J. gen. Virol. (1988), 69, 573-581.
Printed in Great Britain
573
Key words: BTV/translation in vitro/peptide mapping
In vitro Transcription and Translation of Bluetongue Virus mRNA
ByALBERDINA
A. V A N D I J K 1. A N D H . H U I S M A N S 2
JBiochemistrv Section, Veterinary Research Institute, Onderstepoort 0110 and 2Department o,1"
Genetics, Universi O, o f Pretoria, Pretoria 0002, South AJrica
(Accepted 18 November 1987)
SUMMARY
Fractionation of in vitro transcribed bluetongue virus (BTV) m R N A by agarose gel
electrophoresis resulted in the separation of eight of the 10 species. The relative molar
ratio of the m R N A s confirmed that m R N A 5 was transcribed more frequently than
would be predicted from the size of the $5 genome segment, while m R N A 10 was
transcribed less frequently. In vitro translation ofunfractionated BTV mRNAs resulted
in the synthesis of the seven known structural proteins (P1 to P7) and two known nonstructural proteins (NSI and NS2). Two additional non-structural proteins (NS3 and
NS3A) with Mr of 28K and 25K respectively were identified. The protein coding
assignments for the medium- and small-sized double-stranded RNA genome segments
ofBTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17.
The peptide maps of NSI, NS2, NS3 and NS3A synthesized in vitro corresponded to
those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a
truncated form of NS3, since its peptide map completely overlapped that of NS3.
Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of
the cytoplasm of infected cells, and were synthesized in variable amounts in vitro,
whereas the other nine viral proteins were synthesized in constant molar ratios. A
difference in the relative molar ratios in which some of the BTV proteins were
synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the
largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely,
protein P6 was translated much more efficiently in vitro than in vivo.
INTRODUCTION
Bluetongue virus (BTV) is a member of the genus orbivirus in the family Reoviridae. Its
genome consists of 10 segments of dsRNA that range in size from approximately 0.3 × 106 to
2.5 x 100 Mr (Verwoerd et al., 1972). Each genome segment is thought to code for the synthesis
of at least one viral polypeptide, Seven structural proteins (P1 to P7) and two non-structural
proteins (NS1 and NS2) have been identified (Huismans, 1979). The protein coding
assignments for two of the 24 bluetongue virus serotypes have been reported (Grubman et al.,
1983; Mertens et aL, 1984). The results of Grubman et al. (1983) on BTV serotype 17 are difficult
to relate to known structural and non-structural proteins. Mertens et al. (1984) have assigned the
first nine dsRNA segments of BTV-1 to the seven known structural and two known nonstructural proteins. Segment 10 was shown to code for two almost identical proteins, 8 and 8a
respectively, and it was initially postulated that they could be structural outer capsid
polypeptides. Proteins 8 and 8a were, however, not detected in BTV-1 or BTV-4 purified
according to a recently published method (Mertens et al., 1987), arguing against them being
structural components. These proteins have Mr of 20K and 15K respectively which is smaller
than the 25K protein predicted from the base sequence of cloned segment 10 (Lee & Roy, 1986).
Both the in vitro translation studies and coding assignments of the BTV genome segments
have so far been carried out using individually purified denatured dsRNA segments. The one
aspect that has not as yet been investigated is the in vitro translation ofBTV mRNAs. Huismans
& Verwoerd (1973) reported that the genome segments are not all transcribed in vivo at a
0000-7829 © 1988 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
574
A.A.
VAN DIJK AND H. HUISMANS
c o n s t a n t rate o f c h a i n e l o n g a t i o n , r e s u l t i n g i n d i f f e r e n c e s i n t h e a m o u n t s o f B T V m R N A s in
i n f e c t e d cells. H o w e v e r , t h e m R N A s are t r a n s l a t e d in vivo a t a l m o s t t h e s a m e f r e q u e n c y a t w h i c h
t h e y are t r a n s c r i b e d ( H u i s m a n s , 1979). T h i s is in c o n t r a s t to w h a t h a s b e e n o b s e r v e d for r e o v i r u s
w h e r e c o n s i d e r a b l e d i f f e r e n c e s h a v e b e e n f o u n d in t h e t r a n s l a t i o n f r e q u e n c i e s o f t h e s class
m R N A s ( L e v i n & S a m u e l , 1980). I t w a s f o u n d t h a t t h e r e o v i r u s s4 m R N A w a s t r a n s l a t e d a t least
e i g h t t i m e s as efficiently as t h e sl m R N A . It still n e e d s to be i n v e s t i g a t e d w h e t h e r s i m i l a r results
a p p l y to t h e in vitro t r a n s l a t i o n o f B T V m R N A .
