J. gen. Virol. (1987), 68, 2723-2728.
Printed in Great Britain
2723
Key words: P YD V/nucleus/ immunogold labelling
Identification of Viral Structural Proteins in the Nucleoplasm of Potato
Yellow Dwarf Virus-infected Cells
By N A - S H E N G L I N , 1. Y A U - H E I U H S U 2 A N D R E N - J O N G C H I U 2
1Institute of Botany, Academia Sinica, Nankang. Taipei, Taiwan 11529 and "-Agriculture
Biotechnology Laboratories, National Chung Hsing University, Taichung, Taiwan 40227,
Republic of China
(Accepted 22 June 1987)
SUMMARY
Antiserum raised against purified potato yellow dwarf virus (PYDV) reacted
specifically with all five viral structural proteins in immunoblots and also with ultrathin
sections of infected tobacco cells. In these sections viral proteins were detected with
gold-labelled anti-IgG antibodies and gold particles were predominantly located over
the nucleoplasm and over PYDV virus particles either at the periphery of nuclei or in
the cytoplasm. Control sections of uninfected or healthy cells were not labelled. The
results suggest that the nucleoplasm is the only site of accumulation of PYDV structural
proteins.
Potato yellow dwarf virus (PYDV) has bacilliform particles and belongs to subgroup II of the
rhabdoviruses, particles of which characteristically bud at the inner nuclear membrane and
accumulate mainly in the perinuclear space (Peters, 1981; Francki et al., 1981, 1985).
Rhabdoviruses in this subgroup cause disorganization of host cell nuclei, such as the reduction
or disappearance of chromatin material, swelling of the nucleolus and the appearance of
viroplasm-like structures (Martelli & Russo, 1977).
Although the ultrastructure of PYDV-infected tissue has been thoroughly studied (Black et
al., 1965; Black, 1970; Francki et al., 1981, 1985), there has been no immunocytochemical study
of viral proteins in situ. We have examined the cellular location of viral structural proteins in
infected cells by immunogold staining of ultrathin sections.
The Sanguinolata isolate of PYDV (a gift from Dr H. T. Hsu, Beltsville Agricultural Research
Center, USDA, Beltsville, Md., U.S.A.) was mechanically inoculated to, and maintained in,
Nicotiana rustica or N. benthamiana in a greenhouse kept at between 25 and 30 °C.
PYDV was purified by a procedure similar to that described by Jackson & Christie (1977).
Leaves were triturated in extraction buffer (0.1 M-Tris-HC1 pH 8.4, 0-001 M-magnesium acetate,
0-04 M-Na2SO3, 0.001 M-MnC12). After low speed centrifugation of the extract, K,HPO~ and
CaC12 were added to 50 mM each with stirring and the mixture was centrifuged at low speed. The
supernatant fluid was then layered on a 5 ml 20~o sucrose (w/v) in extraction buffer and
centrifuged at 27000 r.p.m, for 45 min in a Beckman type 30 rotor. Virus particles were
suspended in maintenance buffer (as extraction buffer but pH 7.5) and further purified by
centrifugation in 5 to 30~o (w/v) sucrose rate-zonal and 30 to 60% sucrose (w/v) quasiequilibrium gradients. Purified virus particles were stored in maintenance buffer at - 7 0 °C.
Antiserum against PYDV was produced in BALB/c mice by five weekly intraperitoneal
injections of SDS-disrupted virus preparations. Approximately t00 ttg protein (estimated from
the intensity of Coomassie Brilliant Blue staining of a polyacrylamide gel of a sample) suspended
in 0.1% SDS was used in each injection. An equal volume of Freund's complete adjuvant was
added to antigen only in the first injection. Sera were collected 4 days after the last injection.
