JouRNAL OF VIROLOGY, Apr. 1979, p. 56-63 0022-538X/79/04-0056/08$02.00/0
Vol. 30, No. 1
Proteins of Vesicular Stomatitis Virus V. Identification of a Precursor to the Phosphoprotein of Piry Virus JOHN C. BELL AND LUDVIK PREVEC*
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Received for publication 24 August 1978
A metabolic precursor to the major phosphoprotein of Piry virus (NS,) has been identified in extracts of Piry virus-infected L cells. The conversion of the precursor NS, to NS, occurs with a half-life of 20 min and is independent of continued protein synthesis. N5. has a greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than does the product NSv, suggesting an increase in molecular weight during maturation. The conversion is unaffected by cyclic AMP, cyclic GMP, or by theophilline and cordycepin. No decrease in isoelectric point of NS, relative to NSi was observed on isoelectric focusing acrylamide gels. These latter observations suggest that NSi and NS, do not differ in extent of phosphorylation. We also report, without further characterization, the identification of another phosphoprotein in Piry virus-infected cells having an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis just slightly greater than the nucleocapsid N protein.
imposed on the system. Both cyclic nucleotidedependent and cyclic nucleotide-independent phosphorylation/dephosphorylation reactions are known to act as regulation mechanisms in a number of important biochemical pathways (12, 23). Watanabe et al. (24) have demonstrated an apparent requirement for protein kinase activity during in vitro transcriptase activity of VSV. The large excess of NS protein present in the infected cell relative to the amounts actually associated with ribonucleoprotein complexes also points to the possible regulatory function of NS in the infected cell (10). As a first approach to the functional role of phosphoprotein NS in VSV infection, we examined cells infected with the VSV group for the presence of possible modified forms of this protein. We here report the discovery and the partial characterization of a modified NS protein in Piry virus-infected mouse L cells.
The vesicular stomatitis virus (VSV) group of the rhabdoviruses is comprised of a number of structurally and antigenically related virus types. Detailed comparisons between members of this group of the oligonucleotide fingerprints of the single-stranded RNA genomes (3) and of tryptic fingerprints of constituent viral proteins (2a, 4) have recently been published. All members of the group possess five structural polypeptides, two of which, the glycoprotein (G) and the matrix protein (M), are readily solubilized and dissociated from the ribonucleoprotein core with nonionic detergent and salt (5, 22). The ribonucleoprotein complex consisting of viral RNA enclosed in its associated nucleocapsid protein (N) and proteins L and NS can function as a transcription complex in vitro. The addition of ribonucleoside triphosphates and magnesium ion to the complex results in sequential synthesis of functional messenger RNA species, each possessing a polyadenylated 3' terminus and a capped, methylated 5' terminus (1, 2). Reversible protein dissociation studies (6) and antibody inhibition studies (9) strongly suggest that protein NS as well as protein L are essential for the transcription of the RNA-N protein complex, and that NS and L may function in equimolar amounts (15). Although the mechanisms of the functional interactions of proteins L, NS, and N in the transcription complex are unknown, the phosphoprotein nature of NS (21) provides a possible route through which external regulation may be
MATERIALS AND METHODS Material. N,N,N',N'-tetramethylethylenediamine (TEMED), N,N'-methylenebisacrylauide and ammonium peroxydisulfate were purchased from Eastman Organic Chemical Div., Eastman Kodak Co.; acrylamide was from Bio-Rad Laboratories; ampholytes were from LKB; trypsin (TPCK) was from Worthington Biochemical Corp.; oxaloacetate and citrate synthase were from Sigma Chemical Corp.; MN-400 thinlayer cellulose plates were from Brinkman Instruments Inc.; ultrapure urea was from Schwartz-Mann; and [3S]methionine and [32P]orthophosphoric acid were from New England Nuclear Corp. 56
VOL. 30, 1979
Cells and viruses. Mouse L cells were maintained in spinning culture of Joklik modified minimum essential medium (MEM) supplemented with 5% newborn calf serum (NBCS) at 370C. VSV Indiana and Piry serotypes, grown in L cells, all as previously described (2a), were used throughout this work. Pulse-chase experiments. Virus was adsorbed to monolayers of 5 x 10" L cells for 0.5 h at a multiplicity of infection (MOI) of 50 PFU (3700). After infection, cells were incubated for 3 h in MEM (supplemented with 5% NBCS) and then for an additional 0.5 h in MEM growth medium containing 1/20 the normal amino acid complement and 2% NBCS. [3S]methionine was added to the culture for a pulse period of 5 to 15 min at a final concentration of 250 ACi/ml in 1:20 amino acid medium plus 2% NBCS. After the pulse the radioactive medium was removed and monolayers were either rapidly frozen in a methanol/dry ice mixture or washed twice with phosphate-buffered saline, once with MEM, and then chased with MEM plus 5% NBCS for varying lengths of time. In some experiments during the chase period, cycloheximide was added to a final concentration of 50 ,g/ml. "P labeling. Monolayers of 5 x 10' L cells infected with virus at an MOI of 50 PFU/cell were incubated, 3 h postinfection, in MEM lacking phosphate (-PO4 MEM) for 0.5 h. The medium was then removed from the cells, and the monolayers were washed once with -PO0 MEM and then incubated in -PO MEM plus [32P]orthophosphate at a final concentration of 200 pCi/ml. The cells were harvested 90 min later. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (11). Samples were applied to acrylamide gels (10% acrylamide, 0.13% N,N'-methylenebisacrylamide) and electrophoresis at 200 V until the bromophenol blue marker reached the bottom of the gel. The gel was removed and fixed in a methanol-water-acetic acid (50:50:7) mixture, dried onto Whatman filter paper by low-pressure desiccation at 1000C, and autoradiographed on Kodak RPR14 X-ray film (13).
