Journal
of General Virology (2001), 82, 385–396. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
The RNA structures engaged in replication and transcription of
the A59 strain of mouse hepatitis virus
Dorothea L. Sawicki, Tao Wang and Stanley G. Sawicki
Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614, USA
In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse
hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing
subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive
forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt
solutions, approximately 70 % of the replicating and transcribing structures that accumulated in
infected cells by 5–6 h post-infection were multi-stranded intermediates, the RI/TIs. The other
30 % were in double-stranded structures, the native RF/TFs. These replicating and transcribing
structures were separated by velocity sedimentation on sucrose gradients or by gel filtration
chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during
electrophoresis, according to their size. Digestion with RNase T1 at 1–10 units/µg RNA resolved
RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of
0n02 µg/µg RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs
bound to magnetic beads containing oligo(dT)25, suggesting that the poly(A) sequence on the 3h
end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of
incorporation of [3H]uridine showed that both the RI and TIs were transcriptionally active and the
labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure
originally to find TIs and TF cores was probably due to overdigestion with RNase A.
Introduction
Coronaviruses belong to the Order Nidovirales, whose
members utilize a unique discontinuous RNA synthetic process
to produce a 3h co-terminal, nested set of mRNA molecules,
each with the same 5h leader sequence that is found only at
the 5h end of the genome (Snijder & Meulenberg, 1998).
Discontinuous transcription, rather than splicing, most likely is
the mechanism responsible for generating the subgenomic
mRNA (Spaan et al., 1988). The proposal that discontinuous
transcription occurred during positive-strand synthesis, the
‘ leader-primed transcription model ’, was based on finding only
genome-length negative strands and double-stranded cores
(RF) of the viral replicative intermediates (RI) in cells infected
with the A59 strain of mouse hepatitis virus (MHV) (Baric et al.,
1983 ; Lai et al., 1982 b). The RF cores were stated as having an
unusual migration during gel electrophoresis because they
Author for correspondence : Stanley Sawicki.
Fax j1 419 383 3002. e-mail ssawicki!mco.edu
0001-7385 # 2001 SGM
migrated faster than the genome. Subsequently, we found
transcriptionally active RNA intermediates containing a complementary (negative strand) copy of each of the subgenomic mRNAs in coronavirus-infected cells (Sawicki et al.,
1990 ; Sawicki & Sawicki, 1995). The synthesis in coronavirusinfected cells of negative-strand templates corresponding to
the subgenomic mRNAs was first reported by David Brian’s
laboratory (Sethna et al., 1989) with swine transmissible
gastroenteritis virus. Others have also demonstrated that
MHV A59 (Baric & Yount, 2000 ; Schaad & Baric, 1994) and
equine arteritis virus (EAV) (den Boon et al., 1996), which is a
member of the Arteriviridae, produced a series of RF cores
following RNase treatment of infected cell RNA.
We proposed (Sawicki & Sawicki, 1995) a different model,
in which the discontinuous step occurred during negativestrand synthesis. This model also proposed that there were
two classes of RNA intermediates in MHV-infected cells. One
had genome-length templates engaged in replicating the
genome or in the production of subgenome-length negativestrand templates, i.e. anti-subgenomes. The other had anti-
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DIF
D. L. Sawicki, T. Wang and S. G. Sawicki
subgenomes being transcribed into subgenomic mRNA. Because the RNA intermediates active in transcribing subgenomic
mRNA are not replicating, we call them TIs and TFs, for
transcriptive intermediate and form RNA, respectively. We
reserve the terms RI and RF for viral RNA involved in
replication.
RI\TIs and RF\TFs would differ in their relative proportion
of single-stranded and double-stranded character. Native
RF\TFs would be completely or nearly completely doublestranded as a result of having only one or a few polymerases
engaged in transcription on each template. Thus, positive and
negative strands in native RF\TFs would be mostly equal in
length. Native RFs are soluble in high salt solutions such as
2 M LiCl or 1 M NaCl, as are tRNA and DNA (Ammann et al.,
1964 ; Montagnier & Sanders, 1963). RF gives a defined Tm
with a sharp melting point (Ammann et al., 1964 ; Bishop &
Koch, 1967). The biological significance of native RFs in
poliovirus and RNA phages includes functions as short-lived
intermediates and as end-products that accumulate when RNA
synthesis ceases and the last positive strand is not released
(reviewed in Koch & Koch, 1985). On the other hand, RI was
a multi-stranded intermediate active in positive-strand synthesis. Poliovirus RI contained an average of three, or as many
as eight to ten, nascent, single-stranded RNA tails (reviewed in
Koch & Koch, 1985 ; Richards et al., 1984) and one poly(A)
sequence (Yogo & Wimmer, 1975). This high degree of singlestrandedness makes the RI insoluble in high salt solutions, as
are messenger and ribosomal RNA (Baltimore, 1966 ; Bishop et
al., 1969 ; Erikson & Gordon, 1966 ; Fenwick et al., 1964).
Digestion of RI with controlled levels of RNase degraded the
single-stranded regions composed of nascent chains and left an
RF core now soluble in 2 M LiCl (reviewed in Koch & Koch,
1985). While in vivo RI was shown to be predominantly singlestranded (Richards et al., 1984), isolated poliovirus RF cores
contained an intact template strand and short to almost fulllength nascent chains base-paired along the length of the
template (Baltimore, 1968). Inhibiting transcription initiation
converted poliovirus RI to RF cores.