I n t h i s p a p e r we h a v e i n v e s t i g a t e d in vitro t r a n s l a t i o n w i t h B T V m R N A a n d h a v e c o m p a r e d
t h e m o l a r r a t i o in w h i c h t h e viral m R N A species are t r a n s c r i b e d to t h e r a t i o in w h i c h t h e
c o r r e s p o n d i n g p r o t e i n s are s y n t h e s i z e d . W e also r e p o r t o n t h e c o d i n g a s s i g n m e n t for B T V - 1 0
d s R N A g e n o m e s e g m e n t s . S e g m e n t 10 c o d e s for t w o n o n - s t r u c t u r a l p r o t e i n s N S 3 a n d N S 3 A
which have identical peptide maps.
METHODS
Cells and virus. BHK-21 cells from the American Type Culture Collection were grown as monolayers in Roux
flasks or roller bottles in modified Eagle's medium containing 5% bovine serum (Verwoerd et al., 1967). An
avirulent strain of BTV-10 was used. It was propagated in BHK cells and purified as described by Huismans et al.
(1987a).
In vitro translation. Rabbit reticulocyte lysates were prepared from anaemic rabbits as described by Ranu &
London (1979) and were treated with nuclease to eliminate endogenous mRNAs according to the method of
Pelham & Jackson (1976) as modified by Neeleman & Van Vloten-Doting (1978). Lysates were frozen in and
stored under liquid nitrogen. The translation reactions were carried out as described by Pelham & Jackson (1976),
using 0-5 ~tCi/p.1[35S]methionine (New England Nuclear, sp.act. 1000 Ci/mmol; approx. 12 mCi/ml), and 50 ttg/ml
mRNA or denatured dsRNA as indicated. Incubation was carried out at 30 °C for 60 rain unless indicated
otherwise.
Polyacrylamide gel electrophoresis andfluorography. SDS-PAGE was carried out in discontinuous 15% gels as
described by Laemmli (1970). For preparative gels the well-forming comb was replaced with a 10 cm single-well
comb. After electrophoresis the gels were fixed, destained and fluorographed according to the method of Bonner &
Laskey (1974). Dried gels were exposed to Cronex MRF31 X-ray film (DuPont) at - 7 0 °C.
[3sS]Methionine labelling of BTVproteins in infected cells. Confluent BHK-21 monolayer cells were infected with
BTV at 5 p.f.u./cell and pulse-labelled at different intervals after infection, indicated in the text, as described by
Huismans et al. (1987b). Methionine-free Eagle's medium, containing 15 ~tCi/ml [35S]methionine (approx. 12
mCi/ml) was added at 10 ml/10 s cells and incubated for 3 h at 37 °C. Subcellular cytoplasmic particulate (P100)
and soluble (Sl00) fractions were prepared as described by Huismans et al. (1987b).
Preparation oJindit,idually purified dsRNA segments. BTV was grown in BHK-21 cells and dsRNA was isolated
by the phenol extraction method as described by Huismans & Verwoerd (1973). The dsRNA preparation was
separated into 10 segments by PAGE on either 4% or 8% preparative slab gels (15 cm × 18 cm x 0.3 cm) using a
buffer (Loening, 1967) containing 0.01% SDS. The relative mobility of genome segments $5 and $6 are reversed if
the acrylamide concentration is increased from 4% to 8 %
Individual genome segments were obtained by excision from SDS-PAGE gels and characterized by agarose gel
electrophoresis. Contaminated segments were each re-run on a second gel and extracted in the same way. Several
methods were investigated for denaturing the dsRNA segments. The best results were obtained with 10 mMmethylmercuric hydroxide as described by Mertens et al. (1984).
Preparation of BTVmRNA synthesized in vitro. BTV cores were prepared as described by Van Dijk & Huismans
(1980)• For large scale mRNA preparation at least 40 Az6o units of BTV cores were incubated in a transcription
reaction mixture as described by Van Dijk & Huismans (1980). The mixture also contained 0.85 m~-UTP.
