Viral RNAs synthesized in cells infected with germiston bunyavirus

VIROLOGY

157,43

l-439

(1967)

Viral RNAs Synthesized

in Cells Infected

C. CUNNINGHAM MRC

Virology

Unit, University

of Glasgow,

Received

Church

June 25, 1986;

AND J. Street,

with Germiston F. SZILAGYI’

Glasgow

accepted

Bunyavirus

December

G 11 S/R, Scotland,

United

Kingdom

3, 1986

A rapidly growing strain of Germiston virus was used to study intracellular viral RNA synthesis in BHK cells. The RNAs were separated by electrophoresis into seven bands which fell into three size classes: large (bands Ll and L2), medium (bands Ml and M2), and small (bands Sl, S2, and S3). Blot hybridisation established that bands Ll , Ml, and Sl contained the negative-sense genomic RNAs, while bands L2, M2, S2, and S3 contained positive-sense RNAs complementary to the genomic RNAs within the same size class. After glyoxal treatment the RNAs separated into a large, a medium, and two small bands, indicating that the positive-sense RNAs originally present in bands L2, M2, and S2 are similar in size to their genomic RNAs, while the RNA in S3 is shorter than the small genomic segment. These results suggest that band S2 contains the replicative intermediate RNA and band S3 the messenger RNA of the small genomic segment and also that bands L2 and M2 contain both replicative intermediate and messenger RNAs. Long after virus development had ceased in the infected cells the amounts of RNAs in bands Ll, Ml, Sl , and S2 remained the same, those in bands L2 and M2 were reduced, while only trace amounts of RNAs were observed in band S3, suggesting that the genomic RNAs and the replicative intermediate RNAs form ribonuclease-resistant ribonucleoprotein complexes while the messenger RNAs do not form such complexes. Synthesis of RNA in the infected cells was first evident in bands S3 and M2, after which synthesis was soon observed in all seven bands reaching a maximum rate at the logarithmic phase of growth, suggesting that the pattern of Germiston virus development resembles that of other negative-strand RNA viruses. The presence of defective-interfering particles was indicated by the observation that purified virus preparations contained a minor RNA component originating from the large RNA segment. 0 1997 Academic

Press. Inc.

INTRODUCTION

Cells infected with Bunyaviruses contain at least two nonstructural polypeptides, NSs and NSM, as well as the four structural polypeptides L, Gl, G2, and N (Fuller and Bishop, 1982; Short et al., 1982; Elliott, 1985). Genetic analysis using reasserted viruses established that the medium genomic RNA codes for polypeptides Gl , G2, and NSM, the small genomic RNA for polypeptides N and NSs, and by elimination it is presumed that the large genomic RNA codes for polypeptide L (Gentsch and Bishop, 1978, 1979; Cash el a/., 1979; Fuller and Bishop, 1982; Bishop et al., 1984; Pringle eta/., 1984; Elliott, 1985). Duplex analysis showed that cells infected with snowshoe hare, Bunyamwera or Akabane viruses contained polycistronic messenger RNAs (Cash et al., 1979; Abraham and Pattnaik, 1983, 1984). Intracellular viral RNAs in cells infected with Uukuniemi or Germiston viruses were resolved into four bands (L, M, Sl, and S2), the first three having electrophoretic mobilities identical to the three genomic RNAs, while the RNA in S2 migrated faster than the small genomic RNA (Ulmanen et al., 1981; Bouloy et al., 1984a, b). Hybridisation and in vitro translation studies established that bands L, M, and Sl contained both negative- and postive-sense RNAs, that the RNA in band S2 was complementary to but approximately

Germiston virus is a member of the Bunyavirus genus of the family Bunyaviridae (Berge, 1975). Bunyaviruses have a segmented genome consisting of three singlestranded negative-sense RNA molecules designated L (large), M (medium), and S (small) which are present in the virion as ribonucleoprotein complexes in association with polypeptide N and, presumably, polypeptide L (Pettersson et al., 1971, 1977; Pettersson and Kaariainen, 1973; Bouloy et al,, 1973; Pettersson and von Bonsdorff, 1975; Obijeski et al., 197613; Clewley et al., 1977; Kascsak and Lyons, 1977; Ozden and Hannoun, 1980; Pardigon et a/., 1982; Pattnaik and Abraham, 1983). Bunyaviruses also possess an envelope consisting of a phospholipid bilayer and two glycoproteins, Gl and G2 (Renkonen et al., 1972; von Bonsdorff and Pettersson, 1975; Obijeski et al., 1976a; Pennington et a/., 1977; Ozden and Hannoun, 1980). Polypeptide L is presumed to be a component of the virion-associated RNA transcriptase, the activity of which can be assayed in vitro (Ranki and Pettersson, 1975; Bouloy et al., 1975; Bouloy and Hannoun, 1976; Patterson er a/., 1984). ’ To whom

requests

for reprints

should

be addressed. 431

0042.6822187 Copyright All rights

$3.00

@ 1987 by Academac Press. Inc. of reproductron I” any form reserved.