Tryptic peptide mapping. Appropriate protein bands located by autoradiography were excised and eluted from the gel in 50 mM NH.HC03 buffer (pH 8.5), 0.1% in SDS and with bovine serum albumin as carrier (final protein concentration, 100 pg/ml) concentrated by trichloroacetic acid precipitation (20% trichloroacetic acid) at 12,000 x g for 20 min. Trichloroacetic acid was removed from the pellet by repeated acetone washes, and the resultant protein was oxidized for 3 h at 00C in performic acid. After three cycles of water washing and lyophilization, the protein was digested by adding 12.5 pm of TPCK trypsin in 100 pl of 0.05 M NH4HCO3 (pH 8.5) for 4 h at 370C. The resultant peptides were analyzed in two dimensions as previously described (2a). Acid hydrolysis of 89P-labeled proteins. 3P-labeled protein purified by PAGE, and eluted in 50 mM Tris-chloride (pH 6.8)-1% SDS-1% 2-mercaptoethanol was precipitated (with 250 pg of bovine serum albumin as carrier) by the addition of 95% ethanol-0.33% saturated sodium acetate. The protein was then suspended in 2 N HCI and hydrolyzed under nitrogen in
VSV PHOSPHOPROTEINS
57
a sealed vial for 5 h at 1000C. HCI was removed by lyophilization, and the hydrolysate was run in thinlayer electrophoresis as described by Pnvalsky and
Penhoet (19). NEPHGE. For non-equilibrium pH gradient electrophoresis (NEPHGE), samples were prepared as described by O'Farrell (17), and NEPHGE was conducted following the protocol of O'Farrell et al. (18). Isoelectric slab gels containing 3.78% acrylamide, 0.22% bisacrylamide, 9 M urea, 2% NP-40, 4% ampholines (30%, pH range 3.5 to 10; 70%, pH 3 to 6), 0.1% ammonium persulfate and 0.05% TEMED were cast between glass plates separated by 1.5-mm Plexiglass spacers. The lower (cathode) chamber of the electrophoresis apparatus was filled with 0.02 N NaOH, and 400 ml of double-distilled water was added to the upper (anode) chamber. The samples were then applied to the top of the gel and then overlaid with 2% ampholytes in 9 M urea. To the upper (anode) chamber was added 4 ml of 2 N phosphoric acid, and then electrophoresis was carried out for 4 h at 500 V. The pH gradient in the gel was determined by soaking 0.5cm fractions in 1.5 ml of double-distilled water overnight and then measuring the pH of each fraction by using a Fisher Accumet pH meter. Individual channels were sliced from the isoelectric slab and run in the second dimension on SDS-PAGE as described by O'Farrell (17).
RESULTS Existence of a precursor to Piry NS, protein. [3S]methionine-labeled extracts from Piry virus-infected cells contain two distinct protein bands migrating in the region of NS protein on SDS-PAGE. Under similar experimental conditions, Indiana VSV-infected cells show only a single band in this region. [3S]methionine-labeled protein from purified Piry virions showed that the more slowly migrating (upper) band (NS,) is the predominant, if not the only one of this pair of proteins present in the virus (Fig. 1). The structural relationships of the two Piry NS polypeptides were confirmed by fingerprinting of the methionine-labeled tryptic peptides of each band after separation, excision, and elution from a preparative SDS-gel. Most of the features of the tryptic fingerprint are common to both proteins (Fig. 2). The possible precursor-product relationship of the two proteins was demonstrated by pulsechase experiments. Piry virus infected celLs were pulsed with [3S]methionine followed by incubation in the presence of excess unlabeled methionine. Samples removed at various times during the chase period were analyzed on SDSPAGE. Chasing after either 5 or 15 min of labeing resulted in a progressive decrease in radioactivity in the faster band (NSJ) and a corresponding increase in the radioactivity of the NS, protein (Fig. 3). This experiment suggests that
58
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BELL AND PREVEC
J. VIROL.
Conversion of NSi to NS,. The conversion of NSi to NS, is probably not due to phospho: rylation. Following the demonstration by Sokol and Clark (21) that the NS proteins of rhabdoviruses were phosphorylated, the possibility that Lt NSi and NS, differed in extent of phosphorylation was an interesting possibility. Extracts of .,w'mwI . ..... .r Piry-infected cells labeled with inorganic 32PO4 and analyzed on SDS-PAGE, demonstrate the association of phosphate label with the Piry NS, protein (Fig. 5). It has not been possible to conclusively demonstrate that 32P label is associated with protein *. :: ,: .:, NS1 in these experimnents. Although some radioG activity is found in this region, it is always overshadowed by the much larger amount of label in NSv. Short pulse-labeling with 32PO4 does not enhance the ratio of label in NSi/NSv. NSV We do not believe that these experiments demNSji = onstrate the absence or even the reduction on a N 140' 1 442 molar basis of the amount of phosphate in NSi relative to NS,. More likely, our inability to see phosphate label in NS, is simply the result of a L very large excess of NS, and the relatively slow f utilization of exogenous radioactive phosphate s: due to large internal precursor pools. i : Acid hydrolysis of phosphate-labeled NS, showed that phosphoserine was the principle i MAw phosphorylated component. Phosphothreonine was undetected, though a significant amount of unidentified material remained at the origin of Li chromatography. NF VIRWON A second phosphorylated protein, present in PIRY I ND CELL Piry-infected cells and absent in uninfected cells, FIG. 1. Comparison of viral proteins in Piry and migrates with a mobility just slightly faster than Indiana VSV-infected cells and Piry virions. Pitry Piry N protein. We have been unable, to date, virus and Indiana (IND) virus-infected cells (as in- to obtain this material in sufficient quantity and dicated) were labeled with [rS]methionine for30min purity for further analysis. some 4 h postinfection. The infected (INF) -cellprotein If the conversion of NS, to NS, were dependwas analyzed on SDS-PA GE. The location of the two NS bands in the Piry extract is indicated in the left- ent on phosphorylation, inhibitors of protein hand panel. Piry virions purified on sucrose gra- kinase activity might reduce or prevent this dients after overnight growth with [rS]methionine conversion. On this basis, we examined the eflabel were analyzed on SDS-PAGE in the right-hand fects of theophylline, cyclic AMP, and cyclic panel, with a control marker of ribonucleoprotein GMP, all of which act through cyclic nucleotideextract from pulse-labeled Piry-infected cells. mediated kinases as well as cordycepin, an inhibitor of both cyclic nucleotide-dependent and the conversion of NSi to NS, occurs with a half- -independent kinases (8). The theophilline result life of approximately 20 min under the condi- (Fig. 6) demonstrates that, though there was a tions employed. general reduction both in the amount of radioThe apparent conversion of NS, to NS, is active phosphate and [3S]methionine incorpoindependent of continued protein synthesis since rated into proteins at increased concentrations (Fig. 4) the conversion occurs at the same rate of these inhibitors, there was no concomitant in the presence of cycloheximide as in its ab- change in the relative rate of conversion of NSi sence. to NS, as evidenced by [3S]methionine label. From the preceding results we conclude that Cyclic AMP and GMP at concentrations of 10' the protein NSi is converted by post-transla- to 10-6 M also failed to have any effect on the tional modification to a protein of greater ap- conversion of NSi to NSv. The effect of cordyparent molecular weight, NS,. cepin was essentially identical to that of theoI,
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VOL. 30, 1979
VSV PHOSPHOPROTEINS
59
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FIG. 2. Tryptic fingerprint of methionine-labeled proteins NSi and NS,. [3S]methionine-labeled NSi and were purified by preparative SDS-PAGE from Piry-infected cell extracts labeled for 5 min and 30 min, respectively, some 4 h postinfection. The protein bands were excised from the gel, oxidized, and trypsinized as described in the text. Two-dimensional analysis by electrophoresis and chromatography was carried out on cellulose thin layer.
