations of recombinant proteins were incubated
with tobacco mosaic virus RNA (0.5 mg) at room temperature.
Samples were analysed in ethidium bromide-containing 1 % agarose
gels. For the dsRNA in gel-shift analysis, two annealed T7 transcripts
complementary along their entire length (377 nt) were used. As
templates for in vitro transcription, PCR products obtained from
pCK-GFPC3 (Yelina et al., 2005) were used. PCR was carried out with
two pairs of primers, C3-ds-T7-59 (59-GTAATACGACTCACTATAGGGAGAGGGTGAAGGTGATGCAAC-39) and C3-ds-39 (59-GGGCTGCCGTGATGTATACATTGTGT-39), and C3-ds-T7-39 (59-GTAATACGACTCACTATAGGGCTGCCGTGATGTATACATTGTGT-39)
and C3-ds-59 (59-GGGAGAGGGTGAAGGTGATGCAAC-39).
Ultracentrifugation in a sucrose concentration gradient. Protein
preparations were layered onto a 10–40 % sucrose gradient prepared
in buffer containing 50 mM Tris/HCl (pH 7.8), 100 mM NaCl,
5 mM MgCl2 and 2 mM dithiothreitol, and centrifuged at 4 uC for
21 h at 36 000 r.p.m. in an SW41 rotor in a Beckman L-2
ultracentrifuge. Proteins from the collected fractions were precipitated with 12 % trichloroacetic acid. The precipitates were collected
by centrifugation, washed in acetone, dried, dissolved in Laemmli
sample buffer and analysed by SDS-PAGE and immunoblotting.
Dynamic laser light scattering (DLS). A DLS device (Zetasizer
Nano ZS; Malvern Instruments) with a helium–neon laser (633 nm)
was used. Measurements were performed in 1 cm cells in 10 mM
Tris/HCl (pH 7.5) at protein concentrations of 0.05–0.15 mg ml21. A
Peltier thermostat system maintained the temperature at +25 uC. All
curves were fitted using Dispersion Technology Software v5.10.
RESULTS
Construction of recombinant clones and expression in
Escherichia coli. The vector pQE30-N63K used for expression of
Limited proteolysis of recombinant PSLV TGBp1
in E. coli
N63K, comprising aa 1–290 of the 63 kDa (63K) protein, has been
described previously (Kalinina et al., 2001). Based on pQE30-N63K
(Kalinina et al., 2001), plasmids pQE30-NTD and pQE30-ID were
Expression of the full-length 63K protein in E. coli after
IPTG induction (Kalinina et al., 2001) often resulted in its
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
3023
V. V. Makarov and others
fragmentation into several cleaved products, the yield of
which increased after prolonged induction. Such protein
fragmentation may result from a depletion of media
components that mimic minimal medium conditions
known to enhance proteolytic attack by intracellular
proteases (Schlotmann & Beyreuther, 1979). Two major
proteolytic fragments co-purified with the full-length 63K
protein during Ni-NTA agarose chromatography (Fig. 1a),
showing that these fragments contained the 63K N
terminus carrying the 6-His tag.
To identify the cleavage sites in the 63K protein, purified 6His-tagged proteolytic fragments were analysed by mass
spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis showed that
the larger product that migrated in the gel as a 50 kDa protein
corresponded to the N-terminal half of the 576 aa 63K protein
with its C terminus located between aa 279 and 294. The
smaller product that migrated in the gel as a 30 kDa protein
represented the extreme N terminus of the 63K protein whose
C terminus was located between aa 188 and 202. Thus, both of
these 63K fragments, as well as the full-length 63K protein
(Fig. 1a), had gel mobilities lower than expected based on their
molecular masses. These reduced mobilities might be
attributed to stretches of positively charged amino acid
residues at their N terminus. In a total extract of E. coli
expressing the 63K protein, one more proteolytic fragment of
approximately 35 kDa was revealed by Western blotting with
antibodies against the C-terminal region of the 63K protein
comprising the HELD (Fig. 1b). This fragment therefore
represented a C-terminal portion of the 63K protein and
lacked the 6-His tag located at the N terminus of the
recombinant 63K protein, preventing co-purification with the
full-length polypeptide. It should be noted that in vitro
protease treatment of multi-domain cellular and viral proteins
containing RNA/DNA helicase domains also resulted in
release of these domains as distinct protein fragments resistant
to further proteolysis (O’Reilly et al., 1995; Bae et al., 2001).
In E. coli cells grown on minimal medium, proteolytic
attack presumably takes place during synthesis of the
protein, and proteases preferentially attack inter-domain
linkers in multi-domain polypeptides (Schlotmann &
Beyreuther, 1979; Corchero et al., 1996). Thus, we
hypothesized that the 63K protein contains at least three
domains, namely the extreme NTD, an ID and the Cterminal HELD, which are separated by protease-sensitive
linkers at aa 188–202 and 279–294. Interestingly, the ID
could not be detected in our experiments as an individual
polypeptide, presumably reflecting its low stability in
bacteria.
Secondary structure prediction for the TBGp1
extension domain
The 63K amino acid sequence was analysed to predict
secondary structure elements in the N-terminal extension
domain (aa 1–300) preceding the conserved HELD.
