Temperature-sensitivity of the replication of rabies virus (HEP-flury strain) in BHK-21 cells I. Alteration of viral rna synthesis at the elevated temperature

VIROLOGY

188,524-532 (1992)

Temperature-Sensitivity

of the Replication of Rabies Virus (HEP-Flury Strain) in BHK-21 Cells

I. Alteration of Viral RNA Synthesis at the Elevated Temperature AKIHIKO KAWAI’ AND KENJITAKEUCH12 Faculty of Pharmaceutical

Sciences, Kyoto University,

Received April 29, 199 1; accepted

Sakyou-ku 606, Kyoto, Japan

October 25, 199 1

We investigated the nature of temperature sensitivity of the HEP strain of rabies virus. After initial incubation for appropriate period (more than 12 hr) at the permissive temperature (36-37’), incubation temperature of the rabies virus infected cultures was shifted to a nonpermissive temperature (39.5-40.5”). Upon the upshift, virion production was ceased, but the rate of viral RNA synthesis was greatly increased and reached almost 10 times that of 36”infection within 8-l 0 hr, and then the activity quickly decreased together with the onset of accelerated CPE. Little or no 42s genome-sized RNA was produced at the elevated temperature, and almost all RNAs produced in large amounts seemed to be viral mRNAs and were shown to be functional in the cell-free translation system. Consistent with these observations, the viral ribonucleoprotein complex isolated from the temperature-upshifted culture was associated with relatively large amounts of small sized RNAs, which might reflect their increased transcriptive activity. These observations suggest that the viral RNA polymerase itself is not temperature-sensitive and the temperature-induced defect may reside in the regulatory factor which plays a role in turning on the synthesis of viral genome-sized RNA. @1992 Academic

Press, Inc.

When compared with VSV, the rabies virus has some disadvantages for being studied more extensively on the molecular level, that is, it takes much more time and greater effort for assaying the infectivity and propagating the virus in large amounts owing to its very slow growth and much lower yield of progeny. Besides, handling of rabies virus requires more care and caution than for VSV because of its inherent lethal neurovirulent nature. Analysis of temperature-sensitive (ts) mutants of VSV has proved to be a helpful method for understanding the roles of viral proteins in the replicative process (Wagner, 1975; Pringle, 1982, 1987). Such an approach, however, has been unsuccessful in the case of rabies virus. A lot of ts-mutants were isolated from the CVS strain of rabies virus by Wistar and French researchers and were classified biochemically or immunologically into three groups. The complementation tests, however, have not yet been successful between these mutants (Clark and Koprowski, 197 1; Saghi and Flamand, 1979; Bussereau et al., 1982). As to the temperature sensitivity of the HEP-Flury strain of rabies virus, Kawai and Matsumoto (1982) observed peculiar aspects of the virus; when BHK-21 cells infected with the HEP strain were initially incubated at the permissive temperature for an appropriate period (more than 12 hr), upshift of the incubation temperature to a nonpermissive temperature (39.5 to 40.5”) induced the accelerated cytopathic effect (CPE), although the yield of infectivity was greatly reduced. They also found that this acceleration of CPE was blocked by defective interfering particle (Dl)-me-

