Synthesis by Vesicular Stomatitis Virus

Transcription

1 JOURNAL OF VIROLOGY, June, 1975, p Copyright 1975 American Society for Microbiology Vol. 15, No. 6 Printed in U.S.A. Both NS and L Proteins Are Required for In Vitro RNA Synthesis by Vesicular Stomatitis Virus SUZANNE U. EMERSON* AND YU-HWA YU Department of Microbiology, The University of Virginia School of Medicine, Charlottesville, Virginia 2291 Received for publication 22 January 1975 Vesicular stomatitis virions, Indiana serotype, were solubilized with high salt solubilizer and separated by ultracentrifugation into a supernatant fraction containing L, G, NS, and M proteins and pellet fraction containing the RNA complexed with N protein. NS protein was purified from the supernatant fluid by sequential chromatography on phosphocellulose and diethylaminoethyl cellulose columns. The purified NS protein was assayed in a standard transcription system in combination with purified L protein and purified template (pellet fraction) prepared by renografin or CsCl banding. Results of the polymerase assays indicate that both L and NS proteins are required to reconstitute transcription activity with a highly purified template composed of only RNA and N protein. The NS protein polymerase activity is destroyed by trypsin but withstands 9 C temperatures for 1 min. Cytoplasmic NS protein can substitute for virion NS protein in the in vitro transcription assay. Vesicular stomatitis (VS) virus is a negativestrand RNA virus composed of five proteins and a lipid envelope (27). The VS virions contain a transcriptase which synthesizes monocistronic mrna both in vivo and in vitro (1, 5, 12); the entire genome is transcribed and polyadenylic acid sequences are attached to the transcripts by an unknown process (2, 3, 9, 1). Viral proteins are required for infectivity since deproteinized RNA is not infectious (13). A viral subparticle, the ribonucleocapsid, which is composed of the RNA, N (nucleocapsid), L (large), and NS proteins synthesizes messagelike RNA in vitro and is infectious if DEAEdextran is included to promote cellular uptake (6, 7, 24). Therefore, these three ribonucleoproteins are sufficient for both transcription and initiation of the viral reproductive cycle, whereas the two envelope proteins G (glycoprotein) and M (membrane) are not essential. High concentrations of NaCl solubilize the L, G, M, and NS proteins while leaving the N protein complexed with the RNA (7). The RNA-N protein complex can be separated from the solubilized proteins by ultracentrifugation. Neither the pellet (RNA and N protein) nor the supernatant (L, G, NS, and M proteins) fraction can synthesize RNA by itself, but transcription is readily demonstrated if the pellet and supernatant fluid are recombined (4, 7, 14). Through solubilization and reconstitution assays, we previously demonstrated that the L protein and the N protein-rna complex are required for transcription (8). In this report, we propose that NS protein is also required for transcription. In other words, all three nucleoproteins appear to be necessary for efficient synthesis of mrna. MATERIALS AND METHODS Chemicals and radiochemicals. The sources of most chemicals and radiochemicals have been previously identified (8). [3H]UTP (19 Ci/mM) and [3H]leucine (51 Ci/mM) were purchased from Schwarz/Mann, Orangeburg, N.Y. Renografin (76% stock) was from E. R. Squibb and Sons, New York, N.Y. Microgranular DEAE-cellulose was supplied by Whatman Chemical, Ltd., Maidstone, Kent, England. Trypsin-TPCK (211.9 U/mg) was from Worthington Biochemical Corp., Freehold, N.J. Viruses and cells. The Indiana serotype of VS virus was grown in monolayer cultures of BHK-21 cells inoculated at a multiplicity of infection of 1. Incubation was at 37 C for 18 h in Eagle basal medium diluted 1:5 with Earle's balanced salt solution and containing 1% tryptose phosphate broth and.5 jsci of [8H]leucine per ml. Unlabeled virus grown under similar conditions was used to prepare template. Cytoplasmic NS protein was prepared from L cells grown as previously described (7) and was labeled by adding.5 uci of [3H]leucine per ml to Eagle basal medium, diluted 1:5 with Earle's balanced salt solution. Virus purification. The first part of the virus purification was as previously described (8). Virions were pelleted through 5% glycerol and centrifuged in 1348