We undertook an analysis of RNA structures formed in
MHV-infected cells to identify the number and abundance of
RI\TIs and native RF\TFs and to develop methods to purify
individual RI\TIs. A second goal was to attempt to explain the
initial failure to find TF cores after RNase treatment and gel
filtration chromatography of MHV RI\TIs.
Methods
Virus and cells. Seventeen clone 1 (17Cl-1) mouse fibroblast cells
and the A59 strain of MHV (Sturman & Takemoto, 1972) were originally
provided by L. Sturman (Wadsworth Center, Albany, NY, USA). 17Cl-1
cells were grown in Dulbecco’s minimum essential medium (DMEM)
supplemented with 6 % foetal bovine serum (FBS) and 5 % tryptose
phosphate broth, and high titre stocks of MHV were prepared using low
pH medium (Sawicki & Sawicki, 1986 b).
Labelling of viral RNA and isolation of high-salt-soluble
DIG
and -insoluble RNA species. 17Cl-1 cells (5i100 mm Petri dishes
with 10–15i10' cells per dish) were infected with MHV at 50–100 p.f.u.
per cell at 37 mC. Following infection, the cells were labelled with
[$H]uridine or [$#P]orthophosphate. For [$H]uridine, the cells were
incubated in medium containing 200 µCi\ml of [$H]uridine and 20 µg\ml
of dactinomycin in DMEM supplemented with 6 % FBS. For $#P, they
were labelled with 200 µCi\ml of [$#P]orthophosphate and 20 µg\ml of
dactinomycin in phosphate-reduced MEM supplemented with dialysed
FBS. Cells were solubilized with 5 % lithium dodecyl sulfate and 200 µg
of proteinase K\ml and deproteinized by extraction with low pH phenol
(pH 4n3) followed by chloroform. The RNA was collected by ethanol
precipitation. For separation of high-salt-soluble and -insoluble species,
the aqueous phase obtained by phenol and chloroform extraction was
adjusted to 2 M LiCl, placed on ice for 18 h, and centrifuged at
10 000 r.p.m. (15 000 g) for 1 h to obtain the supernatant (LiCl-soluble)
and precipitated (LiCl-insoluble) fractions (Baltimore, 1968 ; Sawicki &
Gomatos, 1976). LiCl-soluble RNA was collected by ethanol precipitation.
Ribonuclease protection assays to measure negativestrand synthesis. Viral RNA was denatured by heating at 100 mC in
1 mM EDTA, quick cooled at 0 mC, and reannealed in the presence of an
excess of virion positive strands and 0n4 M NaCl at 68 mC for 30 min
followed by 25 mC for 30 min, as described (Sawicki & Sawicki, 1986 a).
One half of each sample was analysed directly for acid-precipitable
radioactivity ; the other half was digested with 5 µg\ml of RNase A in
0n4 M NaCl for 30 min at 37 mC before acid-precipitation.
Chromatography. Columns (1n5i90 cm) of Sepharose 2B or
Sephacryl S-1000 (Pharmacia) were equilibrated in 0n1 M NaCl, 10 mM
Tris–HCl, pH 7n4, 20 mM EDTA and 0n2 % SDS, as described earlier
for Sepharose 2B (Sawicki & Gomatos, 1976). Briefly, RNA to be
chromatographed was dissolved in the column buffer and applied to the
column in small volumes. The buffer head was reapplied and the column
allowed to run under the recommended pressure and at a flow rate of
10–13 ml\h. Fractions of 1n0–1n3 ml (Sepharose 2B total RNA and LiClsoluble RNA) or 0n65 ml (Sepharose 2B LiCl-insoluble RNA) or 0n5 ml
(Sephacryl S-1000) were collected. The RNA was precipitated in the
presence of 50–100 µg of carrier yeast tRNA with ethanol.
Ribonuclease digestion. For determination of the ribonucleaseresistance of RI\TIs and native RF\TFs and fractions, RNA samples were
digested with RNase T1 (Ambion) at 30 units per sample in 0n3 M NaCl
at 30 mC for 30 min or with RNase A (affinity purified ; Ambion) at
5 µg\ml in 0n3 M NaCl, 30 mM sodium citrate (2i SSC) at 37 mC for
30 min. Trichloroacetic acid was added to 5 %, and the precipitated RNA
collected on glass fibre filters and counted by liquid scintillation
spectroscopy. We routinely used 30 units of RNase T1 to digest " 30 µg
of RNA, the amount in 1i10' cells.
Velocity sedimentation and agarose gel electrophoresis.
Solubilized cell extracts were passed through a 27 gauge needle and
layered onto 15–30 % sucrose gradients and centrifuged at 20 mC in a
Beckmann SW28 rotor at 28 000 r.p.m. for 18 h, which pelleted the 60S
viral genome RNA. Gel electrophoresis was in 0n8 % or 1 % agarose in
TBE buffer with 0n2 % SDS for 370 V-h, after which the gels were washed
in water to remove excess SDS, stained with ethidium bromide, and
processed for fluorography.