Strikingly, several algorithms of sequence analysis, such
as those implemented in the web-based services SCRATCH
(Cheng et al., 2005) and FoldIndex (Prilusky et al., 2005),
confidently predicted that the N-terminal part of the 63K
protein (from aa 1 to 210–223) represented a natively
unfolded protein region (Fig. 2a). Therefore, according to
this prediction, the NTD could be considered an
unstructured protein region. Conversely, the region
corresponding to the ID was predicted as the folded part
of the 63K protein (Fig. 2a). Thus, the NTD and ID of
PSLV TGBp1 that were proposed to be distinct protein
domains based on proteolysis data appeared to have
different predicted structural features. Further analysis of
TGBp1 encoded by other viruses with hordei-like TGBs,
namely BSMV, potato mop-top virus (PMTV; genus
Pomovirus) and peanut clump virus (PCV; genus
Pecluvirus), resulted in similar structure predictions for
the N-terminal extension domains (Fig. 2b).
Fig. 1. Spontaneous proteolysis of the recombinant 63K protein in E. coli. (a) Equal volumes of E. coli culture expressing 63K
were taken at different time points (0.5–6 h) after induction with IPTG. Proteins were purified by Ni-NTA chromatography under
denaturing conditions, separated by 15 % SDS-PAGE and stained with Coomassie blue. The sizes of the molecular mass
markers are indicated on the left (kDa). Major degradation products were cut out of the gel and subjected to enzymic proteolysis
and MS analysis on a MALDI-TOF MS system. The C-terminal amino acid sequences are shown on the right. (b) Total protein
extract of E. coli cells after 2 h induction with IPTG (lane 1) and a preparation of the recombinant HELD (lane 2) were separated
by 15 % SDS-PAGE and analysed by Western blotting with antibodies against the HELD. The sizes of the molecular mass
markers are indicated on the left (kDa). HELD was termed C-63K in our previous study (Kalinina et al., 2001).
3024
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Journal of General Virology 90
Domain organization of hordeivirus TGBp1
expressed as separate polypeptides in bacteria, purified and
renatured. The CD spectrum of the NTD had a strong
minimum at 202 nm ([h]2025214 000u) and a weak signal
at longer wavelengths (Fig. 3), typical for completely
disordered proteins (Adler et al., 1973; Johnson, 1988;
Uversky, 2002). The ID gave a spectrum characteristic of
proteins with significant b-structure content and some
fraction of a-helices (Fig. 3) (Sreerama & Woody, 2004).
According to current opinion, only a-helix content can be
determined quantitatively from the 200–250 nm CD
spectra (Sreerama & Woody, 2004). Therefore, we
estimated the a-helix content in the ID using the
Greenfield–Fasman equation (Greenfield & Fasman,
1969). This calculation gave a value of 10–12 %. Thus,
the CD spectra of TGBp1 deletion mutants confirmed the
suggestion that the NTD and ID differ drastically in their
structure: the NTD is unstructured whilst the ID contains a
significant amount of b-structure elements and a fraction
of a-helices.
RNA-binding activity of NTD and ID domains
The full-length 63K protein and its separated N-terminal
extension region were shown previously by a NorthWestern assay to bind ssRNA efficiently and nonspecifically at NaCl concentrations ranging from 50 to
500 mM (Kalinina et al., 2001), which is in agreement with
previous results on the RNA-binding properties of BSMV
TGBp1 (Donald et al., 1997). To determine whether the
proposed domains of the 63K N-terminal region exhibited
a ssRNA-binding activity, the NTD and ID expressed in E.
coli as separate polypeptides were immobilized on a
nitrocellulose membrane and incubated with a non-specific
32
P-labelled RNA transcript. As a control, we used the
TGBp1 deletion mutant N63K (comprising aa 1–290 of
63K) (Kalinina et al., 2001). Both the NTD and N63K
bound ssRNA with a similar efficiency at NaCl concentrations ranging from 50 to 300 mM and exhibited no
Fig. 2. Prediction of the multi-domain organization of the 63K
protein and FoldIndex prediction for hordei-like TGBp1s. (a)
Putative domain structure of the 63K protein of PSLV. Numbers
indicate the borders of domains and possible inter-domain
spacers. Domains are indicated by boxes. (b) FoldIndex prediction
of folded and unfolded regions in TGBp1s encoded by BMSV
(genus Hordeivirus), PMTV (genus Pomovirus) and PCV (genus
Pecluvirus). The box indicates the HELD of the TGB1 proteins.
CD spectra of TGBp1 deletion mutants
To analyse the structural properties of the proposed
domains of the 63K N-terminal extension region, the CD
spectra in the far UV region (198–250 nm) were measured
for the NTD (aa 1–190 of the 63K protein) and the central
protein region comprising the ID (aa 190–290), which were
http://vir.sgmjournals.org
Fig. 3. CD spectra of the 63K NTD and ID expressed in E. coli as
separate polypeptides. Far-UV CD spectra of the NTD and ID were
recorded at room temperature.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
3025
V. V. Makarov and others
detectable binding at 500 mM NaCl, whereas the ID
interacted with RNA only at 50–150 mM NaCl (Fig. 4a).
Increases in NaCl concentration up to 300 mM blocked the
ID RNA-binding activity (Fig. 4a).
The ability of the NTD, ID and N63K to bind ssRNA was
further compared in a gel-shift assay. In these experiments,
the NTD, similarly to N63K, formed protein–RNA
complexes migrating in the gel (Fig. 4b), whilst ID-formed
complexes were unable to enter the gel (Fig. 4b).
Application of a Hill transformation to quantified gel-shift
data (Marcos et al., 1999) gave a Hill coefficient of
approximately 2.9, strongly suggesting that the ID bound
RNA in a cooperative manner. The ability of ID to bind
RNA cooperatively was confirmed in a gel-shift assay using
the ID region of BSMV TGBp1 (data not shown). As
reported previously, N63K (which included both the NTD
and ID) formed protein–RNA complexes capable of
migrating into the gel (Fig. 4b; Kalinina et al., 2001).
