Journal of General Virology (1993), 74, 937-941.
937
Printed in Great Britain
Function of rotavirus VP3 polypeptide in viral morphogenesis
M 6 n i c a Vdsquez, Ana M a r i a Sandino, Jacqueline M . Pizarro, Jorge Fermindez, S o f i a Valenzuela
and Eugenio Spencer*
Unidad de Virolog&, Instituto de Nutricidn y Tecnologia de los Alimentos ( I N T A ) , Universidad de Chile,
Macul 5540, Casilla 138-11, Santiago-ll, Chile
The phenotype of the rotavirus SA-11 mutant tsB
carrying a thermosensitive mutation in gene 3, which
encodes VP3, was characterized further from both
infected cells and purified viral particles. The mutant
phenotype was initially identified as negative for in v&o
double- and single-stranded RNA synthesis. Our results
show that the in vitro transcriptional properties of the tsB
mutant at the restrictive temperature were identical to
those of the wild-type strain. Similar results were
obtained with respect to the VP3-associated guanylyl-
transferase activity. Analysis of viral particles made
by mutant-infected cells at the restrictive temperature
showed that only empty single-shelled particles were
assembled. This indicates that viral morphogenesis is
halted after the initial viral transcription and before
RNA replication, suggesting that VP3 may be required
as part of the replicase system but not for subviral
particle assembly. These data suggest that such a
phenotype is not due to alteration of a VP3 function
related to transcription.
Rotaviruses, a genus of the Reoviridae family, are
composed of a double-shelled protein capsid surrounding
a central core that contains the viral genome consisting
of 11 dsRNA segments (Arias et al., 1982; Mason et al.,
1983). The outer capsid is made of a glycoprotein, VP7,
and dimers of VP4, the haemagglutinin, which has a
trypsin-sensitive site (Espejo et al., 1981; Suzuki et al.,
1986). The inner capsid is made of VP6 and the viral core
consists of polypeptides VP1, VP2 and VP3 and the
genome (Estes & Cohen, 1 9 8 9 ; McCrae &
McCorquodale, 1982).
The function of the viral polypeptides during viral
RNA replication and transcription has been studied
recently (Sandino et al., 1988; Patton & Gallegos, 1988,
1990; Mansell & Patton, 1990; Valenzuela et al., 1991).
Studies on in vitro transcription have allowed the
identification of VP1 as the viral RNA polymerase
(Valenzuela et al., 1991), VP3 as the guanylyltransferase,
and VP2 as a single- and double-stranded RNA-binding
protein (Pizarro et al., 1991; Liu et al., 1992; Boyle &
Holmes, 1986). VP6, the major inner capsid polypeptide,
is strictly required for transcription although the polypeptide seems to lack any enzymatic activity directly
involved in transcription. These results suggest that viral
transcription is catalysed by a multienzyme complex,
present in the single-shelled particle, which requires
enzymatic activities other than the viral RNA polymerase
VP1 (Sandino et al., 1986; Valenzuela et aL, 1991). Viral
RNA replication has also been found to occur in precore
particles containing polypeptides VP 1, VP2 and VP3 and
some of the non-structural proteins (Patton & Gallegos,
1988).
The purpose of the present communication is to
investigate the involvement of VP3 in RNA replication
and transcription, using a temperature-sensitive (ts)
mutant of rotavirus strain SA-11 with a lesion in gene 3,
which encodes the polypeptide VPY The mutant, tsB,
was obtained from F. Ramig (Ramig, 1982, 1983;
Gombold et al., 1985). It was phenotypically
characterized in infected cells as an ss- and dsRNAmutant (Chen et al., 1990; Ramig & Petrie, 1984).