Chromatography on oligo(dT) beads. Oligo(dT) beads were
#&
obtained from Dynal (Dynabeads) or from Novagen (Magnetight
particles) and were used according to the instructions of the manufacturers. Immediately before use, the beads were washed in binding buffer
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Structures transcribing MHV genomes and mRNA
twice before addition of the sample. Usually 100 µl of cell lysate or RNA
in water was heated for 2 min at 75 mC and then added to 200 µl of
washed oligo(dT) beads resuspended in 100 µl of binding buffer. After
#&
rotating at 25 mC for 5 min, the magnet was applied and the supernatant
(unbound fraction) was removed. The beads were washed twice with
washing buffer (wash fractions) before incubation in 10–20 µl of 10 mM
Tris–HCl, pH 7n4, and heating at 75 mC for 2 min to elute the poly(A)+
RNA or bound fraction.
Results
Gel filtration chromatography and velocity
sedimentation
MHV RI\TIs are separable by velocity sedimentation.
MHV-infected cells were labelled with [$H]uridine from 2n5 to
6 h post-infection (p.i.) and the RNA applied to a 15–30 %
sucrose gradient. Fig. 1 (A) shows the distribution of labelled,
viral single-stranded RNA (the genome RNA was pelleted) and
Fig. 1 (B) shows the distribution of the RI\TIs after conversion
to RF\TFs with RNase T1. RI\TIs represented about 5 % of the
viral RNA and, while separable from the smaller TIs, the RI
overlapped TIs for RNA-2 and -3 and the TIs for RNA-2 and
-3 overlapped TIs for RNA-4, -5, -6 and -7. For the gel shown
in Fig. 1 (C), the experiment was repeated using $#P and
labelling from 1n5 to 5 h p.i. and fractions were combined into
four pools, numbered starting from the bottom of the gradient :
pool 1 (fractions 1–4) was enriched in the RI ; pool 2 (fractions
6–9) in RI and TIs II and III ; pool 3 (fractions 10–13) in TIs II
and III ; and pool 4 (fractions 16–19) in the smaller TIs IV, V,
VI and VII. After conversion to RF\TFs, pools 2 and 4 were
subjected to chromatography on Sephacryl S-1000 columns
(Fig. 2 A). Sephacryl S-1000 is a hydrophobic, rigid, allyl
dextran\N-Nh-methylenebisacrylamide matrix with a 20 kbp
DNA exclusion limit. RF\TFs in pool 2 were mostly excluded
by the column or at the boundary of the excluded\included
region and consisted of RF cores and some TF-II and TF-III
cores (Fig. 2 B). Only RF was found in the earliest fractions to
exit the column, providing some separation of the RF from the
smaller TF species. Pool 4 (Fig. 2 C) contained low amounts of
the RF and of TF-II and TF-III, and large amounts of the four
smallest TF cores, which eluted very narrowly and by size and
overlapped one another.
Identification of seven native RF/TF RNA species
Native RF\TFs are recovered in the 2 M LiCl-soluble
fraction whereas RI\TIs are mostly single-stranded and are
precipitated by 2 M LiCl. When chromatographed on Sepharose 2B directly, LiCl-soluble RNA fractionates into three
peaks, the largest of which is excluded and in the void volume
(Fig. 3 A). Fractions across the excluded and included (fractionated) regions were combined into eight pools that were
analysed by gel electrophoresis without nuclease treatment
(Fig. 3 B). Pool 1 contained the entire native RF and native TFs
II and III, and small amounts of the native TFs IV, V and VI.
Pool 2, the region between the excluded and included fractions,
contained mostly TFs IV, V, VI and VII. Pool 3 contained
mostly TFs V, VI and VII. Pool 4 was enriched in TF-VII.
Labelled RNA in pools 6–8 was much smaller than mRNA 7,
migrating off the gel during electrophoresis. Most likely, this
represented short oligoribonucleotides created by blocking
transcription with dactinomycin. Thus, MHV-infected cells
contained seven species of native RF\TFs.
Fig. 3 (C) shows the results of the chromatography on
Sepharose 2B of the RNA that was precipitated by 2 M LiCl.
At least 13 times more viral RNA was found in the excluded
volume from the LiCl-insoluble fraction compared to the LiClsoluble fraction (Fig. 3 A) and the RNA in the included volume
eluted broadly. Starting at fraction 56 and ending at fraction
155, every second or third fraction was analysed by gel
electrophoresis (Fig. 3 D). 28S and 18S ribosomal RNA
(detected by ethidium bromide staining) was in fractions
90–145 and 120–160, respectively. Genome (RNA-1) started
to elute in the excluded volume and just after RI, which was
visible at the top of the gel in fractions 60–65 (Fig. 3 D). Eluting
later and according to their relative size was single-stranded
subgenomic mRNA-2 to -7 (RNA-2 to -7). As observed earlier
(Sawicki & Sawicki, 1990), the RI migrated slower than RNA1 on agarose gels.
About 100 fractions were collected into 20 pools of about
five fractions each and numbered in the order of elution. RNA
in pools 1 to 18 was treated with RNase T1 and analysed on
gels to identify the location of RI\TIs (Fig. 3 E). Pool 1
(fractions 56–62) contained most of the RI, TIs II and III, and
pools 6–7 (fractions 81–85 and 86–90) were enriched in TIs IV,
V, VI and VII. The presence of large amounts of RNA in pool
1, and possibly larger fragments derived from RNA-1 by
RNase T1 digestion, may account for the smear of labelled
material. We found the seven species of RI\TIs fractionated
similarly to native RF\TFs on Sepharose 2B and each eluted
ahead of its single-stranded RNA counterpart. To approximate
the relative numbers of RI\TIs to native RF\TFs, radiolabelled
RNA recovered as RNase A-resistant cores after Sepharose 2B
for each was calculated. The 2 M LiCl-insoluble RI\TIs
accounted for 67 % of all labelled, double-stranded RNA
accumulating in infected cells early (1n5–5n5 h p.i.). This is the
period when rates of syntheses of both negative-strand and
positive-stranded RNA were increasing and viral RNA was
accumulating exponentially (Sawicki & Sawicki, 1986 a).