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Purified PYDV was disrupted in protein cracking buffer (Lane, 1978) and electrophoresed in
a 10 ~ polyacrylamide gel using a discontinuous buffer system (Laemmli, 1970). Proteins in the
gel were transferred to nitrocellulose with a Semidry Electroblotter (Sartorius) at a current
density of 0.8 mA/cm 2 for 1 hr (Kyhse-Andersen, 1984). The buffers were 25 mM-Tris/40 mM-6amino-n-hexanoic acid/20~ (v/v) methanol pH 9.4 as a cathode buffer and 300 mM-Tris/20~o
(v/v) methanol pH 10.4; 25 mM-Tris/20~ (v/v) methanol pH 10.4 as the anode buffer.
The protein blot containing transferred proteins was cut into vertical strips. Protein bands
were detected by staining with colloidal gold (Moeremans et al., 1985). After extensive washing
with 0-01 M-phosphate-buffered saline (PBS) pH 7.2 containing 0.3~ (w/v) Tween 20 and 1 mMEDTA at 37 °C for 30 min, a further wash with PBS-Tween at room temperature and a rinse in
excess distilled water, blots were incubated with a stabilized solution of gold particles (15 nm
diam.), prepared as described by Horisberger (1979), containing 0.1~ Tween 20 in 10 mMcitrate buffer pH 3.0, until a pink colour developed.
For immunolabelling, blot strips were first blocked with 3 ~o bovine serum albumin in PBS (B
buffer) for 1 h at 37 °C. Mouse anti-PYDV serum was then added to the blocking solution to a
final dilution of 1/500 and strips were incubated for 2 h at room temperature. After washing
extensively in PBS-Tween, blots were again blocked with B buffer and subsequently incubated
with horseradish peroxidase conjugated to affinity-purified goat anti-mouse IgG (Bio-Rad) for 2
h. The immunoblots were then washed in PBS-Tween, rinsed with distilled water and treated
with a peroxidase substrate solution (60 mg of 4-chloro-1-naphthol dissolved in 20 ml methanol,
added to 100 ml PBS, containing 40 ~tl of 30~ H,O2).
PYDV-infected plants were kept dark overnight before sampling. Two weeks after
inoculation, young leaf tissues showing systemic symptoms were cut into small pieces and
vacuum-infiltrated with 0.1 M-phosphate-citrate buffer pH 7.2 (PC buffer) and chilled for 2 h at
4 °C (Langenberg, 1979). Tissue pieces were then fixed in 1 ~ PC-buffered glutaraldehyde at
4 °C overnight. The fixative solution was then replaced with PC-buffered 0.1 M-ammonium
chloride and after 15 min at 4 °C tissues were dehydrated with a graded methanol series and
embedded in Lowicryl HM20 as described by Lin & Langenberg (1983).
Affinity-purified goat anti-mouse IgG (Sigma) was used to prepare gold-labelled goat antimouse IgG complexes (Lin & Langenberg, 1983). Ultrathin sections of Lowicryl HM20embedded tissue were treated with mouse anti-PYDV antiserum diluted 1/200 as a primary label
followed by incubation with gold-labelled goat anti-mouse IgG as a secondary label according to
Lin & Langenberg (1983). After immunogold staining, sections were stained in uranyl acetate
and lead citrate (Reynolds, 1963) and viewed in a Zeiss 109 electron microscope at 80 kV.
Purified PYDV preparations used to immunize mice were analysed by SDS-polyacrylamide
gel electrophoresis. All preparations contained five major structural proteins with mol. wt. of
190000 (190K), 84K, 52K, 31K, and 26K corresponding respectively to proteins L, G, N, M1
and M2 (Knudson & Macleod, 1972; Falk & Weathers, 1983; Adam & Hsu, 1984) (Fig. la).
Comparison of colloidal gold-stained blots (Fig. 1 b) and immunolabelled blots (Fig. 1 c) showed
that all five major proteins were labelled, indicating that the anti-PYDV antiserum reacted with
all five structural proteins of PYDV. Protein L was seen on the original blots, but does not
appear in Fig. l(b) and (c).