Using the capability of the virus to transcribe RNA
products in vitro similar to those in infected cells, the
mutant phenotype for transcription was determined at
both restrictive and permissive temperatures. In Fig. 1
the mRNA products transcribed by SA-11 and tsB virus
particles are shown. Transcription assays were carried
out using purified virus particles subjected to a thermal
shock (1 rain at 55 °C) (controls were not subjected) and
further incubated at 31 °C or 45 °C. As seen in Fig. 1,
after 30 min of incubation at 31 °C there are considerably
fewer ssRNA products than at 45 °C. Similar results
were obtained when the virus was subjected to a thermal
shock before the transcription assay, suggesting that in
vitro the mutant does not exhibit a ts phenotype and that
its in vitro mRNA synthesis is the same as the wild-type
strain. These results are obtained when similar amounts
of virus were assayed (2 ~tg of viral protein).
Since the mutation is located in the gene encoding the
VP3 guanylyltransferase, the effect of S-adenosyl-
0001-1258 © 1993 SGM
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938
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5
1
1"--"~
6
7
8
•
2--..~
5--~
F
6.----~
p -
¸
Q
7-.-..!~
8--~
9---~
10 -----~ '
~
11----~
Fig. 1. Electrophoretic analysis of transcripts synthesized in an in vitro assay by tsB mutant and SA-11 virus particles at 31 °C (lanes
1 to 4) and 45 °C (lanes 5 to 8). Purified SA-11 (lanes 1, 3, 5 and 7) and tsB (lanes 2, 4, 6 and 8) virus particles (2 gg each) were assayed
for R N A polymerase activity as previously described but using [c~-a2P]GTP of a high specific activity (3000 Ci/mmol) (Valenzuela et
al., 1991). The mixture was incubated for 30 rain at 31 °C or 45 °C; previously the virus particles were incubated, or not, at 55 °C for
1 min. The products were analysed by gel electrophoresis in 8 M-urea5 % polyacrylamide (Sandino et al., 1986). The assays were done
at 31 °C, and the particles subjected (lanes 1 to 2) or not (lanes 3 to 4) to a thermal shock (1 min at 55 °C). The assay was also done
at 45 °C as shown in lanes 5 to 8, and they were heat-activated (lanes 5 to 6) or not (lanes 7 to 8).
methionine (SAM) addition on viral RNA synthesis was
also studied (Pizarro et al., 1991; Liu et al., 1992). The
addition of SAM to the transcription mixture at a final
concentration of 0.3 mM increases the yield of RNA
synthesis, by four- to fivefold for SA-11 and the mutant,
at all the temperatures studied. When similar experiments
were carried out at 31 °C the yield and the rate of RNA
synthesis were less than 20 % of that obtained at 45 °C
for both the mutant and the wild-type strain (data not
shown).
Another assay performed to characterize the guanylyltransferase activity was based on the unique property of
the enzyme to form a covalent complex with GMP. This
complex is an intermediate during the synthesis of the
cap at the 5' end of the mRNA (Shuman, 1982). Assays
were carried out at two temperatures, 31 °C and 37 °C.
Purified viral particles were assayed for GMP-enzyme
complex formation in a 12 gl reaction mixture as previously described using 1 ~Ci of [~-32P]GTP (specific
activity of 3000 Ci/mmol) (Pizarro et al., 1991). The
formation of a covalent complex between GMP and VP3
was determined by electrophoresis in a 12% polyacrylamide gel (Laemmli, 1970). Gels were dried, strips
of approximately 0.25 cm were cut and the associated
radioactivity was determined in a liquid scintillation
counter. In Fig. 2 the formation of the covalent complex [0~-32p]GMP-enzyme, for both the tsB mutant
and the wild-type strain at both restrictive and permissive temperatures are shown. As seen in this figure,
at 37 °C there is a peak of radioactivity associated
with viral proteins with a relative mobility corresponding
to VP3, for both SA-11 and tsB. When the same assay
was done at 31 °C, the VP3-associated radioactivity was
similar to the background, for SA-11 as well as for tsB.
These results imply that the guanylyltransferase properties of the tsB mutant and SA- 11 are similar with respect
to the formation of the covalent GMP-enzyme complex.
These results suggest that once the mutant viral proteins
are assembled in vivo at a permissive temperature (31 °C),
the mutation in VP3 is not expressed in the viral progeny
thus showing similar characteristics to the wild-type
strain for in vitro mRNA synthesis.