INTRODUCTION Rabies and vesicular stomatitis viruses are typical members of the rhabdoviridae, but they differ from each other in many points, such as in their host ranges, pathogenesis, and others. Replicative processes of the rhabdovirus have been extensively studied for many years, but those studies were mostly of vesicular stomatitis virus (VSV) (Wagner, 1987). Although it has been supposed that the replicative mechanism in the cell of rabies virus resembles that of VSV, details of their replicative processes in the cell may be different, and several features in the replicative process are known which are not shared in common by both viruses. For instance, only rabies virus, but not VSV (1) requires as much longer period as 50-60 hr to replicate in the cell; (2) has a gene-like region (or a remnant gene) between the G and L genes in the genome, which might regulate expression of the L gene (Tordo et a/., 1986; Morimoto et a/., 1989); (3) has phosphorylated nucleoproteins (N protein); and (4) constantly forms large cytoplasmic inclusions which have been assumed to be the accumulation of nucleocapsids. Molecular events in rabies virus-infected cells have not been so extensively studied as VSV, accordingly, the detailed intracellular processes of rabies virus replication still remain obscure. ’ To whom reprint requests should be addressed. ’ Present address: National Institute of Health, Kamiosaki. Shinagawa-ku, Tokyo, Japan. 0042-6822192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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diated autointerference, and they successfully applied this phenomenon to the development of a sensitive assay system for rabies virus DI particles (Kawai and Matsumoto, 1982). But the temperature-sensitive nature of this strain is not yet clearly understood. In this report, we describe studies on the molecular events in HEP virus-infected BHK-21 cells at the high temperature, especially concerning the effects of temperature upshift on the viral RNA synthesis, and discuss the possible temperature-sensitive function(s) of the virus. MATERIALS AND METHODS Viruses Rabies virus: the HEP strain (clone 2150-14; Kawai et al., 1975) was used throughout this study. Practically DI-free virus stocks were prepared by one passage of plaque isolates through a BHK-21 cell culture. Culture fluids were harvested after 2-3 days of incubation at 35.5 to 36’, clarified by low speed centrifugation (2000 rpm) for 15 min, and were stored at -20” until use. The New Jersey serotype of VSV was used for comparative studies on the effect of heat shock. VSV stocks were also prepared by one passage of plaque isolates through BHK-21 cells. Cell culture BHK-21 cells were used throughout this study. Cells were usually propagated in Eagle’s MEM supplemented with 10% tryptose phosphate broth (TPB; Difco Co., Ltd.) and 5% bovine serum. For labeling cells with [3H]uridine, HEPES-buffered Eagle’s MEM was used, which contained 25 mlt/l of HEPES (instead, the concentration of bicarbonate-Na was reduced to 1.4 mM). Infectivity assay Infectivity of rabies virus was assayed by plaque formation using agarose-suspended BHK cells (Sedwick and Wiktor, 1967) with some modifications; first, agarose-suspended BHK-21 cell layers were prepared on the preformed bottom layer of agarose-containing Eagle’s medium in 6-cm plastic dishes (Falcon dish 1007). Then, 0.2 ml of virus inoculum was poured onto the top of each plate and spread over the cell layer by gentle tilting to all quarters. Inoculated plates were incubated at 36” in a CO, incubator. On the 7th day, neutral red-containing agarose medium (3 ml) was overlayed, and plaques were counted after further incubation for 4 hr. Instead of BHK-21113s which Sedwick and Wiktor used (Sedwick and Wiktor, 1967) we adopted BHK-21 cells which had not been acclimated to the suspension culture, because the BHK-21 cells

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used in our laboratory were more effective for plaque formation by the rabies virus, that is, the morphology of the plaques was clearer and plaquing efficiency was somewhat higher. Virus purification The procedures for viral purification were the same as those described before (Kawai, 1977). Measurement of the rate of viral RNA synthesis The rate of viral RNA synthesis was determined by measuring the incorporation of [5-3H]uridine in the presence of actinomycin D into the acid-insoluble fraction. Cells were pretreated with actinomycin D (5 pg/ ml) for 30 min and then [5-3H]uridine (15-25 &i/ml) was added to the culture medium and incubated for 60 min. Radioactivity incorporated into the acid-insoluble fraction was determined as follows: after removal of the culture medium, cells were washed with PBS three times and were lysed by 2% sodium dodecyl sulfate (SDS) and precipitated with 7% trichloroacetic acid (TCA). The acid precipitates were collected onto GF/C glass filters (Whatman Ltd., England) and washed with cold 7% TCA three times and then with 99% ethanol, after which the filter papers were dried. The filter papers were then put into vials containing a toluene scintillator and subjected to radioactivity counting by a liquid scintillation spectrophotometer (Tri-Carb 3OOC, Packard scintillation counter; Packard Instrument Company, Inc., IL). Velocity sedimentation analysis of the viral RNAs extracted from infected cells Infected cells were labeled with [5-3H]uridine in the presence of actinomycin D (5 pg/ml). After cells were washed with PBS twice, whole RNA was extracted as follows: the cells were treated with 0.5 ml NAE buffer supplemented with SDS (0.5%) and polyvinylsulfate (5 Fg/ml), centrifuged in a Microfuge (Beckman model B) at 10,000 g for 5 min, and then the supernatants (RNA extract) were harvested. The extracts were then analyzed by velocity sedimentation as follows: the RNA extract was put on a 5-20% (wt/vol) sucrose gradient in SDS-NAE buffer (4.5 ml) and centrifuged in an RPS-40 rotor (Hitachi Co., Ltd., Tokyo) at 120,000 g for 120 min. The sucrose density gradients were fractionated by the drop collection method. To each fraction was added an equal volume of 10% TCA, and the TCA-insoluble precipitate was collected on 2.4-cm Whatman GF/C filter papers, washed three times with 5% TCA, and dried. The radioactivity of each filter was determined as described before. As markers for the velocity of sedimen-

