Effects of Phosphorylation and ph on the Association of NS Protein with Vesicular Stomatitis Virus Cores

Transcription

1 JOURNAL OF VIROLOGY, Aug. 1978, p X/78/ $2.00/0 Copyright 1978 American Society for Microbiology Vol. 27, No. 2 Printed in U.S.A. Effects of Phosphorylation and ph on the Association of NS Protein with Vesicular Stomatitis Virus Cores GAIL M. CLINTON,* BOYCE W. BURGE, AND ALICE S. HUANG Department ofmicrobiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts Received for publication 24 January 1978 The proteins of vesicular stomatitis virus (VSV) were analyzed on the basis of charge as well as size in polyacrylamide gels containing urea and acetic acid. The phosphoprotein NS was resolved into two major species. The less phosphorylated NS1 species contained about 10% fewer phosphate residues than the second species, NS2. These two phosphorylated forms were compartmentalized both in the virus and in the infected cell cytoplasm. Cores from virions and the corecontaining fraction of the infected cell cytoplasm contained only the NS1 form. All of the more highly phosphorylated NS2 form and some of the NS1 form were found to be free of cores, whether they were derived from virions or from the infected cell. Therefore, the degree of phosphorylation appeared to determine whether or not the NS protein became bound to VSV cores. Moreover, the amount of bound NS1 protein relative to nucleocapsids increased as the ph of the culture medium was raised from 6.6 to 7.4. Because an increase in ph increases VSV replication (Fiszman et al., J. Virol. 13: , 1974; Palma and Huang, in W. S. Robinson and C. F. Fox, ed., Mechanisms of Virus Disease, ICN-UCLA Symposia, p , 1974), the NS, protein may either regulate overall VSV RNA synthesis or regulate the switch between transcription and replication. Both DNA and RNA viruses, enveloped and nonenveloped, contain structural proteins which are phosphorylated (16, 20, 21, 23, 27). Frequently these phosphoproteins are associated with the viral nucleic acid (7, 11, 15, 22, 23, 28). These observations have led to suggestions that phosphorylation may play a regulatory role in nucleic acid biosynthesis and gene expression. Vesicular stomatitis virus (VSV), an RNAcontaining enveloped virus, as well as several other rhabdoviruses, contains both kinase activity and phosphorylated proteins (6, 13, 23). In an in vitro system, the VSV-associated kinase will hyperphosphorylate two structural proteins (6) which are normally found to be phosphorylated in the infected cell. These two proteins are the nucleocapsid-associated NS protein and the membrane-associated M protein. The NS protein, a polymerase protein (the L protein), and an RNA-binding N protein constitute the core and are required for in vitro RNA-dependent RNA polymerase activity (3, 4). Intracellularly, the NS protein is also found in cores, as well as in a soluble fraction (8, 14, 29). 340 To determine whether phosphorylation affected the function of the VSV NS protein, the NS protein was analyzed for heterogeneity in the degree of phosphorylation. Two different phosphorylated species were resolved. The distribution of these species and the effects of ph on the distribution indicate that the extent of interaction of the less phosphorylated NS protein with nucleocapsid templates may regulate transcription and replication during VSV RNA synthesis. (A preliminary presentation of these results was made at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La., 8-13 May 1977.) MATERIALS AND METHODS Cells and virus. The growth of Chinese hamster ovary (CHO) cells in Joklik minimal essential medium and of the San Juan strain of the Indiana serotype of VSV has been described previously (25). Only standard wild-type B virions were used (26). Radioactive labeling of cells and virus. The medium was buffered at ph 7.4 with 25 mm N-2- hydroxyethyl piperazine-n'-2-ethanesulfonic acid and 10 mm N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. For the ph experiments, 10 mm piperazine-n,n'-bis(2-ethanesulfonic acid) was added. All organic buffers were from Sigma Chemical Co., St. Louis, Mo. When cells were labeled with radioactive amino acids, they were exposed to either [14C]leucine or [35S]methionine at 5 juci/ml, with the medium adjusted to contain 1/10 the usual concentration of that amino acid. When phosphorylated proteins were la-