In thin sections of PYDV-infected tobacco cells, aggregates of virus particles were commonly
observed at the periphery of nuclei. As described by Black (l 970) and Francki et al. (1981, 1985),
virus particles aligned with their axes perpendicular to the nuclear membrane were often
observed at an early stage of virus multiplication. At a later stage of infection, the particles
increased in number to form virus aggregates that surrounded the nucleus (Fig. 2 a). Perinuclear
spaces were often distorted by accumulations of virus particles. Chromatin was less abundant in
these nuclei than in healthy nuclei. There was a decreased intensity of staining and the fine,
uniform appearance of nucleoplasm was absent. Instead, a granular matrix almost filled each
nucleus (Fig. 2a). However, nucleoli did not degenerate in infected cells even when large
aggregates of virus particles were observed.
After immunogold staining, gold particles were located over virus particles at the periphery of
nuclei and predominantly over the granular matrix in the nucleoplasm (Fig. 2b), even at early
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2725
(a)
2
l
(,b)
200K --
~L
116K~
(c)
--L
--L
'--G
--G
tlW--N
O--N
--M 1
I[#--M1
IlIIp--M1
_M 2
~--M2
.,~--M 2
~,,
92K-66K-- , ~
[lt~ " m
N
45K-- . ~
31K-- " ~
q liP
21K-Fig. 1. (a). Coomassie Brilliant Blue-stained SDS-PAGE pattern of purified potato yellow dwarf
virion polypeptides. Lane 1, marker proteins: myosin, fl-galactosidase, phosphorylase b, bovine serum
albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and ct-lactalbumin. Lane 2,
proteins from purified intact virions. (b, c) Nitrocellulose blots of proteins from purified PYDV stained
with a colloidal gold (b) or immunolabelled (c).
stages of infection (data not shown). Neither heterochromatin nor nucleoli were ever labelled.
Occasionally, virus particles were present in nuclei, presumably because of invagination of the
perinuclear space, and were also labelled. In apparently non-infected cells adjacent to infected
cells, as well as in healthy uninfected controls, nuclei appeared to contain normal chromatin
structures such as interdispersed heterochromatin and euchromatin. N o virus particles were
observed and no gold label was detected (Fig. 2c). Control sections were also not labelled when
diluted preimmune serum was used (data not shown). Similar results were obtained with the two
species of Nicotiana used in this study.
Our results clearly show that the nucleoplasm of PYDV-infected cells was composed largely of
viral structural proteins and becomes the site of protein accumulation. This result supports the
notion that the nucleocapsids, after acquiring their proteins in the nucleoplasm, acquire their
envelopes from the inner nuclear membrane while passing from the nucleoplasm into the
perinuclear space where assembled virus particles appear (Francki et al., 1985). In some
instances small virus aggregates were also observed in the cytoplasm. However, no
accumulation site other than the nucleoplasm has been found for viral structural proteins.
Enveloped particles after acquiring their proteins in the nucleoplasm may enter the lumen of the
endoplasmic reticulum (Francki, 1973) which is considered to be a continuation of the
perinuclear space (Clowes & Juniper, 1968). Therefore, the nucleoplasm seems to be the only
accumulation site for P Y D V structural proteins in infected cells. At the present time we are
unable to examine the sequence of acquisition of P Y D V structural proteins in the assembly of
virus particles. Monoclonal or monospecific antibodies prepared to individual structural
proteins will be necessary for selective identification of the protein pool in the nucleoplasm of
infected nuclei.
We are grateful to W. G. Langenberg for a critical reading of the manuscript, Mr Yuh-Kun Chen-and Mrs
Nancy Wang for their technical assistance. Published as paper No. 322, Scientific Journal Series of the Institute of
Botany, Academia Sinica, Taipei, Taiwan, Republic of China.
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2727
Fig. 2. Electron micrographs of immunogold-stained sections of N. rustica leaf cells. Abbreviations
used: Ch, chloroplast; Cr, chromatin; Cy, cytoplasm; Np, nucleoplasm; Nu, nucleus; Nuo, nucleolus;
V, virus particles; Va, vacuole. All bar markers represent 200 nm. (a, b) PYDV-infected cells, (b) is an
enlargement of part (a). (c) Non-infected cell near the infected one in (a).
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