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Virology 393 (2009) 272–285

Contents lists available at ScienceDirect

Virology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o

Analysis of potato virus X replicase and TGBp3 subcellular locations

Devinka Bamunusinghe a,1, Cynthia L. Hemenway b, Richard. S. Nelson c, Anton A. Sanderfoot d,
Chang M. Ye a, Muniwarage A.T. Silva a, M. Payton e, Jeanmarie Verchot-Lubicz a,⁎
a
Department of Entomology and Plant Pathology, Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA
b
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA
c
The Samuel Roberts Noble Foundation 2510 Sam Noble Pky., Ardmore, OK 73401, USA
d
Biology Department, University of Wisconsin-LaCrosse 1725 State St. La Crosse, WI 54601, USA
e
Department of Statistics, Oklahoma State University, Stillwater, OK 74078, USA

a r t i c l e i n f o a b s t r a c t

Article history: Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane
Received 13 May 2009 system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein
Returned to author for revision 9 July 2009 was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent
Accepted 2 August 2009
protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the
Available online 3 September 2009
subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic
Keywords:
observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A
Potexvirus subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation
Potato virus X showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization
Triple gene block represents a region where both proteins may be synthesized and/or function. There is no evidence to
Replicase indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo
membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies
on ER-derived membrane recruitment and membrane proliferation.
Published by Elsevier Inc.

Introduction
Successful virus infection is a consequence of specific interactions
with host subcellular machinery to enable essential events such as
viral protein synthesis, replication, and cell-to-cell spread. The ability
of viruses to interact with host components underlies the appropriate
subcellular targeting of viral proteins and nucleic acids (Ahlquist,
2006; Buck, 1999a; Hwang et al., 2008). It is often presumed that a
viral movement protein (MP) carries infectious material into
uninfected cells in the form of a ribonucleoprotein complex, although
the exact mechanism by which viral genomes are transferred from the
site of synthesis to the plasmodesmata has not been clearly shown
(Haywood et al., 2002; Kiselyova et al., 2001; Lucas, 2006; Nelson,
2005; Verchot-Lubicz et al., 2007).
For most positive strand RNA viruses, viral replicases cause
membrane rearrangements to create a container or protected
environment for genome replication (Mackenzie, 2005; Salonen et
⁎ Corresponding author. Fax: +1 405 744 6039.
E-mail addresses: dbamu001@ucr.edu (D. Bamunusinghe),
cindy_hemenway@ncsu.edu (C.L. Hemenway), rsnelson@noble.org (R.S. Nelson),
sanderfo.anto@uwlax.edu (A.A. Sanderfoot), Verchot.lubicz@okstate.edu
(J. Verchot-Lubicz).
1
Current address for Devinka Bamunusinghe is University of California- Riverside,
Department of Plant Pathology and Microbiology, 3401, Watkins Drive, Boyce Hall,
Riverside, CA 92521.
al., 2005; Sanfacon and Zhang, 2008; Schwartz et al., 2004; Villanueva
et al., 2005). There are wide ranging reports of plant-infecting RNA
viruses associating with invaginations of the endoplasmic reticulum
(ER), chloroplast outer membrane, vacuolar membranes, mitochon-
dria, nuclear envelope, or peroxisomes (Goodin et al., 2007; Hwang et
al., 2008; Jonczyk et al., 2007; Kim et al., 2006; Prod'homme et al.,
2003; Rubino et al., 2001; Turner et al., 2004; Wei and Wang, 2008).
Grapevine fanleaf virus (GFLV), Cowpea mosaic virus (CPMV), Red
clover necrotic mosaic virus (RCNMV) and Tobacco mosaic virus (TMV)
are a few recent examples of viruses whose replication requires
proliferation and/or invagination of ER membranes to sustain virus
replication (Carette et al., 2000; Heinlein et al., 1998; Mas and Beachy,
1999; Ritzenthaler et al., 2002; Turner et al., 2004). CPMV in particular
provides an example of a virus that stimulates host cells to increase
membrane synthesis resulting in specific expansion of the ER with no
obvious change in the Golgi apparatus (Carette et al., 2000), although
little is known about how viruses are able to induce synthesis of
distinct types of cellular membranes.
Tobacco mosaic virus (TMV) replicase is located on the ER or ER
modifications (Buck, 1999b; Ishikawa and Okada, 2004). For TMV,
irregular shaped membrane bound structures containing the 183-
and 126-kDa replication-associated proteins and viral genomes serve
as a vehicle for trafficking virus within and between neighboring
cells (Kawakami et al., 2004; Liu et al., 2005). A current model is that
the TMV replication associated proteins and/or MP, through an

0042-6822/$ – see front matter. Published by Elsevier Inc.

doi:10.1016/j.virol.2009.08.002
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285
association with a membrane bound complex, traffics viral RNA to the
cell wall near plasmodesmata, where it passes through the desmotu-
bule (Asurmendi et al., 2004; Guenoune-Gelbart et al., 2008; Reichel
and Beachy, 1998).
Potato virus X (PVX) is the type member of the Potexvirus genus
and its genome contains five open reading frames (ORFs) (Huisman et
al., 1988). The PVX replicase is a single 166 kDa protein which
contains methyl transferase, helicase, and polymerase domains
required for virus replication (Davenport and Baulcombe, 1997). A
specific PVX protein or protein domain that anchors the replicase to
cellular membranes has not been identified. In addition, the site for
PVX replication has not been described. The remaining four PVX
proteins include the coat protein (CP) and triple gene block proteins
(TGB) that are required for cell-to-cell movement and encapsidation
(Huisman et al., 1988). The TGB proteins are derived from three
partially overlapping ORFs and are named TGBp1, TGBp2, and TGBp3
(Beck et al., 1991; Verchot et al., 1998). TGBp1 is a multifunctional
protein that suppresses gene silencing, has RNA binding and helicase
activity, unwinds virion particles to promote translation of genomic
RNA, and is responsible for increasing plasmodesmal permeability
enabling viruses to pass from an infected cell into an uninfected cell
(Bayne et al., 2005; Howard et al., 2004; Hsu et al., 2004; Kalinina et
al., 2002; Kiselyova et al., 2003; Lough et al., 2000; Lough et al., 1998;
Rodionova et al., 2003). While a typical plasmodesma restricts
diffusion of molecules less than 1 kDa, TGBp1 causes expansion of
the pore to enable transfer of larger macromolecules between cells.
PVX TGBp2 and TGBp3 are low molecular weight (12 and 8 kDa,
respectively) membrane-binding proteins (Ju et al., 2005, 2008;
Krishnamurthy et al., 2003; Mitra et al., 2003). Since antibodies are
not available for immunodetection of TGBp2 or TGBp3, prior
investigations employed noninvasive imaging of intrinsically fluores-
cent proteins (GFP, YFP, CFP) fused to TGBp2 or TGBp3 to visualize
protein accumulation in leaves and protoplasts (Samuels et al., 2007;
Schepetilnikov et al., 2005; Zamyatnin et al., 2006). In addition,
electron microscopic analysis of transgenic plants expressing GFP
fused to TGBp2 or TGBp3 identified GFP–TGBp2 associating with the
ER and novel vesicles, whereas TGBp3–GFP fusions associated mainly
with ER strands. The ER lumenal binding protein BiP was identified in
all TGBp2 and TGBp3 containing membrane compartments (Ju et al.,
Fig. 1. Infectious clones of PVX.GFP and PVX.TGBp3–GFP show green fluorescent foci. (A) Schematics of the viral genomes with GFP included. PVX.GFP has GFP expressed as a free
protein from a duplicated subgenomic RNA. PVX.TGBp3–GFP has GFP fused to the 3′ end of the TGBp3 coding sequence. (B, C) Infection foci containing PVX-GFP and PVX.TGBp3–GFP,
respectively, were photographed at 3–5 dpi and were similar in dimension. Both viruses spread systemically in N. benthamiana. Scale bar is 1 mm.
273
2005; Krishnamurthy et al., 2003; Samuels et al., 2007). Importantly,
we have noted the presence of spherical bodies containing TGBp3–
GFP fusions in PVX infected cells and the nature of these TGBp3-
containing structures remains unclear. These might be the same
vesicles reported for TGBp2 or they represent another membrane
compartment induced during PVX infection. More importantly,
TGBp3–GFP is seen in spherical bodies only during virus infection
pointing to the possibility that another viral protein(s) are responsible
for directing TGBp3–GFP from the polygonal ER into these structures.
The ER is vital for PVX cell-to-cell movement. Mutations blocking
the ability of TGBp2 or TGBp3 to bind to the ER inhibit virus cell-to-
cell movement (Krishnamurthy et al., 2003). Recent attempts to
dissect the activities of TGBp2 led to the discovery of TGBp2-induced
vesicles that bud from the ER and are essential for virus trafficking
between cells (Ju et al., 2005). The contents of these vesicles beyond
the presence of TGBp2 and BiP are unknown.
This study was undertaken to dissect the spatial relationships of
PVX replicase and the membrane bound TGBp3. We hypothesize
that, should PVX TGBp3 and the replicase lie in proximity, TGBp3
may aid in the acquisition of newly synthesized viral genomes from
the viral replicase. Considering that the ER and ER-derived vesicles
are required for virus movement, it is possible that the viral replicase
may also associate with the ER or ER-derived bodies, as was observed
for TMV. We used infectious clones of PVX expressing the reporter
GFP, and antibodies to monitor the subcellular distribution of the
PVX replicase and TGBp3 in relationship to host constituents during
infection.
Results
Spherical membrane bound bodies contain PVX replicase and TGBp3 in
virus infected leaves
In this investigation we explored the spatial relationship of the
PVX replicase and TGBp3–GFP during PVX infection of N. benthamiana
leaves. PVX.TGBp3–GFP is an infectious clone of PVX that contains GFP
fused to the TGBp3 coding sequence in a manner that does not hinder
virus infection (Fig. 1). We have previously reported that TGBp3–GFP
fluorescence is seen in the tubular ER network with fluorescent
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274 D. Bamunusinghe et al. / Virology 393 (2009) 272–285

