Synthesis of VSV RNPs in vitro by cellular VSV RNPs added to uninfected HeLa cell extracts: VSV protein requirements for replication in vitro

VIROLOGY123,407-419 (1982)

Synthesis of VSV RNPs in Vitro by Cellular VSV RNPs Added to Uninfected HeLa Cell Extracts: VSV Protein Requirements for Replication in Vitro VIRGINIA

M. HILL

AND

DONALD F. SUMMERS

Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City, Utah 8413.2 Received June S, 1982;accepted July 30, 1982 Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa SlO extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to e-fold, although the synthesis of mRNA was inhibited.

pools of RNP proteins in the I-S10 extracts sufficient to sustain replication in vitro for HeLa cell extracts from cells infected an hour or more (Hill et al., 1981). with vesicular stomatitis virus (VSV) (ISince one of our initial goals in these SlO) have been shown to support the rep- studies was to establish an in vitro system lication and assembly in vitro of VSV ri- in which RNA replication and RNP assembonucleoproteins (RNPs) (Hill et al., 1981). bly were totally dependent upon transcripde WUO, we turned to a The newly synthesized RNPs contained tion-translation (-)- and (+)-strand genome-length RNA nuclease-treated uninfected HeLa SlO exmolecules (42 S) and were found in about tract (U-SlO) which was supplemented the same ratio as that observed in viva with VSV cellular RNPs (cRNPs) isolated (Simonsen et al., 1979b). Genome-length from VSV-infected HeLa cells. RNA synthesized in vitro was assembled Here we report that this system is deinto RNPs containing N protein, was nu- pendent on VSV-specific protein synthesis clease-resistant, and had the same buoy- for RNP synthesis and assembly in vitro. ant density in CsCl as virion RNPs. How- Through the use of monospecific antisera ever, replication in vitro continued in the directed against each VSV gene product presence of pactamycin in spite of the fact we have established that the presence of that virus-specific protein synthesis was VSV N and NS protein are required for inhibited; this was probably due to the replication in this coupled in vitro reacpresence of a pool of VSV-specific RNP tion. proteins present in the concentrated I-S10 extracts. If cycloheximide was added to MATERIALS AND METHODS cells before the I-S10 extracts were prepared, replication in vitro did not occur or Cells, virus, and preparation of infected SlO extracts (145’10). HeLa S3cells and VSV was markedly reduced, an observation which indicated the presence of existing Indiana were grown as previously described (Hunt and Summers, 1976), except 1 To whom reprint requests should be addressed. 10% heat-inactivated (56” for 1 hr) calf INTRODUCTION

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serum was used instead of fetal calf serum. VSV-infected HeLa cell SlO extracts were prepared as described (Hill et aZ., 1981) except that the Dounce homogenate was clarified and the nuclei pelleted in one step by centrifugation at 10,000 g for 15 min. Uninfected HeLa SlO extracts (UWO). HeLa S3 cells were collected by centrifugation at 400 g for 5 min and washed once with cold Earle’s salts, and the packed volume was determined by pelleting the cells in a graduated centrifuge tube at 1000 g for 5 min. The supernatant was discarded and a volume of lysis buffer (10 mM KCl, 1.5 mM Mg-acetate, 20 mM HEPES, pH 7.4, and 0.5 mM DTT) equal to that of the cell pellet was added. The cells were resuspended in the lysis buffer, allowed to swell for 5 min at 4” and then disrupted with 30 strokes of a Dounce homogenizer. The homogenate was clarified by centrifugation at 10,000 g for 15 min at 4”. The supernatant was removed and treated with micrococcal nuclease (Sigma) in the following manner to remove the endogenous messenger RNA: SlO extracts were made 1.5 mM CaCla and incubated with 7.5 pg/ml micrococcal nuclease for 15 min at 18”. The extract was then made 6 mM EGTA to chelate the calcium (Pelham and Jackson, 1976). Glycerol was added to 10% (v/v) and the SlO was frozen immediately in small aliquots at -70”. Preparation of VW cellular and virion RNPs (cRNPs and vRNPs). VSV-infected