Ribonuclease-sensitivity of coronavirus RI/TI and
RF/TF species
Essentially all of the [$H]uridine incorporated into the LiClsoluble native RF\TFs was acid-precipitable after digestion
with RNase T1 or A (Table 1). In contrast, RNase A digestion
of LiCl-insoluble, Sepharose 2B-fractionated RNA showed
RNA in pool 1 was mostly double-stranded (49 %), and RNA
in pools 5–20 was mostly single-stranded, 1n5 % or less was
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D. L. Sawicki, T. Wang and S. G. Sawicki
Fig. 1. Separation of MHV RI/TIs by velocity sedimentation. After low pH phenol and chloroform extraction, RNA labelled with
200 µCi/ml [3H]uridine from 2n5 to 6 h p.i. was layered onto 15 % to 30 % sucrose gradients and centrifuged at 28 000 r.p.m.
for 18 h at 20 mC. Fractions (numbered from the bottom to the top of the gradient) of about 1 ml were collected.
(A) Sedimentation of the viral single-stranded RNA. 5 % of each gradient fraction was analysed by electrophoresis on a 1 %
agarose gel that was stained with ethidium bromide to locate the ribosomal RNA. (B) RNA (95 %) in each gradient fraction was
precipitated with ethanol and digested with RNase T1 to remove single-stranded mRNAs and convert RI/TIs to their doublestranded RF/TF cores that are readily visualized by electrophoresis on a 1 % agarose gel. (C) From identical velocity
sedimentation conditions reported above for [3H]uridine, 32P-labelled RNA, from cells labelled 1n5 to 5 h p.i., in pool 1
(fractions 1–4), pool 2 (fraction 6–9), pool 3 (fractions 10–13) and pool 4 (fractions 16–19), was precipitated with ethanol.
After RNase T1 digestion, a small sample of each was analysed by electrophoresis.
resistant to RNase (Table 1). Our next experiment showed
both native form and intermediate structures, while doublestranded, can be degraded with excessive RNase A. We treated
the purified, LiCl-soluble, native RF\TFs (Fig. 3 B, pools 1 and
3 were combined to restore the population of seven species)
and LiCl-insoluble RI\TIs (Fig. 3 E, pools 1 and 5 were
combined together) with RNase T1 or A, using several
concentrations of each. RNase T1 at concentrations of 10–100
units per sample converted RI\TIs to RF\TF cores and left
intact all seven native RF\TFs as judged by their electrophoretic migration in agarose gels (Fig. 4 A ; data not shown).
However, treatment with as little as 1 µg\ml of RNase A
destroyed the intact structure of larger, native RF\TFs species
DII
and higher amounts also degraded the smaller TFs (Fig. 4 B).
The RI\TIs were also degraded by these concentrations of
RNase A (Fig. 4 C). For the volumes used, a concentration of
1 µg\ml represents on a µg basis a 400 : 1 RNA to RNase ratio.
Presence of poly(A) in coronavirus RI/TI and RF/TF
populations
Preparations of LiCl-soluble and -insoluble RNA were
selected on oligo(dT) beads to monitor for the presence of
#&
poly(A) sequences that were not base-paired with a complementary poly(U) sequence (Fig. 5). Presence of a free poly(A)
sequence on nascent strands in the RI\TIs was suggested
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Structures transcribing MHV genomes and mRNA
Fig. 2. Sephacryl S-1000 gel filtration chromatography of RNase-T1-derived RF/TFs. (A) Distribution of radiolabelled RF/TFs.
Pools 2 and 4 from Fig. 1 (C) were individually applied after RNase T1 treatment to Sephacryl S-1000 columns as described in
Methods. Fractions of 0n5 ml were collected and 20 µl samples were acid-precipitated for scintillation spectroscopy. Presence
of individual RF/TFs core species in pool 2, fractions 81–100 (B) and pool 4, fractions 80–130 (C) was determined. The RNA
in each fraction was collected by ethanol precipitation and electrophoresed on agarose gels.
because all seven species of RI\TIs bound (lane 5), in addition
to all the single-stranded mRNA species (lane 2). Although it
appears here that the RI did not bind to oligo(dT) beads (lane
#&
5), other experiments using cell lysates directly, without
phenol–chloroform extraction, demonstrated that almost all
the RI, in addition to the TIs, bound. Essentially all of the
poly(A) RNA that bound to and was eluted from oligo(dT)
#&
rebound when challenged with a second round of poly(A)
selection ; none of the unbound RNA bound when incubated
with fresh oligo(dT) beads (data not shown). Most if not all
#&
of the native RF\TFs had a free poly(A) sequence long enough
to bind oligo(dT) (Fig. 5, lane 8) ; all of the smallest, and
#&
decreasing amounts of the RF\TFs of increasing sizes bound.