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tation, [14C]uridine-labeled ribosomal RNAs were mixed with the samples before centrifugation. Velocity sedimentation analysis of ribonucleoprotein (RNP)-associated RNAs Infected cells (5 X lo6 cells) were harvested with a rubber scraper and homogenized in a Dounce-type stainless steel homogenizer (Kontes Glass Company, NJ) using 4 ml of RSB buffer containing 0.5% NP-40 (Bethesda Research Laboratories, MD) centrifuged at 2000 rpm for 5 min, the supernatant was harvested, and then sodium deoxycholate (DOC) was added at a final concentration of 1% and centrifuged at 10,000 g for 20 min. The supernatant was again ultracentrifuged at 180,000 g for 90 min using a Spinco SW50.1 rotor (Beckman Instruments Inc., CA). The pellet was dissolved into 0.5 ml NAE buffer supplemented with 5 mM EDTA and 0.5% SDS. The analysis of RNAs by velocity sedimentation was the same as that described above with one exception; centrifugation was performed at 149,000 g for 100 min using a Spinco SW50.1 rotor. Fractionation of the sucrose density gradient was also carried out as described above, and the fractions obtained were TCAprecipitated and assayed for radioactivity. [14C]uridinelabeled ribosomal RNAs were used as a marker of sedimentation velocity. Preparation of mRNA from infected and uninfected BHK cells The mRNA used in the cell-free translation system was extracted from both infected and uninfected BHK cell cultures according to the combination of methods by Chirgwin et a/. (1979) and Glisin et a/. (1974). The cell pellet (2 x lo* cells) was lysed with 4 ml of cold guanidinium isothiocyanate solution in a Kontes glass Dounce-type homogenizer (7-ml) and was subjected to 10 strokes of homogenization with a loose-type (Atype) pestol to disrupt the DNA. The homogenate was layered onto 1.O ml of a CsCI-0.1 M EDTA cushion in a cellulose nitrate centrifuge tube (the tube was previously washed with 20% SDS solution and cleaned with autoclaved distilled water), and centrifuged with an RPS-40 rotor at 120,000 g overnight at 20”. The pellet was then dissolved in 2 ml of guanidine hydrochloride solution plus 0.1 mhll EDTA with the aid of a magnetic stirrer and then centrifuged at 10,000 g for 10 min, and the supernatant was mixed with 0.025 vol(50 ~1)of 1 /M acetic acid and then with 0.5 vol (1 ml) of absolute ethanol, mixed by a Vortex mixer, and left overnight at -20”. The RNA precipitate was collected by high speed centrifugation (10,000 g for 10 min at - 10’) and resuspended in 2 ml of guanidine-HCI solution. The RNA was reprecipitated again by adding 0.025 vol of 1

Macetic acid and 0.5 vol of absolute ethanol. The final precipitate was washed with cold ethanol, dried in vacue, and was dissolved in 100 ~1of autoclaved distilled water and was stored at -70” until use. The concentration of RNA was estimated by measuring the absorbance of the appropriately diluted sample at 260 nm (l&, = 45 pg RNA/ml). Cell-free protein synthesis The activity of the mRNA was assayed by the cellfree translation system using a rabbit reticulocyte lysate which was obtained from rabbits recovering from anaemia (Schreier and Staehelin, 1973). Incubation mixtures were prepared according to Shida and Dales (1982) with slight modifications: (1) the concentration of magnesium acetate was increased from 30 PM to 2 mM, the addition of 3’,5’-cyclic AMP was omitted, and [4,5-3H]leucine was used as the labeled compound instead of [36S]methionine. Following incubation for 2 hr at 30”, the polypeptide products were solubilized with the lysis buffer for SDS-PAGE and analyzed by SDSpolyacrylamide gel electrophoresis (PAGE)and fluorography. SDS-PAGE and fluorographic analysis SDS-PAGE was performed as described elsewhere (Kawai, 1977). After the electrophoresis, acrylamide gels were processed for fluorography (Bonner and Laskey, 1974; Laskey and Mills, 1975). In brief, the gel was impregnated with PPO (2,5-diphenyloxazole) in dimethyl sulfoxide (DMSO), washed with distilled water (to remove DMSO), and was dried in vacua. The dried gel was placed in contact with X-ray film (Kodak X0 Mat RP or S film) for an appropriate number of days at -70”. Chemicals and solutions Guanidinium thiocyanate solution contained: 4 IM guanidinium thiocyanate, 0.5% sodium N-lauroylsarcosine, 25 rnM sodium citrate (pH 7.0) 0.1 M Z-mercaptoethanol, and 0.1% antifoam (Sigma Antifoam A). The guanidinium thiocyanate used was Fluka purum grade guanidinium thiocyanate (Tridom, Inc., Hauppage, NY). Guanidine hydrochloride solution contained: 7.5 h/l guanidine-HCI, 25 mM sodium citrate and 5 mM DTT, pH 7.0. RSB buffer contained: 1 mM NaCI, 10 mll/l Tris-HCI, and 1.5 m&I MgCI, (pH 7.4). Phosphate-buffered saline was composed of: NaCI, 0.137 IU; KCI, 0.0027 l\/I; Na,HPO,, 0.008 M; KH,PO,, 0.0015 M; CaCI,, 0.009 M; MgCI,, 0.005 M; pH 7.4 at 25”. NAE buffer was composed of: NaCI, 0.05 n/r; sodium acetate, 0.01 M; EDTA, 0.001 M; pH 5.1. SDSNAE buffer was NAE buffer which was supplemented