2 VOL. 15, 1975 VS VIRION TRANSCRIPTASE 1349 a linear sucrose gradient ( to 4%) to separate B and T particles; in contrast to the initial procedure, the sucrose gradients and remaining solutions used in the purification contained 1 M NaCl and 1 mm disodium EDTA. The B band was collected, diluted with Tris-NaCl-EDTA, and the virions were pelleted. The virions were then centrifuged to equilibrium in a preformed sucrose gradient ( to 6%), containing Tris-NaCl-EDTA, by centrifuging overnight at 5 C at 11, x g. The viral band was collected, and the virions were repelleted. The final pellet was suspended in.1 M Tris-hydrochloride, ph 7.4, containing 15% glycerol, and virions were stored at 4 C (for supernatant fluid preparations) or -7 C (for template). The NaCl-EDTA and the equilibrium gradient are required to obtain a system dependent on NS protein, although a tartrate density gradient can be substituted for the sucrose density gradient. Solutions. High salt solubilizer (HSS) and polymerase reaction mixture were prepared as previously described (8). Water, glycerol, and Tris-hydrochloride were autoclaved. HSS contained glycerol, dithiothreitol, Triton X-1, and.72 M NaCl in.1 M Tris-hydrochloride (ph 7.4). The polymerase reaction mixture was identical to that used before, except the ['HJUTP was of slightly higher specific activity and the Tris was adjusted to ph 8. instead of 7.4. Column buffer was prepared by mixing 75 ml of glycerol, 1 ml of Triton X-1 (2% stock), 9 mg of dithiothreitol, and.5 M Tris-hydrochloride, ph 7.4, to 3 ml; appropriate quantities of NaCl were then added to yield the indicated concentration. Protein solubilization and column chromatography. Supernatant proteins were prepared by gently mixing 5 ml of virus (yield from thirty 75-cm' Falcon flasks each with 2 x 17 cells) with 5 ml of 2x HSS, and by removing the nucleocapsids by centrifugation at 125, x g for 2 h as previously described. Phosphocellulose chromatography was performed as described (8), except the pre-column sample was dialyzed overnight at 4 C against.1 M NaCl column buffer prior to column fractionation. All fractions from the phosphocellulose column were collected manually (8). The wash-through from the phosphocellulose column, containing G and NS proteins, was further fractionated on a DEAE-cellulose column. DEAE-cellulose was equilibrated with.1 M Tris-hydrochloride, ph 7.4, and approximately 3 ml of packed DEAE-cellulose was used per column. The column was equilibrated with.1 M NaCl column buffer, and the wash-through from the phosphocellulose column was loaded after batch readsorption with excess phosphocellulose. The DEAE column was developed with a linear salt gradient of.1 to.3 M NaCl column buffer, and 8-drop fractions were collected by using a drop counter. All fractions had 1 pg of human hemoglobin added as carrier. In one case L and NS fractions were dialyzed against.288 M NaCl column buffer and then assayed. However, we have found that better reconstitution occurs if the labile L protein, which is eluted from the phosphocellulose column with 1 M NaCl column buffer, is stored at -18 C in this buffer, and the more stable NS protein is dialyzed against.1 M NaCl column buffer; a mixture of 1 volume each of template and L protein, 2 volumes of NS protein, and 4 volumes of polymerase reaction mixture yields the correct NaCl concentration for optimal transcription. Template preparation. Renografin template was prepared by diluting the virus purified from 3 flasks to 11 ml with Tris-hydrochloride, ph 7.4 by adding 11 ml of 2x HSS, and by gently swirling periodically in an ice bath for 1 h. Any large aggregates were removed by centrifugation at 1 x g, and the supernatant fluid was layered over.5 ml of renografin stock overlaid with 1 ml of renografin diluted 1:5 with HSS in 5-ml tubes. Centrifugation was for 2 h (5 C) at 125, x g. The light-scattering nucleocapsid band at renografin interfaces was removed with a Pasteur pipette, and samples were pooled, diluted to 12 ml with HSS, and layered over two concentrations of glycerol in 5-ml tubes (.2 ml of 1% glycerol followed by 1 ml of 3% glycerol-7% Tris-hydrochloride). The sample was centrifuged for 9 min (5 C) at 125, x g. The supernatant fluid and most of the 3% glycerol was discarded; the remaining glycerol containing the nucleocapsids was stored at -7 C until used. CsCl-purified template was prepared as above through the renografin step. The renografin interface, corresponding to the virus yield from five flasks, was diluted to 4.63 ml with Tris-hydrochloride (ph 7.4), and 1.46 g of CsCl was added. Centrifugation was for 48 h (22 C) at 125, x g. The renografin crystallized out and was pelleted, while a light-scattering nucleocapsid band was visible in the bottom third of the tube. The band was removed with a pipette, dialyzed extensively against column buffer at 4 C, and frozen at -7 