In some experiments, as for the RI, only about 10 % of native
RF was observed to bind oligo(dT) . This indicated that the
#&
RF poly(A) sequence was not free or the extreme size of the
32 kb double-stranded RF interfered with stable binding to
short oligo(dT) sequences under the assay conditions. In
#&
contrast to the inconsistency of recovery of RI\RF by
oligo(dT) beads, we observed consistent binding by all of the
#&
TIs\TFs.
Kinetics of labelling of MHV RNA synthetic structures
If TIs were merely dead-end products of the synthesis of
negative strands as was suggested by Jeong & Makino (1994),
radiolabelled uridylate would accumulate continuously in these
molecules as seen for poliovirus RF (Baltimore, 1968). On the
other hand, if they were active intermediates in subgenomic
mRNA synthesis, their labelling would resemble the labelling
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D. L. Sawicki, T. Wang and S. G. Sawicki
Fig. 3. Sepharose 2B gel filtration chromatography of MHV LiCl-soluble, native RF/TFs and LiCl-insoluble RI/TIs. MHV-infected
cells were labelled from 1 to 5n5 h p.i. with 200 µCi/ml [3H]uridine, extracted with low pH phenol and chloroform, and the
aqueous phase was adjusted to 2 M LiCl and incubated overnight on ice. (A) The LiCl-soluble, native RF/TFs were precipitated
DJA
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Structures transcribing MHV genomes and mRNA
Table 1. Ribonuclease resistance of coronavirus RI and RF RNA
Ribonuclease-resistance (%)*
Nature of RNA
Native RF† (LiCl-soluble)
Pool 1
Pool 2
Pool 3
Pool 4
Pool 5
RI/TIs/ssRNA† (LiCl-insoluble)
Pool 1
Pool 2
Pool 3
Pool 4
Pool 5
Pool 6
Pool 7
Pool 8
Pool 9
Pool 10
Pool 15
Pool 20
RNase T1
(30 units ; 0n3 M NaCl)
111
98
119
99
105
63
46
29
26
26
32
26
26
–
–
–
–
RNase A
(5 µg/ml ; 2i SSC)
107
116
108
103
107
49n4
26n5
10n8
4n6
1n3
1n0
0n8
0n7
0n7
0n6
0n8
1n2
* RNase-resistance is the per cent of the total, untreated [$H]uridine RNA c.p.m. that remains acid-precipitable.
† Native RF pools 1–5 and RI\TIsjsingle-stranded RNA (RI\TIs\ssRNA) pools 1–20 are those obtained
after Sepharose 2B chromatography (Fig. 3 B and E, respectively).
of RI. Fig. 6 shows the results of this type of analysis. Infected
cells were pulse-labelled for 2–90 min with [$H]uridine at
6n5 h p.i., when less than 10–15 % of the [$H]uridine that is
incorporated into RI\TIs is in negative strands. The RF\TFs
RNA derived by RNase T1 from the RI and TIs was separated
by electrophoresis on gels and the radiolabel incorporated into
each species at each time-point was determined by cutting out
and counting the specific gel area that contained each RF\TFs.
The kinetics of labelling into all of the TFs cores showed the
expected properties for true transcription intermediates (Fig.
6). They rapidly incorporated radiolabelled uridylate into
growing chains at rates indistinguishable from that of RF, and
the amount of incorporation reached a maximum (saturated)
after about 30 min of labelling. Thus, the six species of MHV
TIs, in addition to the RI, are authentic transcription intermediates.
The second type of analysis is shown in Table 2. If
subgenomic mRNA was copied into negative strands that
were not templates for subgenomic mRNA synthesis, the TI
negative strands would be enriched in [$H]uridine. Infected
cells were labelled for 30 min periods from 1 to 6 h p.i., the
large (18S–40S) and small (5S–18S) RI and TIs were obtained
by velocity sedimentation (Fig. 1), and the percentage of
labelled negative strands in their purified, double-stranded
with ethanol and subjected to chromatography on Sepharose 2B, as described in Methods. 20 µl (" 2 %) of each fraction was
TCA-precipitated for scintillation spectroscopy. (B) Fractions of the LiCl-soluble material were pooled and a sample was
electrophoresed directly on agarose gels. (C) The LiCl-insoluble single-stranded RNA and RI/TIs were pelleted directly by
centrifugation (15 000 g for 60 min), resuspended and applied to a Sepharose 2B column, as described in Methods. 10 µl
(" 2 %) of each fraction was analysed for acid-precipitable radioactivity. (D) Distribution of the LiCl-insoluble, mostly singlestranded RNA species. A small part of every second or third fraction was subjected to agarose gel electrophoresis. Fractions
60–65 contained the viral RI that migrates near the top of the gel. Most of the genomes were degraded during sample
preparation, but small quantities of intact genomes were present in fractions 60–65 and they migrated faster than the RI.
Fractions were pooled for further analysis as shown in (E). (E) Distribution on Sepharose 2B of the LiCl-insoluble RI/TIs. The
fractions from the chromatograph shown in (C) were collected into 20 pools, starting with fraction 56 of the excluded fractions
(fractions 56–66). Pool 1 contained the first seven fractions, pool 2 the next three fractions, and thereafter each pool
contained the next five fractions. The RNA in each pool was collected by ethanol precipitation and a sample was treated with
RNase T1. The resultant RF/TF cores were separated by electrophoresis. Only 18 lanes are shown in (E) ; no radioactive bands
were present in pools 19 and 20.