432

CUNNINGHAM

100 nucleotides shorter than the small genomic RNA, and that this RNA directed the in vitro synthesis of polypeptides N and NSs (Ulmanen et a/., 1981; Bouloy et a/., 1984a, b). Ribonucleoprotein complexes isolated from Germiston virus infected cells were resolved into three classes, each containing both negative- and positive-sense RNAs migrating in bands L, M, and Sl , while the polysomes isolated from these cells contained only positive-sense RNAs migrating in bands L, M, and S2 (Bouloy et a/., 1984a). Our aim was to characterise more fully the intracellular viral RNAs synthesised in BHK cells infected with Germiston virus and to study the kinetics of their synthesis throughout the growth cycle. MATERIALS

AND

METHODS

Virus strain The “giant plaque” Germiston virus was isolated from the mutant rs391, originally obtained from Dr. S. Ozden, Institute Pasteur, Paris. It retains the temperature-sensitive character of the parent strain, but grows much faster: after a short lag phase it grows at a logarithmic rate between 3 and 8 hr after infection and the stationary phase is reached by about 11 hr. Growth, purification,

and titration

of the virions

Monolayers of BHK-2 1 Cl 3 cells in plastic roller bottles were infected with giant plaque Germiston virus (1 PFU/l O5 cells) and were incubated at 3 lo in 50 ml Eagle’s medium (Macpherson and Stoker, 1962) containing 2% calf serum. Labelled virus was obtained by adding 0.5 mCi [5,6-3H]uridine (Amersham International plc) to the medium. After 2 or 3 days of incubation (when marked CPE was observed) the virions present in the culture medium were purified by gradient centrifugation (Cunningham and SzilBgyi, to be published) and titrated at 31’ in BHK-21 Cl3 cell monolayers under Eagle’s medium (without phenol red) containing 2.8% calf serum and 0.6% agarose (C. R. Pringle, personal communication). Synthesis

of intracellular

RNAs

Purified giant plaque Germiston virus suspended in 1 ml modified Eagle’s medium (containing only 25% of the usual methionine content, 2% calf serum and IO pg/ml actinomycin D) was added to BHK21 Cl3 cell monolayers in 25-cm2 flasks (50 PFU/cell). After adsorption of the virus at 4’ for 60 min the remaining suspension was removed, 1.5 ml fresh medium was added, and the culture was incubated at 31 O. The intracellular RNAs were labelled with 0.2 mCi/

AND

SZltiGYl

flask [3H]uridine added to the culture either at the start of incubation or at specified times during incubation. Extraction

of RNAs

The RNAs from purified virions and infected tissue culture cells were extracted at 60’ using the phenolcresol method of Kirby et a/. (1967) after which they were precipitated with 66% ethanol, washed with cold 3 M sodium acetate then 66% ethanol, and finally resuspended in a small volume of H20. Electrophoretic

separation

of RNAs

Acid-urea agarose gel electrophoresis of the RNAs followed the method of Wertz and Davis (1979). The gel contained 1.2% agarose, 6 M urea, and 25 mM sodium citrate buffer, pH 3.5. Electrophoresis was at 4’ for 16 hr at 80V with constant recirculation of the tank buffer (25 mM citrate buffer, pH 3.5). The RNA bands were detected by fluorography using Kodak XSl film. Hybridisation

of RNAs

Unlabelled intracellular RNAs were synthesised and separated by acid-urea agarose gel electrophoresis as described above. [3H]Uridine-labelled intracellular RNAs were applied to adjacent wells as markers. After electrophoresis the gel was washed with tank buffer and the RNAs transferred to GeneScreen Plus membrane, NEN Products, DuPont (Southern, 1975). The membrane-bound unlabelled RNAs were hybridised with 32Plabelled probes at 65” (Singh and Jones, 1984), and the labelled RNA bands were detected by autoradiowwb Genomic RNA probes. Probes were prepared by endlabelling genomic RNAs from purified giant plaque Germiston virus with [32P]pCp using T4 RNA ligase (England and Uhlenbeck, 1978). Single-stranded complementary DNA probes. Probes were synthesised in vitro in the presence of [32P]dlTp and [32P]dCTP by reverse transcriptase using as templates the genomic RNAs extracted from purified virions of giant plaque Germiston virus (Gubter and Hoffman, 1983). Double-stranded DNA probes. Double-stranded DNA probes were synthesised using genomic RNAs of giant plaque Germiston virus as templates and cloned into pBR322 plasmids following the method of Gubler and Hoffman (1983). The cloned DNAs were identified by hybridisation to individual genomic RNA segments obtained by separation of the virion RNAs on a Jaw melting point agarose gel and extraction of the individual RNAs from the gel by the hot phenol-cresol method. Finally the DNA probes were 32P-labelled by nick-translation.