C. Transcriptase assays and sodium dodecyl sulfate gel electrophoresis. These procedures were performed exactly as previously described (8). The data for the polymerase assays is given as the average of duplicate or triplicate time points. The time zero of the polymerase assays represented ['H ]leucine incorporated into the supernatant proteins plus about 5 counts/min due to free ['H JUTP. A separate time zero background was determined for each sample, and this value was subtracted from the total trichloroacetic acid-precipitable radioactivity at each time. The time zero control averaged 1,5 counts/min for samples containing L and/or NS proteins and 3, counts/ min for samples containing G protein. RESULTS Renografin purification of template. Template prepared by previously published methods has always manifested a low level of endogenous polymerase activity which is greatly increased by addition of L (large) protein (8). We have found that this residual activity can be eliminated by treating the virus with HSS, centrifuging the nucleocapsid onto a renografin pad, then retreating the nucleocapsid with HSS and centrifuging the nucleocapsid onto a

3 135 EMERSON AND YU J.- VIROL. glycerol pad. This template (referred to as renografin template) has been stored frozen in glycerol for as long as two weeks at -7 C with no loss of activity in reconstitution assays. No detectable RNA was synthesized by such a template preparation incubated under standard polymerase assay conditions for up to 4 h. Figure 1 shows the sodium dodecyl sulfatepolyacrylamide gel patterns of a renografinpurified template compared with that of the high salt-solubilized fraction prior to phosphocellulose chromatography. Protein N is virtually the only protein present in the nucleocapsid template after repeated exposure to HSS (Fig. 1A). All the other virion proteins are recovered in the solubilized fraction (Fig. 1B), lo- A. TEMPLATE 8-6- r? 4-2- L G NS M C I, "N,. B. PRE-COLUMN SUPERNATANT 16- G L 4- N Gel Fraction FIG. 1. Electropherograms of 3H-labeled proteins in the renografin-purified template (A) and in the high salt-solubilized fraction prior to phosphocellulose chromatography (B). VS virions labeled with [9H]leucine were grown in BHK cells and purified (see Materials and Methods). Template was prepared by solubilizing virions with HSS and by centrifuging the nucleocapsid first through renografin and then through glycerol. The pre-column sample was prepared by mixing the virions with HSS and by collecting the supernatant fluid from a 125, x g centrifugation. Samples were trichloroacetic acidprecipitated, mixed with "4C-labeled amino acidlabeled marker virions, and analyzed on 7.5% sodium dodecyl sulfate-acrylamide gels. Arrows indicate the peaks of marker proteins. which served as the source of the L and NS proteins. Two different phosphocellulose column fractions are required to reconstitute polymerase activity. Most of the NS and G proteins do not bind to phosphocellulose, whereas M and L proteins bind but are eluted at different NaCl concentrations (8). Therefore, the soluble virion proteins can be partially purified by phosphocellulose column chromatography. The proteins solubilized by HSS (Fig. 1B) were fractionated on a phosphocellulose column by exclusion (wash-through) of G and NS proteins followed by salt elution of bound L and M proteins as previously described (8). The washthrough from the column was mixed with phosphocellulose to remove any residual L or M proteins which had not bound to the column. The phosphocellulose column was next washed with.5 M NaCl column buffer to remove M protein, and the L protein was eluted with 1 M NaCl column buffer. The L protein and washedthrough fraction (NS and G proteins) were dialyzed overnight at 4 C against.288 M NaCl column buffer and assayed for polymerase activity with varying dilutions of template. A fiveor tenfold dilution of template did not significantly lower the levels of [3IH]UTP incorporation; therefore, template concentrations were not limiting. The renografin template displayed no endogenous polymerase activity but polymerase activity was reconstituted to approximately the same low level by addition of two different concentrations of L protein (Table 1); therefore, the L protein fraction was not limiting under these conditions. Addition of the wash-through fraction to the template did not reconstitute polymerase activity by itself, but markedly increased [3H]UTP incorporation when added concomitantly with L protein. The washthrough fraction served to stimulate transcription in proportion to the amount added, and total incorporation was maximum when both L and the wash-through fractions were increased. From these results, we conclude that both the L protein and a nondialyzable component of the wash-through fraction from a phosphocellulose column are required for maximum polymerase activity. DEAE-cellulose-purified NS protein stimulates transcription. L protein was purified by phosphocellulose chromatography as described above and stored overnight at -18 C in 1 M NaCl column buffer. The wash-through from the phosphocellulose column, containing G and NS proteins, was readsorbed with phosphocel-

