We compared the ability of the leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus to inhibit transcription in infected host cells. The level of cellular RNA synthesis in cells infected with either serotype was drastically reduced by 5 h after infection. Studies with UV-inactivated virus demonstrated that shutoff of cellular RNA synthesis directly correlated with the ability of the infecting virus to transcribe its plus-stranded leader RNA. Although both serotypes

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... nhibited cellular RNA synthesis, the Indiana serotype reduced synthesis to lower levels. In addition, an examination of the kinetics of leader RNA synthesis in vivo indicated that up to four times more leader RNA was produced in cells infected with the Indiana serotype than in those infected with the New Jersey serotype. However, in vivo studies also suggested that the leader RNA of the New Jersey serotype was a more efficient RNA inhibitor than was the Indiana serotype leader RNA. Although up to 2,900 copies of the leader RNA per cell could be detected in infected cells, only 550 copies of the Indiana and 100 copies of the New Jersey leader RNAs per cell were present in infected cells that were demonstrating 50% of the maximal inhibition of RNA synthesis. In an in vitro system, leader RNAs of both serotypes inhibited DNA-dependent transcription of the adenovirus late promoter and adenovirus-associated RNA genes, but the New Jersey serotype leader was also a better inhibitor in this reconstituted system. Data from the dose response of inhibition by each leader suggest that polymerase III transcription was more sensitive to inhibition by viral leaders than was polymerase II transcription. Polyadenylated viral mRNAs and the NS and N gene starts transcribed by both serotypes did not significantly inhibit transcription at levels at which the corresponding leader RNAs were inhibitory. Overall, our results strongly suggest a role for the plus-stranded leader RNAs of the New Jersey and Indiana serotypes of vesicular stomatitis virus in inhibiting cellular transcription in vivo. We discuss differences in the nucleotide sequences of the two leader RNAs in relation to their potential regulatory sequences. differencs in biological activity and to Infection of many cell types with vesicular stomatitis virus (VSV), the prototype rhabdovirus, results in a drastic reduction in the ability of the host cell to synthesize RNA (6, 39, 40, 48) . This effect, along with the reduced ability to synthesize DNA (30, 47) and protein (6, 26, 28, 44) , eventually leads to cell death (25) (26) (27) . The ability of VSV to inhibit cellular RNA synthesis requires transcription by the virion polymerase (41, 46) . However, studies with UV-inactivated virus (38) indicate that only a small part of the genome must be transcribed. Weck and Wagner (41) suggested that the RNA shutoff was at the level of initiation of transcription, primarily by decreasing the number of functional units of polymerase. Degradation of cell mRNA was not enhanced in infected cells (31), and there has been no evidence for modification of post-transcriptional events such as polyadenylation or transport of RNA (39). The VSV genome is transcribed sequentially (1, 4), producing a 47to 48-nucleotide leader RNA and five monocistronic mRNAs. The leader RNA is transcribed from the exact 3'-end of the genome both in vitro (8, 9) and in vivo (20) and is produced in molar amounts higher than any of the viral mRNAs. Because only a small portion of the genome must be transcribed to affect cell RNA synthesis, this leader RNA has been suggested as the inhibitor molecule (38 ). Recently, Kurilla et al. (19) demonstrated that the leader RNA was present in the nucleus of 88 on May 7, 2020 by guest http://jvi.asm.org/ Downloaded from VSV LEADERS INHIBIT CELL TRANSCRIPTION infected cells, and studies by McGowan et al. (29) showed that the leader RNA could inhibit DNA-dependent RNA synthesis in an in vitro transcription