INTRACELLULAR

Glyoxal treatment

GERMISTON

VIRUS

of RNAs

RESULTS

Glyoxal treatment of RNAs followed the method of Carmichael and McMaster (1980). Purified RNAs (4 ~1) were mixed with DMSO (5 ~1) and freshly deionised glyoxal (1 ~1) and incubated at 50” for 60 min. After incubation 44 ~1 buffer (10 M urea, 2.5 mM citrate buffer, pH 3.5) was added and the RNAs were separated by acid-urea agarose gel electrophoresis.

V

MI

433

RNAs

INF

V

RNAs synthesised in cells infected with giant plaque Germiston virus RNAs synthesised during 4 hr of incubation at 3 lo in the presence of 10 pg/ml actinomycin D in BHK cells infected with giant plaque Germiston virus were analysed by acid-urea agarose gel electrophoresis (Fig. 1). Genomic RNAs, isolated from purified virions, and RNAs synthesised in mock-infected cells were used as markers. The virion (V) contained the large (L), medium (M), and small (S) genomic RNA segments and a minor RNA component (mc), while the mock-infected cells (Ml) contained only a single large [3H]uridine-labelled RNA species (h). The RNAs present in the infected cells (INF) were separated into seven major RNA bands which fall into three size classes: large (bands Ll and L2), medium (Ml and M2), and small (Sl, S2, and S3). Approximately equal amounts of RNAs were present in the two large (Ll and L2) and in the two medium (Ml and M2) bands, while band S2 contained significantly less RNAs than the two other small RNA bands (Sl and S3). The infected cells also contained two minor RNA bands (mcl and mc2) and the host RNA h. Due to the displacement of some of the radioactively labelled background material the positions of the unlabelled ribosomal RNAs (r) were discernible. The synthesis of only one RNA in the presence of actinomycin D in the mock-infected cells indicates that the RNAs in the seven major and two minor bands are virus specified, and that 10 pg/ml actinomycin D does not inhibit the synthesis of Germiston virus RNAs. Since bands Ll , Ml, and Sl have electrophoretic mobilities identical to the large (L), medium (M), and small (S) genomic RNA segments of the virion they presumably contain the corresponding genomic RNAs. The minor RNA component (mc) of the virion and the two minor RNA bands (mcl and mc2) of the infected cells are discussed later. Characterisation

FIG. 1. RNAs synthesised in BHK cells infected with giant plaque Germiston virus. Monolayers of BHK cells, in the presence of actinomycin D and rH]uridine, were either mock-infected (Ml) or infected with 50 PFU/cell of giant plaque Germiston virus (INF) and incubated for 4 hr at 31”. The RNAs were then extracted and analysed by acidurea agarose gel electrophoresis. Genomic RNA segments, extracted from [3H]uridine-labelled purified Germiston virions (V), were used as markers. The position of the following are indicated: the large (L), medium (M), and small (S) genomic RNA segments and the minor RNA component (mc) of the virion, the [3H]uridine-labelied host RNA (h), the seven major (Ll , L2, Ml, M2, St, S2, and S3) and two minor (mcl and mc2) RNA bands present in the infected cells and the unlabelled ribosomal RNAs (r).

of the intracellular

viral RNAs

The intracellular viral RNAs were characterised by blot hybridisation using as probes genomic RNAs isolated from purified virions, in vitro synthesised singlestranded DNAs complementary to the genomic RNAs, and three double-stranded cloned DNAs, each derived from one of the genomic segments of the virion. The genomic RNA probes (Fig. 2) hybridised to the RNAs in bands S3, S2, M2, and, to a lesser extent, L2 (visible only after long exposure). The probes also hybridised with one of the minor components, mc2, and with the ribosomal RNAs. Since negative-sense ge-