NS,
philline at corresponding concentrations. Lack of separation of NS, and NS, on nonequilibrium isoelectric focusing gels. The NS proteins of the VSV groups are highly acidic and do not generally focus on the conventional 0
-
isoelectric focusing system of O'Farrell (17). For
4O lbL
this reason, we employed modified non-equilibrium techniques of O'Farrell et al. (18) as described above. Figure 7 shows the relative position of the Piry NS, protein with respect to N, G, and M after two-dimensional analysis em-
FG
ploying non-equilibrium isoelectric focusing followed by SDS-PAGE in the second dimension. Infected cell extracts labeled for 30 min and, therefore, having NS, as the major labeled NS protein, or labeled for 5 min and, therefore, labeled predominantly in NS,, as well as a mixture of these two extracts, are shown. The individual extracts clearly show only a single labeled NS protein, whereas the mixture shows resolution of NS, and NS, in the SDS-PAGE dimension but no significant separation of these proteins in the isoelectric focusing dimnension. One-
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min, the radioactive medium was removed and of the cultures were incubated excess unlabeled methionine, as described in the text. Samples were taken at the indicated times. The infected cells were harvested and analyzed on SDS-PAGE. The figure describes the length of the radioactive pulse plus the kngth of the subsequent chase. The reason for the double-M protein in the 15-min label experiment is not known. An extract of labeled uninfected cells is presented in the central well (CNT). or 15 some
L.~~~~~
5 5+5 5+10 5+20 C
dimensional isoelectric focusing analysis of these
1S 15+15 15+30 15+60
N T
FIG. 3. Pulse chase of protein NSi to NS,, [3SJmethionine label was added to Piry virus-infected L cels at 4 h postinfection. After a labeling period of 5
in
60
BELL AND PREVEC
J*.2Qz,:rXSt
J. VIROL.
greater than the coding capacity of the RNA for this protein (7, 20), makes any attempt to correlate electrophoretic mobility and molecular weight for this protein of doubtful significance. Since shape factors and/or degree of binding of
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CYCLOHEXIMIDE FIG. 4. Conversion of NSi to NS, in the presence and absence of cycloheximide. Piry virus-infected cells at 4 h postinfection were pulse labeled with [3S]methionine for 5 min and chased with excess unlabeled methionine. The chase medium for one experiment contained 50 pg of cycloheximide per ml. Samples were taken at the indicated times and analyzed by SDS-PAGE. The sample wells labeled (P) contain infected cell extracts kept in [3S]methionine
for the fuU 20-min period.
proteins as sharply focused bands failed to show any shift of NS, to a more negatively charged protein after conversion to NS, (data not presented). DISCUSSION This work clearly identifies a metabolic intermediate in the maturation of the NS, protein of Piry virus. The intermediate, NS,, readily demonstrable after short labeling periods in infected-cell extracts, is either absent from or a minor component of the mature virion. The decrease in electrophoretic mobility associated with the conversion on NSi to NS, is consistent under our electrophoretic conditions with an increase in molecular weight of about 1,000. The mobility of the VSV NS proteins relative to the other virion proteins may be altered by changing the percentage of N,N'methylenebisacrylamide in the resolving gel, resulting in apparent molecular weight differences of up to 10,000 (unpublished data). This, together with the fact that all molecular weight estimates of NS are almost a factor of two
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35s 32p 32p FIG. 5. Phosphate-labeled proteins in Piuy virusinfected ceUs. Piry virus-infected cell cultures were labeled with [rPJorthophosphate from 3.5 to 5 h postinfection as described in the text (central well). An infected culture labeled over the same time interval with [3S]methionine (left well) and an uninfected ceU culture labeled for 90 min with [3Plorthophosphate (right well) are included as controls. The labeled ceU extracts were analyzed by SDS-PAGE.
VSV PHOSPHOPROTEINS
VOL. 30, 1979
. . 4, .4_
S
P
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P
5 10
S
P -4
10
Si
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-3
THEOPHILLINE
FIG. 6. Treatment of Piry-infected L cells with theophilline. Monolayers of 5 x 106 L cells were infected with Piry virus at an MOI of 100 PFU/cell as described in the text and then overlaid with MEM containing 1:20 normal amino acid complement for [3Slmethionine labeling or with -PO0 MEM for labeling with [nP]orthophosphate. At this time, theophilline was added to each plate to a final concentration of 10-3 M, 10-4 M, or 10- M. At 3 hpostinfection, the overlay was removed and medium containing either [I35]methionine (30 yCi/ml) or [nP]orthophosphate (200X Ci/ml) was added to appropriate plates along with the inhibitor theophilline. Labeling was allowed to continue for 1.5 h at which time the samples were prepared for electr"phoresis as described in the text. P, labeled with [ Plorthophosphate; S, labeled with [IS]methionine.