system. In view of these observations, the VSV leader RNA appears to provide a unique model for studying regulation of transcription by a small RNA. With the exception of the limited study by Yaoi et al. (48) , information on inhibition of cellular macromolecular synthesis by VSV has been obtained for the Indiana serotype. As an approach to further studying the role of the VSV leader RNA in transcription inhibition, we examined the inhibitory effect of two leader RNAs that have slightly different RNA sequences. We report certain similarities and differences in the ability of these two leader RNAs to inhibit transcription, and by examining the kinetics of leader production in vivo, we provide additional evidence that leader RNAs play a key role in inhibiting RNA synthesis in VSV-infected cells. MATERIALS AND METHODS Viruses and cells. The VSVs used in this study were the San Juan strain of the Indiana serotype (VSV,nId), originally obtained from the U.S. Agricultural Research Center, Beltsville, Md. (37), and the Hazelhurst strain of the New Jersey serotype (VSVNJ), obtained from the American Type Culture Collection, Rockville, Md. (ATCC 159). Clones of each virus were selected from plaques on L-cell monolayers. Virus stocks were prepared by growing virus in BHK-21 cells at a multiplicity of infection (MOI) of =0.1 PFU per cell and were titrated by assay of PFU on monoelayers of L cells. Virus was purified by sucrose and potassium tartrate gradient centrifugation as described by Barenholz et al. (5). The mouse myeloma cells (MPC-11), BHK-21 cells, and L cells were grown as described previously (28) . The culture medium, Dulbecco Modified Eagle Medium (DMEM), calf serum. and horse serum were obtained from GIBCO Laboratories Grand Island, N.Y. Tritiated uridine labeling of RNA in infected and uninfected cells. MPC-11 cells were grown in suspension culture to a cell density of 5 x 105 cells per ml and were centrifuged at 1,000 x g for 5 min. The cell pellets were suspended in DMEM, containing 1% horse serum, to a cell density of 1 x 107 to 2 x 107 cells per ml. Samples were infected at an MOI of either 1, 10, or 50 PFU per cell and were incubated for 30 min at 37°C. After the adsorption period, cells were diluted in DMEM and inoculated into 24-well culture plates at 5 X 105 cells per well. For labeling of BHK-21 cells, monolayer cultures were prepared in 24-well plates and infected at the above multiplicities. Cells were pulse-labeled for 10 min by the addition of [-H]uridine to a final concentration of S ,uCi/ml. After labeling, the cells were lysed by the addition of one-tenth volume of 10% sodium dodecyl sulfate (SDS), and after 5 min, an equal volume of 10% trichloroacetic acid was added. The acid-insoluble material was collected by filtration through glass fiber filters (GSA; Whatman, Inc., Clifton, N.J.) and the radioactive content was determined by counting dried filters by scintillation spectroscopy. The protein content of labeled cells was determined by the method of Lowry et al. (23) . UV irradiation of virus. As described previously (38), 25-,ul samples of purified VSV (5 x 109 PFU/ml) were dispensed into petri dishes (diameter. 100 mm) and placed at a distance of 10 cm from a UV light source. Virus samples, at 4°C, were exposed at various times to irradiation at a wavelength of 254 nm and a dose rate of 85 ergs per mm2 per s. Cells were infected with UV-irradiated virus at an MOI of =10, based on the original titer of the virus before irradiation. In vitro VSV transcription assay. VSV plus-stranded RNAs were produced in a transcription assay (12) containing 0.1 mg of VSV per ml, 0.14 M NaCI, 0.2% Triton X-100. 7.5 mM MgCI. 10 mM Tris-hydrochloride (pH 8.0). 