434

CUNNINGHAM

L2

M2

r

AND

SZILAGYI

The results obtained with the double-stranded cloned DNAs (Fig. 4) showed that the DNA derived from the large genomic RNA hybridised with the RNAs in the large bands (Ll and L2) and with the two minor RNA components (mcl and mc2) (lane 2) the DNA derived from the medium genomic RNA hybridised with the RNAs in the medium bands Ml and M2 (lane 3) and the DNA derived from the small genomic RNA segment hybridised with the RNAs in the three small bands, Sl , S2, and S3 (lane 4). These hybridisation experiments confirm that the RNAs in all seven major bands are virus specified and show that bands Ll, Ml, and Sl contain the negativestrand genomic RNA segments, band L2 contains positive-sense RNAs complementary to the large genomic

r mc2

s2

s3

FIG. 2. Hybridisation of intracellular RNAs with genomic RNA. Unlabelled (lane 2) and [3H]uridine-labelled (lane 1) RNAs, synthesised during 4 hr of incubation at 31’ in the presence of actinomycin D in BHK cells infected with giant plaque Germiston virus (50 PFUkell) were separated by acid-urea agarose gel electrophoresis and transferred to GeneScreen Plus membrane. The unlabelled RNAs (lane 2) were then hybridised with 32P-labelled genomic RNAs extracted from purified Germiston virions. Symbols are as in Fig. 1.

nomic RNAs were used for the hybridisation these resuits indicate that bands L2, M2, S2, and S3 contain positive-sense RNAs complementary to the genomic RNAs, while bands Ll , Ml, and Sl do not. The limited hybridisation of the probes to the ribosomal RNAs is presumably non-specific. The single-stranded complementary DNA probes (Fig. 3) strongly hybridised to the RNAs in bands Sl, M 1, and Ll . They also hybridised to the RNAs in band M2 and, to a slight extent, to those in bands L2, S2, and S3. These results indicate that the RNAs in bands Ll , M 1, and Sl are complementary to the DNA probes and therefore are negative-strand RNAs. The limited hybridisation with the RNAs in the other bands may be due to the synthesis of some negative-sense DNAs by the reverse transcriptase using the complementary DNAs as templates.

FIG. 3. Hybridisation of intracellular RNAs with single-stranded complementary DNAs. Membrane-bound [3H]uridine-labelled (lane 1) and unlabelled (lane 2) intracellular RNAs were obtained as in Fig. 2. The unlabelled RNAs (lane 2) were hybridised with 32P-labelled single-stranded cDNAs synthesised in vitro by reverse transcriptase using genomic RNAs of purified Germiston virions as templates. Symbols are as in Fig. 1.

INTRACELLULAR

GERMISTON

4

VIRUS

RNAs

435

bands L2 and M2 were significantly less, and only trace amounts of RNAs were observed in band S3. Variations in the resistance to cellular ribonucleases of the RNAs in the different bands probably accounts for these results. The RNAs in bands Ll, Ml, Sl, and S2 are resistant to ribonucleases, and those in S3 are sensitive to them, presumably because the former form ribonucleoprotein complexes with polypeptides N and L while the latter do not. It follows therefore that bands L2 and M2 will contain both resistant ribonucleoprotein complexes and sensitive RNAs. Since the genomic RNA segments (in bands Ll , Ml, and Sl) form ribonucleoprotein complexes inside the cells it is possible that those positive-sense RNAs which form similar complexes (in bands L2, M2, and S2) are the replicative

mcl mc2

FIG. 4. Hybridisation of intracellular RNAs with double-stranded cloned DNAs. Membrane-bound [3H]uridine-labelled (lane 1) and unlabelled (lanes 2, 3, and 4) intracellular RNAs were obtained as in Fig. 2. The unlabelled RNAs were hybridised with 3zP-labeiled doublestranded cloned DNAs derived either from the large (lane 2) medium (lane 3) or small (lane 4) genomic RNA segments of the giant plaque Germiston virion. Symbols are as in Fig. 1.

RNA segment, band M2 contains positive-sense RNAs complementary to the medium genomic RNA segment and S2 and S3 both contain positive-sense RNAs complementary to the small genomic RNA segment. Stability of the viral RNAs inside the cells By comparing the composition of the RNAs of Germiston virus infected BHK cells after 4 and 24 hr of incubation, i.e., during the logarithmic phase of growth and long after growth had ceased, we determined the stability of the intracellular viral RNAs. Figure 5 shows that significant differences were observed in the relative amounts of RNAs present in the seven bands. After 24 hr the amounts of RNAs in bands Ll , M 1, Sl , and S2 were similar to those present after 4 hr of incubation. However, the amounts of RNAs in

FIG. 5. Composition of the intracellular RNAs after 4 and 24 hr of incubation. Monolayers of BHK cells, in the presence of actinomycin D and [3H]uridine, were infected with giant plaque Germiston virus (50 PFU/cell) and incubated 4 (lane 4) and 24 (lane 24) hr at 31”. The RNAs were then extracted and analysed by acid-urea agarose gel electrophoresis using genomic RNA segments of purified Germiston virions (V) as markers. Symbols are as in Fig. 1.