Incubation was at 28 °C for 16 h. After incubation, polyvinylsulphuric acid (PVS) was added to a final
concentration of 0.05%. Particulate material was removed by centrifugation at 100000 g at 4 °C for 90 rain. An
equM volume of (~-01 STE (0-01 M-NaC1, 0-01 M-Tris-HC1 pH 7-4, 0-001 M-EDTA) containing 0-05% PVS was
added to the supernatant; the solution was deproteinized by phenol extraction, followed by two ether extractions
and ethanol precipitation. Single-stranded RNA was separated from any remaining dsRNA by overnight
precipitation at 4 °C in the presence of 2 M-LiCI. The ssRNA precipitate was resuspended in water, divided into
small samples and stored frozen at - 7 0 °C.
Agarose gel electrophoresis ofssRNA. Submarine horizontal agarose gel electrophoresis was performed using 3 %
low gelling temperature agarose gels (Sea Plaque; FMC, Rockland, Me., U.S.A.). The electrophoresis buffer
contained 5 mM-sodium acetate, 1 mM-EDTA and 10 mM-Tris-HCl pH 7.9. After dissolving the agarose buffer
mixture in a boiling waterbath, it was autoclaved for 10 rain before pouring the gel. Electrophoresis was performed
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
I n v i t r o translation o f B T V m R N A
575
1
2
1!
3
P
2
3
4
5
NSI
NS2
6
7
5
6
4i~ w
|
7
8
9
2
10
m
~lb
m
NS3A
.,,
~"
Fig. 1
Fig. 2
Fig. 1. Proteins synthesized in vitrofrom denatured BTV dsRNA genome segments of the medium- and
small-sized groups: lane 1, segment 4; lane 2, segment 6; lane 3, segment 5; lane 7, segment 7; lane 8,
segment 8: lane 9, segment 9; lane 10, segment 10. Control samples were [35S]methionine-labeUed : lane
4, P100 fraction of infected cells (Huismans, 1979); lane 5, in vitro translation products of
unfractionated BTV mRNAs; lane 6, purified BTV. Discontinuous SDS-PAGE using 5 ~ stacking gels
and 15~ running gels (Laemmli, 1970) was performed, followed by fluorography (Bonner & Laskey,
1974).
Fig. 2. Fractionation of 3 ~tg BTV dsRNA (lane 1) and 5 ~tg BTV mRNA (lane 2) on a 3 ~ low gelling
temperature agarose gel. The gel was stained with ethidium bromide and visualized on a u.v.
transilluminator.
at 100 mA. The gels were stained in electrophoresis buffer containing 1 p.g/ml ethidium bromide and visualized on
a u.v. transilluminator.
Peptide mapping. The procedure was essentially that of Cleveland et al. (1977) as modified by Burge & Huang
(1979). [35S]Methionine-labelled proteins were located in dried gels by means of autoradiography and were
excised. After removing the filter paper backing, the rectangular protein-containing fragments (0-5 cm x 0.3 cm)
were inserted in the sample wells of an SDS-PAGE gel. As far as possible, protein samples containing similar
amounts of radioactivity (as judged by the intensity of the bands on autoradiography) were used for comparative
purposes. The stacking part of the gel (5 ~ acrylamide) was 4 cm long. The gel fragments were overlaid with 20
glycerol in stacking gel buffer. A 10/al volume of 10% glycerol in stacking gel buffer, containing various amounts
of Staphylococcus aureus V8 protease, as indicated in the text, was layered over the 20% glycerol buffer. The
current was switched on immediately and the voltage was adjusted to 150 V. When the tracking dye had moved 3.5
cm into the stacking gel, the current was turned off for 30 min to allow digestion. After this digestion period, the
current was turned on again, the voltage was adjusted to 200 V, the electrophoresis was continued until the
tracking dye had moved to the bottom of the resolving gel (15~ acrylamide), after about 6 h. The peptides were
visualized by means of fluorography.
RESULTS
Coding assignments o f small- and medium-sized genome segments
I n d i v i d u a l l y purified d s R N A s e g m e n t s w e r e p r e p a r e d , d e n a t u r e d by t r e a t m e n t w i t h 10 mMm e t h y l m e r c u r i c h y d r o x i d e a n d t r a n s l a t e d as d e s c r i b e d in M e t h o d s . T h e results are s h o w n in
Fig. 1.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
576
A. A. VAN DIJK
AND
H. HUISMANS
Translation of medium-sized segments $4, $5 and $6 (migration order from a 4~o gel)
produced proteins comigrating with P4, NS1 and P5 respectively. Smaller, incomplete
translation products were also observed but the assignment was made to the largest of the
comigrating polypeptides, except in the case of segment $9 which was contaminated with $8
because genome segments $8 and $9 virtually comigrate on SDS-PAGE. Translation of the
small segments $7, $8 and $9 resulted in the synthesis of proteins comigrating with P7, NS2 and
P6 respectively. Genome segment 10 coded for two proteins designated NS3 and NS3A.
