Nishioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A.74:2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia.
0022-538X/78/0025-0422$02.00/0 JOURNAL OF VIROLOGY, Jan. 1978, p. 422426 Copyright © 1978 American Society for Microbiology
Vol. 25, No. 1 Printed in U.S.A.
Alterations in the Protein Synthetic Apparatus of Friend Erythroleukemia Cells Infected with Vesicular Stomatitis Virus or Herpes Simplex Virus YUTAKA NISHIOKA AND SAUL SILVERSTEIN* Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received for publication 2 September 1977
We have compared the effects of infection with herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) on the protein synthetic apparatus of Friend erythroleukemia cells. Previous studies demonstrated that infection with HSV rapidly shuts off the synthesis of globin and other cellular polypeptides (Y. Nishioka and S. Silverstein, 1977, Proc. Natl. Acad. Sci. U.S.A. 74:2370-2374). In contrast to these findings, globin synthesis persists in Friend erythroleukemia cells infected with VSV. Physical measurements of the size of bulk-infected cell mRNA, using hybridization with polyuridylic acid, demonstrated that there was no detectable change in the size of mRNA's after infection with VSV. A comparison of the kinetics of hybridization of cytoplasmic RNA extracted from cells infected with either HSV or VSV with globin complementary DNA revealed that by 4 h postinfection with HSV only about 15% of the globin mRNA sequences remained, whereas there was no discernible change in the sequence abundance of globin mRNA in VSV-infected cells. Inhibition of cellular protein synthesis and selective translation of animal virus mRNA's occur during the course of productive infection by many different viruses. For example, infection by adenovirus (15), herpesvirus (26), vaccinia virus (10), mengovirus (1), poliovirus (20), and vesicular stomatitis virus (VSV) (29, 32) all lead to inhibition of translation of cellular mRNA to varying degrees. Little is known about the mechanism that selectively represses expression of cellular information without affecting the expression of viral genomic information. To assess the effects of infection with herpes simplex virus (HSV) on cellular mRNA's, we used the Friend erythroleukemia (FL) cell line as host. The addition of dimethyl sulfoxide causes these cells to synthesize large amounts of globin and the mRNA that codes for globin. The globin mRNA is well characterized (5, 17) and can be easily quantitated by hybridization to a complementary DNA (cDNA) copy (24). Likewise the globin polypeptide is easily recognized by its distinct electrophoretic mobility in polyacrylamide gels. In a previous publication, we demonstrated that very rapidly after infection with HSV globin synthesis was effectively shut off and globin mRNA was rapidly degraded (18). Because degradation of host mRNA does not appear to occur in cells infected with vaccinia virus (23) or picornaviruses (6, 8, 31), it is con422
ceivable that the degradation is unique to HSV. Alternatively, it may be a property of infected FL cells that are terminally differentiating and, therefore, may respond to virus infection differently from other cell lines. To examine the latter possibility, it is necessary to study the fate of globin mRNA during propagation of other viruses. We have approached this problem by investigating the effects of infection with VSV on FL cell metabolism because VSV has a broad host range (27) and is very different from HSV in that VSV has a RNA genome (11), a virionassociated RNA polymerase (2), and does not require the cell nucleus for growth (30). In addition, to our knowledge, the effect of VSV infection on host mRNA has not yet been critically examined. Induced or noninduced FL cells were infected with VSV at a multiplicity of infection (MOI) of 10, and growth was monitored by plaque assay on Vero cell monolayers. VSV multiplied poorly in FL cells. The increase in infectious virus was 50- and 10-fold in noninduced and induced FL cells, respectively (data not shown). The poor growth was not due to the existence of defective interfering particles because the same virus preparation showed 500- and 300-fold increases in PFU per milliliter when propagated in Vero cells and L cells, respectively (data not shown). Since HSV also grew poorly in FL cells (18),
NOTES
VOL. 25, 1978
the poor growth of VSV in FL cells is at least partially due to the capacity of FL cells, which is about two-thirds that of Vero cells in terms of protein content (unpublished observations) and becomes progressively smaller as globin induction proceeds (9). To examine the ability of VSV to inhibit cellular protein synthesis, two types of experiments were performed. In the first, FL cells infected at an MOI of 10 were pulse-labeled for 15 min at intervals postinfection. The kinetics of amino acid incorporation demonstrated that protein synthesis declined continually after infection; at 2 h postinfection, the rate of amino acid incorporation was 80% that of mock-infected controls, and by 4 h postinfection it had further declined to a level that was only 50% of the control value (data not shown). The rate of RNA synthesis, as measured by incorporation of [3H]uridine, was measured in the same experiment. The results demonstrated that, at 2 h postinfection, the rate of RNA synthesis of infected cell cultures had declined by 50%, and that, by 4 h postinfection, the rate of RNA synthesis was only 25% of that in control cultures. In the second series of experiments, we examined the
polypeptides synthesized in VSV-infected FL cells. Infected cells were pulse-labeled at 1-h intervals with "C-labeled amino acids for 30 min, and the pattern of protein synthesis was analyzed by electrophoresis through 10 to 16% sodium dodecyl sulfate (SDS)-acrylamide gradient gels (Fig. 1). The five major VSV-specified proteins, G, L, M, N, and NS, were recognized by 1 h postinfection, and their synthesis increased as the infection proceeded. To demonstrate that cellular protein synthesis decreased relative to viral directed protein synthesis as the infection proceeded, the amount of radioactivity applied to each slot was kept constant. The shut off of host protein synthesis was not as dramatic as when FL cells were infected with HSV (Fig. 1). Of special interest to us was the observation that globin synthesis persisted at a much higher. level of VSV-infected cells than in cells infected with HSV. Previously, we demonstrated that globin synthesis was very sensitive to HSV infection and that by 4 h postinfection it had essentially ceased (Fig. 1) (18). When the MOI with VSV was increased to 100, we were still able to detect globin synthesis. A similar observation with regard to synthesis of cellular poly-