Thus, the ID RNA-binding activity resulting in formation
of fully retarded RNA–protein complexes was suppressed
when the ID was part of N63K. To analyse a possible
mutual influence of the ssRNA-binding activities determined by the NTD and ID, these two separate protein
fragments were mixed in an equimolar ratio and tested in a
gel-shift assay. In contrast with N63K, where the NTD and
ID were present as parts of a single polypeptide, the
mixture of the separate NTD and ID gave rise to complexes
that were fully retarded and that migrated into the gel
(Fig. 4b). These data showed that when the ID is a part of
N63K, the ID-specific RNA-binding activity is masked,
probably due to conformational restrictions, rather than
being outcompeted by the NTD-specific binding activity.
Earlier in vitro analysis of BSMV TGBp1 RNA binding
revealed its ability to bind dsRNA (Donald et al., 1997).
Therefore, we tested the ability of the PSLV TGBp1 NTD
and ID to bind dsRNA. In these experiments, two annealed
non-specific in vitro transcripts complementary to each
other along their entire length were used, so that ssRNA
fragments were not present at the ends of the duplex (see
Methods). One of these transcripts was used as a control
ssRNA. In gel-shift experiments, both the NTD and ID
were able to bind dsRNA (Fig. 5). Comparison of the
protein : RNA ratio resulting in a degree of retardation that
was similar for ssRNA and dsRNA revealed that both the
NTD and ID bound the two types of RNA with a similar
efficiency (Fig. 5). These data are in agreement with the
results of BSMV TGBp1 deletion analysis demonstrating
that dsRNA binding involves the N-terminal extension
region (Donald et al., 1997) and showed that both the NTD
and ID can be involved in such an interaction.
Fig. 4. ssRNA-binding activity of the NTD and
ID. (a) North-Western assay for RNA-binding
activity of the NTD, ID and N63K protein. A
nitrocellulose membrane with immobilized proteins was stained with Ponceau red and
incubated with 32P-labelled RNA transcripts
at different NaCl concentration. (b) Gel-shift
assay for RNA-binding of the NTD, ID or an
NTD/ID mixture. Increasing amounts of each
recombinant protein or the NTD and ID mixed
in an equimolar ratio were incubated with
0.5 mg tobacco mosaic virus RNA and subjected to electrophoresis on a 1 % agarose
non-denaturing gel containing ethidium bromide. Protein : RNA molar ratios are indicated
above each lane. The lane marked ‘RNA’ is the
control without protein added. N63K was used
as a control.
3026
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Journal of General Virology 90
Domain organization of hordeivirus TGBp1
Fig. 5. dsRNA-binding activity of the NTD and
ID. Increased amounts of each recombinant
protein were incubated with 0.2 mg of a
377 bp dsRNA or with 0.4 mg of a 377 nt
control ssRNA and subjected to electrophoresis on a 1 % agarose non-denaturing gel
containing ethidium bromide. Protein : RNA
molar ratios are indicated above each lane.
Protein–protein interactions analysed in sucrose
gradients
Previously, we found that the HELD region of the 63K
protein is involved in homologous protein–protein interactions (Leshchiner et al., 2006). To test the self-interaction
potential of the isolated NTD and ID, purified recombinant proteins were fractionated in a 10–40 % sucrose
gradient and the resulting gradient fractions were analysed
by Western blotting with antibodies against the 63K
protein or its N- and C-terminal portions. Separately
expressed HELD, used as a control, formed a dimer that
was stable upon heating in SDS-containing sample buffer
and subsequent SDS-PAGE, and also formed oligomers
with a molecular mass of about 120–200 kDa (Fig. 6). Both
the NTD and ID were found as oligomers of different
orders. The majority of the NTD was found in the gradient
fractions similar to those for HELD, whilst the ID
predominantly formed oligomers of molecular masses up
to 150 kDa (Fig. 6). In contrast to the NTD, the ID formed
considerable amounts of high-molecular-mass complexes
of more than 440 kDa. In addition, and similar to HELD,
the ID was capable of forming a dimer, which was stable
upon sample preparation and SDS-PAGE (Fig. 6).
Interestingly, N63K, similar to the ID, was able to form
high-molecular-mass multimers (Fig. 6), which can
therefore be attributed to its ID moiety. Thus, the sucrose
gradient experiments demonstrated that the NTD, ID and
HELD are capable of self-interactions in solution.
Importantly, only the ID and ID-containing NTD were
able to form large multimeric complexes.
We analysed further the potential interactions between
TGBp1 domains. When an equimolar mixture of the NTD
http://vir.sgmjournals.org
and ID was analysed in a sucrose gradient, the ID
sedimentation profile was similar to that in the experiment
with the ID only, whereas the sedimentation profile of the
NTD in the mixture was considerably changed compared
with the experiment with NTD alone, and became similar
to the ID sedimentation profile (Fig. 6). Similarly,
sedimentation analysis of an equimolar mixture of HELD
and ID revealed that, under these conditions, the HELD
sedimentation pattern was changed and closely resembled
that of the ID. These data demonstrate that the ID could
interact with both the NTD and HELD in solution.
Interactions between the NTD and HELD could not be
analysed in these experiments as these TGBp1 domains
have similar sedimentation profiles (Fig. 6).