Analysis o f m R N A species synthesized in vitro
The relative molar ratio in which BTV m R N A species were synthesized during in vitro
transcription was assessed by direct electrophoretic analysis of the m R N A s synthesized in vitro,
on a 3 ~ agarose gel with a low gelling temperature (Fig. 2).
Resolution of eight of the 10 BTV m R N A species was achieved. There were marked
differences between the relative amounts in which the three m R N A size classes (large, medium
and small; 1, m, and s) were transcribed. This observation was quantified by scanning the
ethidium bromide-stained gel. The results are summarized in Table 1.
The relative molar ratio (~) in which the m R N A species were synthesized varied from about
3"0 to 20. As expected, the large genome segments were transcribed much less frequently than
the smaller segments. A notable exception was segment 10 which was transcribed in a smaller
relative molar amount than many of the larger segments. The most frequently transcribed
genome segment was $5. The results are in agreement with those obtained earlier by Huismans
& Verwoerd (1973) by an indirect hybridization method. The in vivo transcription frequency was
approximately the same as that observed in vitro.
In vitro translation o f B T V m R N A
The synthesis of BTV proteins in vitro from m R N A s synthesized in vitro was investigated by
analysing samples from an in vitro translation reaction mixture on SDS-PAGE at different time
intervals after the start of the reaction. In order to detect both the large- and small-sized proteins,
duplicate samples were analysed. The gel in Fig. 3 (a) was electrophoresed to obtain separation
of the high Mr proteins, and the other gel (Fig. 3 b) was electrophoresed for a shorter time in
order to allow detection of the low Mr proteins.
The results in Fig. 3 (a) indicate in vitro translation of the seven structural (P 1 to P7) and two
non-structural (NS1 and NS2) BTV-specific proteins from BTV m R N A synthesized in vitro.
Protein P1 was synthesized very inefficiently and could only be identified on heavily
overexposed fluorograms (result not shown).
Generally the proteins were completed in order of increasing size. One possible exception was
NS1 which was synthesized more slowly than the larger P5. Synthesis of NS1 was characterized
further by the fact that it did not appear to migrate as a single, tight band but rather as a number
of peptides that differed slightly from one another with respect to electrophoretic mobility (Fig.
3 a lane 6). As the amount of NS 1 increased, the resolution between these peptides was lost (lanes
7 to 10), and NS1 became the rather broad, slightly diffuse band that is normally observed. A
similar observation with respect to NS1 synthesized in vivo has been made previously.
In vitro translation of the smaller non-structural proteins NS3 and NS3A, with Mr of
approximately 28K and 25K respectively, is shown in Fig. 3(b).
It was evident from Fig. 3 that the relative amounts in which the proteins were synthesized in
vitro did not change after 30 min of incubation. Proteins P5, NS1, NS2, P6 and P7 were all
synthesized in comparatively large amounts. In contrast to the in vivo situation, the protein most
frequently translated in vitro appeared to be NS2. Protein P6, which is translated much less
frequently in vivo than many of the other BTV proteins, was translated very well in vitro. Protein
P4, however, was translated far less efficiently than proteins of similar size such as P5, or even
larger ones, such as P3. The relative molar ratio in which the m R N A species are translated in
vitro was determined from the results depicted in Fig. 3 and is summarized in Table 1.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
1
2
3
4
5
6
7
8
9
10
$2
SI
Fig. 3
2
(b)
1
3
4
5
6
7
8
9
10
ll
20.1 --
30--
94-67 --
3
Fig. 4
2
--NS3
--NS3A
--4
~5
"NS1
-NS2
-6 7
Fig. 4. Autoradiogram of subfractionated cytoplasm of [3sS]methionine-labelled BTV-infected cells. A confluent monolayer of BHK cells (150 cm-') was infected
with BTV at approximately 20 p.f.u./cell and incubated at 31 °C for 16 h before the medium was poured offand the cells were rinsed with methionine-free Eagle's
medium. Then 5 ml of methionine-free Eagle's medium containing 15 ~tCi/ml [3SS]methionine (Amersham, 800 Ci/mmol) was added as described in Methods. After
the labelling period the ceils were harvested, concentrated by low speed centrifugation and resuspended at 5 × I07 cells/ml in 0.01 STE-TX buffer (0.01 M-NaCI,
0.01 M-Tris-HCI pH 7.4, 0.001 M-EDTA, 0.5 % Triton X-100). Nuclei were removed by centrifugation at 1500 g for 5 min and washed once with half of the original
volume of 0.01 STE-TX. The supernatants were combined (SI0 fraction) and centrifuged for 2 h at 45000 r.p.m, in an SWS0.1 rotor through a 2 ml layer of 40%
sucrose. The supernatant (S 100) was divided into small samples and kept at - 20 °C. The pellet (P 100) was resuspended in 0-01 STE (20 % of the S 10 volume) and also
kept at - 2 0 °C. Lane 1, purified BTV; lane 2, P100 fraction; lane 3, S100 fraction, and to the left of the figure is the position of Serva blue-stained low Mr
markers x 10-3 (Pharmacia).