1 mM dithiothreitol, 1 mM each of ATP, GTP, and CTP, and 0.1 mM UTP containing 10 to 100 ,uCi of [c-+32P]UTP (400 Ci/mmol). Transcription mixtures were incubated for 3 h at 31°C, and the reactions were stopped by adding one-tenth volume of lOx SET (lx SET is 0.15 M NaCI-5 mM EDTA-50 mM Trishydrochloride [pH 8.0]) and extracting with an equal volume of water-saturated phenol. The resulting aqueous phase was extracted from one to three times with chloroform-isoamyl alcohol (24:1 [vol/vol]) and adjusted to 0.3 M with sodium acetate, and the RNAs were precipitated at -80°C for 2 h after the addition of three volumes of 95% ethanol. The precipitate was collected by centrifugation at 10,000 x g for 10 min and suspended in I x SET. Polyadenylated RNAs, isolated by oligodeoxythymidylic acid cellulose chromatography as described by Kuchler (18). were precipitated with ethanol, and the incorporation of [32P]UMP was determined by Cerenkov counting of the resulting RNA pellets. Leader RNA was separated from other plus-stranded RNAs by electrophoresis at 1,600 V on 8 M urea-20% polyacrylamide gels in buffer containing 10 mM Tris (pH 8.0). 1 mM EDTA. and 5 mM boric acid. The leader RNA, detected by autoradiography, was eluted from the gel overnight in buffer containing 500 mM ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA. and 0.1% SDS. Acrylamide fragments were removed by filtration of the eluate through a 0.22-pLm Milex-GV filter unit, and the leader RNA was precipitated by the addition of three volumes of 95% ethanol. The precipitate was collected by centrifugation at 12,000 x g for 30 min and was suspended in I x SET. After one extraction with phenol and two extractions with chloroform-isoamyl alcohol (24:1 [vol/vol]), the leader RNA was repeatedly precipitated with ethanol (at least three times), and the final RNA pellet was suspended in distilled water. The amount of leader RNA was determined both by relative incorporation of [32P]UMP and by optical density at 260 nm. The ratio of absorbance at 260/280 nm was typically 1.9 to 2.1. The yield was approximately 1 p.g of leader RNA per 2 mg of virus used for transcription. Samples stored at -80°C showed no degradation within 2 months as determined by gel electrophoresis. DNA-dependent transcription with a HeLa cell extract. As described previously (29) , HeLa cells were grown in DMEM supplemented with 5% calf serum and 10% tryptose phosphate broth (GIBCO) to a density of 5 x 105 cells per ml, and a transcriptionally active extract was prepared from these cells by the 89 VOL. 48, 1983 on May 7, 2020 by guest http://jvi.asm.org/ Downloaded from 90 GRINNELL AND WAGNER method of Manley et al. (24). The DNA templates used for transcription were the plasmid pBR322-Bal-1-E recombinant of the adenovirus (Ad-2) DNA late promoter (LP; originally obtained from P. A. Sharp, Massachusetts Institute of Technology) and a pBR322 recombinant of the adenovirus-associated (VA) RNA genes (originally a gift from D. F. Bogenhagen, Carnegie Institute). Both recombinant plasmids were propagated in Escherichia coli HB101 and were purified by CsCI gradient centrifugation. Before transcription, the Ad-2 late promoter was cleaved with the restriction endonuclease Sma I in the buffer specified by the manufacturer (Bethesda Research Laboratories, Bethesda, Md.). The DNA templates were transcribed in a reaction mixture containing 3 mM MgCI2, 15 mM ammonium sulfate, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 1 mM each of ATP, CTP, and GTP, 0.1 mM UTP containing 10 ,uCi of [o-32P]UTP (410 Ci/mmol) and 20% (vol/vol) of HeLa cell extract. After 30 min at 310C, reactions were stopped by adding 10 volumes of 1 x SET and extracting once with SET-saturated phenol. After adding 30 ,ug of yeast carrier RNA, the transcription products were precipitated from the aqueous phase by the addition of sodium acetate to 0.3 M and three volumes of ethanol. The precipitate was collected by centrifugation at 10,000 x g, suspended in 8 M urea containing 0.1% bromophenol blue and xylene cylanol, and the transcription products were separated on an 8 M urea-8% polyacrylamide slab gel in the Tris-borate buffer described above. Isolation of cellular RNA. Total cellular RNA was isolated from infected and uninfected BHK-21 and MPC-11 cells by a