436

CUNNINGHAM

intermediate RNAs and those which do not (in bands L2, M2 and S3) are the messenger RNAs. Relative

sizes of the intracellular

SZltiGYl A. 4

Glv

4

viral RNAs

Intracellular viral RNAs were treated with glyoxal prior to acid-urea agarose gel electrophoresis in order to eliminate the charge differences between the positive and negative strands, and thus separate the RNA molecules on the basis of size alone. The glyoxal-treated intracellular viral RNAs were extracted from Germiston virus infected cells after 4 hours of incubation at 31’ and separated by electrophoresis into four bands, one large, one medium, and two small (Fig. 6A). Comparison of the electrophoretic mobilities of the glyoxal-treated (Gly) and untreated (4) RNAs suggests that the large band contained the RNAs of bands Ll and L2, the medium band contained those of bands Ml and M2, while the two small bands contained the RNAs of bands Sl, S2, and S3. The composition of the RNA species in the upper and lower small bands was determined by extracting the RNAs from infected cells after 4 and 24 hr of incubation and treating them with glyoxal (Fig. 6B). The amounts of RNAs in the upper small bands were similar in both preparations (Gly 4 and Gly 24) while only trace amounts of RNAs were observed in the lower small band after 24 hr of incubation (Gly 24). Since after 24 hr of incubation (24) only trace amounts of RNAs were present in band S3 these results suggest that the lower small band contains the RNA of band S3. Therefore the positive-strand RNAs in bands L2, M2, and S2 are very similar in size to their respective genomic RNAs (in bands Ll , Ml, and Sl) while the positive-sense RNA in band S3 is shorter than its genomic RNA segment. Rates of synthesis

AND

of the intracellular

viral RNAs

The rates of synthesis of the virus-specified RNAs were studied by pulse-labelling Germiston virus infected BHK cells with [3H]uridine (Fig. 7). Between 0.5 and 1 hr of incubation RNAs were synthesised in detectable amounts only in band S3. Although the rates of synthesis between 1 and 1.5 hr were still very low, observable quantities of RNAs were also detected in band M2. During the next 2.5 hr of incubation the synthesis of RNAs in all seven major bands proceeded at a rapidly increasing rate reaching a maximum at 3.5 to 4 hr of incubation. However, by 7.5 to 8 hr of incubation only small amounts of RNAs were synthesised. Although the host RNA, h, was synthesised between 0.5 and 1 hr of incubation its synthesis declined rapidly and was completely inhibited after approximately 2 hr.

MI

SC s2 s3

FIG. 6. Comparison of the electrophoretic mobilities of glyoxaltreated and untreated intracellular RNAs. BHK cells infected with 50 PFU/cell giant plaque Germiston virus, in the presence of actinomycin D and [3H]uridine, were incubated at 31’ for 4 and 24 hr. Intracellular RNAs were extracted, treated with glyoxal, and analysed by acidurea agarose gel electrophoresis. Untreated RNAs were used for comparison. (A) The glyoxal-treated (Gly) and untreated (4) RNAs after 4 hr of incubation. (B) The glyoxal treated (Gly 4 and Gly 24) and the untreated (4 and 24) RNAs after 4 and 24 hr of incubation. After glyoxal treatment the intracellular RNAs separated into a large (O), a medium (+) and two small (an upper, D, and a lower ,) RNA bands. Other symbols are as in Fig. 1.

Therefore the first RNAs to be synthesised are those in bands S3 and M2 followed by rapid synthesis of the RNAs in all seven bands reaching a maximum at the logarithmic phase of growth after which the synthesis declines rapidly as the stationary phase of growth is reached (approximately 7 to 9 hr). The decline and eventual inhibition of the synthesis of the host RNA h is due to actinomycin D since in mock-infected cells the synthesis of this RNA is similarly inhibited during prolonged exposure to this antibiotic (data not shown).

INTRACELLULAR

GERMISTON

VIRUS

437

RNAs

Preparations of purified giant plaque Germiston virus contained a minor RNA component (mc) with an electrophoretic mobility identical to that of the minor RNA component in band mcl (Fig. 1). These results suggest that some of the virus particles contain a negative-sense RNA segment which originated from the large genomic RNA and which is able to replicate in the infected cells presumably using the positive-sense RNA segment in band mc2 as the replicative intermediate RNA. This truncated RNA may have been generated during a defective replication of the large genomic RNA. This is presumably a nonspecific event, since the parental strain of Germiston virus, mutant ts391, possesses a minor RNA component distinct from that of the giant plaque strain (Fig. 8). 12

FIG. 7. Pulse-labeliing of intracellular RNAs. Monolayers of BHK cells, in the presence of actinomycin D, were infected with giant plaque Germiston virus (50 PFU/cell) and incubated at 31”. After various lengths of incubation [3H]uridine was added, and incubation continued for a further 30 min. The RNAs were then extracted and analysed by acid-urea agarose gel electrophoresis, placing equivalent amounts in each well. The times of pulse-labelling (in hours) are indicated. Genomic RNAs, extracted from purified Germiston virions (virus), were used as markers. Symbols are as in Fig. 1.