Fig. 3. Fluorogram showing SDS-PAGE (15~) analysis (Laemmli, 1970) of the kinetics of the in vitro translation of unfractionated BTV mRNAs in the rabbit
reticulocyte lysate system. A 500 lal translation reaction mixture was set up and incubated at 30 °C as described in Methods. Every 5 min during a 45 min reaction
period two 15 l.tl samples were taken and added directly into protein solvent solution (0.0625 M-Tris-glycine pH 6.8, 2 % SDS, 10 ~o glycerol, 5 % 2-mercaptoethanol)
and kept on ice until the last samples were ready for electrophoresis. Samples were taken at 0, 5, 10, 15, 20, 25, 30, 35 and 45 min; lanes 3 to 11, respectively. Control
~amples on the gel were [35S]methionine-labelled : lanes 1 P 100 sample of infected cells to locate the non-structural proteins, and lanes 2 purified BTV to locate the
structural proteins. The arrows at the left of Fig. 3(b) indicate the position of Serva blue-stained low Mr markers x 10-3 (Pharmacia). The gel in (a) was
:lectrophoresed at 200 V for 16 h and that in (b) was electrophoresed at I00 V for 16 h.
(a)
[
e~
578
A . A . VAN DIJK AND H. HUISMANS
T a b l e 1. A comparison o f B T V m R N A species synthesized in v i v o and in v i t r o to the molar
ratio o f the proteins synthesized in vivo and in vitro that they encode
mRNA
Protein
A
r
~
Molar ratio (%)
A
In vitrot
In vivo~
SI
$2
$3
$4
$5
$6
2-8
3.1
3"4
12.9
19'9
7.8
$7
$8
$9
S10
28'3
3.8
5.3
4.2
8.0
18.2
8' 1
12.8
16.5
12.0
11.0
Segment*
12-5
9'3
In vivo:in vitro
1'3
1.7
1-2
0.6
0"9
1.0
1'0
1-0
1'18
Jk
t
Encoded
by
S1-S10
Molar ratio (%)
r~ j '
In vitro§
In vivoll
In vivo :in vitro
PI
P2
P3
P4
NSI
P5
P7
NS2
P6
NS3
1.9
3"2
3'5
10.9
11-8
20"0
31-4
17"1
*¶
1-4
1.9
2.5
3-9
27.5
10.0
27-0
26.0
4.8
*¶
1.0
0.8
1.1
2.5
0.8
1.1
0.8
0.3
,¶
* Order of the segments is derived from a 4% polyacrylamide gel.
t Determined from densitometer scans of electrophoretically separated mRNAs as shown in Fig. 2. Mr used are
from Verwoerd et at. (1972).
:~ From Huismans & Verwoerd (1973).
§ Determined from densitometer scans of electrophoretically separated proteins synthesized in vitro as shown in
Fig. 3. Mr used are from Verwoerd et al. (1972).
IIDetermined from densitometer scans of electrophoretically separated proteins synthesized in vivo labelled as
described in the legend to Fig. 4.
¶ The relative molar amounts in which this protein was synthesized in vivo were too small to determine, whereas
the amounts synthesized in vitro were found to be variable.