SlO (I-SlO) extracts or purified virions were made 0.5 M NaCl and 1% Triton N101 and pelleted through 2.5 ml of 50% glycerol in the lysis buffer described above for 2 hr at 48,000 rpm in a Beckman SW 50.1 rotor at 4”. The pellet containing RNPs was resuspended in a volume of USlO extracts equal to the volume of the ISlO. About 60% of the VSV cellular RNPs and 50% of the cell ribosomes were contained in this pellet. In vitro replication reaction. mix. A volume of HeLa U-S10 extract, reconstituted with cellular or viral RNPs was added to an equal volume of the following reaction mix: 60 mMHEPES (pH 7.4), 120 mMKC1, 6 mM Mg-acetate, 2 mM DTT, 2 mM spermidine, 50 PM amino acids minus methi-

onine, 2 mM GTP, CTP, and ATP, 0.2 mM UTP, 20 mM creatine phosphate, 80 pg/ml creatine phosphokinase, and 50 PM methionine. 500 &i/ml [cx-~P]UTP (ICN) with a specific activity greater than 400 Ci/ mmol and no unlabeled UTP was used when radiolabeling RNA. Nonradioactive methionine was omitted and 0.5 to 1 mCi/ ml of [?S]methionine (Amersham) with a specific activity of 1500 Ci/mmol was added when radiolabeling proteins. Ten-microliter samples, taken at various times, were precipitated with trichloroacetic acid (TCA) to assay incorporation of radiolabels into macromolecules as previously described (Hill et al, 1981). Polyacrglamide and agarose gel electrophoresis. [35S]Methionine-labeled samples were prepared for analysis as previously described (Hill et ah, 1981) and were analyzed on 10% discontinuous SDS-polyacrylamide slab gels (Toneguzzo and Ghosh, 1977). Methyl mercury hydroxide agarose gel electrophoresis of RNA (Bailey and Davidson, 1976) was performed as described by Batt-Humphries et al. (1979) and Hill et aL(1979). RNA was phenol extracted as described (Hill et al., 1981). CsCl equilibrium gradients. VSV RNPs and mRNAs were isolated in CsCl gradients as described (Hill et al., 1981) except that [a-32P]UTP-labeled RNPs and mRNAs were banded only once in an 8-ml20-30% CsCl gradient. Preparation of antisera. Specific antiVSV protein IgGs were prepared as described by Harmon and Summers (1982). RESULTS

Analysis of VSV-Specific RNAs and RNP Proteins Synthesized in Vitro by an Unirlfected HeLa SlO Extract with Added Cellular RNPs

Our previous results (Hill et al, 1981) had shown that a VSV-infected HeLa cell SlO extract would support the replication of genome-length RNA and assemble it into a RNP structure similar, if not identical, to VSV RNPs observed in v&o. This system was efficient for replication in vitro because all of the necessary components, i.e., pools of VSV-specific proteins, RNP

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templates, VSV mRNAs, and probably

host cell factors were present in these very concentrated cell extracts. Although this system was ideal for the characterization of the replicative products synthesized in vitro, its usefulness was limited because of the presence of large pools of VSV RNPspecific proteins capable of supporting replication and RNP assembly, rendering the I-S10 in vitro system insensitive to inhibitors of protein synthesis, e.g., pactamycin. Furthermore, the presence of pools of RNP proteins would make it impossible to identify which one or ones were essential for continued replication. We, therefore, developed an in vitro replication system consisting of uninfected cell extracts (USlO), treated with micrococcal nuclease to remove endogenous messenger RNA, plus added VSV RNPs isolated from infected cells (cRNPs). This nuclease-treated U-S10 lacked VSV-RNP proteins and thus would be dependent upon transcription of added VSV cRNPs and translation of VSV mRNAs. Using this U-S10 system, we assayed VSV RNP replication and assembly as previously reported (Hill et aZ., 1931) using CsCl gradients to separate VSV mRNA from VSV RNPs (Kolakofsky, 1976; Simonsen et aL, 1979b; Hill et al, 1981; Davis and Wertz, 1982) and analyzing these RNAs on methylmercury hydroxide agarose gels. An U-S10 plus VSV cRNPs reaction mix, radiolabeled for 2 hr in vitro with [(Y32P]UTP, was layered onto a 20-35s preformed CsCl gradient with a l-ml overlay of 5% sucrose and centrifuged in order to band the VSV RNPs. The RNP band, containing both the VSV cRNPs used as templates and RNPs synthesized de novo, was removed from the side of the tube with a hypodermic needle and the RNA species were analyzed on a methyl mercury hydroxide agarose gel following phenol extraction. As shown in Fig. 1, an RNA species that comigrated with marker genomesize RNA was seen. Since this RNA was extracted from a protein-RNA complex that banded in CsCl at the same density as cellular-derived RNPs and had a mobility in a denaturing agarose gel identical to that of virion 42 S RNA. we concluded-.