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D. L. Sawicki, T. Wang and S. G. Sawicki
Fig. 4. Sensitivity of native RF/TFs and
RI/TIs to RNase A. (A) The LiCl-soluble,
Sepharose 2B pools 1–4 (Fig. 3 B) were
digested individually with RNase T1 at 10
units per sample in 0n3 M NaCl for
30 min at 30 mC, SDS was added and the
samples were separated by
electrophoresis on 1 % agarose gels.
(B) Native RF/TFs were reconstituted as
a population by combining pools 1 and 3
(Fig. 3 B). An equal amount was digested
with each of the indicated concentrations
of RNase A in 2i SSC at 37 mC for
30 min. In addition to native RF/TFs from
5n7i106 cells, each sample also
contained 10 µg of tRNA that was added
as a carrier during the ethanol
precipitation step and formed the bulk of
substrate RNA. After digestion, SDS and
proteinase K was added and the samples
were subjected to electrophoresis on a
1 % agarose gel. (C) A sample of LiClinsoluble viral RI/TIs, obtained by
combining pools 1 and 5 (Fig. 3 E), was
treated with RNase A identically to that
described for (B).
Fig. 5. MHV RI/TIs and native RF/TFs bind oligo(dT)25. The 2 M LiClinsoluble single-stranded RNA (lanes 1–3, enriched in mRNA 4–7, is RNA
from pools 13j14 of Fig. 3 E), the 2 M LiCl-insoluble RI/TIs (lanes 4–6 is
RNA from pools 1j5 of Fig. 3 E) and 2 M LiCl-soluble RNA (lanes 7–9 is
RNA from pools 1j3 in Fig. 3 B) were analysed for their ability to bind to
magnetic beads coated with oligo(dT)25. Input RNA (lanes 1, 4 and 7)
was equivalent in amount to that applied to the beads and found either as
bound (lanes 2, 5 and 8) or not-bound (lanes 3, 6, and 9) fractions. RNA
in lanes 1–3 was analysed directly ; that in lanes 4–6 and 7–9 was treated
with RNase T1 before electrophoresis on 1 % agarose gels. Lanes 1–3
contained RNA from one-half as many cells as lanes 4–6, and lanes 4–6
were exposed for twice as long as lanes 1–3 (a final fourfold difference).
cores was determined in ribonuclease-protection assays. The
same relative amounts of labelled negative-strand RNA, newly
made during the pulse period, was present in populations
enriched in the genome or in the four smaller TI species (Table
DJC
Fig. 6. Kinetics of incorporation into MHV RI/RF and subgenomic TI/TF
RNA size classes. Infected cells were labelled at 6n5 h p.i. with [3H]uridine
for 2–90 min periods, as indicated. Cell lysates were treated with RNase
T1 to obtain the RF/TF cores, which were separated by electrophoresis on
1 % agarose gels. The area of the gels containing each RF/TF species (#,
RF ; $, TF-II ; 4, TF-III ; >, TF-IV ; X, TF-V ; , TF-VI ; , TF-VII) was cut
out and its amount of radiolabelled RNA was determined by scintillation
spectroscopy.
2). Early in infection, each 30 min labelling period showed
large amounts of negative-strand synthesis and about equal
accumulation in both size populations of RI\TIs. A value of
33–35 % for labelled RF\TF RNA that was negative-stranded
indicated that about 70 % of the total negative strands were
made during this 30 min period, as expected during the
exponentially increasing phase of replication. This decreased
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Structures transcribing MHV genomes and mRNA
Table 2. Percentage of newly synthesized negative strands in TIs IV–VII is the same as in
RI RNA
Time of labelling
with [3H]uridine
(h p.i.)
3–3n5
3n5–4
4–4n5
4n5–5
5–5n5
5n5–6
1–6
RI/TI RNA
size*
Labelled RF/TF
(c.p.m.)†
Labelled
negative
strands
(c.p.m.)†
40S–18S
18S–5S
40S–18S
18S–5S
40S–18S
18S–5S
40S–18S
18S–5S
40S–18S
18S–5S
40S–18S
18S–5S
40S–5S
1251
1514
2171
3104
3159
2759
3015
3320
2975
3794
3396
3113
3481
481
543
783
1054
1084
753
770
758
521
462
486
418
1228
Labelled
negative
strands in
RF/TF RNA (%)
38
36
36
34
34
27
26
23
18
12
14
13
35
* Sucrose gradient fractions containing the 40S–18S size classes (mainly viral RI and TIs II and III) and the
smaller 18S–5S size classes (TIs IV, V, VI and VII) were pooled separately (Fig. 1).
† The pooled RNA was ethanol-collected, digested with 10 ng\ml of RNase A in 0n3 M NaCl at 25 mC and
applied to CF-11 cellulose columns to purify viral RF and TF cores. These were denatured and hybridized to
unlabelled virion positive strands, as described (Sawicki & Sawicki, 1986 b), to determine the amount of
labelled RF\TF core RNA that was in negative strands newly made during each pulse period.
to 12–14 % at 5n5–6 h p.i., a time when the rate of overall
positive-strand synthesis became maximal (Sawicki & Sawicki,
1986 a, 1990). The results indicated that the TIs were not
formed by a single round of negative-strand synthesis on
subgenomic mRNA ‘ templates ’ (dead-end synthesis) but were
transcription intermediates active in the synthesis of subgenomic mRNAs.