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FIG. 1. Temperature-sensitivity of rabies virus replication. BHK-21 cell monolayers prepared in 35.mm plastic dishes were infected with rabies virus (HEP strain) at an m.o.i. of 3 PFWcell and were incubated at 36”. At 6-or 12.hr intervals as indicated by arrows, a pair of cultures were taken out and transfered to 40.5” incubator without washing the cells. Culture fluids of the temperature-upshifted cultures were harvested at the 48th hr of infection and subjected to the infectivity assay by plaque formation. Culture fluids of the infected control cells incubated at 36” alone were also harvested at each time indicated by an arrow as well as at 0 and 48 hr. Symbols: 0, incubated at 36” alone; 0, exposed to 40.5”.

with 0.2% SDS. Actinomycin D and Antifoam A were acquired from Sigma Chemical Co. (St. Louis, MO). Radioactive compounds [5-3H]uridine (sp act 28-30 Ci/mmol), [2-14C]uridine (sp act 55 mCi/mmol), and L[4,5-3H]leucine (sp act 130-145 Ci/mmol) were purchased from Amersham International plc (Buckinghamshire, England). RESULTS Temperature-sensitivity

of rabies virus replication

We investigated effects of temperature upshift on the rabies virus infection in culture under the one step virus growth conditions. When infected cells were incubated at 40.5” from the beginning of infection, neither virus replication nor the cytopathic effect (CPE) was observed. After initial incubation for 12 to 15 hr at the permissive temperature (36”) exposing the infected cells to the high temperature (39.5 to 40.5”) resulted in a great reduction in the yield of infectivity (Fig. l), but the CPE was accelerated and increased (data not shown). The acceleration of CPE was dependent on duration of preincubation at 36”. When infected cells were preincubated, for instance, for 36 hr at 36”, the onset of cytolysis began around 12 hr after

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the temperature shift and complete cytolysis was observed within 24 hr. On the other hand, in the 36”incubated cultures the onset of CPE began mostly 48 to 60 hr after virus infection, but complete CPE was not observed even on the 5th day. Uninfected BHK-21 cells could be propagated normally at 40.5” for a period of at least three successive subcultures. The New Jersey serotype of VSV used in our laboratory replicated normally in BHK-21 cells at 40.5” (data not shown). A thermal inactivation experiment showed that the half-life of rabies virus at 36 and 40.5” was about 18 and 7.5 hr, respectively, suggesting that thermal inactivation was not only a major factor which contributed to the great reduction in the yield of infectivity at 40.5”. Next, we compared virion production at the permissive and nonpermissive temperatures by the velocity sedimentation as described under Materials and Methods. Figure 2 shows a representative of the experiments. No viral peak was produced by the virus sample which was recovered from the cultures incubated at 39.5”. On the other hand, a clear single viral peak was observed when the virus samples were examined which were obtained from the cultures incubated at the permissive temperatures. These observations suggest that the reduced virion production at the elevated temperature was a major cause of the great decrease in the yield of infectivity, and the production of abnormal or incomplete noninfectious particles was not the case. Effects of temperature upshift on viral RNA synthesis Next, we studied the temperature-sensitive function(s) required for rabies virus replication, mainly by analyzing the effects of temperature upshift on viral RNA synthesis. In our HEP virus-infected BHK-21 cell cultures, virus growth curves reached a plateau level at 50-60 hr after infection, and the rate of viral RNA synthesis increased at 36-40 hr, reaching a plateau at around 45 to 50 hr (unpublished data). Accordingly, the temperature upshift was done mostly at around the 40th hr of infection in the following experiments. First, we studied the effect of temperature upshift on the viral RNA synthesis. Both infected and control cultures were treated in advance with actinomycin D (5 pg/ml) for 30 min before the temperature shift. The incubation temperature was shifted to 40.5”, and both the yield of infectivity and the rate of rH]uridine incorporation into the acid-insoluble fraction were determined at each time as indicated in Fig. 3. Soon after the temperature upshift, production of infectivity was ceased. The rate of [3H]uridine incorporation into the acid-insoluble fraction, however, was greatly in-