spheres at the vertices of the connecting tubules (Ju et al., 2005, 2007;
Krishnamurthy et al., 2003; Samuels et al., 2007). Immunofluores-
cence and immunogold labeling in this study was carried out using
infection foci that were similar in size to those presented in Fig. 1.
To clearly represent the polygonal nature of the ER network, N.
benthamiana leaves were bombarded with plasmids expressing GFP-
er, which has GFP fused to ER targeting and retention signals (Haseloff
and Amos, 1995). Fig. 2A shows GFP-er fluorescence highlights
interconnecting tubules that form a meshwork lattice around the cell.
Furthermore, leaf epidermal cells bombarded with plasmids expres-
sing TGBp3–GFP (Haseloff and Amos, 1995; Krishnamurthy et al.,
2003) show a similar network, although the tubules appear shorter in
comparison to GFP-er expressing cells, and there are cisternae at the
intersection of connecting tubules (Fig. 2B). Intriguingly, the pattern
of spherical bodies seen in PVX.TGBp3–GFP infected cells is not
exactly reproduced in cells expressing TGBp3–GFP alone (compare
Figs. 2B and C), suggesting that there may be other PVX factors
contributing to the build-up of these bodies. Since we were interested
to examine the membrane association of the PVX replicase, we
hypothesized that the PVX replicase and TGBp3–GFP reside in
proximity to facilitate transfer of newly synthesized genomic RNAs
into complexes with the viral movement proteins to facilitate viral
egress.
Therefore we compared the spatial patterns of PVX replicase and
TGBp3–GFP accumulation using immunofluorescence labeling and
confocal microscopy. PVX.TGBp3–GFP infected fluorescent foci were
harvested from N. benthamiana leaves at 7 dpi, when infection foci
were typically 10–15 cells in diameter. Transverse sections were fixed
and subjected to immunolabeling using PVX replicase antisera
followed by Alexafluor 633 conjugated secondary antisera. GFP
fluorescence was not diminished by the immunolabeling process
allowing comparison of the green and red fluorescence patterns in
epidermal and mesophyll cells. Initial observations of TGBp3–GFP
fluorescence confirmed the general integrity of the ER following