DLS analysis
The DLS method is generally used to determine the size
distribution of small particles dispersed in solution and is
often applied to protein complexes (Schmitz, 1990; Barilla
et al., 2005; Tönges et al., 2006). We employed DLS analysis
to determine the hydrodynamic diameter, or size, of
multimers formed by the NTD, ID and N63K. Analysis of
the NTD preparations revealed protein particles of
15±3 nm diameter (Fig. 7). In ID preparations, the major
population of particles ranged from 20 to 55 nm with a
mean size of 40 nm. Distribution profiles found for the
hydrodynamic diameter of N63K had a maximum at
60 nm and a range of 45–80 nm (Fig. 7). It should be
emphasized that the detection of small-sized particles in
the presence of high-molecular-mass multimers was
impossible due to the technical restrictions of the DLS
method (Schmitz, 1990). Thus, after treatment of ID and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
3027
V. V. Makarov and others
Fig. 6. Fractionation of 63K protein deletion
mutants by ultracentrifugation in sucrose
density gradients. Preparations of the recombinant proteins were centrifuged in a 10–40 %
sucrose density gradient. The proteins of the
gradient fractions were analysed by SDSPAGE and Western blotting. The positions of
marker proteins are indicated by arrows: BSA
(67 kDa), aldolase (158 kDa), catalase
(232 kDa), and apoferritin (440 kDa). D,
Dimer; M, monomer.
N63K preparations with SDS at a concentration as low as
0.01 %, a population of large-sized particles was not found,
whereas small particles with sizes of 5.0±2.0 nm were
easily detected (data not shown). Therefore, the DLS data
demonstrated that the ID and N63K, unlike the NTD, are
capable of forming large multimeric structures.
Fig. 7. Size distribution of the hydrodynamic diameter of particles
in the NTD, ID and N63K preparations by the DLS method.
Renatured proteins were analysed by DLS. Size (nm) is the
hydrodynamic diameter. Note, no values fell outside the range 10–
100 nm. Size distribution by number of particles (%) is shown for
the NTD, ID and N63K preparations.
3028
DISCUSSION
Hordeiviral genomic RNAs form RNP complexes containing TGBp1 as the major if not the sole protein component
(Brakke et al., 1988; Lim et al., 2008; Jackson et al., 2009).
As shown in vitro, two RNA-binding activities of
hordeivirus TGBp1 can be involved in the formation of
such RNPs, namely those specified by the C-terminal
NTPase/helicase domain and the two clusters of positively
charged amino acid residues in the protein N-terminal
extension region (Donald et al., 1997; Kalinina et al., 2001).
These clusters are crucial for both the in vitro RNA-binding
ability of the extension region and the long-distance
transport of virus during infection, but are not necessary
for viral cell-to-cell movement (Kalinina et al., 2001).
However, the role of the TGBp1 N-terminal region in viral
vascular transport remains unknown.
In this paper, we have reported that the N-terminal
extension region of the 63 kDa PSLV TGBp1 consists of
two domains termed the NTD and the ID. The NTD was
found to be a disordered, or natively unfolded, protein
domain. Intrinsically unstructured or natively unfolded
proteins and protein domains are characterized by a lack of
specific structure and represent a distinct protein element
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Journal of General Virology 90
Domain organization of hordeivirus TGBp1
involved, in most cases, in molecular interactions (Fink,
2005). It is generally accepted that functions of natively
unfolded proteins or protein regions involve their binding
to ligands such as proteins or nucleic acids, and that
interaction with a target ligand often induces a transition
from the previously disordered structure to a folded form,
which represents the functional state of the polypeptide
(Uversky, 2002; Fink, 2005). Two RNA-binding basic
clusters of the 63K N-terminal extension region (Kalinina
et al., 2001) fall into the natively unfolded NTD; therefore,
one can speculate that NTD interaction with RNA can
render this region structured and, as a consequence,
competent for functional interactions involved, for
example, in virus long-distance transport. On the other
hand, natively unfolded domains are often found in
proteins capable of self-assembly into large multimeric
complexes such as viral capsids and nucleocapsids (Namba,
2001). Natively unfolded domains both prevent unwanted
spontaneous assembly of such proteins, as has been shown
for the measles virus nucleoprotein (Longhi et al., 2003),
and stabilize multimeric complexes due to folding upon
interaction with the correct partners (Namba, 2001).
The ID represents a protein region with a pronounced
secondary structure. The ID is capable of self-interaction,
resulting in the formation of stable dimers and highmolecular-mass multimeric complexes. Similar properties
are found for the whole N-terminal extension region
(N63K). Although comparative analysis of the ID sequence
failed to reveal any known RNA-binding motifs, the ID
appeared to bind ssRNA in a cooperative manner.
Importantly, the ID-specific cooperative ssRNA binding
was manifested only when the ID was analysed as a separate
polypeptide, and was not detected when the ID was a part of
a larger TGBp1 fragment comprising both the NTD and ID
(Kalinina et al., 2001). Thus, the NTD and ID could not be
considered as functionally independent domains, and one
can speculate that the presence of the NTD either induces ID
structural changes that are incompatible with RNA binding
or binds to the ID to hinder its RNA-interacting interface.
The latter hypothesis was confirmed by the observation that
the isolated NTD and ID were able to interact in solution.