4 VOL. 15, 1975 VS VIRION TRANSCRIPTASE 1351 TABLE 1. Stimulation of transcription by the wash-through fraction from a phosphocellulose column loaded with soluble viral proteins Additiona (ml) ['H]UTP (counts Stimulation L Wash- per min per 5 h) by NSb through O , ,93 2.6x.1.3 5, x.3 1, , x athe standard assay mixture contained.1 ml of template,.7 ml of reaction mixture, and a total of.6 ml of L protein, wash-through fraction, and.288 M column buffer. Duplicates (.1 ml) were incubated at 31 C and precipitated at hourly intervals. Kinetics were linear for the entire 5-h period. b Calculated by dividing activity reconstituted by L with NS protein, by that from L protein alone. lulose, and then the components were fractionated by DEAE-cellulose column chromatography (16). The fractions collected from the DEAE-cellulose column were dialyzed against.1 M NaCl column buffer and tested in the polymerase assay in combination with renografin template and L protein. Only fractions 2 to 22 stimulated polymerase activity when added with L protein to the template (Fig. 2); fractions 2 to 22 were coincident with a labeled peak of viral protein. The template itself exhibited no endogenous activity, so the base line represents the level of transcription reconstituted by addition of the L protein. A twofold dilution of fraction 21 stimulated transcription to approximately the same level as the undiluted fraction 21; therefore, the active component in fraction 21 was assayed at, or very near, saturating concentrations, and stimulated transcription approximately 2-fold above the level produced by L protein alone. Figure 3A shows the sodium dodecyl sulfateacrylamide gel profile of the polymerase active peak from a DEAE-cellulose column. The NS protein is the major component, although there are trace contaminants of G and M proteins. However, since low levels of G and M proteins also contaminate the L fraction (Fig. 3B), NS is the only viral protein unique to the polymerase peak of the DEAE-cellulose column. Fractions 5 and 8, which were inactive in the polymerase assay (Fig. 2), contained only G protein (data not shown). These results indicate that NS protein plays a major role in transcription. Kinetic analysis and effect of preincubating NS protein with template. DEAE-purified NS protein was mixed with renografin template and assayed immediately with or without L protein and nucleoside triphosphates, or preincubated for 2 h at 37 C prior to addition of L protein and nucleoside triphosphates to determine if the kinetics of the reaction were altered by preincubation. The two preparations behaved identically (Fig. 4). The kinetics are linear for at least 4 h, and the lines extrapolate to time zero, suggesting that there is no appreciable lag before reconstitution occurs. NS protein is required for reconstitution of polymerase activity. The L fraction alone reconstituted a low level of polymerase activity when added to renografin template. Two interpretations of this result are possible. (i) L protein could function as a polymerase by itself t T x 2- - D E 8- N'. - L) 4 1 v).e Cl -2 E -1 OC Fraction FIG. 2. Fractionation of G and NS proteins on a DEAE-cellulose column. Tritium-labeled proteins solubilized with HSS were dialyzed against.1 M NaCI column buffer and loaded onto a phosphocellulose column. The wash-through fractions were pooled (total, about 1 ml), retreated with phosphocellulose, and then chromatographed on a DEAEcellulose column. The column was developed with a linear gradient consisting of 15 ml of.1 M NaCl column buffer and 15 ml of.3 MNaCl column buffer. Fractions of 8 drops each were collected into tubes containing hemoglobin. Aliquots (25 ul) of each fractions were counted to locate tritium-labeled protein. Individual fractions were dialyzed against.1 MNaCl column buffer, and.2-ml aliquots were tested in the standard polymerase assay system by using.1 ml of renografin template and.1 ml of L protein in 1 M NaCl column buffer. Samples were trichloroacetic acid-precipitated and counted (see Materials and Methods).