Fig. 2. Localization of PVX replicase and TGBp3 proteins through fluorescence imaging of N. benthamiana leaf epidermal cells after infection. (A and B) Cells were bombarded with
pRTL2-GFPer or -TGBp3–GFP, respectively. Arrows in panel B point to examples of ER cisternae. (C) PVX.TGBp3–GFP infected cell shows similar polygonal tubular network as seen in
panels A and B. Arrowheads point to vesicles. Bars represent 10 μm. (D–I) Immunolabeling of PVX.TGBp3–GFP infected leaf epidermal cell (D–F) and mesophyll cells (G–I) shows
green fluorescence due to TGB3–GFP fusion and red fluorescence indicating PVX replicase. (F, I) represent overlaid images show yellow highlighting where red and green
fluorescence overlap. (D–F) Bars represent 10 μm. (G–I) Bars represent 200 μm. (J–L) Mock inoculated leaf epidermal cell treated with replicase antisera. (J, K) Green and red
fluorescence images, respectively show no indication of signal in mesophyll cell. Outline of mesophyll cell is shown in white. Bars represent 10 μm.
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285
fixation and immunolabeling. In epidermal cells, the tubular network
is more condensed following fixation than the dispersed network
commonly seen in living cells (compare Figs. 2B, C, and D). The green
fluorescent spheres remained associated with the ER network. In
mesophyll cells, which have a greater abundance of chloroplasts, the
ER is compressed by both the vacuole and chloroplasts making it
difficult to characterize ER architecture, although the spherical bodies
are easily identified. Immunolabeling of both epidermal (Figs. 2D–F)
and mesophyll cells (Figs. 2G–I) shows spherical bodies co-labeled
with green and red fluorescence which appear yellow in the merged
images (Figs. 2D–F). These data indicate that, although TGBp3–GFP
broadly associates with the ER network (Fig. 2F), the PVX replicase
and TGBp3 coincide in spherical ER-associated bodies.
PVX replicase antisera have been used extensively in past
biochemical analysis of polymerase activity and its ability to reliably
detect the PVX replicase is well established (Braun and Hemenway,
1992). However, for the purpose of this study, we included controls to
demonstrate that red fluorescence is due to immuno-recognition of
the PVX replicase. Transverse sections of mock inoculated leaf
segments were subjected to the same immunolabeling procedures.
We found no evidence of immunofluorescence labeling of mock
inoculated host tissues (Figs. 2J, K).
PVX replicase localizes to spherical bodies along the ER network
in protoplasts
Given that leaf segments harvested at 7 dpi likely fail to represent
early events during virus infection, and our inability to identify
infected leaf cells at earlier times, we employed a protoplast system
for studying the patterns of viral protein accumulation during the first
48 h following inoculation. Protoplasts were prepared from BY-2
suspension cells and inoculated with PVX–GFP (Figs. 3A–E) or PVX.
TGBp3–GFP (Figs. 3H–M). PVX–GFP shows cytosolic and nuclear
fluorescence that does not change over time. PVX replicase was
detected in vesicles surrounding the nucleus and throughout the
periphery of the cell. At high magnification in Fig. 3D the GFP
fluorescence has a lot of black holes in it. When we superimpose the
red fluorescence image in Fig. 3E it is clear that these holes contain red
fluorescence, indicating that GFP is excluded from the red spheres
containing the PVX replicase.
TGBp3–GFP fluorescence in fixed protoplasts during the early
hours of infection is less intense than in leaf sections that were
infected for seven days. Therefore, we employed dual-immunofluo-
rescence labeling using PVX replicase polyclonal and GFP monoclonal
antiserum. Alexafluor 633 conjugated secondary anti-goat sera was
used to detect replicase with peak emissions at 650 nm, while
Alexafluor 488 conjugated secondary anti-mouse sera was used to
detect GFP, with peak emissions at 520 nm.
For PVX.TGBp3–GFP, green fluorescence was seen throughout the
ER network and in the nucleus (Fig. 3C) as reported previously (Ju et
al., 2008; Samuels et al., 2007). We also reported that the ER changes
shape during PVX infection of protoplasts. The ER is more dense
around the nucleus in virus infected protoplasts and the strands are
shorter (compare Figs. 3F and G with Figs. 3H and J) (Samuels et al.,
2007). Red fluorescence identifies PVX replicase residing in spherical
bodies along the tubular network at 12 h post inoculation (Figs. 3I and
J). These data show the close association between the PVX replicase
and TGBp3, even at early stages of infection. Later at 48 h, the ER
reticulum is less apparent and spherical bodies containing red and
green fluorescence are seen in the cortical regions of the cell (Figs. 3K–
M). Notably in protoplasts and in leaves the PVX replicase and TGBp3–
GFP do not completely coincide. TGBp3–GFP and the replicase are
located in the spherical bodies, but TGBp3 is also observed throughout
the cortical ER.
To verify immunodetection of PVX replicase and TGBp3–GFP,
mock-inoculated samples were treated with PVX replicase or GFP
275
antisera along with the appropriate secondary antisera. These
protoplasts failed to show evidence of green or red fluorescence
(Figs. 3F–H). In addition, virus infected samples treated with buffer
and secondary antisera did not exhibit red or green fluorescence (data
not shown).
PVX replicase related spherical bodies are membrane bound and contain
ER markers
Although TGBp3–GFP and replicase have their own characteristic
patterns of subcellular accumulation, the combined leaf and proto-
plast data indicate that the PVX replicase and TGBp3 do appear in the
spherical bodies. To better visualize the precise membrane compart-
ments containing TGBp3 and replicase we employed immunogold
labeling and transmission electron microscopy. Thin sections of PVX.
TGBp3–GFP-infected and healthy tobacco leaf segments were treated
with GFP antisera alone, or both replicase and BiP antiserum. BiP is an
ER resident chaperone which is not present at high concentration in
the other areas of the endomembrane system and is typically used to
confirm the origin of ER-derived structures in virus infected cells
(Fontes et al., 1991). For dual labeling experiments, secondary
antisera conjugated with 10 nm gold particles were used to detect
BiP while secondary antiserum conjugated with 20 nm gold particles
were used to detect PVX replicase.
Immunogold labeling patterns were broadly analyzed to assess a
variety of subcellular compartments. First, the distribution of gold
particles in major compartments of the cell was examined. Gold
particles were counted in 10 μm2 fields and were scored for their
association with the cytoplasm, vacuole, cell wall (Fig. 4A), chloro-
plast (Figs. 4A, B, and C), and ER (Figs. 4D, E, and F). The average
numbers of gold particles labeling specific structural components of
the cell were reported in Table 1. Tabulated results were analyzed
statistically to provide an essential assessment of the subcellular
locations containing significant quantities of immunogold label.
Both PVX replicase (10 nm particles) and BiP (20 nm particles)
associated mainly with strands of ER network (Table 1 and Figs. 4D–G).
Moreover, the greatest amount of immunogold label detecting
TGBp3–GFP was also along strands of ER (∼ 2 to 4 particles/10 μm2
area). Some replicase and TGBp3–GFP labeling was detected in the
cytoplasm (∼ 1 particle/10 μm2), although the amount was not statis-
tically significant.
PVX replicase, BiP and TGBp3–GFP labeling was not observed
along the cell wall at a significant levels (b1/10 μm2). There was no
significant labeling in mock inoculated tissues (b1/10 μm2) probed
with antisera against PVX replicase or GFP, indicating the specificity of
the respective antisera (Table 1 and data not shown).
A second analysis was conducted to quantify the extent of
immunogold label associated with various organelles and vesicles.
Table 2 presents the average numbers of gold particles associating with
specific vesicles or organelles: mitochondria (Fig. 4C), plasmodesmata
(Fig. 4G), membrane bound bodies (MBB; see below) (Figs. 5A, B, and
C), peroxisomes (Fig. 5D), coated vesicles (Fig. 5F; see below) and Golgi
(Fig. 5G). Many of these structures were less abundant than the
structures noted in Table 1 or were rarely seen, and thus the population
numbers for each antiserum treatment vary in Table 2.
Two unique membrane bound compartments were identified in
PVX.TGBp3–GFP infected samples that were not observed in healthy
leaf segments. These structures may represent virus induced altera-
tions of cellular membranes. First are the MBBs, which have an
average diameter of 1048.14 ± 228.72 nm (Figs. 5A–C) and contain
virions, ribosomes, and membrane strands. Statistically significant
amounts of immunogold label for PVX replicase, TGBp3–GFP and BiP
were identified in the MBBs (Table 2). These structures likely derive
from the ER, since they contain significant quantities of BiP. The
second structures resembled coated vesicles and have an average
diameter of 375.95 ± 82.04 nm (Fig. 5F). These vesicles are
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285 277

Fig. 4. Electron micrographs of thin sections through PVX.TGBp3–GFP infected leaf segments. (A–C) Low magnification images showing a region of neighboring cells, and major
structures: Chloroplasts (Chl), mitochondria (M), cell wall (CW) and vacuole (Vac). (B and C) Samples were treated with PVX replicase and BiP antisera showing few or no gold
particles in these organelles. (D) PVX.TGBp3–GFP inoculated sample treated with PVX replicase and BiP antisera. Lines point to 10 nm gold particles which correspond to BiP antisera
and arrows point to 20 nm gold particles corresponding to PVX replicase. (F) Mock-inoculated samples show rough ER treated with PVX replicase antisera. No gold particles were
detected in mock samples. (G) Higher magnification of area represented by box in panel D shows 10 and 20 nm gold particles. Scale bars represent 500 nm.

Fig. 3. Localization of PVX replicase and TGBp3 proteins through fluorescence imaging of BY-2 protoplasts. (A, D) PVX–GFP infected protoplast shows green cytosolic and nuclear
fluorescence at 24 h post inoculation. The vacuoles (v) and nucleus (n) are indicated. (B) Red fluorescence shows replicase in punctae throughout the protoplast. (C and E) Overlay of
red and green fluorescence shows red spherical bodies occur in sites that are devoid of green fluorescence. (F, G) GFP-er expressing protoplast show tubular network extending from
perinuclear region to the plasma membrane. (H–J) PVX.TGBp3–GFP infected protoplast shows the tubular ER network at 12 h post inoculation. The tubular strands are shorter than in
control samples although the polygonal pattern is maintained. Red fluorescent spherical bodies overlay the network. The yellow orange fluorescence highlights regions containing
both TGBp3–GFP and replicase. (K–M) PVX.TGBp3–GFP infected protoplast shows the tubular ER network at 44 h post inoculation (green tubular haze). Spherical bodies contain
TGBp3–GFP and replicase. (N–P) Mock inoculated protoplast treated with replicase antisera. (N) Transmitted light image of protoplast following immunolabeling procedure. (O and P)
Green and red fluorescence images, respectively show no indication of signal. Bars in all panels represent 10 μm.
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278 D. Bamunusinghe et al. / Virology 393 (2009) 272–285

Table 1
Distribution of immunogold labeling with GFP, RdRp, and BiP antisera in N. benthamiana cells.