SDS appear to play a large role in determining the mobility of this protein, it is conceivable that even a
low-molecular-weight substituent could
61
produce the resultant mobility difference observed between NS, and NSi. Although we originally pursued the idea that NS, and NS, differed in extent of phosphorylation, we have been unable to either support or directly disprove this hypothesis. The lack of effect of cordycepin and the cyclic nucleotides on the rate of conversion of NS, and NS, can be considered only as negative evidence rather than proof that phosphorylation is not involved. Furthermore, our inability to demonstrate an increased negative charge in NS, relative to NSi argues against phosphorylation as the direct cause of the conversion. The biological significance of the NS, to NS, conversion is not known. Unpublished experiments carried out by us show approximately the same relative ratio, after short labeling periods, of NS, to NS, in the polyribosomal as well as the soluble protein fraction of the infected cell. This would suggest that the conversion of NSi to NS, occurs equally in both fractions and that there is no apparent functional discrimination between these proteins in ribonucleoprotein complexes as compared to free protein. The central role of NS protein in the VSV transcriptase system as well as its potential involvement in virus replication and host cell regulation make these post-translational modifications of considerable interest. The finding of a second major phosphoprotein in Piry-infected cells, that protein having a mobility just slightly faster than Piry N protein, distinguishes Piry from the Indiana, Cocal, New Jersey, and Chandipura serotypes which, in our hands, show a single major phosphoprotein band. Two phosphoprotein bands have been reported for the salmonid virus IHN (14) and Kern Canyon virus (21). A more detailed analysis of the number and association of phosphates with proteins in different members ofthe rhabdovirus group must be carried out before any meaningful assessment of the functional or genetic significance of this distribution can be obtained. The possibility that analogs of Piry NSi exist in other members of the VSV group is currently under investigation. ACKNOW LEDGMENTS We thank E. G. Brown, D. Takayesu, and H. P. Ghosh for helpful discussion and advice. Theophilline, cAMP, and dibutryl cGMP were generously supplied by R. Haslam, and P. Branton donated cordycepin used throughout this work. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. LITERATURE CIMD 1. Abraham, G., D. P. Rhodes, and A. K. Baneijee. 1975. The 5' terminal structure of the methylated mRNA
62
BELL AND PREVEC
J. VIROL.
5
A
30 G ilN I-
IF p4
pH ,
V),
-*
0
a
(ft
3
2
5+30
NS
4' i
.N
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FIG. 7. Two-dimensional analysis of Piry-infected L cells. Infected monolayers were pulse labeled with
[nSJmethionine, 4 h postinfection, either for 5 min or 30 min as described in the text. Samples, prepared for NEPHGE, were applied to the top of isoelectric slab gels and run for 4 h at 500 V. Appropriate channels were then immediately prepared for and run in the second dimension in SDS-PAGE, as described by O'Farrell (17). Shown above: 5-min pulse label; 30-min pulse label; mixture of a 5- and 30-min pulse label. synthesized in vitro by vesicular stomatitis virus. Cell 5:51-58. 2. Banerjee, A. K., and D. P. Rhodes. 1973. In vitro synthesis ofRNA that contains polyadenylate by virionassociated RNA polymerase of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 70:3566-3570. 2a.Brown, E. G., and L Prevec. 1978. Proteins of vesicular stomatitis virus. IV. A comparison of tryptic peptides of the VSV subgroup of rhabdovirus. Virology 89:7-21. 3. Clewley, J. P., D. H. L Bishop, C. Y. Kang, J. Coffim, W. M. Schnitzlein, M. E. Reichman, and R. E. Shope. 1977. Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the VSV subgroup. J. Virol. 23:152-166. 4. DoeL T. R., and F. Brown. 1978. Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus. J. Gen. Virol. 38:351-361. 5. Emerson, S. U., and R. R. Wagner. 1972. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J. Virol. 10:297-309. 6. Emerson, S. U., and Y.-H. Yu. 1975. Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. J. Virol. 15:1348-1356. 7. Freeman, G. J., J. K. Rose, G. K. Clinton, and A. S. Huang. 1977. RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation. J. Virol. 21:1094-1104.
8. Hirsch, J., and 0. J. Martello. 1976. Inhibition of nuclear protein kinases by adenosine analogues. Life Sci.
19:85-90. 9. Imblum, R. L., and R. R. Wagner. 1975. Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus. J. Virol. 15:1357-1366. 10. Kang, C. Y., and L Prevec. 1971. Proteins of vesicular stomatitis virus. Virology 46:628-690. 11. Laemlli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Lee, S. G., M. V. Micelli, R. A. Tungman, and P. P. Huang. 1975. Protein kinase and its regulatory effect on reverse transcriptase activity of Rous Sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 72:2945-2949. 13. Legault, D., D. Takayesu, and L Prevec. 1977. Heterotypic exclusion between vesicular stomatitis virus of the New Jersey and Indiana serotypes. J. Gen. Virol.
35:53-65.
14. McAllister, P. E., and R. R. Wagner. 1975. Structural proteins of two Salmonid rhabdoviruses. J. Virol. 15: 733-738. 15. Naito, S., and A. Ishihama. 1976. Function and structure of RNA polymerase from VSV. J. Biol. Chem. 251: 4307-4314. 16. Nowakowski, M., W. Bauer, and J. Kates. 1978. Characterization of a DNA-binding phosphoprotein from vaccinia virus replication complex. Virology 86:217-225.
VOL. 30, 1979 17. O'Farrell, P. H. 1975. High resolution two dimensional electrophoresis by proteins. J. Biol. Chem. 250:40074021. 18. O'Farrel, P. Z., H. M. Goodman, and P. H. O'Farrell. 1977. High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133-1142. 19. Privalsky, M. L, and E. E. Penhoet. 1977. Phosphorylated protein component present in influenza virions. J. Virol. 24:401-405. 20. Rhodes, D. P., G. Abraham, R. J. Colonno, W. Jelinek, and A. K. Banerjee. 1977. Characterization of vesicular stomatitis virus mRNA species synthesized in vitro. J. Virol. 21:1105-1112.
VSV PHOSPHOPROTEINS
63
21. Sokol, F., and H. F. Clark. 1973. Phosphoproteins structural components of rhabdoviruses. Virology 52:246263. 22. Szilagy, J. F., and L. Uryvayev. 1973. Isolation of an infectious ribonucleoprotein from VSV containing an active RNA transciptase. J. Virol. 11:279-286. 23. Taborsky, G. 1974. Phosphoproteins. Adv. Prot. Chem. 28:1-210. 24. Watanabe, Y., S. Sakeura, and S. Tanaka. 1974. A possible biological function of the protein kinase associated with vaccinia and VSV virion. FEBS Lett. 41: 331-334.