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FIG. 1. Pattern ofprotein synthesis in VSV-infected FL cells. At intervals, infected cells were pulse-labeled with 14C-labeled amino acids for 30 min. Cytoplasm was prepared by treating the cells with 0.5% Triton X1(X in 10 mM Hepes buffer (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid [pH 7.8) containing 5 mM NaCI, 3 mM MgC42, and 10% sucrose. Nuclei were removed by centrifugation, and the cytoplasmic proteins were separated by electrophoresis through 10 to 16% gradient SDS-acrylamide gel by the buffer system of Laemmli (14). The gel was processed for fluorography (3), dried, and exposed to Cronex 2DC X-ray film. An arrow indicates globin. L, G, N, NS, and M are VSV proteins named according to Wagner et al. (28). The numbers represent the hour after infection.
424
NOTES
J. VIROL.
peptides was made when Vero celIs were used as the host. These results agree wvith the data of Nuss and Koch (19), who demo)nstrated the persistence of immunoglobulin synthesis in mouse myeloma cells infected with VSV. These findings led us to conclude that iIafection with VSV has a less debilitating effect on synthesis of cellular polypeptides than does i]nfection with HSV. HSV infection severely curtails host protein synthesis, and this effect is accoLanted for by dissociation of polyribosomes earlsy after infection (26). Another example of tiie difference between HSV and VSV was the effect on the distribution of polyribosomes in sucrose gradients after infection (Fig. 2). Mocd i-infected FL cells were actively engaged in glob)in synthesis, which was carried out on the p olyribosomes consisting of 5 to 7 monosomal v ints (Y. Nishioka and S. Silverstein, manuscrript in preparation). At 2 h postinfection withk VSV, when the rate of protein synthesis had declined by 20%, there was very little, if any, cMhange in the amount of ribosomes present as polyribosomes when compared with the mock-infescted control. There is, however, an alteration in the distribution of polyribosomes in the infected cell. The relative amount of small polyribos4 omes had decreased, reflecting what probably r epresents the preferential translation of the larger VSV .4
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FiG. 2. Changes in distribution of :olyribosomes. Cells were either infected at 10 PFUi/cell or mock infected and harvested at the time indio cated. Lysates were prepared by washing with phosj Dhate-buffered saline, suspending in 10 mM Tris (plHr 7.2), 10 mM MgC4, and 10 mM KCI at 5 x 107 celd Is per ml, and disrupting by 30 strokes in a Dounce homogenizer. Nuclei were removed by low-speed c and a portion of the cytoplasm was solution) sucrose gradients (15 to 50% in the l1 and centrifuged at 35,000 rpm for 2.25 h at 4°C in a Spinco SW41 rotor. The absorbance profiles were monitored at 254 nm, using an ISCO de!nsitygradient fractionator.
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mRNA's that is occurring on larger polyribosomes. Alternatively, if, as a consequence of VSV infection, the rate of elongation of cellular polypeptides has decreased, it is conceivable that this accounts for the accumulation of larger polyribosomes. In this vein it is worthwhile noting that, when fB-globin chain elongation is blocked at position 112 by the addition ofO-methylthreonine, very large polyribosome structures containing up to 12 monosomes are formed (21). We analyzed the size distribution of mRNA in VSV-infected FL cells to examine the possibility that the decrease in cell-specific polypeptide synthesis was a result of degradation of cellular mRNA, as when FL cells are infected with HSV (18). Cytoplasmic RNA isolated from infected cells was displayed on a sucrose gradient, and the size distribution of polyadenylic acid [poly(A)]-containing mRNA was determined by hybridizing a portion of each fraction to 3H-labeled polyuridylic acid [poly(U)]. In comparison with HSV-infected cells (Nishioka and Silverstein, in preparation), there was little detectable change in the distribution of poly(A)-containing mRNA (Fig. 3). These results suggest that, unlike infection of FL cells with HSV, VSV infection does not result in degradation of host mRNA. The increase in counts per minute of hybridized poly(U) in the region of high-molecular-weight mRNA reflects the accumulation of VSV-specific mRNA's that are larger than 9S (4, 13, 16) and known to be polyadenylated (7, 25). These studies on the physical state of mRNA after VSV infection suggest that there is no significant degradation of cellular mRNA. The persistence of the peak of 9S mRNA suggests that globin mRNA remains, as would be predicted from the pattern of protein synthesized after VSV infection (Fig. 1). To demonstrate that replication of VSV does not lead to degradation of cellular mRNA, we quantitated the amount of globin mRNA present in the cytoplasm of VSV-infected FL cells at 2 and 4 h postinfection. Cytoplasmic RNA extracted from mock-infected, VSV-infected, and HSV-infected cells was hybridized to labeled globin cDNA. Analysis of the kinetics of hybridization demonstrated the following (Fig. 4): (i) at 2 or 4 h postinfection with VSV, no change in the amount of globin mRNA sequences could be detected as determined by the failure to measure a change in the Crtl/2 of the reaction; (ii) as previously shown (18), at 4 h postinfection with HSV only about 15% of the globin mRNA sequences persists in a hybridizable form. These data demonstrate that, unlike infection of FL cells with HSV, infection with VSV does not cause detectable degradation of globin mRNA.
