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Interference of reovirus strains occurs between the stages of
uncoating and dsRNA accumulation
Michael N. Rozinov and Bernard N. Fields1"
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 0211 5, USA
Interference of wild-type reovirus growth by some
temperature-sensitive (ts) mutant viruses under
non-permissive conditions or by other wild-type
isolates has been demonstrated; however, the
stage of the virus replication cycle at which interference occurs has not been defined. Examination of the time-course of the yields of T1 Lang
(T1 L) dsRNA in the progeny of mixed infections of
T1L with T3 Dearing (T3D) or with a panel of T3D ts
mutants at a non-permissive temperature revealed
that interference takes place by 8 - 1 0 h postinfection and occurs prior to or at the same time as
accumulation of reovirus dsRNA. Taken together
with our previous results, these data indicate that
interference occurs during a window between virus
uncoating and synthesis of dsRNA in the reovirus
replication cycle, probably at the stage of assembly
of primary reovirus particles.
The phenomenon of virus interference or the inhibition of
virus growth by another virus has been well known for a long
time; however, the molecular mechanisms of this phenomenon
remain obscure (for review see Whitaker-Dowling &
Youngner, 1987). Interference has been described in two
different systems: (i) serial passage of viruses at high m.o.i.,
leading to accumulation of defective-interfering viruses
(Huang, 1973; Huang & Baltimore, 1977; Lazzarini et al., 1981;
Von Magnus, 1954); and (ii) mixed infections of wild-type
viruses with certain temperature-sensitive (ts) mutants (Chakraborty et al., 1979; Cooper, 1965; Jordan et al., 1989; Keranen,
1977; Youngner & Quagliani, 197~). Recently, we described a
third situation where interference occurs: co-infection of cells
Author for correspondence:Michael N. Rozinov.Presentaddress:
ExperimentalRetrovirologySection, Medicine Branch, Building 10,
Room 5A11, NationalCancer Institute, 9000 RockvillePike, Bethesda,
MD 20892, USA,
Fax + 1 301 402 0709.
t Deceased
OOO1-3878 © 1996 SGM
with different wild-type virus isolates (Rozinov & Fields,
1994).
Mammalian reoviruses are non-enveloped viruses and their
genomes consist of 10 dsRNAs enclosed in a double protein
shell (reviewed in Schiff & Fields, 1990). One of the outer shell
proteins, p~l, has been linked to the property of interference of
wild-type reovirus isolates (Rozinov & Fields, 1994). Interference of reovirus wild-type isolate T3 Dearing (T3D) by
certain T3D ts mutants (Ahmed et al., 1980; Ahmed & Fields,
1981; Chakraborty et al., 1979) prompted these authors to
speculate that interference was a direct consequence of
incorporation of the mutant product into mixed virus progeny
(Ahmed et al., 1981; Ahmed & Fields, 1981). In other studies it
has been proposed that interference was due to competition for
the virus replication machinery (Huang & Baltimore, 1977;
Maloy et al., 1994). In terms of their ability to interfere with the
growth of other strains, reovirus isolates have demonstrated
the following hierarchical order: T3D = T2 Jones (T2J) > T1
Lang (TIL) = T3 Abney (T3A) (Rozinov & Fields, 1994). In
this work we focused on the question of which step in the
reovirus replication cycle is the probable stage where interference occurs. Previously we have found that interference
did not take place during the early steps of virus infection,
adsorption and uncoating, and therefore interference appeared
to be a later event in the virus replication cycle (Rozinov &
Fields, I994). Reovirus dsRNA segments can be resolved by
SDS--PAGE (10% polyacrylamide) in Tris-glycine buffer
(Laemmli, 1970; Ramig et al., 1977). Most of the corresponding
dsRNAs from the different reovirus isolates have different
electrophoretic mobilities (Ramig et al., 1977), allowing us to
compare the yields of virus dsRNAs in single infection versus
mixed infection with a second virus. L929 cell monolayers
infected by a mixture of TIL plus T3D and TIL plus T3A or
by single viruses TIL, T3D and T3A (initial m.o.i, of 10 for
both single and mixed infections; Rozinov & Fields, 1994)
were incubated at 37 °C for 8 or 10 h (including l h of
adsorption) in the presence of 0"5 ~g/ml actinomycin D, a
concentration that inhibits cellular mRNA synthesis but not
reovirus RNA synthesis (Acs eta]., I971), and [3~P]orthophosphate. Unpurified virus lysates were incubated with 1%
SDS (55 °C for 10 min) to release virus RNA and treated by
DNase I followed by RNase A in 0"4 M-NaC1 (in these
conditions only dsRNA survives). We observed a reduction in
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Fig. 1. SDS-PAGE (10% polyacrylamide) autoradiogram of dsRNAs yield
of 32p-labelled viruses (T1 L, T3D and T3A) in single and mixed infections
after 10 h post-infection at 37 °C. L, M and S to the right of the gel
designate the three size groups of reovirus dsRNAs: large (L1, L2 and
L3), medium (M1, M2 and M3) and small ($1, $2, S3 and $4),
respectively.