modification of the method of Wold et al. (45) . Cells were washed in TE buffer (10 mM Tris-hydrochloride (pH 8.0)-10 mM EDTA) and centrifuged at 1,000 x g for 10 min. The cell pellets were suspended in 10 volumes of TE buffer containing 1% SDS and 500 ,ug of proteinase K per ml and were incubated at 37°C overnight. The cell lysate was extracted twice with phenol-chloroform-isoamyl alcohol (24:24:1 [vol/vol/vol]), and the nucleic acids were precipitated with ethanol in the presence of 0.2 M sodium acetate. After centrifugation, the nucleic acid pellet was lyophilized and suspended in TE buffer containing 50 mM sodium acetate. To remove contaminating DNA, samples were digested for 30 min at 37°C with 50 ,ug of pancreatic DNase (Worthington Diagnostics, Freehold, N.J.; RNase-free) per ml, followed by phenol extraction and precipitation with ethanol. Alternatively, samples were mixed with CsCI to a final density of 1.56 g/ml and centrifuged at 240,000 x g for 24 h. The CsCI-pelleted RNA was suspended in SET containing 0.1% SDS and was precipitated with ethanol. The ratio of absorbance at 260/280 nm was typically 2.0 to 2.1. Preparation of 3'-end-labeled VSV RNA. The 42S minus-stranded genome was extracted from purified virus by incubating with 0.1% Triton X-100-1% SDS-500 j±g of proteinase K per ml for 1.5 h at 310C. The mixture was extracted twice with SET-saturated phenol, and RNA was precipitated with ethanol. The 42S RNA was specifically labeled by ligation of [5'-32P]cytidine-3',5'-bisphosphate ([32P]pCp) to its 3'-end by T4 RNA ligase (Bethesda Research Laboratories). The 20-,ul reaction mixture contained 3 to 5 jig of 42S RNA, 50 mM N-2-hydroxyethylpiperazine-N'-2-eth-J. VIROL. anesulfonic acid (HEPES) (pH 8.1), 10 mM MgCl, 3 mM dithiothreitol, 10 ,uM ATP, 10% dimethyl sulfoxide, 10 ,ug of bovine serum albumin (Boehringer Mannheim Corp., New York, N.Y.; nuclease-free) per ml, 250 ,uCi of [32P]pCp (3,000 Ci/mmol), and 10 U of T4 RNA ligase. After 18 h at 4°C, 30 ,ug of yeast RNA carrier was added, and the 42S RNA was precipitated with ethanol. The precipitated RNA was collected, dissolved in SET, and centrifuged through a 5 to 25% sucrose gradient (10 mM Tris-hydrochloride-1 mM EDTA-0.15 M NaCI-0.5% SDS) for 105 min at 125,000 x g. The peak at 42S was collected, and the RNA was recovered by ethanol precipitation. The specific activity of the end-labeled probe was typically 2 x 10' to 5 x 105 cpm/,ug of 42S RNA. Detection of VSV leader RNA by hybridization. The 3'-end-labeled virion RNA was hybridized to leader RNA essentially as described by Leppert et al. (20). Briefly, samples of cellular RNA (approximately 12 ,ug) from infected and uninfected cells were adjusted to contain 10 mM Tris-hydrochloride (pH 8.0) and were boiled for 5 min in the presence of approximately 2 x 104 cpm of 42S RNA probe. In preparing the RNA samples, identical numbers of cells and volumes were used to ensure that differences in hybridization would reflect differences in leader RNA content. After boiling, samples were cooled on ice, adjusted to 0.5 M NaCI, and incubated at 65°C. After 4 h, the samples were digested with 30 ,ug of pancreatic RNase A (Worthington) per ml and 60 U of RNase Ti (Bethesda Research Laboratories) per ml for 30 min at 37°C. SDS and pronase were then added to final concentrations of 0.4% and 200 ,ug/ml, respectively, and the incubation was continued for 30 min. After adding 30 jig of yeast carrier RNA and adjusting the mixture to 0.3 M with sodium acetate, the double-stranded RNA was precipitated at -80°C with three volumes of 95% ethanol. The resulting precipitate was collected and lyophil lized, and the RNAs were separated by electrophoresis on a 10% polyacrylamide gel. The number of genome equivalents of leader in infected cell RNA samples was determined from the specific activity of the probe and the counts per minute in the doublestranded leader. In all experiments, the amount of endlabeled probe used for hybridization was determined to be in excess.