VOL. 25, 1978
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FIG. 3. Distribution ofpoly(A) sequence in sucrose gradients. About 300 pug of cytoplasmic RNA was layered onto precooled 15 to 30% (wt/vol) sucrose gradients in 10 mM Tris (pH 7.0) containing 0.1 N NaCI and 1 mMEDTA. After centrifugation at35,000 rpm for 24 h in an SW41 rotor, the gradients were fractionated. From each fraction a 0.3-ml fraction was removed and hybridized to 3,000 cpm of 3Hlabeled poly(U) [400 cpm is equivalent to 1 ng of poly(A)] in 0.5 ml of 0.3 N NaCl, 0.1% SDS, and 30 mM Tris (pH 7.4). After 10 min of incubation at 45°C, the reaction mixtures were quickly cooled to 0°C, diluted with 2.5 ml of 10 mM Tris containing 0.3 N NaCI, and nonhybridized 3H-labeled poly(U) was digested with pancreatic RNase (20 pg/ml) for 10 min at 0°C. Trichloroacetic acid-precipitable material was collected onto membrane filters (Millipore Corp), and the radioactivity was determined in a scintillation counter. 0, Mock-infected control; A, 4 h after infection with VSV.
The experiments presented here demonstrate that infection with VSV, as with vaccinia virus (23) and picornaviruses (6, 8, 31), does not result in degradation of cellular mRNA. In comparison with HSV, which arrests host protein synthesis by disaggregating polyribosomes (26; Silverstein and Engelhardt, manuscript in preparation) and subsequently recruiting ribosomes into polyribosome structures that preferentially synthesize HSV polypeptides, infection with VSV does not significantly alter the polyribosome pattern during the course of infection, nor does it dramatically affect the amount of globin synthesized. Nevertheless, VSV polypeptides are the major species synthesized in infected cells by 1 h postinfection. Therefore, there must be a selective translation control that operates to permit VSV mRNA to be translated without increasing the amount of ribosomes present as polyribosomes.
0.9
1
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2
FIG. 4. Hybridization of globin mRNA extracted from FL cells after infection with VSV. Globin mRNA was isolated from reticulocytes of phenylhydrazinetreated DBA mice by phenol extraction, oligodeoxythymidylic acid-cellulose chromatography, and velocity sedimentation through 15 to 30% sucrose gradients. cDNA was prepared with RNA-dependent DNA polymerase from avian myeloblastosis virus as described by Kacian and Myers (12). This cDNA back-hybridized to globin mRNA with a C4ia of 1 x 103 mol * s/liter. Cytoplasmic RNA from VSV-infected FL cells was hybridized to the cDNA under the conditions described by Ramirez et al. (22). Unhybridized cDNA was digested with Sl nuclease for 30 min at 45°C in 30 mM sodium acetate buffer (pH 4.5) containing 1.8 mM ZnCI, 0.3 M NaCI, and 10 ,Ag of heat-denatured calf thymus DNA per ml 0, Control; *, 2 h after infection; A, 4 h after infection with VSV; O, 4 h after infection with HSV.
The observations reported here favor the possibility that degradation of cellular mRNA is a unique consequence of HSV infection. The mechanism responsible for degradation is now under investigation in our laboratory. We are attempting to determine if degradation is mediated by a virion component, or if it requires expression of the HSV genome. We thank D. L. Engelhardt for VSV and Dr. C. Milcarek for 3H-labeled poly(U). This investigation was supported by Public Health Service grant CA17477 to S.J.S. from the National Cancer Institute. LITERATURE CITED 1. Baltimore, D., R. M. Franklin, and J. Callender. 1963. Mengovirus-induced inhibition of host ribonucleic acid and protein synthesis. Biochim. Biophys. Acta 76:425-430. 2. Baltimore, D., A. S. Huang, and M. Stampfer. 1970. Ribonucleic acid synthesis of vesicular stomatitis virus. H. An RNA polymerase in the virion. Proc. Natl. Acad. Sci. U.S.A. 66:572-576. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-8. 4. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975. Translation and identification of the viral mRNA spe-
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