5 1352 EMERSON AND YU J. VIROL. protein was omitted. Therefore, it appears that NS protein is required for transcription and a renografin template retains a low level of residual NS protein. The conclusion that renografin template is impure is also supported by the G E,,,I s B. L FRACTION ~~~~GN Gel Fraction FIG. 3. Electropherograms of 3H-labeled proteins in the active peak from a DEAE column (A) and the L fraction as purified on phosphocellulose (B). The tubes corresponding to the active fractions from a DEAE column loaded with the phosphocellulose wash-through proteins were pooled, trichloroacetic acid-precipitated, and analyzed (see legend to Fig. 1). The L protein was purified from a phosphocellulose column by elution with 1 M NaCI column buffer as previously described and processed (see legend to Fig. 1). while NS merely enhances the rate of transcription, or (ii) NS protein might be absolutely required for polymerase activity, in which case, the low level of transcription resulted from the interaction of L protein with residual NS protein contaminating the renografin template. An attempt to further purify the template was undertaken to distinguish between these possibilities. Template was prepared as usual through the renografin step; next, the renografin interface was diluted with CsCl in Tris-hydrochloride, and the mixture was centrifuged to equilibrium. The nucleocapsid template was removed, dialyzed extensively against column buffer, and tested in a reconstitution assay by using purified L and NS proteins. L protein was completely inactive when assayed alone with the CsCl template, whereas a mixture of L and NS reconstituted a high level of polymerase activity (Fig. 5). The NS protein was tested in a separate experiment and was inactive if L M M 4- ~ X / L + N S D C E / A A NS Hours FIG. 4. Kinetic analysis of in vitro transcription demonstrating the stimulatory effect of NS protein. The labeled fractions corresponding to the L and NS peaks from phosphocellulose and DEAE-cellulose columns, respectively, were individually pooled. The L protein was stored overnight in the 1 M NaCI column buffer at -18 C, while the NS protein was dialyzed against.1 M NaCI column buffer. The proteins were assayed with renografin template under standard polymerase conditions. Neither the template fraction nor template with NS protein added synthesized detectable RNA. Symbols: template alone (), template and NS protein (), template and L (), template and L and NS proteins (A), template preincubated with NS then L protein added (x). L K

6 VOL. 15, 1975 VS VIRION TRANSCRIPTASE r- ~~~~~L+NS x 2 L+ S - D 16- c *\8-/ Template + L Template Alone Hours FIG. 5. 7ranscription assay demonstrating that both L and NS protein are required to reconstitute activity with a CsCI template. The template was purified by CsCI equilibrium banding (see Materials and Methods). A standard polymerase assay was performed. Symbols: template alone (x), template and L (x), template and L and NS (A). results of a NS antibody neutralization experiment. Anti-NS gamma globulin (described in the accompanying paper) inhibits transcription (16). [3H ]UTP incorporation by a reconstituted renografin template was inhibited by antibody to NS protein whether exogenous NS protein was added or not, suggesting some NS protein was already present on the template; 77% of the incorporation was eliminated if only L protein was added, and 85% was eliminated if both L and NS proteins were added (data not shown). The viral nucleocapsids aggregated into a compact mass during CsCl equilibrium centrifugation, so that only small quantities of material could be prepared by this method. Therefore, even though the renografin template was not as pure as the CsCl-purified template, the renografin template was used for most experiments because it could be prepared in sufficient amounts, and the background reconstitution of polymerase activity by L protein with renografin template was always insignificant compared to the level reconstituted by a combination of L and NS proteins. Heat stability and trypsin sensitivity of NS protein. To demonstrate that the second active component was NS protein rather than a nonprotein compound, purified NS protein was digested with trypsin. L protein was purified as described above, while template was prepared by glycerol gradients and CsCl banding. NS protein was purified as previously stated and dialyzed against.1 M NaCl column buffer. NS protein retains its activity when heated (Table 