-Origin

-42s RNA Z-L mRNA

-G mRNA -N mRNA -NS+M

mRNA

FIG. 1. Methyl mercury hydroxide agarose gel analysis of the RNA contained in in vitro synthesized RNPs purified in a CsCl gradient. RNPs, synthesized over 2 hr in an US10 with added cellular VSV RNPs (1200 ~1 reaction mix) and radiolabeled with [a52P]UTP, were phenol extracted, after CsCl gradient purification, and the RNA species were analyzed on a methyl mercury hydroxide agarose gel. The markers to the right of the figure were derived from separate gel lanes containing radiolabeled mRNA and virion RNA.

that this large RNA species was the product of replication in vitro. There was a large amount of VSV messenger-size RNA and other RNA species, both larger and smaller than VSV mRNA, associated with the nucleocapsid band. Generally small

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A

6

c

-L

-G -NS -N

-M

FIG. 2. Autoradiogram of a 10% polyacrylamide gel of [%]methionine radiolabeled proteins associated with RNPs synthesized in vitro. Lanes A and C: VW virion proteins. Lane B: Proteins associated with RNPs synthesized in vitro by VSV cellular RNPs added to U-S10 extracts and isolated by CsCl gradient centrifugation.

RNA molecules and RNAs that were the size of NS and M mRNA predominated. In our previous studies with the I-S10 in vitro system (Hill et aZ.,1981), we also observed the association of newly synthesized VSV mRNAs and other RNA species with RNPs which had been banded twice in CsCl and showed that these RNA species were sen-

sitive to micrococcal nuclease digestion in contrast to the genome-size RNA which was resistant. As seen in Fig. 7, most of these smaller RNA species were sensitive to the digestion by micrococcal nuclease. Our previous results showed that the genome-sized RNA synthesized in vitro by I-SlOs was associated with N protein plus a slower migrating minor polypeptide species not observed in vivo (Hill et al., 1981). In order to analyze the proteins contained in in vitro assembled RNPs using the USlO plus VSV cRNPs, we labeled a reaction mix with r5S]methionine for 2 hr and purified the RNPs by twice banding them in CsCl gradients, as previously described (Hill et ah, 1981). The RNPs banded at a density of 1.3 g/cm3 (data not shown). The nucleocapsid band was collected and precipitated with 2 vol of ethanol and analyzed on a SDS-polyacrylamide gel. An autoradiogram of this gel is shown in Fig. 2. The radiolabeled proteins associated with the RNPs extracted from the second CsCl gradient are shown in lane B. The major radiolabeled protein comigrated with VSV N protein and as observed in the I-S10 system, a second minor protein species migrating slightly slower than N was also present. Preliminary results, using high-pressure liquid chromatography of tryptic peptides, has shown that this minor species was a variant of N protein (data not shown). Thus, it was clear that the U-S10 plus VSV-cRNP system synthesized a genome-size RNA assembled with VSV N protein in vitro that banded at 1.3 g/cm3 in CsCl similar to the products found in the I-S10 system. Kinetics of RNP Synthesis by U-SlO’s plus Cellular RNPs We next examined the kinetics of RNP synthesis by analyzing aliquots with time after synthesis in vitro. RNPs were labeled with [%]methionine and banded twice in CsCl gradients. The second CsCl gradients were fractionated and analyzed for TCAprecipitable radioactivity, the RNP areas were pooled and precipitated for SDSpolyacrylamide gel analysis. A representative experiment examining the kinetics