Minor RNA components In cells infected with giant plaque Germ&on virus two minor RNA bands (mcl and mc2) were frequently observed above the small RNA bands (Figs. 1, 2, and 6). During blot hybridisation studies it was observed that the genomic RNA probes hybridised with the RNAs in band mc2 only (Fig. 2) and the cloned doublestranded DNA probe derived from the large genomic RNA segment hybridised with the RNAs in both bands (Fig. 4). These results show that mcl contains negativesense RNA segments derived from the large genomic RNA of the virion, and mc2 contains a positive-sense RNA complementary to the RNAs in mcl.

FIG. 8. Genomic RNA segments of two strains of Germiston virus. The mutant fs391 (lane 1) and the giant plaque (lane 2) strains of Germiston virus were grown in BHK cells in the presence of [3H]uridine. From purified virions the RNAs were extracted and analysed by acid-urea agarose gel electrophoresis. The virions contained the large (L), medium (M), and the small (S) genomic RNA segments and a minor RNA (mc) component.

CUNNINGHAM

438

DISCUSSION Intracellular viral RNAs synthesised in BHK cells by a rapidly growing strain of Germiston virus were separated by acid-urea agarose gel electrophoresis into seven bands which fell into three size classes: large, medium and small. The uppermost band in each size class (Ll , Ml, and Sl) contained one of the segments of the negative-sense genomic RNA, while the lower bands (L2, M2, S2, and S3) contained positive-sense RNAs complementary to the genomic RNA segment within the same size class. Thus, because separation of RNAs by acid-urea agarose gel electrophoresis is charge related, we have succeeded in separating the intracellular viral RNAs on the basis of both size and polarity, whereas previously they were separated by size alone (Ulmanen et al., 1981; Bouloy et a/., 1984a, b). Since the RNAs were separated into four bands by size alone following glyoxal treatment, the positivesense RNAs originally present in L2, M2, and S2 are presumably similar in size to their respective genomic RNA segments, while the RNA in S3 is significantly shorter than the small genomic RNA. These results are in good agreement with those of Bouloy et al., (1984a, b) who also found that on denaturing gels the large, medium, and upper small band contained both positiveand negative-sense RNAs while the lower small band contained the positive-sense messenger RNA for polypeptides N and NSs. Thus we conclude that RNA in band S3 is the messenger RNA for polypeptides N and NSs, while the positive-sense RNA in band S2 is probably the full size replicative intermediate RNA of the small genomic RNA segment. Therefore we believe that the positive-sense RNAs in bands L2 and M2 are mixtures of similar size replicative intermediate and messenger RNAs derived respectively from the large and medium genomic RNA segments. The stability of RNAs in bands Ll , Ml, and Sl indicates that the genomic RNA segments exist inside the cells as ribonucleoprotein complexes presumably in conjunction with polypeptides N and L. The resistance of the RNA in S2 and some of the RNAs in bands L2 and M2 indicates that the full-size positive-strand replicative intermediate RNAs also form such complexes inside the cells. The degradation of the RNAs in S3 and some of the RNAs in bands L2 and M2 during long incubation indicates that the messenger RNAs in these bands do not form such ribonucleoprotein complexes. These results accord well with those of Bouloy et al., (1984a) who isolated from infected cells ribonucleoprotein complexes containing both positive- and negative-sense large, medium, and small RNAs, and polysomes containing only positive-sense RNAs of all three size classes.

AND SZltiGYl

RNA synthesis in the infected cells commenced with the synthesis of RNAs in bands S3, M2, and presumably also L2. Because these positive-sense RNAs appear to be synthesised prior to replication of the genomic RNAs they are almost certainly messenger RNAs synthesised by the RNA transcriptase of the infecting virions. After approximately 1.5 to 2 hr of incubation small amounts of RNAs in all seven bands were observed, their rates of synthesis increasing rapidly to reach a maximum at the logarithmic phase of growth. These results indicate that Germiston virus resembles other negative-strand viruses, i.e., RNA synthesis starts with primary transcription, while most of the intracellular viral RNAs are synthesised by gene amplification and secondary transcription (Flamand and Bishop, 1974). Since actinomycin D was present in all the experiments, the results indicate that transcription and replication of viral RNAs cannot be strongly inhibited by this antibiotic and that de nova synthesis of host RNAs may not be required. Although plaque-purifed virus preparations were used in our experiments, they contained a negativesense minor RNA component which was derived from the large genomic RNA segment, presumably by mutation. This component, which represents about 20% of the large genomic RNA, was able to replicate inside the infected cell. The virus particles carrying this segment are presumably defective-interfering (DI) particles such as those observed in the case of Bunyamwera and Marituba viruses (Kascsak and Lyons, 1978; Volkmer et al., 1983). ACKNOWLEDGMENTS We thank J. H. Subak-Sharpe for his encouragement and critical reading of the manuscript, S. Ozden for his mutant ts391, R. M. Elliott, K. M. Jones, and J. C. M. Macnab for their advice on hybridisation and DNA cloning, and J. T. Poyner for helping with the preparation of the manuscript.