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VP1
VP2 VP3 VP4
1
2
3
4
5
6
Subviral
particles
/
500
Double
shell
400
300
200
100
0
1
4
2
VP1
5
6
7
Single
shell
VP2 VP3 VP4
*d
.o
"0
Core--
500
400
300
200
100
0
1
2
3
4
5
6
7
Strips
Fig. 2. Quantification of the formation of the covalent VP3-GMP
complex. The virus particles SA-11 (a) and tsB (b) were assayed at
permissive temperature (31 °C; hatched columns) and at 37 °C (solid
columns). The formation of the covalent complex VP3-GMP was
assayed in a 12 gl reaction mixture as previously described using 1 gCi
of [c~-~2P]GTP(specific activity 3000 Ci/mmol) and 1 gg of purified
SA-11 or tsB virus particles (Pizarro et al., 1991). The virus particles
showed similar transcriptional activity as seen in Fig. 1. The formation
of a covalent complex between GMP and VP3 was determined as
described in the text. The graphics represent the radioactivity associated
with proteins in the different strips. The position of the viral proteins
was determined according to the migration of 3SS-labelled rotavirus
proteins.
Based on the above results and others previously
reported (Chen et al., 1990), the m u t a n t phenotype
initially described as in vivo ss- and d s R N A - could be
accounted for by the following alternatives: (i) VP3 is the
viral replicase; (ii) the VP3 m u t a t i o n affects the assembly
of a subviral particle required for the encapsidation o f
the viral d s R N A ; (iii) VP3 interacts with other proteins
in a multienzyme complex or subviral particle which has
the replicase activity. The first theory is unlikely because
it has been reported that VP1 is the viral polymerase
(Valenzuela et al., 1991). The second is also improbable
since at restrictive temperatures no d s R N A is detected in
infected cells (Chen et al., 1990; E. Spencer, unpublished
::•i;:••••• :: •::••: ••••:••.•.
••:i
•• •
Fig. 3. Electrophoresis of subviral particles recovered from MA-104
infected cells using the tsB mutant at permissive (31 °C) and restrictive
(39 °C) temperatures. Viral particles were isolated from cells grown at
31 °C (lanes 1 to 3) and 39 °C (lanes 4 to 6), at 8 h (lanes 1 and 4), 12 h
(lanes 2 and 5) and 24 h p.i. (lanes 3 and 6). 3aS-labelled subviral
particles were obtained as previously described from confluent
monolayers of MA-104 cells infected with 5 p.f.u. (Patton & Gallegos,
1990). The pellets containing the subviral particles were suspended in
25 ~1 of buffer HGD (10 mM-HEPES pH 7"6, 10% glycerol, 2mMdithioerythritol) and subjected to electrophoresis on a 0'6%
Tris-glycine agarose gel, then dried and analysed by autoradiography.
The migration of purified double-shelled, single-shelled and core
particles are indicated in the figure.
results). The last possibility appears m o s t likely, because
in isolated subviral particles engaged in viral R N A
replication the presence o f V P 1, VP2 and VP3 and some
o f the non-structural polypeptides has been detected
(Helmberger-Jones & Patton, 1986; Mansell & Patton,
1990).
To study the association between the f o r m a t i o n o f
subviral particles and viral R N A replication, the kinetics
o f assembly o f subviral particles was studied at restrictive
and permissive temperatures. F o r this purpose, M A - 1 0 4
cells were infected with tsB at b o t h 31 °C and 39 °C and
subviral particles were isolated f r o m these cells 8, 12
and 24 h post-infection (p.i.). Fig. 3 shows the autor a d i o g r a m obtained after electrophoresis on Tris-glycine
agarose gels o f the different subviral particles recovered
f r o m the cytoplasm o f infected cells (Gallegos & Patton,
1989). To identify the different virus particles, markers
were prepared f r o m purified virus particles as described
elsewhere (Sandino et at., 1986). At 12 h p.i. at the
permissive temperature (lanes 1 to 3) the synthesis o f
single-shelled particles reached a m a x i m u m level.