2 VOL. 27, 1978 beled, phosphate-free medium containing 50,Ci of carrier-free 32P per ml was used. All radioisotopes were from New England Nuclear Corp., Boston, Mass. Suspension cultures of CHO cells were concentrated and infected with VSV at a multiplicity of 20. After adsorption at 4 C for 30 min, the cells were diluted to a concentration of 2 x 106 cells per ml in the appropriate medium containing actinomycin D (Merck Sharp & Dohme, Rahway, N. J.) at 5,ug/ml. After 2 h of incubation at 34 C, the appropriate radioisotope was added. Cytoplasmic extracts were prepared at 4 h and progeny virions were prepared at 8 h after the beginning of incubation at 34 C. Preparation of bound and free viral proteins from the cytoplasm. Cytoplasmic extracts were prepared by swelling cells in hypotonic buffer followed by Dounce homogenization (19). The cytoplasm was separated from nuclei, plasma membranes, and unbroken cells by centrifugation. The cytoplasmic extract was brought to 20 mm EDTA and centrifuged at 125,000 x g for 90 min by the method of Wagner et al. (29). This fractionation resulted in the separation of soluble NS protein from the sedimentable NS protein associated with viral cores. The pelleted fraction also contained the polymerase protein, L, and nucleocapsid protein, N, associated with viral cores, as well as the M protein and glycoprotein, G, associated with sedimentable membranes (9, 12, 29; Clinton and Huang, unpublished data). The proteins were concentrated into 100 pl of a solution containing 10 M urea, 0.9 N glacial acetic acid, 1%,B-mercaptoethanol, and 1 mg of protamine sulfate (Sigma Chemical Co.) per ml and put into suspension by sonic treatment using a microtip of a Branson sonic oscillator (Branson Instruments Co., Stanford, Conn.). To denature the proteins, the samples were held at room temperature for at least 16 h. Additional,B-mercaptoethanol was added to each sample to a final concentration of 2%. 32P-labeled bound or free proteins, each containing 500,000 cpm, and "Cor 3S-labeled bound or free proteins, each containing 250,000 cpm, were analyzed on polyacrylamide gels. Preparation of bound and free proteins from virions. VSV (500,000 cpm of 32P-labeled virions or 300,000 cpm of 3S-labeled virions) was suspended in 1 ml of 0.05 M Tris M EDTA, ph 7.5, containing 1% Nonidet P-40 (Particle Data Laboratories, Elmhurst, Ill.). The mixture was kept on ice for 10 min and diluted into 5 ml of the above buffer containing 0.5% Nonidet P-40. The cores were separated from the solubilized components by centrifugation at 125,000 x g for 90 min. The supernatant was decanted, and the proteins were concentrated by precipitation with 4 volumes of ice-cold acetone. The core and solubilized samples were prepared for electrophoresis in acid-urea gels as described for the cytoplasmic bound and free viral proteins. Acid-urea polyacrylamide gels. The technique of acid-urea gel electrophoresis was derived from Panyim and Chaildey (18). Slab gels 1.5 mm thick were made with 15% acrylamide, 0.1% bisacrylamide, 0.9 N acetic acid, 4 M urea, 0.1% ammonium persulfate, and 1.25% N,N,N',N'-tetramethylethylenediamine (Eastman Organic Chemical Div., Eastman Kodak Co., Rochester, N.Y.). A spacer gel of 5% acrylamide was ASSOCIATION OF NS PROTEIN WITH VSV CORES 341 added to the top. The running buffer was 0.9 N acetic acid. Gels were prerun for 1,000 V-h with the addition of 4 M urea to the running buffer in the upper reservoir. Samples were layered and subjected to electrophoresis at 150 V for 26 to 30 h. Gels were dried in a Hoefer slab dryer (Hoefer Scientific Instruments, San Francisco, Calif.) and exposed to Dupont Cronex-4 X-ray film (E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.) for about 48 h. In some cases the autoradiograms were scanned by a densitometer (E-C Apparatus, St. Petersburg, Fla.), using a filter which transmits at 511 nm and plotted by a model 330 chart recorder (Laboratory Data Control, Riviera Beach, Fla.). SDS-polyacrylamide gel electrophoresis. By using an autoradiogram of the acetic acid-urea gel as a template, the bands were sliced from the dried gel and placed individually in the wells of a 10% acrylamide gel containing sodium dodecyl sulfate (SDS) (10) and subjected to electrophoresis at 50 V for 12 h. These gels were dried and autoradiographed as described for the acid-urea gels. RESULTS Migration of VSV proteins in acid-urea polyacrylamide gels. To determine the migration pattem of virus-specific proteins under conditions when charge as well as size contribute to the rate of migration, VSV proteins were subjected to electrophoresis in 15% acrylamide gels containing 4 M urea and 0.9 M acetic acid (18). Figure 1 compares the distribution of virion proteins labeled with 32P and [35S]methionine (slots a and b, respectively). 