Vol. 30, No. 1
Proteins of Vesicular Stomatitis Virus V. Identification of a Precursor to the Phosphoprotein of Piry Virus JOHN C. BELL AND LUDVIK PREVEC*
Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Received for publication 24 August 1978
A metabolic precursor to the major phosphoprotein of Piry virus (NS,) has been identified in extracts of Piry virus-infected L cells. The conversion of the precursor NS, to NS, occurs with a half-life of 20 min and is independent of continued protein synthesis. N5. has a greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than does the product NSv, suggesting an increase in molecular weight during maturation. The conversion is unaffected by cyclic AMP, cyclic GMP, or by theophilline and cordycepin. No decrease in isoelectric point of NS, relative to NSi was observed on isoelectric focusing acrylamide gels. These latter observations suggest that NSi and NS, do not differ in extent of phosphorylation. We also report, without further characterization, the identification of another phosphoprotein in Piry virus-infected cells having an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis just slightly greater than the nucleocapsid N protein.
imposed on the system. Both cyclic nucleotidedependent and cyclic nucleotide-independent phosphorylation/dephosphorylation reactions are known to act as regulation mechanisms in a number of important biochemical pathways (12, 23). Watanabe et al. (24) have demonstrated an apparent requirement for protein kinase activity during in vitro transcriptase activity of VSV. The large excess of NS protein present in the infected cell relative to the amounts actually associated with ribonucleoprotein complexes also points to the possible regulatory function of NS in the infected cell (10). As a first approach to the functional role of phosphoprotein NS in VSV infection, we examined cells infected with the VSV group for the presence of possible modified forms of this protein. We here report the discovery and the partial characterization of a modified NS protein in Piry virus-infected mouse L cells.
The vesicular stomatitis virus (VSV) group of the rhabdoviruses is comprised of a number of structurally and antigenically related virus types. Detailed comparisons between members of this group of the oligonucleotide fingerprints of the single-stranded RNA genomes (3) and of tryptic fingerprints of constituent viral proteins (2a, 4) have recently been published. All members of the group possess five structural polypeptides, two of which, the glycoprotein (G) and the matrix protein (M), are readily solubilized and dissociated from the ribonucleoprotein core with nonionic detergent and salt (5, 22). The ribonucleoprotein complex consisting of viral RNA enclosed in its associated nucleocapsid protein (N) and proteins L and NS can function as a transcription complex in vitro. The addition of ribonucleoside triphosphates and magnesium ion to the complex results in sequential synthesis of functional messenger RNA species, each possessing a polyadenylated 3' terminus and a capped, methylated 5' terminus (1, 2). Reversible protein dissociation studies (6) and antibody inhibition studies (9) strongly suggest that protein NS as well as protein L are essential for the transcription of the RNA-N protein complex, and that NS and L may function in equimolar amounts (15). Although the mechanisms of the functional interactions of proteins L, NS, and N in the transcription complex are unknown, the phosphoprotein nature of NS (21) provides a possible route through which external regulation may be
MATERIALS AND METHODS Material. N,N,N',N'-tetramethylethylenediamine (TEMED), N,N'-methylenebisacrylauide and ammonium peroxydisulfate were purchased from Eastman Organic Chemical Div., Eastman Kodak Co.; acrylamide was from Bio-Rad Laboratories; ampholytes were from LKB; trypsin (TPCK) was from Worthington Biochemical Corp.; oxaloacetate and citrate synthase were from Sigma Chemical Corp.; MN-400 thinlayer cellulose plates were from Brinkman Instruments Inc.; ultrapure urea was from Schwartz-Mann; and [3S]methionine and [32P]orthophosphoric acid were from New England Nuclear Corp. 56
VOL. 30, 1979
Cells and viruses. Mouse L cells were maintained in spinning culture of Joklik modified minimum essential medium (MEM) supplemented with 5% newborn calf serum (NBCS) at 370C. VSV Indiana and Piry serotypes, grown in L cells, all as previously described (2a), were used throughout this work. Pulse-chase experiments. Virus was adsorbed to monolayers of 5 x 10" L cells for 0.5 h at a multiplicity of infection (MOI) of 50 PFU (3700). After infection, cells were incubated for 3 h in MEM (supplemented with 5% NBCS) and then for an additional 0.5 h in MEM growth medium containing 1/20 the normal amino acid complement and 2% NBCS. [3S]methionine was added to the culture for a pulse period of 5 to 15 min at a final concentration of 250 ACi/ml in 1:20 amino acid medium plus 2% NBCS. After the pulse the radioactive medium was removed and monolayers were either rapidly frozen in a methanol/dry ice mixture or washed twice with phosphate-buffered saline, once with MEM, and then chased with MEM plus 5% NBCS for varying lengths of time. In some experiments during the chase period, cycloheximide was added to a final concentration of 50 ,g/ml. "P labeling. Monolayers of 5 x 10' L cells infected with virus at an MOI of 50 PFU/cell were incubated, 3 h postinfection, in MEM lacking phosphate (-PO4 MEM) for 0.5 h. The medium was then removed from the cells, and the monolayers were washed once with -PO0 MEM and then incubated in -PO MEM plus [32P]orthophosphate at a final concentration of 200 pCi/ml. The cells were harvested 90 min later. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (11). Samples were applied to acrylamide gels (10% acrylamide, 0.13% N,N'-methylenebisacrylamide) and electrophoresis at 200 V until the bromophenol blue marker reached the bottom of the gel. The gel was removed and fixed in a methanol-water-acetic acid (50:50:7) mixture, dried onto Whatman filter paper by low-pressure desiccation at 1000C, and autoradiographed on Kodak RPR14 X-ray film (13).