This finding demonstrated that the ID can be considered as
yet another NTD ligand that might induce an NTD
conformational transition from the unfolded to an ordered
state. This hypothesis implies that, in the native TGBp1 or in
TGBp1-formed RNP complexes, the NTD could represent
an ordered structure. Additionally, we found that the ID is
capable of interaction with HELD. One can speculate that ID
interaction with two other TGBp1 structural domains might
be the basis for remodelling of the TGBp1-formed RNP
complexes during different phases of cell-to-cell and longdistance transport. It should also be kept in mind that the
TGBp1 NTPase/helicase domain has its own RNA-binding
activity. Therefore, RNP formation by TGBp1 can involve
RNA interaction with three different protein domains.
Although TGBp1-formed RNP complexes isolated from
BSMV-infected plants contain only virus-specific ssRNAs
http://vir.sgmjournals.org
of positive polarity (Lim et al., 2008), BSMV TGBp1 also
appears to bind dsRNA in vitro (Donald et al., 1997).
Moreover, both BSMV and PSLV TGBp1 are able to
unwind RNA duplexes in vitro, thus demonstrating the
RNA helicase activity (Kalinina et al., 2002). In this paper,
we showed that both the NTD and ID had dsRNA-binding
activity, which is in good agreement with the results of
BSMV TGBp1 deletion analysis reported by Donald et al.
(1997). Indeed, the truncated BSMV TGBp1 with the Nterminal 195 aa residues removed (the region including the
NTD and most of the ID according to BSMV and PSLV
sequence alignment) lost the ability to bind dsRNA
(Donald et al., 1997). Interestingly, an internal deletion
in BSMV TGBp1 spanning most of the ID sequence
resulted in the inability of excess heterologous ssRNA or
dsRNA to displace homologous ssRNA from complexes
with such mutant protein in competition experiments
(Donald et al., 1997), suggesting that the mutant lost –
probably due to improper protein conformation – the
native RNA-binding properties of the two other RNAbinding TGBp1 domains, the NTD and HELD. A similar
effect was shown by an internal deletion in the NTPase/
helicase domain (Donald et al., 1997) that could potentially
destroy its structural subdomain 2A (Morozov & Solovyev,
2003). These data suggest that the structural integrity of
TGBp1 domains is required for their correct interactions
and, in turn, their RNA-binding properties are dependent
on such interactions. It has been suggested that the ability
of TGBp1 to bind and unwind dsRNA can be important
for unwinding of secondary structure elements in viral
genomic RNAs to form RNP complexes capable of
translocation through plasmodesmata (Morozov &
Solovyev, 2003) or of vectorial diffusion, in a complex
with TGBp2/TGBp3 rafts, along the endoplasmic reticulum
(Epel, 2009).
Many RNA chaperones – proteins able to bind nonspecifically to RNA and resolve its secondary structure in
an ATP-independent manner (Rajkowitsch et al., 2007;
Russell, 2008) – possess natively unfolded regions serving
as molecular recognition elements typically required for
interaction with RNA substrates (Tompa & Csermely,
2004). Upon such interaction, according to an entropy
transfer model, unfolding of RNA secondary structure may
be accompanied by folding of natively unfolded regions of
RNA chaperones (Tompa & Csermely, 2004). Several wellstudied RNA chaperones such as YB-1, the major protein
of messenger RNP complexes, and E. coli regulatory
protein StpA have a bipartite structure composed of two
domains, one of which is stably folded, exhibiting RNAbinding and/or protein oligomerization activity, whereas
another is natively unfolded, containing sites of RNA
chaperone activity (Matsumoto & Wolffe, 1998; Kloks et
al., 2002; Mayer et al., 2007; Rajkowitsch et al., 2007). The
PSLV TGBp1 N-terminal extension region is organized in a
similar way, with RNA binding and protein oligomerization functions associated with the structured ID and
another RNA-binding activity associated with the natively
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
3029
V. V. Makarov and others
unfolded NTD. Additionally, similarly to YB-1, which has
been found to form homomultimeric complexes with
molecular masses of up to 800 kDa (Evdokimova et al.,
1995), N63K forms high-molecular-mass multimers. Taken
together, the available data allow us to propose that the
PSLV TGBp1 N-terminal extension region can share some
properties with RNA chaperones. Further research is
required to verify this hypothesis and to determine the
step(s) of viral transport in plants at which this activity is
required.
Like many plant viruses, most TGB-containing virus
species require viral coat protein (CP) for cell-to-cell and
vascular movement. Comparisons of virus-coded protein
arrays required for transport of different TGB-containing
viruses has revealed an inverse correlation between the size/
complexity of the TGBp1 N-terminal extension region and
the necessity for viral CP in viral spread in plants. One side
is represented by potexviruses, where TGBp1 contains no
N-terminal extension region and the CP is essential for
both cell-to-cell and long-distance transport (Beck et al.,
1991; Chapman et al., 1992). It is assumed that virions
rather than viral RNA or a viral RNP complex is the
transport form of the virus genome (Santa Cruz et al.,
1998; Morozov & Solovyev, 2003), which is in agreement
with the discovery of specific complexes between TGBp1
and potexvirus virions (Atabekov et al., 2000; Kiselyova
et al., 2003; Rodionova et al., 2003; Karpova et al., 2006).