Sample Treatment # Fields Cell wall Cytoplasm ER Chloro

PVX.TGBp3–GFP RdRp + BiP 35 0.57 ± 0.10 cd 1.00 ± 0.17 cd 2.40 ± 0.41 ab 0.00 ± 0.00 d
RdRp + BiP 35 0.86 + 0.14 c 0.31 ± 0.05 c 4.43 ± 0.75 b 0.29 ± 0.05 c
buffer 35 0.10 ± 0.02 a 0.54 ± 0.09 a 0.06 ± 0.01 a NO
GFP 35 0.43 ± 0.08 c 0.97 ± 0.16 bc 2.80 ± 0.47 a NO

Mock RdRp + BiP 35 0.00 + 0.00 a 0.00 + 0.00 a 0.14 + 0.02 a 0.00 + 0.00 a
RdRp + BiP 35 0.03 + 0.01 b 0.00 + 0.00 b 2.57 + 0.43 a 0.32 + 0.06 b
buffer 35 0.09 + 0.01 a 0.00 + 0.00 a 0.06 + 0.01 a 0.00 + 0.00 a
GFP 35 0.36 + 0.1 a 0.10 ± 0.02 a 0.00 ± 0.00 a NO

PVX.TGBp3–GFP infected N. benthamiana leaf segments were embedded, sectioned, and analyzed by immunogold labeling and electron microscopy to assess the subcellular
accumulation of TGBp3–GFP and replicase. Control samples included mock inoculated healthy leaves. Immunogold labeling was conducted using commercially available full-length
mouse monoclonal AV antiserum to detect GFP, BiP antiserum, PVX RdRp antisera, or no primary antiserum (buffer). Samples were treated with 10-nm gold-conjugated anti-mouse
to detect GFP. Samples were dual-labeled using antiserum to detect RdRp and BiP and were treated with 10 and 20 nm of gold-conjugated anti-goat and anti-rabbit sera. Fields are
defined as areas of 1 μm2 (using an ultrastructure size calculator) that contain gold particles. Gold particles in each field were counted manually. The total numbers of fields analyzed
for each plant sample are indicated. The numbers of gold particles detected in the cell wall (CW), cytoplasm, ER, vacuole, and chloroplast (Chloro) were determined for all fields
treated with each antiserum. Since certain samples were treated with two antisera, we highlighted the antisera treatment that was scored in the row in bold italics to under the
treatment heading. Zeros indicate subcellular domains with no label. NO indicates that the structure was not observed. Statistical analysis was conducted as detailed in Materials and
methods. Bolded values represent means and standard errors within the same row that show the greatest amount of immunogold label and were significantly above zero. Statistical
significant differences are represented by different letters next to each standard error.

reminiscent of the TGBp2-induced vesicles reported previously in
cells transgenically expressing GFP–TGBp2 fusions (Ju et al., 2005),
however we lack antisera to TGBp2 which is necessary to evaluate
their identity. There was minimal replicase- or TGBp3-label associated
with these vesicles (Table 2), suggesting that these are not essential
centers for PVX replicase or TGBp3 related activities (data not shown).
There was little immunogold labeling of mitochondria, peroxi-
somes, Golgi and plasmodesmata, indicating that these are not sites
for TGBp3–GFP or PVX replicase accumulation during virus infection
(Table 2). Surprisingly, Golgi stacks were not easily identified in PVX.
TGBp3–GFP infected samples compared to healthy samples. This
could indicate that Golgi stacks are depleted during virus infection, or
that we had similar difficulties in detecting Golgi as for detecting
coated vesicles in healthy samples. Healthy samples had minimal
labeling with GFP, replicase, and BiP antisera.
The combined immunofluorescence and immunogold data indi-
cate that PVX replicase and TGBp3 resides in ER-derived compart-
ments such as MBBs. The evidence of such unique membranous
structures in virus-infected samples indicates that PVX causes
modifications of the ER.
PVX replicase and TGBp3 co-fractionate with ER markers but not Golgi,
endosome or vacuolar markers
To more clearly identify the source of the MBB structures, we used
buoyant density gradients to determine the proportions of ER, Golgi,
endosome, and vacuolar markers that are found in PVX-induced
MBBs. Infected (10 day post inoculation) and uninfected leaf extracts
were subjected to homogenization and centrifugation to achieve
separation of organelles and nuclei from other cellular components.
Post nuclear extracts from PVX.TGBp3–GFP infected and mock-
inoculated N. benthamiana leaves were separated by ultracentrifuga-
tion of continuous sucrose gradients. Sixteen 0.5 ml-fractions were
collected from the top of the gradient and then examined for the
presence of relevant marker proteins by immunoblot analysis.
Membranes associated with PVX replicase and TGBp3–GFP proteins,
respectively, were identified using replicase and GFP antisera and
other membranes were examined using antiserum to previously
established marker proteins. For most marker proteins, fractions were
examined by SDS-PAGE, though the replicase fractions were exam-
ined by slot blots and then treated with PVX replicase antisera.
Because of the low levels of replicase in these fractions, we used slot
blots which allowed us to load greater quantities of each fraction into
each lane prior to immunodetection. There was no non-specific
labeling observed from healthy tissue extracts run through sucrose
gradients and probed with PVX replicase and GFP antisera (data not
shown).
Immunoblot analysis of PVX.TGBp3–GFP infected leaf extracts
determined that PVX replicase, TGBp3–GFP, and BiP (ER lumenal
binding protein) co-fractionated near the top of the sucrose gradient
(fractions 1–6; Fig. 6A), although replicase and TGBp3 do not
completely coincide in their peak fractions. These results reflect a
broader association of TGBp3 with the ER while the PVX replicase
resides in a subcompartment of ER membranes. Slot blot analysis

Table 2
Association of immunogold labeling vesicles and organelles in N. benthamiana leaves.

Sample Treatment Fields Coated vesicles MBBs Mitochondria Peroxisomes Golgi Plasmodesmata

PVX-TGBp3 infected RdRp + BiP 6 to 35 0.70 ± 0.12bc 3.10 ± 0.58a 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c
RdRp + BiP 6 to 35 0.80 ± 0.14bc 6.17 ± 1.15a 0.09 ± 0.02c 0.20 ± 0.03c 0.00 ± 0.00c 0.00 ± 0.00c
buffer NO NO NO NO NO NO 0.00 ± 0.00
GFP 20–29 0.08 ± 0.02b 1.90 + 0.42a NO 0.07 ± 0.01b NO NO

Healthy RdRp + BiP 27 to 35 NO NO 0.09 ± 0.01a 0.00 ± 0.00a 0.00 ± 0.00a NO

RdRp + BiP 27 to 35 NO NO 0.09 ± 0.01a 0.17 + 0.00a 0.00 ± 0.00a NO
buffer NO NO NO NO NO NO NO
GFP 17 NO NO NO 0.00 + 0.00 NO NO

PVX.TGBp3–GFP infected and non-inoculated healthy N. benthamiana leaf segments were embedded in LR White, sectioned, and analyzed by immunogold labeling and electron
microscopy as in Table 1. Fields are defined as numbers of vesicles or organelles identified in a broad range of samples and blocks. Gold particles in each field were counted manually.
The total numbers of fields analyzed for each plant sample are indicated. Zeros indicate subcellular domains with no label. NO, none observed. Statistical analysis was conducted as
detailed in Materials and methods. Bolded values represent means and standard errors, within the same row that show the greatest amount of immunogold label and were
profoundly above zero. Statistical relatedness is represented by letters next to each standard error.
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285 279

Fig. 5. Electron micrographs of PVX.TGBp3–GFP and mock inoculated leaf segments. (A–C) MBBs found in PVX.TGBp3–GFP infected leaf tissues but are absent from mock-inoculated
samples. These are PVX-induced structures which contain virions, ER membranes, and granules which resemble ribosomes. Bodies label with PVX replicase, BiP and GFP antisera.
Images presented here show lines pointing to 10 nm gold particles which correspond to BiP antisera and arrows pointing to 20 nm gold particles corresponding to PVX replicase.
Arrowheads point to virion particles. Boxed areas are shown as insets at higher magnification to show virions and gold particles (D) Example of peroxisomes. These failed to label
with antisera. (E, F) Examples of coated vesicles which were more abundant in PVX.TGBp3–GFP infected samples but were rarely seen in mock inoculated samples. Statistically, the
amount of replicase and GFP labeling these vesicles was not significant (Table 2). These resemble TGBp2 containing structures reported in Ju et al., (2005). (G) Example of Golgi
apparatus. Scale bars represent 500 nm.