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1
2
3
4
5
6
aen
R~
8-
Fig. 4. PAGE of dsRNA obtained from tsB mutant viral particles recoveredfrom MA-104 infectedcells at restrictiveand permissive
temperatures. The cell suspension containing the subviral particles at the times described below was phenol-extractedand ethanolprecipitated and then analysed by electrophoresison a 10% polyacrylamidegel and further stained by silver nitrate. Lanes 1 to 3
correspond to the infection done at 31 °C, lanes 4 to 6 correspond to the infectiondone at 39 °C, at 8, 12 and 24 h p.i. respectively.
Double-shelled particles seemed to accumulate after 12 h
p.i. during viral morphogenesis. These results were
identical to those obtained with the wild-type strain, SA11 (data not shown). When the same experiment was
carried out at the restrictive temperature (lanes 4 to 6),
mostly single-shelled particles were synthesized, even at
late times p.i. but the assembly of double-shelled particles
seemed to be inhibited. Moreover, single-shelled particles
appeared to be unstable since their amount decreased
during the course of infection. This suggests that the
single-shelled particles made at the restrictive temperature may correspond to particles lacking the genome
RNA.
To test this hypothesis, the R N A of tsB subviral
particles was phenol-chloroform-extracted and then
resolved by electrophoresis on a polyacrylamide gel. Fig.
4 shows the electrophoretic analysis of the R N A content
of such particles after silver staining. Lanes 1 to 3
correspond to the viral R N A extracted from subviral
particles obtained at 31 °C at 8, 12 and 24 h p.i. An
increase in the dsRNA content can be observed during
the course of infection as expected for viral morphogenesis. On the other hand, when the R N A content
of subviral particles obtained at restrictive temperature
was analysed (lanes 4 to 6), no dsRNA was detected at
any time after infection, suggesting that the defect lies in
the minus-strand R N A synthesis, and not in the assembly
of subviral particles. Furthermore, P A G E analysis
revealed the presence of VP1, VP2, VP3 and VP6 in the
purified subviral particle fraction before separation by
electrophoresis in Tris-glycine agarose gels (data not
shown).
These results make the role of VP3 in R N A replication
very difficult to understand, since during transcription
VP3 is responsible for the capping of viral mRNA, a
reaction not carried out during replication. Furthermore
the cap fragment is already present in the template
(capped mRNA), suggesting that VP3 may function
differently depending on the stage of viral infection, i.e.
transcription or replication.
The results also show that there is some level of
transcription, which may correspond to m R N A synthesis, catalysed by the originally infecting virions, which
may allow the initial synthesis of sufficient structural and
non-structural viral polypeptides, since the mutation
does not affect that function of VP3. Therefore it is
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Short communication
possible to argue that the mutation affects only the
process of RNA replication in such a way that the
subviral particles made during the infective cycle would
not be able to replicate the genome and therefore the
newly made particles will be unable to transcribe.
It is of interest to point out that the formation of the
single-shelled particle does not require viral replication.
However, this result could be artefactual, not
representing a real step in the morphogenetic pathway of
the virus, and due only to the high affinity of viral
polypeptides that accumulate in the absence of dsRNA
and form particles under the restrictive conditions.
Formation of virus-like particles had been observed
when VP2 and VP6 were coexpressed in the baculovirus
system (Tosser et al., 1992). The study of the formation
of empty particles by a mutant may be of interest for
possible rotavirus vaccine development.
The mutant tsB was a gift from Dr Frank Ramig. We would like to
thank Dr Arturo Yudelevich and Pierre Pothier for their critical review
of the manuscript and Mr P. Ortiz for his technical assistance. This
work was supported by grants from FONDECYT (1067-92) and
SAREC (Sweden) to E. Spencer. M.V. is a recipient of a FONDECYT
Fellowship.
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(Received 27 July 1992," Accepted 31 December 1992)
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