Tryptic peptide mapping. Appropriate protein bands located by autoradiography were excised and eluted from the gel in 50 mM NH.HC03 buffer (pH 8.5), 0.1% in SDS and with bovine serum albumin as carrier (final protein concentration, 100 pg/ml) concentrated by trichloroacetic acid precipitation (20% trichloroacetic acid) at 12,000 x g for 20 min. Trichloroacetic acid was removed from the pellet by repeated acetone washes, and the resultant protein was oxidized for 3 h at 00C in performic acid. After three cycles of water washing and lyophilization, the protein was digested by adding 12.5 pm of TPCK trypsin in 100 pl of 0.05 M NH4HCO3 (pH 8.5) for 4 h at 370C. The resultant peptides were analyzed in two dimensions as previously described (2a). Acid hydrolysis of 89P-labeled proteins. 3P-labeled protein purified by PAGE, and eluted in 50 mM Tris-chloride (pH 6.8)-1% SDS-1% 2-mercaptoethanol was precipitated (with 250 pg of bovine serum albumin as carrier) by the addition of 95% ethanol-0.33% saturated sodium acetate. The protein was then suspended in 2 N HCI and hydrolyzed under nitrogen in
VSV PHOSPHOPROTEINS
57
a sealed vial for 5 h at 1000C. HCI was removed by lyophilization, and the hydrolysate was run in thinlayer electrophoresis as described by Pnvalsky and
Penhoet (19). NEPHGE. For non-equilibrium pH gradient electrophoresis (NEPHGE), samples were prepared as described by O'Farrell (17), and NEPHGE was conducted following the protocol of O'Farrell et al. (18). Isoelectric slab gels containing 3.78% acrylamide, 0.22% bisacrylamide, 9 M urea, 2% NP-40, 4% ampholines (30%, pH range 3.5 to 10; 70%, pH 3 to 6), 0.1% ammonium persulfate and 0.05% TEMED were cast between glass plates separated by 1.5-mm Plexiglass spacers. The lower (cathode) chamber of the electrophoresis apparatus was filled with 0.02 N NaOH, and 400 ml of double-distilled water was added to the upper (anode) chamber. The samples were then applied to the top of the gel and then overlaid with 2% ampholytes in 9 M urea. To the upper (anode) chamber was added 4 ml of 2 N phosphoric acid, and then electrophoresis was carried out for 4 h at 500 V. The pH gradient in the gel was determined by soaking 0.5cm fractions in 1.5 ml of double-distilled water overnight and then measuring the pH of each fraction by using a Fisher Accumet pH meter. Individual channels were sliced from the isoelectric slab and run in the second dimension on SDS-PAGE as described by O'Farrell (17).
RESULTS Existence of a precursor to Piry NS, protein. [3S]methionine-labeled extracts from Piry virus-infected cells contain two distinct protein bands migrating in the region of NS protein on SDS-PAGE. Under similar experimental conditions, Indiana VSV-infected cells show only a single band in this region. [3S]methionine-labeled protein from purified Piry virions showed that the more slowly migrating (upper) band (NS,) is the predominant, if not the only one of this pair of proteins present in the virus (Fig. 1). The structural relationships of the two Piry NS polypeptides were confirmed by fingerprinting of the methionine-labeled tryptic peptides of each band after separation, excision, and elution from a preparative SDS-gel. Most of the features of the tryptic fingerprint are common to both proteins (Fig. 2). The possible precursor-product relationship of the two proteins was demonstrated by pulsechase experiments. Piry virus infected celLs were pulsed with [3S]methionine followed by incubation in the presence of excess unlabeled methionine. Samples removed at various times during the chase period were analyzed on SDSPAGE. Chasing after either 5 or 15 min of labeing resulted in a progressive decrease in radioactivity in the faster band (NSJ) and a corresponding increase in the radioactivity of the NS, protein (Fig. 3). This experiment suggests that
58
t . .t:.e : .
BELL AND PREVEC
J. VIROL.
Conversion of NSi to NS,. The conversion of NSi to NS, is probably not due to phospho: rylation. Following the demonstration by Sokol and Clark (21) that the NS proteins of rhabdoviruses were phosphorylated, the possibility that Lt NSi and NS, differed in extent of phosphorylation was an interesting possibility. Extracts of .,w'mwI . ..... .r Piry-infected cells labeled with inorganic 32PO4 and analyzed on SDS-PAGE, demonstrate the association of phosphate label with the Piry NS, protein (Fig. 5). It has not been possible to conclusively demonstrate that 32P label is associated with protein *. :: ,: .:, NS1 in these experimnents. Although some radioG activity is found in this region, it is always overshadowed by the much larger amount of label in NSv. Short pulse-labeling with 32PO4 does not enhance the ratio of label in NSi/NSv. NSV We do not believe that these experiments demNSji = onstrate the absence or even the reduction on a N 140' 1 442 molar basis of the amount of phosphate in NSi relative to NS,. More likely, our inability to see phosphate label in NS, is simply the result of a L very large excess of NS, and the relatively slow f utilization of exogenous radioactive phosphate s: due to large internal precursor pools. i : Acid hydrolysis of phosphate-labeled NS, showed that phosphoserine was the principle i MAw phosphorylated component. Phosphothreonine was undetected, though a significant amount of unidentified material remained at the origin of Li chromatography. NF VIRWON A second phosphorylated protein, present in PIRY I ND CELL Piry-infected cells and absent in uninfected cells, FIG. 1. Comparison of viral proteins in Piry and migrates with a mobility just slightly faster than Indiana VSV-infected cells and Piry virions. Pitry Piry N protein. We have been unable, to date, virus and Indiana (IND) virus-infected cells (as in- to obtain this material in sufficient quantity and dicated) were labeled with [rS]methionine for30min purity for further analysis. some 4 h postinfection. The infected (INF) -cellprotein If the conversion of NS, to NS, were dependwas analyzed on SDS-PA GE. The location of the two NS bands in the Piry extract is indicated in the left- ent on phosphorylation, inhibitors of protein hand panel. Piry virions purified on sucrose gra- kinase activity might reduce or prevent this dients after overnight growth with [rS]methionine conversion. On this basis, we examined the eflabel were analyzed on SDS-PAGE in the right-hand fects of theophylline, cyclic AMP, and cyclic panel, with a control marker of ribonucleoprotein GMP, all of which act through cyclic nucleotideextract from pulse-labeled Piry-infected cells. mediated kinases as well as cordycepin, an inhibitor of both cyclic nucleotide-dependent and the conversion of NSi to NS, occurs with a half- -independent kinases (8). The theophilline result life of approximately 20 min under the condi- (Fig. 6) demonstrates that, though there was a tions employed. general reduction both in the amount of radioThe apparent conversion of NS, to NS, is active phosphate and [3S]methionine incorpoindependent of continued protein synthesis since rated into proteins at increased concentrations (Fig. 4) the conversion occurs at the same rate of these inhibitors, there was no concomitant in the presence of cycloheximide as in its ab- change in the relative rate of conversion of NSi sence. to NS, as evidenced by [3S]methionine label. From the preceding results we conclude that Cyclic AMP and GMP at concentrations of 10' the protein NSi is converted by post-transla- to 10-6 M also failed to have any effect on the tional modification to a protein of greater ap- conversion of NSi to NSv. The effect of cordyparent molecular weight, NS,. cepin was essentially identical to that of theoI,
I
.: :::.
* '' .pP.:.
.,: :i-,z7::
:: .:
:.
*:
.. ':
:
'
... ...
:.
..
..
*:.
i.:
4
.: :::
*',.;.;'
t X
_M..
i
' .:;! _ Tr.
VOL. 30, 1979
VSV PHOSPHOPROTEINS
59
a I C a: U 0 rs a.