The opposite pole is occupied by hordeiviruses characterized by the longest known TGBp1 N-terminal extension
region. In this case, the CP is dispensable for both cell-tocell and systemic viral movement, which is believed to
occur in the form of RNP complexes containing TGBp1
and genomic RNAs (Brakke et al., 1988; Petty & Jackson,
1990; Lim et al., 2008; Jackson et al., 2009). Between the
two extremes are BNYVV (genus Benyvirus) and PCV
(genus Pecluvirus) with TGBp1 characterized by a shorter
N-terminal extension region comprising the ID and a
truncated NTD (Morozov & Solovyev, 2003). In BNYVV
and PCV, the CP is dispensable for viral cell-to-cell
movement (as in hordeiviruses) but is required for vascular
transport (as in potexviruses) (Schmitt et al., 1992; Tamada
et al., 1996; Herzog et al., 1998). Apparently, transport
forms of BNYVV/PCV genomes involved in local and
systemic movement are structurally different. One can
presume that the hordeivirus NTD may play the role of
RNA chaperone specifically involved in phloem transport
by stabilizing/protecting genomic RNAs, therefore functioning similarly to the CP in other viruses. However, the
CP and NTD fall into distinct categories in spite of a
common function in virus spread: CPs stabilize RNA
folding via specific binding and formation of a stable coat,
whereas the NTD, like other RNA chaperones, may unfold
RNA by means of transient and non-specific binding.
Another rather unusual example of an intermediate TGB
transport system is provided by PMTV. PMTV TGBp1 has
an NTD similar in size to that of PCV (Morozov &
Solovyev, 2003). Nevertheless, PMTV TGBp1 is able to
3030
function similarly to hordeivirus TGBp1: two PMTV
genomic RNAs coding for a replicase and TGB proteins
can move locally and systemically in the absence of the viral
CP, presumably in the form of TGBp1-formed RNP
complexes (Savenkov et al., 2003). However, PMTV
TGBp1 has an additional function in viral systemic spread:
in wild-type infections, the genomic RNA component
encoding the CP and a CP-readthrough moves long
distances in the form of a virus particle, one end of which
is believed to be associated with the CP-readthrough and
TGBp1 (Torrance et al., 2009). Thus, PMTV TGBp1 can
function in systemic transport in two ways, either forming
RNP complexes like hordeivirus TGBp1 or interacting with
one end of the virion similar to potexvirus TGBp1.
Conceivably, in the latter mode, the PMTV TGBp1 Nterminal extension region can function as a protein region
responsible for protein–protein interaction, possibly due to
the natively unfolded NTD.
ACKNOWLEDGEMENTS
The work was supported by grant no. 07-04-00061-a of the Russian
Foundation for Basic Research, the Royal Society (M. E. T. and
N. O. K.) and the Scottish Government (M. E. T.).
REFERENCES
Adler, A. J., Greenfield, N. J. & Fasman, G. D. (1973). Circular
dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol 27, 675–735.
Atabekov, J. G., Rodionova, N. P., Karpova, O. V., Kozlovsky, S. V. &
Poljakov, V. Y. (2000). The movement protein-triggered in situ
conversion of potato virus X virion RNA from a nontranslatable into
a translatable form. Virology 271, 259–263.
Bae, S. H., Kim, J. A., Choi, E., Lee, K. H., Kang, H. Y., Kim, H. D., Kim,
J. H., Bae, K. H., Cho, Y. & other authors (2001). Tripartite structure
of Saccharomyces cerevisiae Dna2 helicase/endonuclease. Nucleic Acids
Res 29, 3069–3079.
Barilla, D., Rosenberg, M. F., Nobbmann, U. & Hayes, F. (2005).
Bacterial DNA segregation dynamics mediated by the polymerizing
protein ParF. EMBO J 24, 1453–1464.
Beck, D. L., Guilford, P. J., Voot, D. M., Andersen, M. T. & Forster, R. L.
(1991). Triple gene block proteins of white clover mosaic potexvirus
are required for transport. Virology 183, 695–702.
Bleykasten, C., Gilmer, D., Guilley, H., Richards, K. E. & Jonard, G.
(1996). Beet necrotic yellow vein virus 42 kDa triple gene block
protein binds nucleic acid in vitro. J Gen Virol 77, 889–897.
Boevink, P. & Oparka, K. J. (2005). Virus–host interactions during
movement processes. Plant Physiol 138, 1815–1821.
Brakke, M. K., Ball, E. M. & Langenberg, W. G. (1988). A non-capsid
protein associated with unencapsidated virus RNA in barley infected
with barley stripe mosaic virus. J Gen Virol 69, 481–491.
Chapman, S., Hills, G., Watts, J. & Baulcombe, D. (1992). Mutational
analysis of the coat protein gene of potato virus X: effects on virion
morphology and viral pathogenicity. Virology 191, 223–230.
Cheng, J., Randall, A., Sweredoski, M. & Baldi, P. (2005). SCRATCH: a
protein structure and structural feature prediction server. Nucleic
Acids Res 33, W72–W76.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Journal of General Virology 90
Domain organization of hordeivirus TGBp1
Corchero, J. L., Viaplana, E., Benito, A. & Villaverde, A. (1996). The
position of the heterologous domain can influence the solubility and
proteolysis of b-galactosidase fusion proteins in E. coli. J Biotechnol
48, 191–200.
Kloks, C. P., Spronk, C. A., Lasonder, E., Hoffmann, A., Vuister, G. W.,
Grzesiek, S. & Hilbers, C. W. (2002). The solution structure and
DNA-binding properties of the cold-shock domain of the human Ybox protein YB-1. J Mol Biol 316, 317–326.
Donald, R. G., Lawrence, D. M. & Jackson, A. O. (1997). The barley
stripe mosaic virus 58-kilodalton b(b) protein is a multifunctional
Leshchiner, A. D., Solovyev, A. G., Morozov, S. Yu. & Kalinina, N. O.
(2006). A minimal region in the NTPase/helicase domain of the
RNA binding protein. J Virol 71, 1538–1546.