32P appeared in the NS and M bands, whereas 35S appeared in the N and M bands and only faintly in the NS band. Because virions contain much less NS protein than do infected cells, the virus-specific corecontaining fraction from infected cells labeled with [3S]methionine was also examined (slot c). Here, the radioactivity appeared quite clearly in the N, NS, and M bands. Identification of VSV proteins from acidurea polyacrylamide gels in SDS-polyacrylamide gels. To positively identify VSV proteins, the separated bands on the acid-urea gel shown in Fig. 1 were cut out and the proteins in them were subjected to electrophoresis in an SDS gel together with virion markers. Both NS, and NS2 proteins (Fig. 1, slot a) comigrated with NS protein in the SDS gel (Fig. 2, slots d and e). This demonstrates that neither of the two phosphorylated species is a degradation product of the NS protein. The other phosphorylated virion protein, M, comigrated in the SDS gel with authentic M protein (Fig. 2, slots a and fl. The putative N protein migrated as authentic N protein on SDS gels (data not shown). The glycoprotein, G, and the large transcriptase protein, L, were not resolved into identifiable bands in

3 342 CLINTON, BURGE, HUANG J. VIROL. NS2- NS1 m.-n N1 - N - M FIG. 2. Autoradiogram of NS and Mproteins subjected to electrophoresis in an SDS-polyacrylamide gel after separation in an acid-urea polyacrylamide gel. (a) 32P-labeled marker VSV; (b) [XiS]methioninelabeled marker VSV; (c) NS from slot c of Fig. 1; (d) NS, from slot a of Fig. 1; (e) NS2 from slot a of Fig. 1; (t) Mfrom slot a of Fig. 1. erated by limited proteolysis with chymotrypsin, were analyzed. Figure 3 demonstrates that the four major 32P-labeled peptides generated from the NS1 and NS2 phosphoproteins migrated identically in SDS-polyacrylamide gels, ruling out the possibility that one of these forms might be a contaminating host phosphoprotein. Phosphorylated state of the NS1 and NS2 proteins. Because NS1 and NS2 proteins migrated identically in SDS gels, their differential migration rates in acid-urea gels were most likely due to differences in phosphorylation. To determine relative levels of phosphorylation, 35S- and 32P-labeled VSV were subjected to coelectrophoresis in acid-urea gels, and the radioactivity in FIG. 1. Autoradiogram of VSV proteins subjected the NS1 and NS2 proteins was quantitated by to electrophoresis in an acid-urea polyacrylamide scanning the autoradiogram Figure 4showsthat gel. The direction ofmigration was from top to bottom there wee auto10%iore phspae residus toward the negative pole. (a) 32P-labeled VSV; (b) there were about 10% more phosphate residues [35S]methionine-labeled VSV; (c) [3S]methionine-la- in the NS2 form than in the NS1 form. Figure 4 beled bound viral proteins obtained from VSV-in- also demonstrates the presence of two major fected cells. phosphorylated species of the membrane-associated M protein. By comparison with the NS acid-urea gels, presumably due to their charge protein, the M protein contained only about 1/10 the amount of phosphate residues. Bound versus free NS protein in the vi- rus. To determine whether the level of phos- heterogeneity. To further compare the NS1 and NS2 proteins separated in acid-urea gels, their peptides, gen-

4 VOL. 27, 1978 (~~~~~~~~~)~~~S ~~~~~~~~~~~~NS, D/STANCE MIGRATED ASSOCIATION OF NS PROTEIN WITH VSV CORES 343 N- a b c d FIG. 3. Identity of the NS1 and NS2 phosphoproteins by analysis of their peptides. 3P-labeled NS1 and NS2 proteins from virions were separated in an acid-urea polyacrylamide gel, excised, and then subjected to limitedproteolysis by chymotrypsin in a 15% polyacrylamide gel by the method of Cleveland et al. (2). The gel containing the separated chymotryptic peptides was dried and exposed to X-ray film. The autoradiogram was scanned. DISTANCE MIGRATED FIG. 4. Autoradiogram ofproteins from virions of VSV subjected to electrophoresis in an acid-urea polyacrylamide gel. The autoradiogram of the dried gel was scanned., 3P-labeled VSV;.