I
I _*''
CHROMATOGRAPHY
NSV
NS;
FIG. 2. Tryptic fingerprint of methionine-labeled proteins NSi and NS,. [3S]methionine-labeled NSi and were purified by preparative SDS-PAGE from Piry-infected cell extracts labeled for 5 min and 30 min, respectively, some 4 h postinfection. The protein bands were excised from the gel, oxidized, and trypsinized as described in the text. Two-dimensional analysis by electrophoresis and chromatography was carried out on cellulose thin layer.
NS,
philline at corresponding concentrations. Lack of separation of NS, and NS, on nonequilibrium isoelectric focusing gels. The NS proteins of the VSV groups are highly acidic and do not generally focus on the conventional 0
-
isoelectric focusing system of O'Farrell (17). For
4O lbL
this reason, we employed modified non-equilibrium techniques of O'Farrell et al. (18) as described above. Figure 7 shows the relative position of the Piry NS, protein with respect to N, G, and M after two-dimensional analysis em-
FG
ploying non-equilibrium isoelectric focusing followed by SDS-PAGE in the second dimension. Infected cell extracts labeled for 30 min and, therefore, having NS, as the major labeled NS protein, or labeled for 5 min and, therefore, labeled predominantly in NS,, as well as a mixture of these two extracts, are shown. The individual extracts clearly show only a single labeled NS protein, whereas the mixture shows resolution of NS, and NS, in the SDS-PAGE dimension but no significant separation of these proteins in the isoelectric focusing dimnension. One-
*b:
NSv
4. ..~
* --i
'O'o ow.
W,-!~~~~~-:-
00
-~ n.11,..
A,4..-
NS,
I..
F.
to
w-
:;
go
N
M
. .40. .
min, the radioactive medium was removed and of the cultures were incubated excess unlabeled methionine, as described in the text. Samples were taken at the indicated times. The infected cells were harvested and analyzed on SDS-PAGE. The figure describes the length of the radioactive pulse plus the kngth of the subsequent chase. The reason for the double-M protein in the 15-min label experiment is not known. An extract of labeled uninfected cells is presented in the central well (CNT). or 15 some
L.~~~~~
5 5+5 5+10 5+20 C
dimensional isoelectric focusing analysis of these
1S 15+15 15+30 15+60
N T
FIG. 3. Pulse chase of protein NSi to NS,, [3SJmethionine label was added to Piry virus-infected L cels at 4 h postinfection. After a labeling period of 5
in
60
BELL AND PREVEC
J*.2Qz,:rXSt
J. VIROL.
greater than the coding capacity of the RNA for this protein (7, 20), makes any attempt to correlate electrophoretic mobility and molecular weight for this protein of doubtful significance. Since shape factors and/or degree of binding of
em.~~~~~~O
P
.50 5.20 5 +5 C O NTROL
5 5.5
510
5.20
CYCLOHEXIMIDE FIG. 4. Conversion of NSi to NS, in the presence and absence of cycloheximide. Piry virus-infected cells at 4 h postinfection were pulse labeled with [3S]methionine for 5 min and chased with excess unlabeled methionine. The chase medium for one experiment contained 50 pg of cycloheximide per ml. Samples were taken at the indicated times and analyzed by SDS-PAGE. The sample wells labeled (P) contain infected cell extracts kept in [3S]methionine
for the fuU 20-min period.
proteins as sharply focused bands failed to show any shift of NS, to a more negatively charged protein after conversion to NS, (data not presented). DISCUSSION This work clearly identifies a metabolic intermediate in the maturation of the NS, protein of Piry virus. The intermediate, NS,, readily demonstrable after short labeling periods in infected-cell extracts, is either absent from or a minor component of the mature virion. The decrease in electrophoretic mobility associated with the conversion on NSi to NS, is consistent under our electrophoretic conditions with an increase in molecular weight of about 1,000. The mobility of the VSV NS proteins relative to the other virion proteins may be altered by changing the percentage of N,N'methylenebisacrylamide in the resolving gel, resulting in apparent molecular weight differences of up to 10,000 (unpublished data). This, together with the fact that all molecular weight estimates of NS are almost a factor of two
ii>,...,-s: .....
_.
D:
NS v NSj N
.^:
....}
..
.: .:.
......
..
::.e:W
RYe:
*s., w .::
..,
,fi,
i 7r.
:.
*
_.W. _r
..
,js.
:.
...
...
.....
_|k;-
_ k
i ;' :}, - F
." _ Ei
35s 32p 32p FIG. 5. Phosphate-labeled proteins in Piuy virusinfected ceUs. Piry virus-infected cell cultures were labeled with [rPJorthophosphate from 3.5 to 5 h postinfection as described in the text (central well). An infected culture labeled over the same time interval with [3S]methionine (left well) and an uninfected ceU culture labeled for 90 min with [3Plorthophosphate (right well) are included as controls. The labeled ceU extracts were analyzed by SDS-PAGE.
VSV PHOSPHOPROTEINS
VOL. 30, 1979
. . 4, .4_
S
P
CNT
S
P
5 10
S
P -4
10
Si
M-
10
-3
THEOPHILLINE
FIG. 6. Treatment of Piry-infected L cells with theophilline. Monolayers of 5 x 106 L cells were infected with Piry virus at an MOI of 100 PFU/cell as described in the text and then overlaid with MEM containing 1:20 normal amino acid complement for [3Slmethionine labeling or with -PO0 MEM for labeling with [nP]orthophosphate. At this time, theophilline was added to each plate to a final concentration of 10-3 M, 10-4 M, or 10- M. At 3 hpostinfection, the overlay was removed and medium containing either [I35]methionine (30 yCi/ml) or [nP]orthophosphate (200X Ci/ml) was added to appropriate plates along with the inhibitor theophilline. Labeling was allowed to continue for 1.5 h at which time the samples were prepared for electr"phoresis as described in the text. P, labeled with [ Plorthophosphate; S, labeled with [IS]methionine.