REFERENCES ABRAHAM,G., and PAI-~NAIK,A. K. (1983). Early RNA synthesis in Bunyamwera virus-infected cells. J. Gen. Viral. 64, 1277-l 290. ABRAHAM,G., and PAI-~NAIK,A. K. (1984). The S segment of Bunyaviruses codes for two complementary RNAs. In “Segmented Negative Strand Viruses” (R. W. Compans and D. H. L. Bishop, Eds.), pp. 37-44. Academic Press, New York/London. BERGE, T. 0. (1975). “International Catalogue of Arboviruses.” U.S. DHEW Publication No. (CDC) 75-8301, Washington, DC. BISHOP,D. H. L., RUD,E., BELLONCIK,S.. AKASHI. H., FULLER,F., IHARA, T., MATSUOKA,Y., and ESHITA,Y. (1984). Coding analyses of Bunyavirus RNA species. ln “Segmented Negative Strand Viruses” (R. W. Compans and D. H. L. Bishop, Eds.), pp. 3-l 1. Academic Press, New York/London. BOULOY, M., COLBERE, F., KRAMS-OZDEN, S., VIA~T, P., GAFIAPIN, A. C., and HANNOUN.C. (1975). ActivitB-RNA polymerasique associbe a un bunyavirus (Lumbo). C.f?.Acad. Sci. Paris 280, 213. BOULOY, M., and HANNOUN,C. (1976). Studies on Lumbo virus rep-

INTRACELLULAR

GERMISTON

lication. I. RNA-dependent RNA polymerase associated with virions. Virology 69, 258-264. BOULOY, M., KRAMS-OZDEN, S., HORODNICEANU, F., and HANNOUN, C. (1973). Three-segment RNA genome of Lumbo virus (Bunyavirus). Intervirology 2, 173-l 80. BOULOY, M., PARDIGON, N., GERBAUD, S.. VIALAT, P., and GIRARD, M. (1984a). Transcription of the S RNA segment of Germiston Bunyavirus. /n “Segmented Negative Strand Viruses” (R. W. Compans and D. H. L. Bishop, Eds.), pp. 29-35. Academic Press, New York/ London. BOULOY, M., VIALAT, P., GIRARD, M., and PARDIGON, N. (198413). A transcript from the S segment of the Germiston Bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein. J. Viral. 49, 717-723. CARMICHAEL, G. G., and MCMASTER, G. K. (1980). The analysis of nucleic acids in gels using glyoxal and acridine orange. In “Methods in Enzymology” (L. Grossman and K. Moldave. Eds.), Vol. 65, pp. 380-391. Academic Press, New York/London. CASH, P., VEU~, A. C., GENTSCH, J. R., and BISHOP, D. H. L. (1979). Genome complexities of the three mRNA species of snowshoe hare Bunyavirus and in vitro translation of S mRNA to viral N polypeptide. J. Viral. 31, 685-694. CLEWLEY, J., GENTSCH, J., and BISHOP, D. H. L. (1977). Three unique viral RNA species of snowshoe hare and La Crosse bunyaviruses. J. Viral. 22, 459-468. ELLIOTT, R. M. (1985). Identification of nonstructural proteins encoded by viruses of the Bunyamwera serogroup (family Bunyaviridae). Virology 143, 119-l 26. ENGLAND, T. E., and UHLENBECK, 0. C. (1978). 3’-Terminal labelling of RNA with T4 RNA ligase. Nature (London) 275, 560-561. FLAMAND, A., and BISHOP, D. H. L. (1974). /n viva synthesis of RNA by vesicular stomatitis virus and its mutants. J. Mol. Biol. 87, 3153. FULLER, F., and BISHOP, D. H. L. (1982). Identification of virus-coded nonstructural polypeptides in Bunyavirus-infected cells. J. Viral. 41, 643-648. GENTSCH, 1. R., and BISHOP, D. H. L. (1978). Small viral RNA segment of Bunyaviruses codes for viral nucleocapsid protein. J. Viral. 28, 417-419. GENTSCH, J. R., and BISHOP, D. H. L. (1979). M viral RNA segment of Bunyaviruses codes for two glycoproteins, Gl and G2. J. Viral. 30, 767-770. GUSLER, U., and HOFFMAN, B. J. (1983). A simple and very efficient method of generating cDNA libraries. Gene 25, 263-269. KASCSAK, R. J., and LYONS, M. J. (1977). Bunyamwera virus. I. The molecular complexity of the virion RNA. Virology 82, 37-47. KASCSAK, R. J., and LYONS, M. J. (1978). Bunyamwera virus. II. The generation and nature of defective interfering particles. Virology 89,539-546. KIRBY, K. S., FOX-CARTER, E., and GUEST, M. (1967). Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria. Biochem. J. 104,258-282. MACPHERSON, I. A., and STOKER, M. G. P. (1962). Polyoma transformation of hamster cell clones-an investigation of genetic factors affecting cell competence. Virology 16, 147-151. OBIJESKI, J. F., BISHOP, D. H. L., MURPHY, F. A., and PALMER, E. L. (1976a). Structural proteins of La Crosse virus. J. Viral. 19, 985997.