shows the greatest amount of PVX replicase in fraction 1 with lesser
amounts in fractions 2–5. Western blots show peak levels of TGBp3–
GFP in fractions 1, 2, 5, and 6 with lesser amounts in fractions 3–4.
Peak levels of BiP were seen in fractions 2 and 5. Identification of BiP in
the same fractions at TGBp3–GFP and replicase support immunogold
and immunofluorescence data indicating these proteins localize with
ER membranes, although there is not complete identity in their
fractionation patterns.
Further antisera were employed to characterize the distribution of
the trans Golgi network, endosome, and vacuolar membranes across
the sucrose gradient. The additional antisera tested recognize: SYP61
(trans Golgi network [TGN] and endosome); SYP41 (TGN); and SYP21
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280 D. Bamunusinghe et al. / Virology 393 (2009) 272–285
Fig. 6. (A) Presence of viral proteins in sucrose gradient fractions of membranous rich
extracts from PVX.TGBp3–GFP infected plants. Fractions were analyzed by slot or
Western blot using a variety of antisera. The identity of each antiserum is on the top left
of each panel. Fractions 1–16 are identified next to each lane. Fraction 1 is from the top
of the gradient and fraction 16 is near the bottom. The bar on top of each Western blot
indicates fractions 1–6 which contain PVX replicase, TGBp3–GFP and BiP. The
remaining fractions contain post-ER membranes. (B) Box plots representing qRT-PCR
analysis of host transcript levels in healthy and virus infected leaves. PVX-3D and PVX-
9D indicate PVX.TGBp3–GFP infected leaves which were harvested at 3 and 9 day post
inoculation. Box plots represent the range of values obtained for 20 samples at each
time point and represent the variability of gene expression. The boundaries of each box
represent the 25th and 75th percentiles, and the horizontal line within the box
represents the median values (i.e. 50 percentile). The spacing of components within the
box indicates the degree of dispersal or skewness in the data. The lines at the top and
bottom of the box (whiskers) represent the sample minimum and maximum. Longer
lines at the top indicate a positive skewness. Outliers are indicated by “x”.
(late endosome and vacuole), (Bar-Peled and Raikhel, 1997; Bassham
et al., 2000; Sanderfoot et al., 1999). These Golgi, endosomal, and
vacuolar proteins were mainly distributed across fractions 6–16.
These data show that the ER resident proteins clearly separate from
later components of the endomembrane network, and demonstrate
that the PVX replicase and TGBp3–GFP do not associate with the Golgi
or post-Golgi networks.
We further investigated whether the changes in endomembrane
architecture, that are necessary for formation of MBBs, coincided with
changes in the expression of essential membrane resident proteins.
We conducted qRT-PCR to determine if mRNAs representing BiP,
SYP61, SYP41 and SYP21 are specifically upregulated in response to
PVX infection. Total RNA was extracted from PVX.TGBp3–GFP-infected
or healthy leaves harvested at 3 and 9 day post inoculation. Initial
experiments using a small set of plants revealed that the mRNA levels
in PVX.TGBp3–GFP infected leaves, for some genes, ranged from 0.5 to
5-fold increase in expression compared to healthy samples. Such wide
variation could result if virus infection is asynchronous and gene
expression is altered for a short time and then subsides at some later
time. Therefore we undertook a more complex qRT-PCR analysis by
isolating RNA from 20 plants representing healthy or PVX.TGBp3–GFP
inoculated leaves. qRT-PCR provides an advantage over northern
analysis because it allows for simultaneous analysis of gene expression
trends in larger populations than can be reasonably analyzed in a gel-
based assay. Following qRT-PCR analysis the data were statistically
analyzed and presented in a box plot diagram (Fig. 6B). Box plots are
employed for nonparametric analysis of populations.
For healthy samples the qRT-PCR results show BiP, SYP61, SYP41
and SYP21 values ranged from 0.25 to 1.8 with a median of
approximately 1.0 (Fig. 6B). The boxes and whiskers were generally
small indicating low dispersion of values among the 20 plants
analyzed. Similarly, the median values for SYP61, SYP41, SYP21,
using mRNA isolated from PVX.TGBp3–GFP infected leaves ranged
from 0.25 to 1.5. Thus maximum and minimum values were similar for
healthy and PVX.TGBp3–GFP infected samples. This is contrasted by
qRT-PCR results detecting BiP expression in PVX.TGBp3–GFP infected
samples at 3 and 9 dpi. The median value reflecting BiP gene
expression increased 2.25 fold. At 9 dpi, in particular, the range of
values was positively skewed showing a 3.5 fold increase in expression
for plants in the 75th percentile and maximum increase of 4.35 fold.
These data indicate that PVX.TGBp3–GFP generally causes as much as a
4.35-fold increase in BiP expression over a 9 day period, while
expression of Golgi and post-Golgi resident proteins is unaltered.
Cerulenin treatment reduces PVX replication
Many positive strand RNA viruses induce proliferation of ER
membranes to sustain virus replication (Mackenzie, 2005; Salonen et
al., 2005). Cerulenin treatment causes cessation of phospholipid
biosynthesis and has been employed to assess the role of membrane
synthesis in the replication of Cowpea mosaic virus (CPMV), Brome
mosaic virus (BMV), Grapevine fanleaf virus (GFLV) (Carette et al.,
2000; Lee and Ahlquist, 2003; Ritzenthaler et al., 2002). These virus
encoded replication complexes associate with the ER and studies
revealed that virus-induced membrane biosynthesis plays an essen-
tial role in the viral replication cycles.
For the purpose of the study, tobacco BY-2 protoplasts were
infected with PVX–GFP and fluorometry was employed to monitor
GFP expression as a measure of successful virus replication. Proto-
plasts were treated with a range of cerulenin concentrations (0, 15,
30, and 45 μM cerulenin) for 24 h and the percentage of fluorescent
protoplasts was recorded (Fig. 7). Seventy-eight percent of proto-
plasts were infected with PVX–GFP with no cerulenin treatment.
Infection decreased to 54% of cells treated with 15 μM cerulenin, and
10–13% of cells treated with 30–45 μM cerulenin. The effects of higher
cerulenin concentrations on PVX–GFP accumulation was comparable
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285
Fig. 7. Effect of cerulenin on PVX–GFP accumulation in infected protoplasts. Protoplast
viability was measured immediately after electroporation with viral RNA using 0.1%
FDA. Protoplasts were placed in culture medium containing 0, 15, 30, or 45 μM
cerulenin. Protoplasts were incubated for 24 h and then assayed for the proportion
expressing GFP. GFP is derived from a viral subgenomic RNA and its expression is
dependent upon successful virus infection. Controls were transfected with pRTL2-GFP
plasmids which transcribe GFP in the nucleus. GFP expression is unaffected by cerulenin
treatment. The numbers of GFP expressing protoplasts in each sample were counted.
The results represent the proportion of viable protoplasts that express GFP
fluorescence.
to related reports of GFLV and CPMV(Carette et al., 2000; Ritzenthaler
et al., 2002) indicating a similar requirement for membrane synthesis.
To exclude the possibility that cerulenin decreased the viability of
protoplasts and thereby hampered virus infection, the effect of
cerulenin on protoplasts transfected with pRTL2-GFP plasmids was
examined. The percentage of protoplasts expressing GFP ranged
between 40% and 50% in untreated and cerulenin treated samples
(Fig. 7). Identical concentrations of cerulenin had no obvious impact
on GFP expression from CaMV 35S promoter. The negative impact of
cerulenin on PVX-GFP infection indicates that formation of new ER
membranes is required for virus replication.
Discussion
In this report we have shown that the PVX replicase and TGBp3
proteins associate with the ER during infection. Previous studies have
reported the localization patterns of the TGB proteins when expressed
ectopically, alone or when co-expressed (Chang et al., 1997; Davies et
al., 1993; Samuels et al., 2007), but not in relation to the replicase. The