2); therefore, high temperatures were employed to inactivate the trypsin without destroying the NS protein polymerase activity. Aliquots of NS protein were mixed with trypsin or column buffer and incubated first at 37C to allow proteolysis and then at 9 C to denature the trypsin. The activities of heated samples, trypsintreated samples, and unheated NS protein samples were compared in a standard polymerase assay after addition of L protein and template. NS protein is very heat stable (Table 2); the sample with NS heated to 9 C incorporated 89% as much label as the sample with unheated NS. The active factor is a protein, since proteolytic digestion of the NS fraction with trypsin decreased the amount of incorporation by 94%. Cytoplasmic NS protein can replace virion NS protein in the polymerase assay. NS is the only VS viral protein which can be found in infected cells both in the soluble cytoplasmic fraction and bound to nucleocapsids (17, 2, 28). The other viral proteins interact with either nucleocapsids or membranes and can be separated from cytoplasmic NS protein by centrifugation (16, 26). It is not known whether cytoplasmic NS protein is identical to nucleocapsid TABLE 2. Heat stability and trypsin sensitivity of the transcriptase activity of the NS protein fractiona ['H JUTP % Activity NSNSsample incorporated (counts per of heated NS min per h) sample Untreated 3, Heated 3,66 1 Trypsinized, heatedb Heated NS-trypsin mixc 2, Heated NS and heated 2, trypsind athe template and template plus either L or NS protein incorporated less than 85 counts per min per h. Aliquots (.2 ml) of NS protein were incubated at 37 C for 2 min with either 1 gl of trypsin (.5 mg/ml) or an equivalent volume of.1 M NaCl column buffer. All samples but the untreated NS protein were then incubated at 9 C for 1 min (heated samples) prior to being tested. Assays were performed after adding.1 ml of L protein in 1 M NaCl column buffer to the NS protein samples. btrypsin was added prior to 37 C incubation. csample was incubated at 9 C for 1 min, then trypsin was added for the last 9 min at 9 C. dns protein and trypsin were individually heated to 9 C, and then mixed prior to testing.

7 1354 EMERSON AND YU J. VIROL. NS protein. Therefore, it was of interest to determine if the cytoplasmic NS protein could replace virion NS protein in a polymerase assay. Tritium-labeled cytoplasmic NS protein was prepared by centrifuging a lysate of VS virus infected L cells at 125, x g for 2 h, and the supernatant NS fraction was purified by phosphocellulose treatment and DEAE-cellulose chromatography. Only 5% of the 3H label applied to the DEAE-cellulose bound to the column; this label eluted as a single peak at the NaCl concentration which also elutes virion NS protein. The gel pattern of the proteins in this peak (Fig. 6) confirms that NS protein was the major labeled component isolated from the cytoplasm. The fractions corresponding to NS protein were pooled and dialyzed against.1 M NaCl column buffer, and then assayed for polymerase activity. Cytoplasmic NS protein was inactive in the absence of added L protein but reconstituted polymerase activity when L protein was included (Fig. 7). The sample with cytoplasmic NS protein incorporated as much radiolabel as did the sample with virion NS protein (data not shown). However, since the virion NS protein was assayed at close to saturating concentrations, no conclusions can be drawn regarding the relative efficiencies of cytoplasmic and virionderived NS proteins in the in vitro polymerase assay. DISCUSSION We had previously demonstrated that L protein was required for in vitro transcription of VS virus but were unable to show a dependence on NS protein (8). By purifying virions through 1 x c._e NS I N L G I M I I I Gel Fraction FIG. 6. Electropherograms of the cytoplasmic NS protein purified on a DEAE-cellulose column. The WHulabeled cytoplasmic NS fraction (assay shown in Fig. 7) was trichloroacetic acid-precipitated and electrophoresed on sodium dodecyl sulfate-acrylamide gels (see legend to Fig. 1). Arrows denote the positions of "4C-labeled marker proteins. b ' <6 X / L+NS :D ^ 4- E '/ L NS 1 2 Hours FIG. 7. Polymerase assay demonstrating that cytoplasmic NS protein can be substituted for virion NS protein in vitro. Twenty bottles of L cells were infected at a multiplicity of approximately 2. [8H]leucine was added 2 h after infection, and the cells were harvested 3 h later. Cell lysates were prepared by Dounce homogenization (see Materials and Methods) and particulate material, including nucleocapsids, was removed by centrifugation. The supernatant fluid (-. 