VSV PROTEIN REQUIREMENTS

of in vitro RNP synthesis is shown in Fig. 3. Because of the requirement for the in vitro synthesis of VSV proteins for replication and assembly, we expected to find a lag before the onset of in vitro RNP synthesis reflecting the time necessary to transcribe the several necessary VSV mRNAs and to translate them subsequently. However, possibly due to the presence of VSV mRNAs and proteins that contaminated the cellular RNP preparations only a minimal lag in RNP synthesis, when compared with reactions containing I-S10 extracts (data not shown and Hill et aa, 1981), was observed. Replication and assembly seemed to occur over a period of several hours as we had observed previously in the I-S10 system (Hill et ak, 1981). Inhibition of Protein Synthesis Inhibits Replication Earlier studies have shown that inhibition of protein synthesis in VSV-infected cells caused cessation of replication in vivo (Wertz and Levine, 1973; Rubio et ah, 1980). When protein synthesis inhibitors were used in the I-S10 system, no block in RNA replication in vitro was found (Hill et ah, 1981). This was likely due to a pool of VSV-specific proteins contained in the concentrated cell extracts which were sufficient to sustain replication. The U-S10 plus cellular RNPs should show a dependence of replication on VSVspecific protein synthesis. Pactamycin (67 CLM)was added to one-half of a reaction mix containing VSV cRNPs and US10 extracts, using [a-32P]UTP as the radiolabel. After 2 hr, the reaction was stopped and the RNPs were isolated in C&l gradients. The RNPs were collected after centrifugation and the RNA species contained therein were analyzed on methyl mercury hydroxide agarose gels. Figure 4 is an autoradiogram of this gel. Lane A shows the RNAs contained in the RNPs of the control without pactamycin while lane B shows the RNAs from the reaction mix treated with pactamycin. It is evident from the autoradiogram that the amount of 42 S RNA in the sample treated with pactamycin was greatly reduced. In dif-

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FIG. 3. Kinetics of RNP synthesis and assembly in vitro. A 2400-~1reaction mix, containing U-S10 plus cellular RNPs, was radiolabeled with FSjmethionine. At indicated times, 350 pl was removed and RNPs were isolated by centrifugation through two successive CsCl gradients. The second gradient was fractionated and 50 ~1 of each fraction was assayed for TCA-precipitable radioactivity. Each point in the figure represents the amount of radioactivity in the peak area per assayed 50711sample.

ferent experiments, this reduction ranged from 75 to 90% (data not shown). It is possible that small amounts of contaminating VSV proteins present in the preparation of VSV cRNPs allowed a low level of genome-length synthesis to occur even in the presence of pactamycin. However, it is quite clear that the U-S10 system is dependent upon ongoing VSV-specific protein synthesis. Comparison of the Products of VSV Virion (vRNPs) and VSVcRNPs Added to the U-S10 Extracts Since it was felt that in vitro replication by our U-S10 reaction was dependent upon preformed replicating complexes contained within the cellular RNP pools, it was of interest to compare the products synthesized in this coupled in vitro system by VSV virion RNPs (vRNPs) and cRNPs. VSV virions were treated as described under Materials and Methods. RNPs were pelleted and added to the U-S10 system using [a-32P]UTP as a radiolabel. After 2

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HILL AND SUMMERS A - Origin

-42s RNA

-L

mRNA

--G mRNA -N mRNA

-NS+M

mRNA

FIG. 4. Autoradiogram of a methylmercury hydroxide agarose of RNA from RNPs synthesized in vitro by U-S10 plus cellular RNPs in the presence and absence of pactamycin. Lane A: RNA from CsCl purified RNPs (from a 1200~~1reaction mix) incubated 2 hr and radiolabeled with [c~-~*PJUTP.Lane B: Same as A but the reaction mix was made 6’7pM pactamycin. Lane C: VSV mRNA from the CsCl pellet from the same experiment as A.

hr, the reaction mix was phenol extracted and the RNAs were analyzed on a methylmercury hydroxide gel. Figure 5, lane B, shows the RNAs synthesized by vRNPs in U-S10 extracts. All of the VSV mRNA species were transcribed and comigrated with the transcripts of cellular RNPs produced

in the same system (Fig. 5, lane A). There was no 42 S genome-length RNA ever detected when these reactions were analyzed by CsCl gradients and methylmercury hydroxide agarose gels. An experiment comparing the translation products of vRNPs and cRNPs added to an U-S10 reaction mixture was performed to compare VSV-specific protein synthesis, using [35S]methionine as the radiolabel. After 3 hr of incubation, to allow time for the RNPs to transcribe the necessary mRNAs and to translate them, the reaction was stopped and the proteins synthesized were analyzed on SDS-polyacrylamide gels. The results of the experiment using vRNPs are shown in the autoradiogram in Fig. 6, lane C; all VSV proteins except L protein are seen (G protein was visible after a longer exposure of the gel). The reaction containing cRNPs translated all of the VSV proteins including L (lane A, Fig. 6). The reason for this difference in L synthesis is not clear since both transcriptional systems produced comparable amounts of L mRNA (see Fig. 5, lanes A and B). There may be preexisting L-mRNP complexes in the cellular RNP preparations which support more efficient translation of this large mRNA. We are examining this finding at the present time since this lack of L protein synthesis may be in part responsible for the lack of detectable RNA replication in a system containin U-S10 plus vRNPs. Anti-N and Anti-NS Replication