-,[. S- methionine-labeled VSV. phorylation might regulate the interaction of the NS protein with the RNA-containing nucleocapsid, virus was disrupted with the detergent Nonidet P-40, and the cores were separated from the solubilized proteins and membranes by differential centrifugation. Figure 5 shows the autoradiogram of the acid-urea gel in which the core proteins (slots a and c) and soluble proteins (slots b and d) were subjected to electrophoresis. NS protein bound to cores was the less phosphorylated NS1 species, whereas the more highly phosphorylated NS2 species was solubilized along with a fraction of the NS1 form. The band seen between the NS1 and the M bands in slot d, labeled with 3S, presumably represents contamination by a host protein (Fig. 2, slot b). Bound versus free NS protein in the cytoplasm. Because there were two degrees of phosphorylation of the NS protein associated NS NS2- S *4. ;. FIG. 5. Autoradiogram of core-associated and solubilized proteins from virions subjected to electrophoresis in an acid-urea polyacrylamide gel (a) 'Plabeled core-associated proteins; (b) 3P-labeled solubilized proteins; (c) [3S]methionine-labeled coreassociated proteins; (d) [5S]methionine-labeled solubilized proteins. with whole virions and only one associated with viral cores (Fig. 5), it was of interest to determine the state of phosphorylation of the NS protein found in the total unfractionated cytoplasm. Both states of NS protein were found in the cytoplasm (data not shown). Further fractionation of the cytoplasm showed compartmentalization of the two states of NS protein (Fig. 6, slots c and f). NS protein in cores consisted of only the less phosphorylated protein, whereas free NS protein existed in both phosphorylated states. The slots containing 3"P-labeled proteins (Fig. 6, slots a and d) indicate that the NS protein, whether bound or free, was the major phosphoprotein in infected cells. In this gel, resolution of the two NS species was only clearly seen for the 35S-labeled material because 32P-labeled host proteins obscured the VSV proteins. The phosphorylation pattern of cellular proteins was largely unchanged by infection with VSV (Fig. 6, slots a, b, d, and e). To maximize resolution in the area of the NS band, the same samples containing fewer 32P

5 344 CLINTON, BURGE, HUANG J. VIROL. VV. -- N -NS NS- -NS i Downloaded from FIG. 6. Autoradiogram of fractions from VSV-infected and uninfected cytoplasm subjected to electrophoresis in an acid-urea polyacrylamide gel. VSVinfected and uninfected CHO cells were fractionated into sedimentable, bound proteins and nonsedimentable, free proteins as described in the text. (a) 32Plabeled free proteins from infected cells; (b) 32P-labeled free proteins from uninfected cells; (c) [14C]- leucine-labeled free proteins from infected cells; (d) 32P-labeled boundproteins from infected cells; (e) 32Plabeled bound proteins from uninfected cells; (f) ['4C]leucine-labeled bound proteins from infected cells; (g) [14C/leucine-labeled marker VSV. counts were subjected to electrophoresis for 30 h instead of 26 h (Fig. 7). Under these conditions, the bound NS protein was only found in the NS1 phosphorylated form which comigrated with the "4C-labeled bound NS protein (slots a and b). The 32P-labeled free NS protein, however, was resolved into NS1 and NS2 proteins which comigrated with the two forms of '4C-labeled free NS proteins (slots d and e). Therefore, NS protein in cores was indeed homogeneous with respect to phosphorylation. However, free NS protein was found in two forms, NS1 and the more highly phosphorylated NS2 species. Therefore, FIG. 7. Autoradiogram of fractions from VSV-infected cytoplasm subjected to electrophoresis in an acid-urea polyacrylamide gel. Portions of the same samples as shown in Fig. 4 were used. (a) ['4CJleucine-labeled bound proteins from infected cells; (b) 32P-labeled bound proteins from uninfected cells; (c) 32P-labeled bound proteins from infected cells; (d) 32P-labeled free proteins from infected cells; (e) ['4C]leucine-labeled free proteins from infected cells. the association of NS molecules with cores cannot be random because some selection appears to exist for only NS, molecules. Effect of ph on the association of NS1 protein with the core-containing fraction of the cytoplasm. Because the amount of NS1 in cores was variable from infection to infection, the extent of binding of NS protein to the corecontaining fraction was measured relative to the ph of the medium. Initial observations were made on the distribution of virus-specific proteins between the free and bound fractions relative to ph. Cells in- on September 20, 2018 by guest