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0.5% SDS, and the lysates were then subjected to the velocity sedimentation analysis. As shown in Fig. 4, the radioactivity incorporated into the acid-insoluble materials of the 40.5”-incubated infected culture was almost 10 times that of the 36”-incubated infected culture. However, sedimentation profile of the radioactive materials produced at 40.5“ resembled that of the 36“ infection, except for the absence of a peak denoting 42s genome-sized RNA. Majority of the radioactivity was found in the 12-328 region which corresponded to viral mRNA’s (Holloway and Obijeski, 1980). Almost the same results were also obtained when the [3H]uridine-labeled RNAs were subjected to the agarose gel electrophoresis and fluorographic analysis, the

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FIG. 2. Comparison of viral particle production at various temperatures. BHK cells cultures prepared in 30 Roux’s culture bottles were infected with rabies virus (at an m.o.i. of 2 PFU/cell). The cultures were divided into three groups; 10 bottles were incubated at 33” and another 10 bottles were incubated at 37” until the 72nd hr. The remaining 10 bottles were incubated first at 37” for 15 hr and then exposed to 39.5” until the 48th hr, when CPE reached grade (3+). Culture fluid collected from each group was treated with 6.5% polyethylene glycol (6000) to precipitate the virus as described elsewhere (Kawai, 1977). Concentrated viral suspensions were then purified by applying them to lo-45% sucrose density gradients. After centrifugation, each sucrose density gradient was fractionated into 30 fractions, whose optical densities were determined by a spectrophotometer at 260 nm. Fractions were numbered from the bottom (left) to the top (right). Symbols: 0 and A, incubated at 33 and 37”, respectively; l , incubated at 39.5” from the 15th hr.

creased in the rabies virus infected cultures, but not in the uninfected control cultures. The [3H]uridine uptake reached a plateau level about 8-10 hr after the temperature shift, and the rate of RNA synthesis at 40.5” was 6-10 times increased compared with that at 36’. Then, the rate quickly decreased with the onset of CPE (data not shown). Similar findings were also observed when the temperature upshift was performed at the earlier or later time of infection (data not shown). Next, the nature of [3H]uridine-labeled materials produced at the elevated temperature was examined. After 41 hr of incubation at 36”, the infected cultures were divided into two groups, half of which were shifted to 40.5” incubation and the other (control group) was incubated at 36” as before. After further incubation for 5 hr, both cultures were labeled with [3H]uridine (10 &i/ml) for 2 hr in the presence of actinomycin D (5 pg/ml) at each temperature. Cells were harvested and lysed with a lysing buffer containing

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a ,-lo E 5 h 5

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FIG. 3. Effects of the temperature upshift on viral RNA synthesis. BHK cells were infected with rabies virus at an m.o.i. of 2.5 PFU/cell and incubated first in ordinary culture medium at 36”. At the 40th hr of infection, culture medium was replaced by HEPES-buffered Eagle’s MEM medium (see Materials and Methods). The cultures were then divided into two groups, one of which was then incubated at 40.5’, and the other was incubated again at 36” as before. Uninfected BHK cell cultures were treated in the same way. At each time as indicated in the figure, two pairs of each culture were used to determine the yield of infectivity and [3H]uridine incorporation into the acid-insoluble materials. The latter was assayed as follows: [53H]uridine (20 &i/ml) was added to the cultures and incubated further for 60 min. Actinomycin D (5 pg/ml) was added to the culture 30 min before the addition of rH]uridine. After removal of the culture medium, cells were washed with PBS three times and then lysed with 2% SDS. The lysates were precipitated with 7% trichloroacetic acid (TCA) and collected onto GF/C glass filter papers, washed with cold 7% TCA three times and once with 99% ethanol, and then dried. Radioactivity was determined using a toluene-based scintillator and a liquid scintillation spectrophotometer. Symbols: 0, l , yield of infectivity at 36 and 40.5’, respectively; A, A, t3H]uridine incorporated by infected cells at 36 and 40.5”, respectively; q , a, [3H]uridine incorporated by uninfected cells at 36 and 40.5’, respectively.