Synthesis o f N S 3 proteins in vivo
It was important to determine whether the non-structural proteins NS3 and NS3A were also
synthesized in infected cells. To investigate this, BTV-infected cells were pulse-labelled for
different 3 h intervals after infection with [35S]methionine. Synthesis of NS3 could only be
detected very late in the infection cycle (results not shown). A subfractionation analysis of the
cytoplasmic extract of cells labelled 16 to 19 h p.i. is shown in Fig. 4. The particulate fraction
did not contain NS3 or NS3A, but a small amount of both NS3 and NS3A was consistently
found in the soluble fraction.
Peptide mapping
To determine whether the non-structural proteins NS1, NS2 and NS3 synthesized in vivo were
identical to their counterparts synthesized in vitro, peptide map analysis of the proteins was
carried out using S. aureus V8 protease digestion (Cleveland et al., 1977; Burge & Huang, 1979).
The respective maps are shown in Fig. 5.
A characteristic pattern was obtained for each protein. NS1 and NS2 proteins synthesized in
vivo were indistinguishable from those synthesized in vitro. Except for the initial difference in Mr
the peptide map of NS3 was almost identical to that of NS3A in the case of both NS3 and NS3A
synthesized in vivo and in vitro. The two proteins are therefore probably translated from the same
in-phase overlapping reading frame on genome segment 10.
In almost all cases in Fig. 5 some digestion of proteins occurred in wells (lanes 2 and 11) that
had not received any protease. This could be attributed to diffusion of the protease during the 30
min digestion period when electrophoresis was interrupted.
DISCUSSION
In vitro translation of the 10 mRNA species of BTV-10 resulted in the synthesis of at least 11
distinct proteins. The coding assignments for these proteins were identical to those reported by
Mertens et al. (1984) for BTV- 1 and Grubman et al. (1983) for BTV- 17. Although preparations of
$8 and $9 were always cross-contaminated, the $9 preparations more so with $8 than vice versa,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
579
In vitro translation o f B T V m R N A
(a)
1
(b)
2
3 4
5 6
7
8
9 10 11
1
2
3 4
5
6
7
8
9 10 11
NS2-
(c)
1
NS
2 3 4
5 6
(a)
1
7 8 9 10 11
2 3 4
5 6 7 8 9 10 11
NS3...
NS3A1
Fig. 5. Peptide mapping of the non-structural proteins of BTV analysed by fluorography after SDSPAGE (15°o). (a) NS1, (b) NS2, (c) NS3 and NS3A synthesized in vivo and (d) NS3 and NS3A
synthesized in vitro. In (a) and (b), lanes 1 contain P100 samples of BTV-infected cells: lanes 2 to 6 are
the maps of the proteins synthesized in vivo and lanes 7 to 11 those of the proteins synthesized in t'itro. In
(c) and (d) lanes 1 contain purified BTV, lanes 2 to 6 are the maps of NS3 and lanes 7 to 11 are those of
NS3A. In all cases the amounts of protease V8 used for digestion were: lanes 2 and 11,0 rig; lanes 3 and
10, 5 ng; lanes 4 and 9, 25 ng; lanes 5 and 8, 500 ng and lanes 6 and 7, 2500 ng.
their coding assignments could nevertheless be made. Synthesis of the non-structural proteins
NS3 and N S 3 A was also observed in vivo. The peptide maps of non-structural proteins NS1 and
NS2 synthesized in vitro were found to be similar to the maps of their counterparts synthesized in
vivo. The peptide m a p for protein NS3 synthesized in vitro was very similar to that of NS3A in
vitro, as was the case for NS3 and NS3A in vivo. However, the peptide m a p of NS3 synthesized in
vivo (Fig. 5 c) does not closely resemble that of NS3 synthesized in vitro (Fig 5 d), and the same
applies to NS3A. Unfortunately not enough in vivo material was available to do the direct
comparison with the proteins synthesized in vitro. Careful examination reveals that the m a p s are
essentially similar, with those of the proteins synthesized in vivo not as fully digested as those of
the in vitro proteins. This explanation accounts for the difference in intensities of the various
peptide bands, although the general peptide pattern is quite similar.
The translation with individually purified d s R N A segments indicated that NS3 (28K) and
NS3A (25K) were both encoded by genome segment 10. Despite the difference in Mr these
proteins seem to be the equivalents of proteins 8 (20K) and 8a (15K) reported by Mertens et al.