6 VOL. 27, 1978 fected with VSV were incubated at ph 7.4, 7.0, and 6.6. At 4 h after infection, cytoplasmic extracts were prepared and separated into free and bound protein fractions. The ratio of total 35Slabeled proteins between the bound and free fractions was found to be about 6:4 at ph 7.4, 4:6 at ph 7.0, and 3:7 at ph 6.6. This indicated a shift in the distribution of total viral proteins from the bound to the free fraction as progeny formation was inhibited. Overall incorporation of 35S was, also, reduced at the lower ph. Figure 8 shows the proportion of NS, molecules to N protein in the bound protein fraction relative to ph. The 35S counts were normalized to the peak containing N protein. There was a reduction in the amount of NS, molecules in cores when the ph was decreased. For comparison, marker virions labeled with 35S were also examined. (This virus sample contained about half the amount of nucleocapsids that the complexed fractions contained.) Virions had the least amount of NS protein relative to N protein (Fig. 8). Therefore, in infected cells, as more virusspecific proteins were synthesized, more protein was distributed into the bound fraction and more Na I. D/STANCE a -ph 74 M/GRATEV-- -ph 70 -ph 66 -VSV Marker FIG. 8. Autoradiogram of bound proteins from VSV-infected cells subjected to electrophoresis in an acid-urea gel. VSV-infected CHO cells buffered atph 6.6, 7.0, and 7.4 were labeled with [3SJmethionine. The cytoplasms were fractionated into bound and free viral proteins. Samples of the bound viral proteins, each at 250,000 cpm, were subjected to electrophoresis. The autoradiogram of the dried gel was scanned by a densitometer. ASSOCIATION OF NS PROTEIN WITH VSV CORES 345 NS protein became associated with nucleocapsids. DISCUSSION The VSV N, NS, and M proteins have been resolved by charge and size in polyacrylamide gels containing acetic acid and urea. The NS protein from whole virions separated into two distinct 32P-containing bands. Both forms of NS were also found intracellularly. A comparison of the ratios of 32P to [35S]methionine incorporation indicated that the NS2 protein was more highly phosphorylated than the NS, protein. When intracellular VSV cores were analyzed, only the less phosphorylated species, NS,, was found. Likewise, when virions were treated with detergent and the cores were separated by centrifugation, the NS2 form and some of the NS, form were released. The remainder of the NS, form was the only species left in association with cores. Because cores and whole virions contained a different spectrum of NS species, the virions must have accumulated the second NS species during morphogenesis or, more likely, hyperphosphorylated some of the NS protein associated with cores. The soluble fraction from the virus and the cytoplasm contained both phosphorylated forms of the NS protein. It is presently unclear why the NS, form was found in the soluble fractions as well as in the bound fraction. One possibility is that within the NS, protein band there was microheterogeneity which could not be resolved in the acid-urea gels. However, the observation that the more highly phosphorylated NS2 species did not bind to nucleocapsids suggests that phosphorylation regulated the interaction of the NS protein with the nucleocapsids. Similarly, the p12 phosphoprotein of Rauscher murine leukemia virus, which binds specifically to its homologous RNA, interacts with the RNA to a lesser extent when it is more highly phosphorylated (22). The finding that the number of NS, molecules associated with the core-containing fraction increased with increasing ph suggests an important physiological role for NS proteins. Fiszman et al. (5) found that optimal growth of VSV is dependent on the ph of the medium in which infected cells are incubated. Incubation at ph 6.6 reduces progeny formation by 99% when compared with the optimal ph of 7.4 (5, 17). This ph effect is reflected intracellularly by increased synthesis of nucleocapsids at the higher ph (17). Nucleocapsids are the templates for both transcription and replication of VSV RNA (24). Although these nucleocapsids have not been physically separated by function, the

7 346 CLINTON, BURGE, HUANG increased numbers of NS molecules correlated with an increase of genome replication. Moreover, virions contained the least amount of NS protein relative to nucleocapsids, and virions appear to have only transcriptase activity in vitro (1). ACKNOWLEDGMENTS We thank Sheila Little and Brian Schaffhausen for useful advice, Norma Hewlett and Trudy Lanman for excellent technical support, and Suzanne Ress for typing the manuscript. This work was supported by Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases and by American Cancer Society research grant VC-63. G.M.C. is an American Cancer Society Postdoctoral Fellow. A.S.H. was a research career development awardee of the Public Health Service. LITERATURE CITED 1. Baltimore, D., A. S. Huang, and M. Stampfer Ribonucleic acid synthesis of vesicular stomatitis virus. II. An RNA polymerase in the virion. Proc. Natl. Acad. Sci. U.S.A. 66: Cleveland, D. W., S. G. 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