1 ml) was absorbed twice with 5 ml of phosphocellulose and stored overnight at 4 C prior to chromatography on DEAE-cellulose. The remaining protocol was identical to that used to purify and assay virion NS. Symbols: template alone (), template and NS (), template and L (), template and L and NS (A). M NaCl to reduce the level of ribonuclease and by modifying the procedures for template preparation, we have been able to demonstrate that template plus L protein alone reconstitutes little or no transcriptase activity, whereas the inclusion of both the NS and L protein fractions reconstitutes a high level of transcription. The trypsin sensitivity of the NS protein fraction indicates that the active component in this NS fraction is a protein even though it retains activity after being heated to 9 C. Unfortu- r

8 VOL. 15, 1975 VS VIRION TRANSCRIPTASE nately, the viral proteins tend to aggregate during the purification procedures, and so far it has not been possible to obtain NS protein absolutely free of G or M proteins. However, since the envelope proteins G and M are unnecessary for transcription or infection (6, 7, 24) and since the L fraction, which also contained traces of G and M proteins, reconstituted only a low level of activity at two different concentrations (Table 1), we believe that NS is the second solubilized protein which is required for transcription. Because no RNA synthesis is observed if L or NS protein is added individually to CsCl-purified template, it would appear that NS protein, as well as L protein, is absolutely required for transcription and is not just increasing the efficiency of transcription. This result suggests that previous attempts to detect an effect of NS protein failed because residual NS protein contaminated the template. The question of why some cytoplasmic NS protein is soluble whereas the remainder is bound to nucleocapsid has not been answered. Although cytoplasmic and virion NS proteins apparently are interchangeable in in vitro polymerase assays, each may perform different functions in vivo, which could explain the observed compartmentalization. Alternatively, cytoplasmic NS protein. may be unable to interact with nucleocapsid due to a trivial reason such as lack of available binding sites on the template. Since NS is a phosphoprotein (15, 19, 23), it will be of interest to determine if the cytoplasmic and virion NS proteins are phosphorylated to the same extent and whether the degree or sites of phosphorylation alter the activity of NS protein. The NS protein we have studied displays a striking heat stability. Therefore, it may be very difficult to isolate temperature-sensitive mutants which have an altered NS protein. Three complementation groups of VS virus Indiana contain mutants deficient in RNA synthesis (18, 22, 29). These three groups could correlate with mutations in each of the three proteins, N, L, and NS, all of which are required for optimal transcription in vitro. The roles of L and NS proteins in transcription remain to be determined. Both could interact as subunits in a heteropolymeric enzyme or each could act as independent proteins. For instance, NS protein might interact with nucleocapsid protein to expose specific sites on the RNA, thus enabling L protein to bind and act as the polymerase or vice-versa. The accompanying paper (16) demonstrates that addition of anti-ns gamma globulin to a 1355 transcribing system immediately terminates [3H]UTP incorporation; this result, in conjunction with the results presented above, suggests that one or more functions of NS protein is continuously required for transcription. Transcription by VS virus is clearly more complicated than originally thought, and much more data are required before the exact functions of either L or NS protein are defined. ACKNOWLEDGMENTS This research was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. We thank Charles Emerson, Margaret Hunt, Robert Wagner, and Jay Brown for many helpful suggestions. 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: Baneijee, A. K., and D. P. Rhodes In vitro synthesis of RNA that contains polyadenylate by virionassociated RNA polymerase of vesicular stomatitis virus. Proc. Natl. Acad. U.S.A. 7: Bishop, D. H. L Complete transcription by the transcriptase of vesicular stomatitis virus. J. Virol. 7: Bishop, D. H. L., S. U. Emerson, and A. 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