(1984). The size differences could be due to differences in electrophoretic conditions. The
correspondence in the peptide maps of NS3 and N S 3 A suggests that they are translated from the
same open reading frame, with NS3A being a truncated form of NS3. Lee & Roy (1986) have
shown that there are two possible translation initiation sites in the same open reading frame of
segment 10 of BTV-10. The 39 base pair difference between these initiation sites appears to be
too small to account for the 3K difference in size o f NS3 (28K) and N S 3 A (25K). However, Rae
& Elliott (1986) have shown that even a single base pair change in the 5' coding region of
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
580
A. A. VAN D I J K A N D H. H U I S M A N S
vesicular stomatitis virus NS protein resulted in a large difference in electrophoretic mobility in
the corresponding proteins, as determined on SDS-PAGE. The apparent 3K size difference
between NS3 and NS3A is therefore not necessarily a reflection of a large difference in the
length of the peptide chain, but could reflect differences in the polarity and structure of the
terminal regions of the two proteins. The size of the 25K, NS3A protein agrees exactly with the
size predicted for the S10 gene product (Lee & Roy, 1986).
We obtained results confirming the report by Mertens et at. (1984) that the NS3 proteins are
only synthesized in very small amounts at a late stage in the infection cycle, which could explain
why they were not identified in infected cells in the initial studies on BTV proteins (Verwoerd et
al., 1972; Huismans, 1979). No NS3 proteins were found in the particulate fraction, a result
which would argue against them being structural proteins that are removed during the virus
purification step as has been suggested by Mertens et al. (1984).
The amounts in which the two NS3 proteins were synthesized in vitro were rather variable
(results not shown) when compared to the synthesis of the other nine proteins which were
synthesized in an almost constant molar ratio. As has been found with reovirus (McCrae &
Joklik, 1978), the large proteins were translated very inefficiently in vitro (Table 1). This is
thought to be due to deficiencies in the in vitro translation system rather than to specific inherent
control elements. The small- and medium-sized BTV proteins are all synthesized quite
efficiently and there is no evidence for large differences in translation of m R N A species as has
been reported for the s m R N A s of reovirus (Levin & Samuel, 1980). The only exception is
possibly BTV m R N A 4 which is translated much less frequently than m R N A 5 even though the
results in Fig. 2 and Table 1 indicate that relatively large amounts of m R N A 4 are transcribed.
In contrast to what has been found in vivo (Huismans, 1979), the relative abundance of BTV
m R N A species does not appear to have an effect on the ratio in which the polypeptides they
encode are synthesized in vitro. This is demonstrated by m R N A species 5 and 6 which are
transcribed in a molar ratio of approximately 5:2 whereas the corresponding NS1 to P5 ratio in
vitro is about 1 : 1. In vivo, however, NS1 is the most frequently translated polypeptide. Another
difference between m vivo and in vitro translation is that P6 is translated very poorly in vivo,
whereas in vitro P6 is translated almost as frequently as protein P7. We obtained similar results
using denatured dsRNAs for in vitro translation. Mertens et al. (1984), however, did not find any
such quantitative differences between proteins synthesized in infected cells and in t~itro in the
reticulocyte lysate system. Therefore, it needs to be investigated whether the differences
between our results and those of Mertens et al. (1984) could be due to artefacts of the in vitro
translation reaction.
Following in vitro transcription, we were able to separate electrophoretically eight of the 10
BTV m R N A species synthesized which allowed an estimate of the molar ratio in which the
m R N A species are transcribed. These results confirmed an earlier report (Huismans &
Verwoerd, 1973) that not all BTV dsRNA segments are transcribed in equal relative amounts.
Genome segment 10 for instance is transcribed at less than half its predicted frequency, whereas
segment 5 is transcribed at more than twice the expected rate. Similar results have been
observed with epizootic haemorrhagic disease virus (Huismans et al., 1979), and Ibaraki virus
(Namiki et al., 1983), and this transcription pattern could well be characteristic for all
orbiviruses.
REFERENCES
BONNER, W. M. & LASKEY, R. A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in
polyacrylamide gels. European Journal of Biochemistry 46, 83-88.
BURGE, B. W. & HUANG, A. S. (1979). Conserved polypeptides in the proteins of vesicular stomatitis virus. Virology
95, 4 4 5 4 5 2 .
CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER, M, W. & LAEMMU, U. K. (1977). Peptide m a p p i n g by limited
proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. Journal of Biological Chemistry 252,
1102-1106.