Protein

IgG Inhibits

It has been proposed that the level of N protein might play a role in determining whether a minus-strand containing RNP template will transcribe mRNAs or replicate genome-length RNA (Blumberg et al, 1981, Blumberg and Kolakofsky, 1981). It was suggested that N protein, when at higher concentrations, binds to the leader RNA sequence and suppresses a termination signal at the end of the leader, initiating replication and RNP assembly, but at lower concentrations RNA synthesis stops at the termination sequence and the polymerase then proceeds to transcribe

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A the monocistronic mRNAs from the rest B of the genome template. Since we had - Origin prepared monospecific antisera directed against each VSV gene product (Harmon and Summers, 1982), we could test this hypothesis in part by using these antisera in our U-S10 in vitro system. If the antiN IgG, which had been shown to have no effect on in vitro mRNA synthesis (Harmon and Summers, 1982), bound to newly synthesized N protein in such a manner that the N protein became unavailable for RNP assembly and for suppression of the termination signal, RNA replication in our LmRNA in vitro system would cease. Other antisera directed against L and NS, the enzyme subunits, could also be assayed for their effects on VSV RNA replication in vitro and compared with their effects on in vitro transcription; two IgG preparations, anti-L IgG and anti-NS IgG, had been shown to completely inhibit transcription in vitro (Harmon and Summers, 1982). VSV cRNPs, resuspended in U-S10 extracts, were incubated either in the presG mRNA ence of 0.01 M phosphate buffer or various - N mRNA IgG preparations. VSV mRNAs and RNPs, synthesized in vitro, were radiolabeled with [~u-~*P]UTPin the reaction mix and were separated in CsCl gradients. The NS+M mIRNA RNAs were then subjected to electrophoresis on a methylmercury agarose gel. Figure 7, an autoradiogram of this gel, shows the effects of the various IgGs on transcription and replication in vitro. Lanes A through D in Fig. ‘7 show the mRNAs synFIG. 5. Autoradiogram of a methyl mercury hythesized by in vitro reactions containing antisera. Lane A (control) and lane B droxide agarose gel of [cY-32P]UTP-labeledRNAs syn(anti-N) show levels of uninhibited in vitro thesized by virion RNPs added to an U-S10 extract. Virion RNPs, isolated from 50 ~1 of a virus stock at transcription, whereas lane C (anti-L) 9 X 10” PFU/ml, were added to an U-S10 (100 rl), showed almost complete inhibition of and incubated for 2 hr with the standard reaction mRNA synthesis. Lane D (anti-NS) showed mix. Total RNA was phenol extracted for analysis a reduced amount of mRNA synthesis in a denaturing agarose gel. Lane A: Marker VSV compared to the control and anti-N (in mRNA synthesized in vitro by cellular RNPs. Lane other experiments this reduction was B: VSV RNA synthesized in vitro by virion RNPs. more pronounced). Measurments of total RNA synthesis in these reactions were consistent with the results in Fig. ‘7 and anti-L, synthesis was reduced to 5% of the with previous findings (Harmon and Sum- control, and in the presence of anti-NS, mers, 1982). Total VSV-specific RNA syn- synthesis ranged from 50 to 95% of the thesis in the presence of anti-N was iden- control (data not shown). The small tical to the control, in the presence of amount of mRNA synthesized in the pres-

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G ,Isjs

M

FIG. 6. Autoradiogram of a polyacrylamide gel of proteins synthesized by VSV cellular and virion RNPs added to an U-SlO. Lane A: [?5]Methionine radiolabeled proteins synthesized in vitro by an U-S10 plus added cellular RNPs. Lane B: VSV virion protein markers. Lane C: [%]Methioninelabeled proteins synthesized in vitro by vRNPs extracted from 60 ~1 (9 X 10” PFU/ml) of purified VSV and added to 100 pl U-SlO. Lane D: [‘?6S]Methionine-labeled proteins synthesized in vitro by an U-S10 without added RNPs. Lane E: VSV virion protein markers.

ence of anti-L (lane C) probably represents the completion of already initiated mRNA transcripts. Lanes E through H in Fig. 7 show the RNA species extracted from the CsCl banded RNPs that were synthesized in vitro in the presence of the antisera and therefore represent the effects of these IgG preparations on in vitro replication.