subcellular location of a potexvirus replicase previously has not been
reported. For other RNA viruses the replicase, or replication-
associated proteins, have been shown to be essential for virus
intracellular activities beyond virus accumulation (Guenoune-Gelbart
et al., 2008; Liu et al., 2005). In particular the TMV 126 and/or 183 kDa
proteins associate with virus accumulation and also function as a
silencing suppressor, associate with microfilaments and are required
for intercellular virus movement (Ding et al., 2004; Liu et al., 2005;
Hirashima and Watanabe, 2003). Some of these findings relied
strongly on cell biological analyses. Considering the importance of
the PVX replicase for virus accumulation and its potential to influence
other activities such as virus movement, it was important to
determine the subcellular location of this protein and its relationship
with the TGB proteins during PVX infection to serve as a basis for
future studies in those areas.
Most positive strand RNA viruses infecting plants and mammals
replicate in association with cellular membranes (Salonen et al.,
2005). Viruses often induce membrane modifications such as
proliferation or invaginations to create available membrane scaffolds
for assembly of the replication machinery (Ahlquist, 2006; Netherton
281
et al., 2007; Suhy et al., 2000; Villanueva et al., 2005). Recent cell
biological investigations with RCNMV and GFLV revealed extensive
redistribution of the ER as a result of virus infection (Ritzenthaler et
al., 2002; Turner et al., 2004). Both viruses cause accumulation of ER
aggregates or vesicles in the perinuclear region. In this study,
immunolabeling of leaf cells and BY-2 protoplasts showed PVX
replicase and TGBp3–GFP both localized to spherical bodies occurring
along the ER network. In protoplasts, MBBs containing PVX replicase
and TGBp3–GFP were observed between 12 and 48 h indicating that
formation of these structures is an early event. The MBBs observed in
leaf cells at 7 dpi appeared larger than in protoplasts, which could
result from increasing modifications and distortion of the ER over
time, due to the presence and accumulation of viral products.
Fractionation experiments provide further indication that both the
PVX replicase and TGBp3–GFP proteins coincide with the ER marker
BiP, and are excluded from Golgi and endosomal vesicles, pointing to
the ER as the primary site for PVX replication.
Localization of a viral MP and replicase in the same membrane
bound complexes was first reported for TMV (Heinlein et al., 1998;
Kahn et al., 1998). The TMV MP is an integral membrane protein that
causes distortions in the ER network (Reichel and Beachy, 1998;
Reichel et al., 1999). Thus, the data reported in this study provide an
example of another virus whose replicase and MP appear in the same
membrane bound complexes. These similarities between TMV and
PVX suggest that certain interactions between plant viral replicases,
or replication-associated proteins, and MPs could be conserved.
Although it is logical that this localization represents a region where
both proteins are synthesized and/or function, evidence presented
here showing the proximity of the PVX replicase and the TGBp3
movement protein in particular subcellular sites provides the basis for
future work to examine whether the PVX replicase and TGBp3 act in
concert to promote virus infection and movement.
In leaf cells and protoplasts, the primary site for TGBp3–GFP
accumulation is the cortical ER (Ju et al., 2005; Krishnamurthy et al.,
2003) and the same spherical bodies containing PVX replicase. Thus,
TGBp3–GFP associates broadly with the ER while the PVX replicase is
restricted to a subcompartment containing ER membranes. Given that
early studies revealed that deleting TGBp3 from the viral genome does
not hamper virus replication, TGBp3 is not likely to function as the
membrane anchor for the PVX replicase (Beck et al., 1991; Verchot et
al., 1998). Typical membrane-anchoring proteins are essential and
knockout mutations would eliminate virus replication. Mutational
analysis has shown that TGBp3 binding to the ER is essential for virus
intercellular transport although there has been little progress toward
clarifying the role for the ER in modulating virus cell-to-cell
movement. Thus, the exact role of TGBp3 in the ER is unknown,
although we have recently proposed that TGBp3 could play a role in
modulating viral protein turnover (Ju et al., 2008). Moreover, we
chose to examine the amount of TGBp3–GFP in the cytoplasm in this
study based on our recently reported results (Ju et al., 2008) which
suggested that TGBp3–GFP is turned over by ER-associated degrada-
tion pathways (ERAD). TGBp3–GFP is dislodged from the ER for
cytosolic degradation by the 26S proteasome (Brandizzi et al., 2003; Ju
et al., 2008; Meusser et al., 2005). Given the role of TGBp3 in viral
protein turnover and virus movement, it is possible that TGBp3 may
function to target the PVX replicase for degradation and acquiring
viral RNAs for transfer to plasmodesmata. Further research is needed
to determine if the stability of the PVX replicase is altered by the
presence of TGBp3.
In this study, electron microscopy of virus-infected tissues
identified MBBs, which were filled with ER strands and virions.
Immunolabeling detected PVX replicase, TGBp3 and the ER marker
protein, BiP, in these bodies. Since no other ER associated bodies were
detected in virus infected cells, it is reasonable to consider these MBBs
to be the spherical bodies seen using confocal microscopy. Virus
infected cells also showed an abundance of coated vesicles that were
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282 D. Bamunusinghe et al. / Virology 393 (2009) 272–285
not detected in healthy tissues. The identity of these vesicles was not
further examined in this study because they failed to show
immunoreactivity with GFP or replicase antisera. However, they
have some resemblance to TGBp2 induced vesicles reported in a
related study (Ju et al., 2005). The TGBp2-related vesicles are similar
in size and show the same granular appearance along the outer
membrane. GFLV polyprotein precursors were reported to associate
with membranous vesicles that resembled the COP-coated vesicles
which traffic from the ER to the Golgi apparatus. Researchers
proposed that GFLV might employ the COPII budding machinery to
recruit membrane scaffolds for the viral replicase (Ritzenthaler et al.,
2002). The COP vesiculation machinery has been shown to be
essential for poliovirus replication (Belov et al., 2005; Cuconati et
al., 1998; Fogg et al., 2003). While we found no evidence that coated
vesicles (Fig. 5E and F) are COP-related structures their abundance in
PVX.TGBp3–GFP infected cells warrants further investigations.
Through treatment with the lipid biosynthesis antagonist, cerule-
nin, we determined that membrane synthesis was necessary for PVX.
TGBp3–GFP replication (Fig. 7). Cerulenin was also shown to inhibit
replication of GFLV, CPMV, and poliovirus (Carette et al., 2000; Fogg et
al., 2003; Ritzenthaler et al., 2002). Thus, PVX belongs to a class of
viruses that require de novo membrane synthesis to support virus
replication. The moderate increase in BiP expression seen in PVX
infected cells may correlate with expansion of the ER or may reflect a
moderate level of stress exerted upon the ER as the result of virus
infection. Viral pathogenesis can sometimes trigger the unfolded
protein response (UPR), leading to increased cellular synthesis of ER
resident chaperones necessary to restore ER homeostasis. BiP is one ER
resident chaperone that is known to play a vital role in UPR (Kamauchi
et al., 2005; Urade, 2007). Similar events have been reported to occur
during mammalian virus infection. Specifically, flaviviral nonstructur-
al proteins trigger UPR signaling, which, for viruses such as Hepatitis C
virus, leads to restoration of ER homeostasis necessary for a persistent
virus infection (Tardif et al., 2005). Increased expression of ER resident
chaperones ensures proper protein folding and processing in the ER, as
a result of the increased demand on the cellular machinery. In addition,