TGBp1 plant virus movement protein is responsible for ATPase
activity and cooperative RNA binding. J Gen Virol 87, 3087–3095.
Epel, B. L. (2009). Plant viruses spread by diffusion on ER-associated
movement-protein-rafts through plasmodesmata gated by viral
induced host b-1,3-glucanases. Semin Cell Dev Biol (in press).
doi:10.1016/j.semcdb.2009.05.010
Evdokimova, V. M., Wei, C. L., Sitikov, A. S., Simonenko, P. N.,
Lazarev, O. A., Vasilenko, K. S., Ustinov, V. A., Hershey, J. W. &
Ovchinnikov, L. P. (1995). The major protein of messenger
ribonucleoprotein particles in somatic cells is a member of the Ybox binding transcription factor family. J Biol Chem 270, 3186–3192.
Lim, H. S., Bragg, J. N., Ganesan, U., Lawrence, D. M., Yu, J., Isogai, M.,
Hammond, J. & Jackson, A. O. (2008). Triple gene block protein
interactions involved in movement of Barley stripe mosaic virus. J Virol
82, 4991–5006.
Longhi, S., Receveur-Bréchot, V., Karlin, D., Johansson, K., Darbon,
H., Bhella, D., Yeo, R., Finet, S. & Canard, B. (2003). The C-terminal
Fink, A. L. (2005). Natively unfolded proteins. Curr Opin Struct Biol
domain of the measles virus nucleoprotein is intrinsically disordered
and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278, 18638–18648.
15, 35–41.
Lucas, W. J. (2006). Plant viral movement proteins: agents for cell-to-
Garnier, J., Osguthorpe, D. J. & Robson, B. (1978). Analysis of the
cell trafficking of viral genomes. Virology 344, 169–184.
accuracy and implications of simple methods for predicting the
secondary structure of globular proteins. J Mol Biol 120, 97–120.
Marcos, J. F., Vilar, M., Pérez-Payá, E. & Pallás, V. (1999). In vivo
Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, V. M.
(1989). Two related superfamilies of putative helicases involved in
replication, recombination, repair and expression of DNA and RNA
genomes. Nucleic Acids Res 17, 4713–4730.
detection, RNA-binding properties and characterization of the RNAbinding domain of the p7 putative movement protein from carnation
mottle carmovirus (CarMV). Virology 255, 354–365.
Matsumoto, K. & Wolffe, A. P. (1998). Gene regulation by Y-box
Greenfield, N. & Fasman, G. D. (1969). Computed circular dichroism
proteins: coupling control of transcription and translation. Trends
Cell Biol 8, 318–323.
spectra for the evaluation of protein conformation. Biochemistry 8,
4108–4116.
Mayer, O., Rajkowitsch, L., Lorenz, C., Konrat, R. & Schroeder, R.
(2007). RNA chaperone activity and RNA-binding properties of the E.
Haupt, S., Cowan, G. H., Zeigler, A., Roberts, A. G., Oparka, K. J. &
Torrance, L. (2005). Two plant–viral movement proteins traffic in the
coli protein StpA. Nucleic Acids Res 35, 1257–1269.
endocytic recycling pathway. Plant Cell 17, 164–181.
design of a multifunctional machine for plant virus movement. J Gen
Virol 84, 1351–1366.
Herzog, E., Hemmer, O., Hauser, S., Meyer, G., Bouzoubaa, S. &
Fritsch, C. (1998). Identification of genes involved in replication and
movement of peanut clump virus. Virology 248, 312–322.
Jackson, A. O., Lim, H.-S., Bragg, J., Ganesan, U. & Lee, M. Y. (2009).
Hordeivirus replication, movement, and pathogenesis. Annu Rev
Phytopathol 47, 385–422.
Johnson, W. C., Jr (1988). Secondary structure of proteins through
circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem 17,
145–166.
Kalinina, N. O., Fedorkin, O. N., Samuilova, O. V., Maiss, E.,
Korpela, T., Morozov, S. Yu. & Atabekov, J. G. (1996). Expression
and biochemical analyses of the recombinant potato virus X 25K
movement protein. FEBS Lett 397, 75–78.
Kalinina, N. O., Rakitina, D. A., Yelina, N. E., Zamyatnin, A. A., Jr,
Stroganova, T. A., Klinov, D. V., Prokhorov, V. V., Ustinova, S. V.,
Chernov, B. K. & other authors (2001). RNA-binding properties of
the 63 kDa protein encoded by the triple gene block of poa semilatent
hordeivirus. J Gen Virol 82, 2569–2578.
Kalinina, N. O., Rakitina, D. V., Solovyev, A. G., Schiemann, J. &
Morozov, S. Y. (2002). RNA helicase activity of the plant virus
Morozov, S. Yu. & Solovyev, A. G. (2003). Triple gene block: modular
Namba, K. (2001). Roles of partly unfolded conformations in
macromolecular self-assembly. Genes Cells 6, 1–12.
O’Reilly, E. K., Tang, N., Ahlquist, P. & Kao, C. C. (1995). Biochemical
and genetic analyses of the interaction between the helicase-like and
polymerase-like proteins of the brome mosaic virus. Virology 214,
59–71.
Petty, I. T. & Jackson, A. O. (1990). Mutational analysis of barley
stripe mosaic virus RNA beta. Virology 179, 712–718.
Prilusky, J., Felder, C. E., Zeev-Ben-Mordehai, T., Rydberg, E. H.,
Man, O., Beckmann, J. S., Silman, I. & Sussman, J. L. (2005).