Discussion
Seven species of MHV RF\TFs and of RI\TIs were readily
isolated and analysed by velocity sedimentation or gel
filtration chromatography. Comparison of gel filtration on
Sephacryl S-1000 (Fig. 2) and Sepharose 2B (Fig. 3) demonstrated that they chromatographed close to their predicted
sizes based on the fractionation properties of each matrix.
Only Sephacryl S-1000 had all TFs\TIs species within its
fractionation range and excluded mainly RI\RF. Not even
Sephacryl S-1000, however, was able to eliminate overlapping
of the two mid-sized TI\TF RNA molecules with each other,
and of the four smallest TIs\TFs with each other. Thus,
electrophoresis on agarose gels remains the best method to
purify individual size classes of RI\TIs and RF\TFs, which was
the method we used previously (Sawicki & Sawicki, 1990). By
combining sedimentation on sucrose gradients and gel filtration, larger RI and TIs II and III were separated from the
smaller TIs IV, V, VI and VII.
At the time of maximum RNA synthesis rates, the multistranded RI and TIs were the vast majority of replication and
transcription intermediates. Less than 30 % were present as
native RF\TFs. This is comparable to other positive-stranded
RNA animal and bacterial viruses (reviewed in Koch & Koch,
1985). RI\TIs represent replication and transcription intermediates on which viral RNA-dependent RNA polymerases
are repeatedly initiating positive-strand synthesis. The relative
abundance of each RI\TIs or native RF\TFs species was similar
and proportional to the relative abundance of the viral positive
strands. Native RF was originally defined by its property of
solubility in high salt, a property shared by both native RF and
TFs of MHV. That this indeed reflected their completely or
nearly completely double-stranded nature was confirmed by
finding essentially all of the labelled MHV native RF\TF RNA
was acid-precipitable after RNase A digestion. No singlestranded labelled RNA was found when this population was
analysed directly by electrophoresis. In contrast, high-saltinsoluble viral RNA contained genomes and subgenomic
mRNA as well as RI\TIs and only 3 % of it was resistant to
RNase A. Viral genome and subgenomic mRNA and positivestrand components of RI\TIs possessed a poly(A) sequence
capable of binding to oligo(dT) sequences, showing that
coronavirus RI\TIs resemble RIs formed by other positivestranded RNA viruses (Ammann et al., 1964 ; Baltimore,
1966, 1968 ; Erikson & Gordon, 1966 ; Koch & Koch,
1985 ; Montagnier & Sanders, 1963 ; Richards et al., 1984 ;
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DJD
D. L. Sawicki, T. Wang and S. G. Sawicki
Sawicki & Gomatos, 1976 ; Yogo et al., 1977). Binding of native
MHV RF\TF species to oligo(dT) suggests that if a
#&
polyuridylate is present at the 5h ends of MHV negative
strands, it is shorter than the poly(A) in its positive-strand
counterpart. Bovine coronavirus was reported to have a
poly(U) of about 9–26 nt, compared to a positive-strand
poly(A) of 100–130 nt (Hofmann & Brian, 1991).
Initial failure to find subgenomic TIs led to the leaderprimed transcription model (Baric et al., 1983). The present
results indicate this failure was due most likely to technical
error and not to any aberrant behaviour of these molecules on
gel filtration chromatography. Each of seven RI\TIs and native
or core RF\TF species fractionated as predicted on each of the
two different matrices. In addition to detecting and characterizing seven species of RI\TIs and RF\TFs in experiments
duplicating that of earlier investigators, it was also important
to attempt to explain their failure to find TI\TFs. While MHV
A59 RI and RF are resistant to concentrations of RNase A of
0n33 µg\ml (Sawicki & Sawicki, 1990), the concentrations of
ribonuclease A of 10 µg\ml (Baric et al., 1983) and 20 µg\ml
(Lai et al., 1982 b) used would have degraded RI\TIs and native
RF\TFs, leaving only short fragments of the original structures.
Moreover, this and other studies (Baric & Yount, 2000 ; Sawicki
& Sawicki, 1990) found native RF\TFs and double-stranded
cores of RI\TIs migrated on gels slower than RNA-1, not
faster as was claimed (Baric et al., 1983). This means that not
even authentic RF was recovered in earlier studies (Baric et al.,
1983 ; Lai et al., 1982 b).
Another issue has to do with recovery of particular RNase
T1 oligonucleotides assigned to viral mRNA and found in
fractions thought to contain only RI (Baric et al., 1983 ; Lai et al.,
1982 b). Finding RNase T1-resistant oligonucleotides F10 and
F19 in RI-containing fractions after Sepharose 2B chromatography led others (Baric et al., 1983) to claim the ‘ genomelength ’ RI was utilized for the synthesis of subgenomic mRNA.
At the time, the authors (Baric et al., 1983) expressed surprise
at not finding oligonucleotide F3a that was unique to
subgenomic mRNA-5 and oligonucleotide F19a that was
unique to subgenomic mRNA-6 (Lai et al., 1982 a, 1983).