the amount of all TIL dsRNAs in the mixed infection with
T3D compared to dsRNAs of TIL in single infection (Fig. 1,
compare lanes 2 and 5). The amount of TIL SI dsRNA in the
progeny of mixed infection with TdD was reduced by
approximately 4"5-fold compared to that in single TIL
infection, as evaluated by autoradiogram scanning. We had
shown previously a similar reduction of $1 TIL dsRNA in a
mixed infection with T3D grown for 43 h (4'3-fold; Rozinov &
Fields, 1994). T3A did not interfere with the yield of TIL
dsRNA in the mixed infection (Fig. 1, compare lanes 2 and 6)
and this is also in accordance with our previous data (Rozinov
& Fields, 1994). Consequently, interference of TIL by T3D
occurred prior to or at the same time as dsRNA synthesis or
accumulation. We were able to detect virus dsRNAs as early as
10 h post-infection (including 1 h of adsorption); prior to this
the level of virus dsRNA was too 'low.
To examine the time-course of interference by a different
approach, we measured interference of TIL by T3D ts mutants
in temperature-shift experiments. We had shown previously
that: (i) the phenotype of interference between reovirus isolates
mapped to the M2 gene (Rozinov & Fields, 1994); and (ii) the
~tl protein, product of the M2 gene, was phenotypicaIIy mixed
in the virus progeny of mixed infection (Rozinov & Fields,
42(
1996). The rationale for temperature down-shift experiments
was as follows: in the mixed infection of TIL and TdD tsA324
under non-permissive conditions (39 °C) the mutant T3D p.1
protein is functionally defective and, thus, interference of TIL
is expected to be reduced significantly; however, shifting to
the permissive temperature (31 °C) will restore the interfering
function of the mutant I~1 protein and interference will occur
again. Consequently, we would expect that the longer the
incubation time at the non-permissive temperature before
transfer to permissive conditions, the higher the yield of TIL
in mixed infection. In addition, if the step in the reovirus
repIication cycle in which interference occurs happens during
incubation at the non-permissive temperature, then increasing
the incubation time will not increase the TIL yield. Thus, these
experiments allow us to determine the time-course of interference. In the down-shift experiments, L929 cell monolayers were infected with a mixture of TIL and TdD tsA324
(laboratory collection) viruses (m.o.i. of 10 for each virus) and
incubated under non-permissive conditions for 2-22 h followed by transfer to the permissive temperature at 2 h intervals
for the remainder of the 68 h period of infection. Subsequently,
the amounts of TIL dsRNAs in the mixed virus progeny were
analysed by gel electrophoresis. The best markers of interference were the TIL $1 and M2 dsRNAs, owing to their
good resolution from the corresponding T3D dsRNAs (Fig. 2).
The amounts of TIL $1 and M2 dsRNAs progressively
increased, reaching a plateau when cells were incubated for at
least 8-10h or longer at 39 °C before shifting to the
permissive temperature. However, these yields were still less
than those of TIL dsRNAs in control a single infection at
31 °C for 68 h (Fig. 2, lane 1). Thus, the interference of TIL by
TdD tsA324 at the non-permissive temperature was reduced
but not completely lost compared to that under permissive
conditions. It should be noted that the yield of TIL dsRNAs in
single TIL infections did not depend on the growth temperature in the range 31-39 °C (data not shown). These results
demonstrated that the interfering events occurred by 8-10 h
post-infection at 39 °C, consistent with the time-course of
interference of wild-type viruses TIL and TdD described
above. However, the interference pattern of TIL at the nonpermissive temperature was the same with two other T3D
mutants: tsB271 (L2 gene) and ~sC447 (SZ gene) (data not
shown). Neither the L2 gene nor the $2 gene was linked to the
phenotype of interference (Rozinov & Fields, 1994). Because
these L2 and $2 mutants behaved in the same manner as the
M2 gene mutant and because the mutant gene segments
encode capsid proteins, this suggests that interference may be
exerted at the level of capsid assembly or function. It is
important to note that the corresponding proteins (~tl, ;~2 and
~r2) have never been shown to possess any functions outside of
assembled virions.
To examine the time-course of interference between wildtype viruses, we delayed infection of the interfering virus for
various times up to 8 h. Because two kinds of experiments
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Tune at 39 °C (h)
T1 T3
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2
4
6
8
10 12 14
16 18 20 22
22 42
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M-
S-
Fig. 2. Time-course of the yield of T1L dsRNAs in a mixed infection of T1L and tsA324 T3D mutant at down-shift temperature
conditions (39 °C to 31 °C). SDS-PAGE (10% polyacrylamide; ethidium bromide staining) of dsRNAs prepared from
unpurified virus lysates. T1L and T3D (m.o.i. of 1O) single infections grown at 31 °C for 68 h are shown in the two left lanes.