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Methods), lysed with SDS-NAE buffer, and then subjected to the velocity sedimentation analysis. Figure 5 shows the sedimentation profiles of the RNP-associated RNAs. RNAs in the RNPs that were isolated from the 36” culture displayed a typical profile, in which the majority of radioactivity was found in the 42s region and only small amounts were detected in the slow sedi5,r

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FIG. 4. Velocity sedimentation profile of viral RNAs. BHK cells (2 X 10?3.5-cm plastic plate) were infected with rabies virus (m.o.i. = 3 PFU/cell) and were incubated at 36”. At the 41st hr, culture medium was replaced by HEPES-buffered Eagle’s MEM, and the cultures were divided into two groups, one of which was then incubated at 40.5”, and the other (control) was incubated again at 36” as before. After further incubation for 4.5 hr, both cultures were treated for 30 min with actinomycin D (5 fig/ml). Then, [5-3H]uridine (10 &i/ml) was added to the cultures, which were then incubated again for 2 hr at each temperature. After being washed twice with PBS, cells were harvested and whole RNA was extracted by simple extraction with 0.5 ml of NAE buffer supplemented with 0.5% SDS and polyvinylsulfate (5 rg/ml). The extracts were then centrifuged at 10,000 g for 5 min, and the supernatants were harvested. The RNA in the lysates was analyzed by velocity sedimentation through a 5-20% (wt/vol) sucrose density gradient as described under Materials and Methods. As markers of the sedimentation velocity assay, [‘4C]uridinelabeled ribosomal RNAs were mixed with the sample before centrifugation. After centrifugation in a Hitachi RPS-40 rotor at 120,000 g for 120 min, the gradients were divided into 31 fractions by drop collection, and the radioactivity in each fraction was determined as described under Materials and Methods. Symbols: 0, 36”; 0, 40.5”.

method which was described by Waterborg and Matthews (1984) (data not shown). These observations indicate that the replication of viral genome-sized 42s RNA was suppressed at the elevated temperature but, instead, viral transcription was greatly enhanced. To confirm the absence of 42s RNA synthesis at the elevated temperature, we next examined the ribonucleoprotein (RNP)-associated[3H]uridine-labeled RNAs. Five hours after the temperature upshift, infected cells were labeled with [3H]uridine in the presence of actinomycin D as described in Fig. 4. After further incubation for 2 hr, RNPs were isolated (see Materials and

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FIG. 5. Velocity sedimentation profile of RNP-associated RNAs. BHK cell cultures (5 X lo6 cells/6cm plastic plate) were infected with rabies HEP virus and were incubated at 36”. At the 40th hr the cultures were divided into two groups, half of which were then exposed to 40.5’, and the remaining half were incubated at 36” as before. Six hours after the temperature upshift, the cultures were labeled with [5-3H]uridine (20 &i/ml) for 2 hr in the presence of actinomycin D, and then RNPs were isolated as described under Materials and Methods. Infected cells were harvested with a rubber policeman and homogenized in a Dounce-type stainless steel homogenizer with 4 ml of RSB buffer containing 0.5% NP-40. The lysates obtained were centrifuged at 2000 rpm for 5 min and the supernatants were harvested and treated with 1% sodium deoxycholate and centrifuged again at 10,000 g for 20 min. The supernatant was then ultracentrifuged in a Spinco SW50.1 rotor at 180,000 g for 90 min. The pellets of RNP were lysed with 0.5 ml of NAE buffer supplemented with 0.5% SDS and 5 mM EDTA. RNAs in the lysates were then analyzed by velocity sedimentation through a 5-20% (wt/ vol) sucrose density gradient. [2-Yluridine-labeled ribosomal RNAs were added to the sample before centrifugation as a marker of velocity sedimentation. After centrifugation in a Spinco SW50.1 rotor at 149,000 g for 100 min, the gradient was fractionated into 32 fractions by a drop collection method. Radioactivity in the acid-insoluble materials of each fraction was determined as described under Materials and Methods. 0, 36”; 0, 40.5”.

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menting fractions. These slow sedimenting fractions (the peak was around 5s) might be comprised of nascent viral mRNAs, since RNP has been supposed to serve for the template of viral RNA synthesis. On the other hand, the RNPs isolated from the temperatureupshifted infected cultures contained little or no 3H-labeled 42s RNA, but contained large amounts of small sized RNAs sedimenting at around 5-105, which might reflect the increased viral transcription.