misfolded proteins can be relocated to the cytoplasm for proteasomal
degradation. The role for TGBp3 or PVX replicase in regulating
accumulation of ER resident chaperones has not yet been examined,
although we have reported TGBp3 accumulation is monitored by the
26S proteasome (Ju et al., 2008).
Taken together, the data demonstrate that the ER-derived MBBs
contain both the PVX replicase and TGBp3 proteins. PVX replicase and
TGBp3 do not exactly coincide throughout the ER or in membrane
fractions. While the top fractions 1–5 contain BiP, which is a marker for
ER membranes, replicase was predominantly in fraction 1 while peak
levels of TGBp3 were in fraction 5. These results are reflective of
evidence that the replicase does not occur throughout the cortical ER but
are restricted to a subcompartment of the ER, such as MBBs. Particles
resembling PVX virions were also seen inside MBBs, although further
studies are needed to learn how many PVX-encoded proteins associate
with these structures. Importantly, this is the first report to demonstrate
a spatial relationship between the PVX TGBp3 and replicase. Given the
close relationship of the TMV replicase and movement protein, it is
worth considering that interactions between the PVX replicase and
TGBp3 may serve analogous functions. However, further dynamic
investigations are necessary to reveal whether TGBp3 is similar to the
TMV movement protein and drives trafficking of membrane bound viral
replication complexes toward the plasmodesmata.
Materials and methods
Infectious clones, in vitro transcription, and plant inoculations
PVX–GFP and PVX.TGBp3–GFP are infectious clones of PVX
described in prior studies (Fig. 1; Krishnamurthy et al., 2003; Mitra
et al., 2003; Verchot et al., 1998). Infectious clones were maintained in
Escherichia coli strain JM109 (Sambrook et al., 1989). One microgram
of SpeI linearized DNA was transcribed using the mMESSAGE
mMACHINE™ kit (Ambion Inc., Austin, TX). The leaves of N.
benthamiana were dusted with cellite powder and then rub inoculated
with 5 μg of transcripts. Mock inoculated plants were treated similarly
with 5 μL of ddH2O.
BY-2 protoplast preparation and transfection
Protoplasts were prepared using tobacco BY-2 suspension cells as
described previously (Ju et al., 2005; Nagata et al., 1992). For RNA
transfection, 500 μL of protoplasts was mixed with 5–10 μg of
transcripts and placed in 0.4 cm gap cuvettes on ice. Electroporation
was carried out at 0.25 kV, 100 Ω and 125 μF using a BioRad Gene
Pulser (Bio-Rad Laboratories, Hercules, CA). Protoplasts were trans-
ferred to 6-well cell culture plates containing BY-2 culture media plus
0.45 M mannitol and 1% agarose (w/v; pH 5.7). Samples were
maintained at 27–28 °C in dark.
Immunofluorescence labeling of virus infected leaf segments
and protoplasts
Leaf segments containing fluorescent infection sites were fixed in
eppendorf tubes with a solution of 3.7% (v/v) formaldehyde, 5% (v/v)
dimethyl sulfoxide, and PHEM buffer (60 mM PIPES, 25 mM HEPES,
5 mM EGTA, 2 mM MgCl2, pH 6.9) for 2 h (Liu et al., 2005). Fixed
segments were placed on microscope slides and digested with cell
wall degrading enzymes (1% Onozuka R10 cellulase, 0.1% pectolyase
[Kikkoman, Tokyo, JP] and 0.1% bovine serum albumin [BSA; fraction
V] in PHEM buffer) for 2 h. Following washes with PHEM (3 min/
wash) the segments were incubated with 1% (v/v) Triton X-100 for
20 min. Ice cold methanol was added, incubated for 10 min at room
temperature, and then washed thrice for 5 min with PHEM.
Protoplast samples were collected by centrifugation at 59 × g for
5 min, washed twice with PHEM buffer, and fixed with a solution of 2%
[v/v] paraformaldehyde and 0.5% [v/v] DMSO for 15 min at room
temperature. Samples were transferred onto a cover glass atop a thin
film of 0.7% Bacto-agar and 200 μL of 1% Triton X-100 (in PHEM) was
added for 10 min. Following three washes with PHEM, NaBH4 was
added to the cover glass, incubated for 10 min, and then washed thrice
with PHEM. Ice cold methanol was added, incubated for 10 min at
room temperature, and then washed thrice for 5 min with PHEM.
For immunolabeling, leaf segments and protoplasts were incubat-
ed overnight in a moisture chamber at 4 °C with PVX replicase goat
antisera (diluted 1:100 or 1:500) in a moisture chamber at 4 °C.
Samples were washed five times for 5 min with PHEM and then
incubated with Alexa Fluor 633 conjugated secondary antisera (1:100
dil) (Molecular Probes, Inc., Eugene, OR) for 2 h in a moisture
chamber at room temperature. Cover glasses were washed five times
for 5 min with PHEM and then mounted on a slide with commercially
available Vectashield™ mounting media (Vector Laboratories, Inc.,
Burlingame, CA).
Fixation and LR-White or Spurr's resin embedding for electron
microscopic analysis
Virus-infected and mock-inoculated leaf segments were harvested
at 5 dpi and subjected to chemical fixation and embedding in Spurr's
resin. Stepwise chemical fixation was carried out as described
(Dunoyer et al., 2002a, 2002b). Leaf segments were pre-incubated
with 1% glutaraldehyde in 25 mM potassium phosphate buffer (pH
7.4) for 1 h in a bench top vacuum chamber and then immersed in a
primary fixative [2% (v/v) glutaraldehyde, 0.1 ml of saturated picric
acid in 25 mM potassium phosphate buffer (pH 7.4)] for 15 min at
room temperature. Segments were incubated at 4 °C for 16 h, washed
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D. Bamunusinghe et al. / Virology 393 (2009) 272–285
four times (5 min per wash) in 25 mM PIPES buffer (pH 7.0) and then
transferred into secondary fixatives [2% (w/v) OsO4 and 0.5% (w/v)
potassium ferrocyanide in 25 mM PIPES buffer (pH 7.0)] for 2 h at
room temperature. Samples were washed twice with 25 mM PIPES
buffer (pH 7.0) for 15 min and then twice with ddH2O for another
15 min. Samples were transferred to 2% (w/v) aqueous uranyl acetate
for 16 h at 4 °C and washed twice with ddH2O for 15 min. Samples
were dehydrated in an acetone series (10%, 20%, 40%, 60%, 80%, and
100%) and infiltrated with acetone/Spurr's (1:1) or LR-White resin
overnight and then embedded in Spurr's or LR-White resin.
Ultra thin sections (700 nm) were cut using a diamond knife on a
Sorvall MT 6000 ultra microtome. Sections were mounted on formvar-
coated nickel grids (Electron Microscopy Science, Hatfield, PA) and
used for immunogold labeling.
Cerulenin treatment
Following electroporation, protoplast viability was measured
using 0.1% fluorescein diacetate (FDA) in 1 mL of 0.05 M phosphate
buffer. Cerulenin (Sigma-Aldrich Co., St. Louis, MO) was dissolved in
dimethyl sulfoxide and added to the protoplast culture medium to a
final concentration of 0, 15, 30, and 45 μM (Carette et al., 2000;
Ritzenthaler et al., 2002). Protoplasts were incubated for 24 h and
harvested. A haemocytometer was employed to calculate the
proportion of GFP expressing protoplasts. The proportions of viable
protoplasts that were virus infected were recorded.
Immunogold labeling of LR-White or Spurr's resin-embedded
plant material
Immunogold labeling of tissues were conducted using monoclonal
GFP (BD Living Colors™; Clontech Laboratories, Mountain View, CA)
and PVX replicase polyclonal goat antisera as previously described
(Mitra et al., 2003). Grids were incubated in blocking solution
consisting of PBS, pH 7.5 (130 mM NaCl, 7.0 mM Na2HPO4, 3.0 mM
NaH2PO4) plus 2% BSA (w/v) for 15 min, and then was incubated with
5% normal sheep sera (Sigma-Aldrich Co.) in PBS plus 2% BSA for
15 min. Then samples were incubated with GFP monoclonal antisera
diluted 1:500 in PBS plus 2% BSA (w/v), PVX replicase goat antisera
diluted 1:500 in PBS plus 0.1% Tween (v/v), or buffer containing no
primary antisera for 2 h. Grids were then washed five times for 5 min
with PBS and then with PBS plus 2% fish gelatin (v/v) for 15 min. Grids
were then incubated for 1 h with either 10 or 20 nm gold-conjugated
goat antisera (EY Labs, San Mateo, CA) diluted 1:10 in PBS plus 2% fish
gelatin.
Some grids were dual labeled with antiserum to detect replicase
and GFP and a longer procedure was followed (Mitra et al., 2003).
Grids were first incubated with replicase goat antisera diluted 1:500
in PBS plus 0.1% Tween (v/v) for 2 h and then with the 20 nm gold