SDS appear to play a large role in determining the mobility of this protein, it is conceivable that even a
low-molecular-weight substituent could
61
produce the resultant mobility difference observed between NS, and NSi. Although we originally pursued the idea that NS, and NS, differed in extent of phosphorylation, we have been unable to either support or directly disprove this hypothesis. The lack of effect of cordycepin and the cyclic nucleotides on the rate of conversion of NS, and NS, can be considered only as negative evidence rather than proof that phosphorylation is not involved. Furthermore, our inability to demonstrate an increased negative charge in NS, relative to NSi argues against phosphorylation as the direct cause of the conversion. The biological significance of the NS, to NS, conversion is not known. Unpublished experiments carried out by us show approximately the same relative ratio, after short labeling periods, of NS, to NS, in the polyribosomal as well as the soluble protein fraction of the infected cell. This would suggest that the conversion of NSi to NS, occurs equally in both fractions and that there is no apparent functional discrimination between these proteins in ribonucleoprotein complexes as compared to free protein. The central role of NS protein in the VSV transcriptase system as well as its potential involvement in virus replication and host cell regulation make these post-translational modifications of considerable interest. The finding of a second major phosphoprotein in Piry-infected cells, that protein having a mobility just slightly faster than Piry N protein, distinguishes Piry from the Indiana, Cocal, New Jersey, and Chandipura serotypes which, in our hands, show a single major phosphoprotein band. Two phosphoprotein bands have been reported for the salmonid virus IHN (14) and Kern Canyon virus (21). A more detailed analysis of the number and association of phosphates with proteins in different members ofthe rhabdovirus group must be carried out before any meaningful assessment of the functional or genetic significance of this distribution can be obtained. The possibility that analogs of Piry NSi exist in other members of the VSV group is currently under investigation. ACKNOW LEDGMENTS We thank E. G. Brown, D. Takayesu, and H. P. Ghosh for helpful discussion and advice. Theophilline, cAMP, and dibutryl cGMP were generously supplied by R. Haslam, and P. Branton donated cordycepin used throughout this work. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. LITERATURE CIMD 1. Abraham, G., D. P. Rhodes, and A. K. Baneijee. 1975. The 5' terminal structure of the methylated mRNA
62
BELL AND PREVEC
J. VIROL.
5
A
30 G ilN I-
IF p4
pH ,
V),
-*
0
a
(ft
3
2
5+30
NS
4' i
.N
.NS
'NS. 2
...p .4.
pH 4
3
2
FIG. 7. Two-dimensional analysis of Piry-infected L cells. Infected monolayers were pulse labeled with
[nSJmethionine, 4 h postinfection, either for 5 min or 30 min as described in the text. Samples, prepared for NEPHGE, were applied to the top of isoelectric slab gels and run for 4 h at 500 V. Appropriate channels were then immediately prepared for and run in the second dimension in SDS-PAGE, as described by O'Farrell (17). Shown above: 5-min pulse label; 30-min pulse label; mixture of a 5- and 30-min pulse label. synthesized in vitro by vesicular stomatitis virus. Cell 5:51-58. 2. Banerjee, A. K., and D. P. Rhodes. 1973. In vitro synthesis ofRNA that contains polyadenylate by virionassociated RNA polymerase of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 70:3566-3570. 2a.Brown, E. G., and L Prevec. 1978. Proteins of vesicular stomatitis virus. IV. A comparison of tryptic peptides of the VSV subgroup of rhabdovirus. Virology 89:7-21. 3. Clewley, J. P., D. H. L Bishop, C. Y. Kang, J. Coffim, W. M. Schnitzlein, M. E. Reichman, and R. E. Shope. 1977. Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the VSV subgroup. J. Virol. 23:152-166. 4. DoeL T. R., and F. Brown. 1978. Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus. J. Gen. Virol. 38:351-361. 5. Emerson, S. U., and R. R. Wagner. 1972. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J. Virol. 10:297-309. 6. Emerson, S. U., and Y.-H. Yu. 1975. Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. J. Virol. 15:1348-1356. 7. Freeman, G. J., J. K. Rose, G. K. Clinton, and A. S. Huang. 1977. RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation. J. Virol. 21:1094-1104.
8. Hirsch, J., and 0. J. Martello. 1976. Inhibition of nuclear protein kinases by adenosine analogues. Life Sci.
19:85-90. 9. Imblum, R. L., and R. R. Wagner. 1975. Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus. J. Virol. 15:1357-1366. 10. Kang, C. Y., and L Prevec. 1971. Proteins of vesicular stomatitis virus. Virology 46:628-690. 11. Laemlli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Lee, S. G., M. V. Micelli, R. A. Tungman, and P. P. Huang. 1975. Protein kinase and its regulatory effect on reverse transcriptase activity of Rous Sarcoma virus. Proc. Natl. Acad. Sci. U.S.A. 72:2945-2949. 13. Legault, D., D. Takayesu, and L Prevec. 1977. Heterotypic exclusion between vesicular stomatitis virus of the New Jersey and Indiana serotypes. J. Gen. Virol.
35:53-65.
14. McAllister, P. E., and R. R. Wagner. 1975. Structural proteins of two Salmonid rhabdoviruses. J. Virol. 15: 733-738. 15. Naito, S., and A. Ishihama. 1976. Function and structure of RNA polymerase from VSV. J. Biol. Chem. 251: 4307-4314. 16. Nowakowski, M., W. Bauer, and J. Kates. 1978. Characterization of a DNA-binding phosphoprotein from vaccinia virus replication complex. Virology 86:217-225.
VOL. 30, 1979 17. O'Farrell, P. H. 1975. High resolution two dimensional electrophoresis by proteins. J. Biol. Chem. 250:40074021. 18. O'Farrel, P. Z., H. M. Goodman, and P. H. O'Farrell. 1977. High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133-1142. 19. Privalsky, M. L, and E. E. Penhoet. 1977. Phosphorylated protein component present in influenza virions. J. Virol. 24:401-405. 20. Rhodes, D. P., G. Abraham, R. J. Colonno, W. Jelinek, and A. K. Banerjee. 1977. Characterization of vesicular stomatitis virus mRNA species synthesized in vitro. J. Virol. 21:1105-1112.
VSV PHOSPHOPROTEINS
63
21. Sokol, F., and H. F. Clark. 1973. Phosphoproteins structural components of rhabdoviruses. Virology 52:246263. 22. Szilagy, J. F., and L. Uryvayev. 1973. Isolation of an infectious ribonucleoprotein from VSV containing an active RNA transciptase. J. Virol. 11:279-286. 23. Taborsky, G. 1974. Phosphoproteins. Adv. Prot. Chem. 28:1-210. 24. Watanabe, Y., S. Sakeura, and S. Tanaka. 1974. A possible biological function of the protein kinase associated with vaccinia and VSV virion. FEBS Lett. 41: 331-334.