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Assay of the mRNA produced under the temperature-upshifted conditions The sedimentation profile of the RNA produced at the elevated temperature indicates that all species of viral mRNAs were produced even at the high temperature (Fig. 4). Accordingly, we tested whether they were functional or not by using the cell-free translation system. Translation products were labeled with [3H]leutine and were subjected to SDS-PAGE and fluorography. As shown in Fig. 6, electrophoretic patterns of the cell-free translation products demonstrate that the mRNAs extracted from the 40.5”-upshifted infected culture were functional and directed the production of all species of the viral proteins as done by mRNAs which were obtained from the 36” culture. The amount of viral proteins in the cell-free translation products of mRNAs from the 40.5“ culture was larger than that from the 36” culture by about 2.5 times. The degree of increase (2.5 times) does not seem to be small, because quite a large amount of viral mRNAs had already been produced in the cells at the time of temperature upshift. In our cell-free translation system, however, G and Go proteins were not as abundantly produced as other viral proteins (N, M, ) and M, proteins), probably due to a lack of a microsomal system required for glycoprotein synthesis. Further, since the high molecular weight viral protein (L) was produced only in a small amount in this system, it was not possible to compare the relative amounts of L mRNA produced at both temperatures. An unusual polypeptide (indicated as p48 in Fig. 6) of estimated molecular weight 48 kDa was also detected in the cell-free translation products by using the mRNA which was obtained from either the 36”incubated or temperature-upshifted infected cultures. p48 was shown to be a truncated form of viral N protein by immunoprecipitation with the anti-N protein antibody(unpublished observation). In this experiment, besides the bands of viral proteins, many polypeptide bands of cellular origin were seen on the background of each lane, whose profile and density were almost the same as those seen on the lanes for the mRNAs from the uninfected 36” culture.

FIG. 6. Cell-free translation assay of viral mRNAs. Infected BHK cells were prepared in 10 Roux’s culture bottles (1 X 10’ cells/bottle). Virus was infected at an m.o.i. of 3.6 PFU/cell, and the cultures were incubated at 36”. At the 40th hr, of infection, 5 bottles were exposed to 40.5” and incubated for 10 hr. The other 5 bottles were incubated at 36” as before until the 50th hr. Cells were harvested with a rubber policeman and subjected to procedures for the extraction and purification of mRNAs as described under Materials and Methods. The concentration of RNA was estimated by absorbance at 260 nm. Purified mRNA samples were assayed for template activity in a cell-free translation system (see Materials and Methods). Reaction mixtures were incubated for 2 hr at 30’. Polypeptide products were labeled with L-[4,5-3H]leucine (5 &i/reaction tube) and were analyzed by SDS-PAGE and fluorography. Lanes 1, 2, and 3, mRNA from infected cells incubated at 36” (1.6, 3.2, and 7.9 rg RNA per reaction tube, respectively); lanes 4, 5, and 6. mRNA from infected cells exposed to 40.5” for 10 hr (1.6,3.2, and 7.9 rg RNA per reaction tube, respectively); 7 and 8, mRNA from uninfected BHK cells (3.2 and 7.9 pg RNA per reaction tube, respectively).

DISCUSSION We have studied temperature sensitivity of the HEP strain of rabies virus. Upshift of incubation temperature to a nonpermissive one (40.5”) brought about, besides stopping of the virion formation, suppression of the viral genome-sized (42s) RNA synthesis, while viral mRNA synthesis was greatly increased. The results shown here indicate that the viral factor which is involved in turning on the viral genomic RNA synthesis is temperature sensitive, but the viral RNA synthetic apparatus itself is not. RNA synthesis of the rhabdovirus has been thought to be performed in close association with RNP, which comprises of a viral genomic RNA and N proteins and serves as the template for both the viral genomic RNA synthesis and transcription (Huang and Manders,