VIRUS

RNAs

439

OSIJESKI, J. F., BISHOP, D. H. L., PALMER, E. L., and MURPHY, F. A. (1976b). Segmented genome and nucleocapsid of La Crosse virus. J. Viral. 20, 664-675. OZDEN, S., and HANNOUN, C. (1980). Biochemical and genetic characteristics of Germiston virus. Virology 103, 232-234. PARDIGON, N., VIALAT, P., GIRARD, M., and BOULOY, M. (1982). Panhandles and hairpin structures at the termini of Germiston virus RNAs (Bunyavirus). Virology 122, 191-l 97. PATERSON, J. L., HOLLOWAY, B., and KOLAKOFSKY, D. (1984). La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease. 1. Viral. 52, 215-222. PA~NAIK, A. K., and ABRAHAM, G. (1983). Identification of four complementary RNA species in Akabane virus-infected cells. J. Viral. 47,452-462. PENNINGTON, T. H., PRINGLE, C. R., and MCCRAE, M. A. (1977). Bunyamwera virus-induced polypeptide synthesis. J. Viral. 24, 397400. PET~ERSSON, R. F., HEWLETT, M. J., and BALTIMORE, D. (1977). The genome of Uukuniemi virus consists of three unique RNA segments. Cell 11, 51-63. PE‘TTERSSON, R., and ~RIAINEN. (1973). The ribonucleic acids of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 56, 608619. PEITERSSON, R., ~RI#INEN, L., VON BONSDORFF, C.-H., and OKER-BLOM, N. (197 1). Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 46, 72 l-729. PEI-~ERSSON, R. F., and VON BONSDORFF, C.-H. (1975). Ribonucleoproteins of Uukuniemi virus are circular. 1. Viral. 15, 386-392. PRINGLE, C. R., LEES, 1. F., CLARK, W., and ELLIOTT, R. M. (1984). Genome subunit reassortment among Bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes. Virology 135, 244-256. RANKI, M., and PEITERSSON, R. F. (1975). Uukuniemi virus contains an RNA polymerase. J. Viral. 16, 1420-l 425. RENKONEN, O., WRIAINEN, L., PET~ERSSON, R., and OKER-BLOM, N. (1972). The phospholipid composition of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 50, 899-901. SHORT, N. J., MEEK, A. D.. and DALGARNO, L. (1982). Seven infectionspecific polypeptides in BHK cells infected with Bunyamwera virus. J. Viral. 43, 840-843. SINGH, L., and JONES, K. W. (1984). The use of heparin as a single cost-effective means of controlling background in nucleic acid hybridization procedures. Nucleic Acid Res. 12, 5627-5638. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503517. ULMANEN, I., SEPP#L& P., and PETTERSSON, R. F. (1981). In vitro translation of Uukuniemi virus-specific RNAs: Identification of a nonstructural protein and a precursor to the membrane glycoproteins. J. Viral. 37, 72-79. VOLKMER, N., SOARES, M. C. M., and RESELLO, M. A. (1983). Autointerference of Marituba virus (Bunyaviridae) in mouse L cells by defective interfering particles. lntervirology 20, 108-l 13. VON BONSDORFF, C-H., and PETTERSSON, R. (1975). Surface structure of Uukuniemi virus. J. Viral. 16, 1296-l 307. WERTZ, G. W., and DAVIS, N. L. (1979). RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral RNA’s J. Viral. 30, 108-l 15.