The control (lane E) showed a prominent genome-length RNA species (see also Fig. l), whereas the reaction containing antiN IgG (lane F) showed that no 42 S RNA was synthesized. This was in striking contrast to the lack of effect of anti-N IgG on mRNA synthesis. The reactions containing anti-L IgG showed increased amounts of 42 S RNA synthesized even though

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ABCDEFGH

- 42s - L mRNA

G mRNA

- N mRNA - NS+M mR NA

FIG. 7. Autoradiogram of a methyl mercury hydroxide agarose gel of VSV mRNAs and RNPs synthesized in vitro in the presence of various antisera. VSV cRNPs from 1600pl I-S10 were added to 1600~1U-S10 extracts; 400 ~1was added to each antisera and incubated % hr at 4’. The reaction mix (400 pl), with 250 pCi [a-3?]UTP as a radiolabel, was then added to each U-SlO-cRNP-antisera combination. After 2 hr, the VSV mRNAs and RNPs were separated by CsCl gradients as described. The VSV mRNA pellets were resuspended in 200 ~1 Hz0 and 2 pl of each pellet was used for the agarose gel. The VSV RNPs were diluted with lysis buffer, pelleted [55,000 rpm (Beckman SW 60) for 1 hr], resuspended in lysis buffer, and treated with micrococcal nuclease as described. Pronase, 100 pg/ml, was added and all of the RNA was phenol extracted and analyzed on agarose gels. Lane A: VSV mRNA synthesized in the presence of ‘75pl of 0.01 M NaP04 buffer, pH 6.6. Lane B: Same but in the presence of 75 ~1 anti-N IgG (35 mg/ml). Lane C: Same but in the presence of ‘75pl antiL IgG (10 mg/ml). Lane D: Same but in the presence of ‘75 ~1 anti-NS IgG (17 mg/ml). Lane E: RNA from CsCl banded RNPs synthesized in the presence of 0.01 M NaPO,. Lane F: Same as E but in the presence of anti-N IgG. Lane G: Same as E but in the presence of anti-L IgG. Lane H: Same as E but in the presence of anti-NS IgG.

transcription was inhibited almost completely. The increase of genome-length RNA was 1.5 to 2-fold by densitometer tracings (data not shown). The reaction plus anti-NS displayed an almost complete inhibition of 42 S RNA synthesis. Other

experiments with anti-G IgG and anti-M IgG showed no changes in the amounts of total RNA or 42 S RNA synthesized in vitro compared with the control (data not shown). In agreement with the Blumberg and

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Kolakofsky model given above which assumes that VSV transcription and replication are carried out by the same viral polymerase whose function is modulated by the amounts of available N protein, it was not wholly unexpected that anti-N would inhibit replication and yet have no effect on transcription. Presumably a sufficient amount of newly synthesized N protein was bound by unreacted antibody in such a way that N was not available for RNP assembly. We have not detected by autoradiography any genome-length RNA in the mRNA CsCl pellets of these reactions that might represent continued synthesis of 42 S RNA which was not assembled into RNPs. Small amounts may have been present, but since 42 S RNA synthesis represented about 1% of the total RNA synthesized in vitro we have not been able to detect any 42 S RNA in the large background of mRNA in the CsCl pellet. It is extremely difficult to obtain an accurate estimate of the percentage of the total RNA synthesized as 42 S RNA because the only reliable assay is the separation of genome-length RNA species from other RNAs on methyl mercury agarose gels. We detected large amounts of smaller RNA species that banded with the RNPs in CsCl gradients and these RNAs are not totally sensitive to digestion by micrococcal nuclease (see Fig. 7). One of the predominant species that we observed was about the size of leader RNA, this could represent encapsidated leader that would band with RNPs in CsCl gradients (Blumberg and Kolakofsky, 1981). The increased amounts of genome-length RNA synthesized in vitro in the presence of anti-L IgG was unexpected and the meaning of this observation is not clear at this time. DISCUSSION