GRUBMAN,M. J., APPLETON, J. A. & LETCHWORTH,G. J., III (1983). Identification of bluetongue virus type 17 genome
segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology 131,
355-366.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48
I n v i t r o translation o f B T V m R N A
581
HUISMANS, H. (1979). Protein synthesis in bluetongue infected cells. Virology 92, 385-396.
HUISMANS, H. & VERWOERD, D. W. (1973). Control of transcription during expression of the bluetongue virus
genome. Virology 52, 81 88.
HUISMANS, H., BREMER, C. W. & BARBER, T. L. (1979). The nucleic acid and proteins of epizootic haemorrhagic
disease virus. Onderstepoort Journal of Veterinary Research 46, 95 104.
nUISMANS,rL, VAN DER WALT, Y. Y., CLOETE, M. & ERASMUS,B. J. (1987 a). Isolation of a capsid protein of bluetongue
virus that induces a protective i m m u n e response in sheep. Virology 157, 172-179.
HUISMANS,H., VAN DIJK, A. A. & BAUSKIN,A. R. (1987 by. In vitro phosphorylation and purification of a nonstructural
protein of bluetongue virus with affinity for single-stranded R N A . Journal of Virology 61, 3589-3595.
LAEraMLI, U_ K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 227, 680-685.
LEe, J. W. & ROY, P. (1986). N ucleotide sequence of a c D N A clone of R N A segment 10 of bluetongue virus (serotype
10). Journal of General Virology 67, 2833-2837.
LEVIN, K. n. & SAMUEL, e. E. (1980). Biosynthesis of reovirus-specified polypeptides. Virology 106, 1-13.
LOENING, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel
electrophoresis. Biochemical Journal 102, 251-257.
McCRAE, M. A. & JOKLIK, W. K. (1978). The nature of the polypeptide encoded by each of the double-stranded R N A
segments of reovirus type 3. Virology 89, 578-593.
MERTENS, P. P. C., BROWN, F. & SANGAR,D. V. (1984). Assignment of the genome segments of bluetongue virus type 1
to the proteins which they encode. Virology" 135, 207-217.
MERTENS, P. P. C., BURROUGHS,J. N. & ANDERSON,J. (1987). Purification and properties of virus particles, infectious
subviral particles, and cores of bluetongue virus serotypes 1 and 4. Virology 157, 375-386.
NAMIKI, M., SUZUKI, Y., NAKAGAWA,S., YOSHIOKA, 1. & SAITO, Y. (1983). Characterization of ribonucleic acid
transcriptase in Ibaraki virus core particles. Journal of General Virology 64, 1397-1400.
NEELEMAN, L. & VAN VLOTEN-DOTING,L. (1978). Excess of micrococcal nuclease m a y h a r m the exogenous m R N A
dependent rabbit reticulocyte cell-free system. FEBS Letters 95, 103-106.
PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient m R N A - d e p e n d e n t translation system from reticulocyte
lysates European Journal of Biochemistry 67, 247-256.
RAE, B. P. & ELLIOTT, g. M. (1986). Characterization of the mutations responsible for the electrophoretic mobility
differences in the NS proteins of vesicular stomatitis virus New Jersey complementation group E mutants.
Journal of General Virology' 67, 2635-2643.
RANU, R. S. & LONDON, I. M. (1979). Regulation of protein synthesis in rabbit reticulocyte lysates; preparation of
efficient protein synthesis lysates and the purification and characterization of the heme regulated
translational inhibitory protein kinase. Methods in Enzymology 60, 4 5 9 4 8 4 .
VAN DIJK, A. A. & HUISMANS,H. (1980). The in vitro activation and further characterization of the bluetongue virusassociated transcriptase. Virology' 104, 347-356.
VERWOERD, D. W., OELLERMANN, R. A., BROEKMAN, J. & WEISS, K. (1967). The serological relationship of South
African bovine enterovirus strains (Ebco SA-I and II) and the growth characteristics in cell culture of the
prototype strain (Ebco SA-I). Onderstepoort Journal of Veterinary Research 34, 41-52.
VERWOERD, D. W., ELS, H. J., DE VILIERS, E.°M. & HUISMANS, H. (1972). Structure of the bluetongue virus capsid.
Journal oJ Virology 10, 783-794.
(Received 28 April 1987)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 14 Jun 2017 13:49:48