Subgenomic mRNA-6 is almost as abundant as subgenomic
mRNA-7. We can explain their results in the light of our
finding the TIs for subgenomic mRNA-2 and -3, together with
the RI, in the excluded fraction after Sepharose 2B chromatography. Oligonucleotide F10, which is derived from the leader
sequence present only once at the 5h end of the genome and of
each subgenomic mRNA, and oligonucleotide F19, present in
sequences from the leader–body junction regions of the
genome and mRNA-2, -3 and -7, were found in the excluded
fraction of Sepharose 2B (Baric et al., 1983). We found this
fraction actually contained the genome and some mRNA-2 and
-3, in addition to the RI and TIs II and III. This readily accounts
for the presence of oligonucleotides F10 and F19. Absence of
oligonucleotides F3a and F19a is explained by our finding TIs
for subgenomic mRNA-5 and -6, respectively, in the included
DJE
volume, not in the excluded volume. If genome-length, RI
negative strands were being used to synthesize subgenomic
mRNA-6, oligonucleotide F19a would have been present in
the excluded column volume. Rather than favouring ‘ leaderprimed ’ transcription, the results of Baric et al. (1983) actually
argued against it.
Is leader-priming used at any time during nidovirus
replication ? Our results and those of Baric & Yount (2000) for
MHV and van Marle et al. (1999) for EAV would argue not.
Recently, a study suggested leader-primed transcription occurred immediately after infection. An et al. (1998) detected
subgenomic defective interfering (DI) mRNA but not its
negative-strand template at the very earliest times after
infection, although shortly thereafter subgenomic DI negative
strands became detectable. Because there are about 100 times
fewer negative-strand templates compared to its products, it
would be difficult to rule out the possibility that formation of
the negative-strand template for the subgenomic DI did not
precede formation of the subgenomic mRNA. An et al. (1998)
did in fact confirm that negative-strand templates for subgenomic DI mRNA were detectable very early after infection
and at levels reflective of subgenomic DI mRNA levels. If
leader-primed transcription was required to produce subgenomic DI mRNA at early but not later times, we are left with the
question of why two, redundant mechanisms, one requiring a
primer and one not, are used to produce subgenomic positive
strands ?
MHV TIs behaved during metabolic labelling as authentic
transcription intermediates. Kinetics of their labelling with
[$H]uridine were similar to that observed for the RI. Also,
kinetics of negative-strand synthesis for smaller TIs were the
same as for the RI, and all populations of RI\TIs incorporated
[$H]uridine mostly into positive strands late in infection when
negative-strand, but not positive-strand, synthesis was declining. Kinetic labelling experiments similar to those reported
in this study have been published by others (Baric & Yount,
2000 ; Schaad & Baric, 1994). These support our hypothesis
that complementary negative strands function as templates for
subgenomic mRNA synthesis during coronavirus infection.
The ability to isolate the full set of seven viral RI\TIs and
seven native RF\TFs from infected cells and to explain the
initial failures (Baric et al., 1983 ; Lai et al., 1982 b) to find them
invalidates the original basis for proposing the leader-primed
model. Furthermore, the initial failure (Lai et al., 1982 b) to find
MHV subgenome-length negative strands was most likely also
due to a technical error. The authors used as a probe to detect
negative strands $#P viral RNA obtained from infected cells
grown in [$#P]orthophosphate. Such a probe would not have
sufficiently high specific activity to detect negative strands by
Northern blot. Contrary to their claim, and with our results, it
is now clear that authentic negative strands were not detected.
At this time, our model (Sawicki & Sawicki, 1995)
identifying the discontinuous transcription event as the step or
process generating the 5h nested set of negative strands that
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Structures transcribing MHV genomes and mRNA
serve as templates for subgenomic mRNA synthesis best
explains all available experimental data. Because subgenomic
negative strands in coronavirus-infected cells contain antileader sequences (Sawicki & Sawicki, 1995 ; Sethna et al., 1991),
they directly serve to template the 3h nested set of viral
mRNA. However, with MHV, in order for positive strands to
serve as templates for negative strands, i.e. to replicate,
sequences downstream of the 5h genomic leader sequence are
required (Masters et al., 1994). Therefore, coronavirus subgenomic mRNA and their negative-strand templates do not
replicate. The exact mechanism by which coronaviruses, and
the related arteriviruses, generate a 5h nested set of negativestrand templates that are complementary copies of the
subgenomic mRNA remains to be elucidated. Whatever the
unique and intriguing mechanism, it is used probably by all
members of the Nidovirales (Snijder & Meulenberg, 1998). It
would determine and regulate relative numbers of each of the
variously sized negative-strand templates. With the Nidovirales,
the synthesis of genomes and subgenomic mRNAs would be
determined by the number of each negative-strand template
formed during infection. Details of this mechanism and
identification of the RNA sequences\structures and essential
viral proteins are only now being investigated at the molecular
level (van Dinten et al., 2000 ; van Marle et al., 1999). The
endeavour will benefit greatly from availability of infectious
clones for arteriviruses (van Dinten et al., 1997) and more
recently for coronaviruses (Almazan et al., 2000 ; V. Thiel, J.
Herold, B. Schelle & S. G. Siddell, unpublished data).
Support for these studies was from the National Institutes of Health,
awarded to S. G. S. (AI-28506) and D. L. S. (AI-15123).
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Received 31 August 2000 ; Accepted 27 October 2000
Published ahead of print (9 November 2000) in JGV Direct as
DOI 10.1099/vir.0.17385-0
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