Mixed infections were performed at an m.o.i, of 10 for both viruses. Growth was at the non-permissive temperature for 0-22 h
as indicated, followed by a shift to permissive conditions and continued incubation to 68 h. The two right lanes represent mixed
infection of T1L and tsA324 T3D mutant grown at permanent non-permissive temperature (39 °C) for the times shown.
T1
T3
0
Delay of T3D (h)
2
4
6
8
T1
T3
0
Delay of T3D (h)
2
4
6
8
-L
-M
$1
$1
-S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fig. :3. DsRNAs yield of mixed infections of T1L and T3D with delaying of T3D infection until the times indicated, SDS-PAGE of
unpurified virus lysates (10% polyacrylamide; ethidium bromide staining). Single infections (lanes 1,2, 8 and 9) and mixed
infections (all other lanes) were performed at an m.o.i, of 10 for both T1 L and T3D except lanes 8 and 1O-14, where the
m,o.i, of T1L was 20. All incubations continued at 37 °C for 43 h.
indicated that interference occurred b y 8 - 1 0 h post-infection
(see above), we determined if the yield of T I L would still be
inhibited after delay of infection of dominant-interfering parent
T3D for a comparable time. If it was, interference could occur
later, indicating that there is another step in the replication
cycle of T I L where in~:efference could take place. M i x e d
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infections of T I L and T3D at an m.o.i, of 10 for each virus were
done, with T3D infection delayed for 2 - 8 h post-infection.
The strongest interference was observed with T3D infection
delayed up to 6 h (Fig. 3, lanes 1-6), a time-course consistent
with the ts mutant experiments described above. However,
even when the T3D infection was delayed as long as 8 h, the
T I L dsRNA yields were somewhat reduced (Fig. 3, compare
lanes 1 and 7). Similar results were obtained when the m.o.i, of
T I L was increased twofold over T3D (m.o.i. of 20 and 10,
respectively; Fig. 3, lanes 8-14). These data showed that the
major interference occurred by 8 h post-infection but partial
interference can occur later in the replication cycle of TIL.
We report here that the majority of the interfering events
took place by 8-10 h post-infection, prior to or at the same
time as dsRNA accumulation. Our previous data showed that
interference was not involved in the early infection steps such
as adsorption and uncoating (Rozinov & Fields, 1994). Taken
together, these results define a window in the reovirus
replication cycle, between virus uncoating and synthesis of the
second strands of virus RNAs, during which interference can
occur. This period in the replication cycle includes the events
of core-derived plus-strand RNA transcription, virus protein
synthesis and assembly of primary virus particles. Reovirus
assembly presumably proceeds via entry of plus-strand virus
ssRNAs into nascent virions followed by synthesis of dsRNAs
(Acs et al., 1971; Joklik, 1974). Support for assembled virus
particles as a site of interference is provided by our recent
finding of phenotypic mixing of the rtl protein in the progeny
of mixed reovirus infections (Rozinov & Fields, 1996). We
assume that interference could be a consequence of differential
viability of the phenotypically mixed primary virus particles
that represent the first step of virus assembly in the infected
cell. Although the possibility that interference could act at
stages other than assembly (e.g. RNA transcription or
translation) cannot be excluded, there is no proof that lal
protein is involved in these stages. Rather, ~tl protein has
properties that suggest a role in the interaction of assembled
reovirus with cellular membranes (Lucia-Jan&is et al., 1993;
Nibert & Fields, 1992; Nibert et aL, 1991). We speculate that
the dominant ~tl protein on the outer shell surface of
phenotypically mixed virus particles determines the differential
survival of particles. This is in accordance with our current
knowledge that ~tl forms a critical protein network involved in
assembly of the virion outer shell and thus ~tl can control
assembly (Dryden et al., I993). Another possibility is selection
for differential entry of isolate-specific ssRNAs into nascent
virions via functioning of either the dominant ~tl protein or
M2 ssRNA itself; however, there is no clear evidence for the
interaction of ~tl protein with virus ssRNAs either alone or in
complex with other reovirus proteins (e.g. non-structural) or
host proteins. The fact that delaying infection with the
interfering wild-type T3D (in the mixed infection with TIL) for
as long as 8 h still caused partial inhibition of the yield of T I L
shows that part of the interfering events can take place later via
42~
another step in the replication cycle of T I L These two steps
may share common features, possible candidates being primary
and secondary assembled virus particles (Acs et al., 197I;
Joklik, 1974). Nevertheless, two different mechanisms may
account for the early and late interference observed with wildtype isolates. Finally, further studies of the mechanisms of
interference and the key role of ~1 protein (or the M2 gene
segment) may shed light on our understanding of reovirus
assembly.
David Knipe and Ann Georgi are acknowledged for critical reading of
manuscript and valuable comments. Jay Hooper and William Lucas are
acknowledged for helpful discussions and Elaine Friemont for excellent
technical assistance. This work was supported by Public Health Service
program project grant 5 P50 NSI6998 from the National Institute of
Neurological and Communicative Disorders and Strokes and Public
Health Service grant 2 R01 AI13178 from the National Institute of
Allergy and Infectious Diseases.
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Received 15 January 1996; Accepted 13 March 1996
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