conjugated rabbit anti-goat serum diluted 1:10 in PBS plus 2% fish
gelatin for 2 h as described above. Samples were washed twice for
10 min with PBS and then incubated for 30 min with GFP monoclonal
antisera diluted 1:500 in PBS. Grids were washed five times for 5 min
with PBS, then for 15 min with PBS with 2% fish gelatin, and were
incubated 30 min with 10 nm gold-conjugated goat anti-mouse
serum (EY Labs) diluted 1:10 in PBS plus 2% fish gelatin. Grids were
washed three times for 5 min with ddH2O, and stained with a solution
of 2.5% uranyl acetate and 70% methanol (v/v) for 30 min, and then
with a solution of 2% Reynold's lead citrate pH 12.0 (in ddH2O) for
20 min. Samples were washed with mildly warm ddH2O three times
for 5 min and then dried. Resin embedded sections were consecu-
tively labeled with PVX replicase goat primary, anti-goat secondary
and GRP78 (BiP) rabbit primary and anti-rabbit secondary antisera
(ABR-Affinity Bioreagents Inc., Rockford, IL) using established proto-
cols (Ju et al., 2005). Grids were stained with uranyl acetate and
Reynolds lead citrate.
283
Laser scanning confocal microscopy and transmission
electron microscopy
A Leica DMRE microscope with Leica TCS SP2 confocal imaging
system was used. Ar/Kr lasers were used for detecting GFP (488 nm
excitation) and He/Ne lasers for detecting Alexa Fluor 633 fluores-
cence. Electron microscopy was carried out using a JEOL JEM-2100
Scanning Transmission Electron Microscope System with an EDAX
Genesis 2000 EDS system (JEOL Ltd., Tokyo, Japan). Electron
microscopic images were taken, and number of gold particles labeling
specific structural components of the cell was scored in 10 μm2 fields
(using an ultrastructure size calculator) or in each organelle. Gold
particles in each field/organelle were counted manually. Average and
standard error was calculated and tabulated. Images were compiled
using Adobe Photoshop CS software (Adobe Systems, San Jose, CA).
Sucrose gradient fraction of plant extracts containing PVX replicase
Forty grams of PVX.TGBp3–GFP infected N. benthamiana leaves
was collected from plants at 14 dpi and then homogenized in 100 ml
of ice-cold buffer A [50 mM Tris–HCl, pH 8.2; 15 mM MgCl2, 120 mM
KCl, 20% glycerol, 1 mM DTT, 5 mM EDTA and 10 μL/mL of Protease
Inhibitor Cocktail for plant cell and tissue extracts (Sigma-Aldrich Co.)
(Hu et al., 2007; Plante et al., 2000). The crude homogenate was
filtered through gauze and miracloth (Calbiochem, La Jolla, CA) into a
fresh tube and centrifuged at 500 × g for 10 min at 4 °C. The
supernatant was removed and centrifuged at 30,000 × g for 30 min
using a SW 32.1 ultracentrifuge rotor and OptimaTM L-XP Series
preparative ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). The
resulting pellet was resuspended in 6 ml buffer B (50 mM Tris–HCl, pH
8.2; 15 mM MgCl2, 5% glycerol, 1 mM DTT, 5 mM EDTA and 10 μL/mL
of Protease Inhibitor Cocktail) and divided into 3 ml samples which
were loaded onto 7.5 mL, 20–60% continuous sucrose gradients. After
centrifugation at 189,000 × g for 1 h at 4 °C, 0.5 mL fractions were
collected from the top of each gradient. Approximately 16 to 18
fractions were collected per gradient (Plante et al., 2000).
Gradient fractions were concentrated five-fold as described
(Wessel and Flugge, 1984). A 0.2 mL aliquot of each fraction was
combined with 0.8 mL of methanol, followed by vortexing and low
speed centrifugation at 9000 × g for 10 s. Samples were extracted
using sequential addition of 0.2 mL of chloroform and 0.6 mL of ddH2O
followed again by centrifugation at 9000 × g for 1 min. Methanol
(0.6 mL) was added to the organic and inter-phase. Proteins were
pelleted by centrifugation at 9000 × g for 2 min, dried under a stream
of air, and then boiled with 40 μL protein dissociation buffer
(Sambrook et al., 1989) before loading onto 12.5% SDS-polyacryl-
amide gels. Vertical transfer of denatured proteins onto nitrocellulose
membranes (Perkin Elmer, Boston, MA) was conducted using a
BioRad Trans-Blot apparatus (Bio-Rad Laboratories). To detect PVX
replicase, 150 μL fractions plus 50 μL of protein dissociation buffer
were applied directly to a nitrocellulose membrane using a Bio-Dot SF
apparatus (Bio-Rad Laboratories).
Table 3
Primers for qRT-PCR.
Genes Accession Primers Primer sequences
18S rRNA AJ236016 Forward 5′-ATGGCCGTTCTTAGTTGGTGGAGC-3′
Reverse 5′-AGTTAGCAGGCTGAGGTCTCGAAC-3′
BiP FJ463755 Forward 5′-AGCTTTGAGCAGTCAACACCAAGT-3′
Reverse 5′-AAAACGTGCCCGAGTAAGTGGTTC-3′
SYP21 L41651 Forward 5′- CCGATTTACGCGACAAGTTGCACA-3′
Reverse 5′- ACTCGATGATCTGTTTCGCTGGCT-3′
SYP41 NM_001036873 Forward 5′- TTCAGTGAACTGCAGACGACCACT-3′
Reverse 5′- TAGAACGACCCGGCATGAATCCAA-3′
SYP61 NM_001036025 Forward 5′- ACTTGTGGAAGCATTGAGTGGCAG-3′
Reverse 5′- TTCACATTCCGCACCTGTGTCCTA-3′
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284 D. Bamunusinghe et al. / Virology 393 (2009) 272–285
Membranes were probed with PVX replicase goat polyclonal or
commercially available
GFP monoclonal or BiP (GRP 78) rabbit polyclonal antiserum. In
addition, membranes were probed with Arabidopsis thaliana (At)
SYP61, AtSYP21, and AtSYP41 rabbit polyclonal antiserum (Bar-Peled
and Raikhel, 1997; Bassham et al., 2000; Sanderfoot and Raikhel,
1999) to determine if these cellular proteins reside in the same
fractions as the PVX proteins. Blots were developed using the
Western Lightning Chemiluminescence reagent plus developer
(Perkin Elmer), and then exposed to Kodak Bio Max Light Film
(Kodak, Rochester, NY).
Density values were calculated using the FluorChem software. For
each autoradiograph, the background was automatically subtracted
by determining the average of the 10 lowest pixel values surrounding
each individual band. For a more rigorous quantification of the data,
the density values for an empty lane was calculated and subtracted
from each density value recorded for lanes 1–16. The final values are
presented as relative density values (RDV) and were plotted using
Microsoft Excel 2003.
Statistical analysis
ANOVA procedures with PC SAS version 8.2 (SAS Institute) and
PROC GLM were used to evaluate differences in location in the
number of gold particles observed in the electron micrographs that
were reported in Tables 1 and 2. The analysis was performed for each
combination of virus-infected or healthy plus antiserum. Due to
problems in homogeneity of variance and distributional assumptions,
a square-root transformation was used prior to conducting the
ANOVAs. When the ANOVA was significant, pairwise comparisons of
the locations were made with a PDIFF option in an LSMEANS
statement. A significance level of 0.05 was used for all comparisons.
Quantitative RT-PCR (qRT-PCR)
N. benthamiana leaves were inoculated with 100 μg/ml purified
PVX.TGBp3–GFP. Total RNA was extracted from inoculated leaves of
20 plants at 3 and 9 day post inoculation with SV Total RNA Isolation
Kit (Promega Corp., Madison, WI). First strand cDNA synthesis was
carried out using Superscript reverse transcriptase III (Invitrogen
Corp., Carlsbad, CA) and hexamer random primers. Primers of SYP21,
SYP41 and SYP61 are designed based on N. benthamiana homologous
ESTs of the Arabidopsis syntaxins (Table 3). 18S rRNA serves as the
endogenous control. Twenty five nanograms cDNA of each sample
and 100 to 900 nM primers were used to perform qPCR with power
SYBR Green II and ABI7500 system (Applied Biosystem). Twenty-five
microliters PCR reactions were incubated at 95 °C 10 min to activate
Taq polymerase, followed by 40 cycles of 95 °C for 15 s and 60 °C for
60 s. Efficiencies of all primers are verified by normal RT-PCR and gel
electrophoresis prior to qRT-PCR. Duplicate PCR reactions for each
sample were carried out and averaged. The comparative CT method
employs the formula 2 -dd CT where the values of the endogenous
control and calibrator (constant quantity of healthy sample
template) and are subtracted from the target sample value to
provide the ddCT value. The 2 -dd CT represents the fold of RNA
accumulation. The raw qRT-PCR data were treated in Excel, and box
plots were drawn in GenStat 11th edition (The Numerical Algo-
rithms Group Ltd, Oxford, UK).
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