FoldIndex: a simple tool to predict whether a given protein sequence
is intrinsically unfolded. Bioinformatics 21, 3435–3438.
Rajkowitsch, L., Chen, D., Stampfl, S., Semrad, K., Waldsich, C.,
Mayer, O., Jantsch, M. F., Konrat, R., Bläsi, U. & Schroeder, R. (2007).
RNA chaperones, RNA annealers and RNA helicases. RNA Biol 4,
118–130.
Rodionova, N. P., Karpova, O. V., Kozlovsky, S. V., Zayakina, O. V.,
Arkhipenko, M. V. & Atabekov, J. G. (2003). Linear remodeling of
helical virus by movement protein binding. J Mol Biol 333, 565–572.
movement proteins encoded by the first gene of the triple gene block.
Virology 296, 321–329.
Russell, R. (2008). RNA misfolding and the action of chaperones.
Karpova, O. V., Zayakina, O. V., Arkhipenko, M. A., Sheval, E. V.,
Kiselyova, O. I., Poljakov, V. Yu., Yaminsky, I. V., Rodionova, N. P. &
Atabekov, J. G. (2006). Potato virus RNA-mediated assembly of
Santa Cruz, S., Roberts, A. G., Prior, D. A. M., Chapman, S. & Oparka,
K. J. (1998). Cell-to-cell and phloem-mediated transport of potato
single-tailed ternary complexes ‘coat protein–RNA–movement protein’. J Gen Virol 87, 2731–2740.
Kiselyova, O. I., Yaminsky, I. V., Karpova, O. V., Rodionova, N. P.,
Kozlovsky, S. V., Arkhipenko, M. V. & Atabekov, J. G. (2003). AFM
study of potato virus X disassembly induced by movement protein.
J Mol Biol 332, 321–325.
http://vir.sgmjournals.org
Front Biosci 13, 1–20.
virus X: the role of virions. Plant Cell 10, 495–510.
Savenkov, E. I., Germundsson, A., Zamyatnin, A. A., Jr, Sandgren, M.
& Valkonen, J. P. (2003). Potato mop-top virus: the coat protein-
encoding RNA and the gene for cysteine-rich protein are dispensable
for systemic virus movement in Nicotiana benthamiana. J Gen Virol
84, 1001–1005.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
3031
V. V. Makarov and others
Schlotmann, M. & Beyreuther, K. (1979). Degradation of the DNA-
Tompa, P. & Csermely, P. (2004). The role of structural disorder in
binding domain of wild-type and i2d lac repressors in Escherichia coli.
Eur J Biochem 95, 39–49.
the function of RNA and protein chaperones. FASEB J 18, 1169–1175.
Schmitt, C., Balmori, E., Jonard, G., Richards, K. E. & Guilley, H.
(1992). In vitro mutagenesis of biologically active transcripts of beet
necrotic yellow vein virus RNA 2: evidence that a domain of the 75kDa readthrough protein is important for efficient virus assembly.
Proc Natl Acad Sci U S A 89, 5715–5719.
Tönges, L., Lingor, P., Egle, R., Dietz, G. P., Fahr, A. & Bähr, M.
(2006). Stearylated octaarginine and artificial virus-like particles for
transfection of siRNA into primary rat neurons. RNA 12, 1431–1438.
Torrance, L., Lukhovitskaya, N. I., Schepetilnikov, M. V., Cowan, G. H.,
Ziegler, A. & Savenkov, E. I. (2009). Unusual long-distance movement
Schmitz, S. K. (1990). An Introduction to Dynamic Light Scattering by
strategies of Potato mop-top virus RNAs in Nicotiana benthamiana. Mol
Plant Microbe Interact 22, 381–390.
Macromolecules. New York: Academic Press.
Uversky, V. N. (2002). Natively unfolded proteins: a point where
Solovyev, A. G., Savenkov, E. I., Agranovsky, A. A. & Morozov, S. Y.
(1996). Comparisons of the genomic cis-elements and coding regions
in RNAb components of the hordeiviruses barley stripe mosaic virus,
lychnis ringspot virus, and poa semilatent virus. Virology 219, 9–18.
Sreerama, N. & Woody, R. W. (2004). Computation and analysis of
biology waits for physics. Protein Sci 11, 739–756.
Yelina, N. E., Erokhina, T. N., Lukhovitskaya, N. I., Minina, E. A.,
Schepetilnikov, M. V., Lesemann, D.-E., Schiemann, J., Solovyev,
A. G. & Morozov, S. Yu. (2005). Localization of Poa semilatent virus
protein circular dichroism spectra. Methods Enzymol 383, 318–351.
cysteine-rich protein in peroxisomes is dispensable for its ability to
suppress RNA silencing. J Gen Virol 86, 479–489.
Tamada, T., Schmitt, C., Saito, M., Guilley, H., Richards, K. & Jonard, G.
(1996). High resolution analysis of the readthrough domain of beet
Zamyatnin, A. A., Jr, Solovyev, A. G., Savenkov, E. I., Germudson, A.,
Sandgren, M., Valkonen, J. P. T. & Morozov, S. Yu. (2004). Transient
necrotic yellow vein virus readthrough protein: a KTER motif is
important for efficient transmission of the virus by Polymyxa betae.
J Gen Virol 77, 1359–1367.
coexpression of individual genes encoded by the triple gene block of
potato mop-top virus reveals requirements for TGBp1 trafficking.
Mol Plant Microbe Interact 17, 921–930.
3032
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 15 May 2017 20:28:58
Journal of General Virology 90