TEMPERATURE SENSITIVITY OF RABIES VIRUS

1972; Soria et al., 1974). Accordingly, to discuss the temperature sensitivity of rabies virus genome RNA synthesis, we should consider at least three viral elements involved: (1) activity of the viral RNA polymerase (or, L and NS proteins), (2) activity of the RNP which serves for the template of genomic RNA synthesis, and (3) availability of functional viral proteins (such as N, and possibly M, (NS) and L proteins) required for continued genomic RNA synthesis. Among these elements, the first and second ones do not seem to be the matter, because the viral transcription of the HEP virus was not inhibited but rather stimulated at the high temperature, and apparently functional mRNAs were produced. But, a certain regulatory domain of the RNA polymerase or other viral regulatory factor which is involved in turning on the genome RNA synthesis seems to be impaired at the high temperature. It has been known that continued supply of viral N protein is necessary for the genomic RNA synthesis of the rhabdovirus. N proteins bind to a newly synthesized viral RNA to form a new RNP (Wertz and Levine, 1973; Soria et al., 1974; Hill et al., 1979). In the VSV-infected cells, cycloheximide inhibited only the replication of genomic RNA, but not the transcription, which is ascribed to the interruption of continued supply of N protein (Perlman and Huang, 1973; Wertz and Levine, 1973; Palma et a/., 1974; Patton et a/., 1984). In the HEP virus-infected cells, all species of viral mRNAs seem to be produced at the elevated temperature (Fig. 4) suggesting that the continued supply of viral proteins (N and possibly L and M, proteins) required for the viral RNA synthesis is apparently kept. Our preliminary experiments suggested that all species of known viral proteins were produced at least for several hours after the temperature upshift (unpublished observation). In addition to the regular viral proteins, however, a truncated form of N protein (referred to as p48 in this paper) was also transiently produced at the elevated temperature. Further investigations are in progress to elucidate the possible role of this unusual polypeptide in the disturbed viral RNA synthesis. Among many studies on the temperature-sensitive VSV mutants, similar observations have been reported for certain ts-mutants which showed the defective viral RNA replication at a nonpermissive temperature (Unger and Reichmann, 1973; Perlman and Huang, 1974; Combard et a/., 1974). VSV tsG41, one of the group IV ts-mutants which has a defect on the N protein, is unable to synthesize genomic RNA at the nonpermissive temperature, while all species of viral mRNAs are synthesized almost normally (Unger and Reichmann, 1973). They also obtained similar results with a group II ts-mutant (tsG22) whose temperatureinduced impairment was located on the NS protein. Combard et a/. (1974) also reported similar observa-

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tions with other ts-mutants of group IV, which showed decreased N protein synthesis at a nonpermissive temperature. In contrast, VSV ts-1 14 (group I), having a temperature-sensitive defect on the L protein molecule, displayed apparently opposite behavior at a nonpermissive temperature, that is, viral transcription ceased at the nonpermissive temperature, while replication of its genomic RNA was not affected (Perlman and Huang, 1973, 1974). Recently, La Ferla and Peluso (1989) reported that an appropriate interaction was required between N and NS proteins for efficient replication of the VSV genome RNA. Accordingly, we could expect that a ts-mutation on either the N or NS protein molecule causes interruption of genomic RNA synthesis at the high temperature. And, from analogy with VSV ts-mutants of group IV (tsG41) and group II (tsG22), we suppose that the temperature-sensitive defect of HEP virus is located in either the N or M, (NS) gene product. Enhancement of viral transcription at the elevated temperature might be caused passively by turning off of the viral genome RNA synthesis (the viral RNA synthetic apparatus wholly works only fortranscription), by temperature-induced inactivation of a certain viral factor which regulates viral transcription, or by activation of the resting or inactive form of RNPs (possibly by mobilization of those in the cytoplasmic inclusion bodies). VSV matrix protein (M) is known to regulate viral transcription, and the cells infected with the virus having a temperature-sensitive lesion on the M protein overproduced viral mRNA at the nonpermissive temperature (Clinton et al., 1978; Martinet et a/., 1979). In addition, amino acids near the N-terminus of the protein are thought to be involved in the regulatory function of the protein (Pal et al., 1985; Ogden eta/., 1986). Matrix protein (MJ of the rabies virus may also play such a regulatory role in the viral RNA synthesis. And, if the HEP strain has also a temperature-sensitive lesion on the M, protein, the impairment of M, protein at the high temperature might be responsible for the increased viral transcription. But, even if one of these possibilities is the case, it would not be everything, because our unpublished observations indicate that the stimulation of viral RNA synthesis at the elevated temperature is dependent on the continued protein synthesis, that is, some proteinaceous factor(s) produced or accumulated at the high temperature is involved in stimulation of viral transcription. It is noteworthy that in the fluorographic analysis of the in vitro translation products, besides the bands of each viral protein, almost normal profiles of cellular protein bands were observed as the background (Fig. 6) even when we tested the mRNAs that were extracted from the infected cells whose cellular protein synthesis was shut off almost completely. This obser-

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vation suggests that the shut off of cellular protein synthesis in the rabies virus infected cells is not due to the breakdown of preformed cellular mRNA. ACKNOWLEDGMENTS Gratitudes are due to Mr. D. Mrozek for critical reading of the manuscript and to Miss I. Kodera for her assistance with manuscript preparation. This work was supported in part by Grant-in-Aid for Cooperative Research A (60304053; Dr. Y. Nagai, director) from the Ministry of Education, Science and Culture, Japan.

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