In a previous report from this laboratory we described an in vitro VW RNA replication system which was capable of synthesizing and assembling VSV RNP particles for a prolonged period of time (Hill et aL, 1981). The proteins in the RNP product of this reaction were analyzed in

SUMMERS

SDS-polyacrylamide gels and shown to contain the RNP protein, N, and a variant of N migrating slightly slower than typical N. The structures assembled in vitro were ribonuclease resistant and contained both plus and minus strands of genome-length RNA, as identified in denaturing agarose gels. This system, which contained infected cell extract (I-SlO), however, was shown to be resistant to pactamycin inhibition of RNA replication and we concluded that the likely explanation for this resistance was that the I-S10 contained pools of RNP proteins which were capable of supporting replication in vitro for an hour or more. Because our initial studies with this infected cell extract showed resistance to pactamycin inhibition, we then turned to a system containing uninfected HeLa cell extracts (U-SlO) so that RNA replication would be completely dependent on mRNA transcription and on the synthesis of VSVspecific proteins. We have shown in this report that the product of this in vitro reaction was identical to the product of the I-S10 system in that it was ribonucleaseresistant, it contained the same VSV-specific proteins and genome-length RNA and banded in cesium chloride at the expected density. But this U-S10 system was indeed sensitive to inhibition by pactamycin. With this U-S10 replication system we could begin to determine the VSV proteins synthesized in vitro upon which replication was dependent. One method would be to add back each individual VSV protein to this system to define the dependence of the system on one or more of the individual proteins. However, the VSV RNP proteins are quite insoluble and this approach was not likely to be effective. One could also add purified specific VSV messenger RNAs to the system so that the translational products of each VSV mRNA would define exactly which protein(s) were required to support replication in vitro; this work is now in progress. Another approach available to us since we had raised specific antisera directed against each VSV gene product (Harmon and Summers, 1982) was to use these sera in our U-S10 system to test the dependence of the in

WV

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vitro replication system on VSV-specific gene products. Our previous report had shown that these antisera were specific for the five VSV proteins and that anti-L and anti-NS caused a complete inhibition of transcription in an in vitro system but anti-N was without effect on transcription in vitro. These same sera were tested in the in vitro replication system for their effects on RNA synthesis in vitro. The results shown in Fig. 7 demonstrated that these antibodies do have effects on in vitro replication. The most striking were that anti-N antibody and anti-NS antibody caused complete or nearly complete inhibition of replication in vitro. As mentioned above, if N protein were required for ongoing replication by acting to suppress a termination signal in the leader sequence in the RNP template or because a modified VSV-RNA polymerase in a replicative mode synthesizes not only genome-length RNA molecules but also assembles RNP particles (Rubio et ah, 1980), then in either case, depletion of available N protein necessary for replication and assembly would cause an inhibition of RNA replication in vitro. Our observations did support such a model since anti-N IgG had no effect on in vitro mRNA synthesis and completely inhibited in vitro replication. Since anti-NS antibody caused inhibition of transcription in vitro, its effect on replication was not totally surprising. One interpretation of this finding would be that newly synthesized NS and N are both required for ongoing replication both in vivo and in vitro, since the product of replication is not a naked RNA molecule but as a matter of fact is a fully assembled RNP particle containing L, NS, and N proteins (Rubio et aL, 1980). This is also consistent with the fact that Wertz and Levine (1973) had shown that VSV RNA replication was dependent on ongoing protein synthesis or at least the presence of certain VSV-specific proteins. Another possible mechanism for the inhibition of in vitro replication and transcription by anti-NS IgG is that this protein, which has been shown to be a subunit

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of the transcriptase (Emerson and Yu, 1975; Naito and Ishihama, 19’76),is also a subunit of the VSV replicase, and antibody bound to this portion of either enzyme inhibits RNA synthesis. The fact that anti-N and anti-NS antisera produced a complete or nearly complete inhibition of RNP replication and assembly in vitro, might not be totally due to their depletion of newly synthesized N and/or NS protein pools upon which replication was dependent, but the observed inhibition might have been due at least in part to interaction of these antibodies with the replicase enzyme-RNP template complex whose configuration might be different from that of the transcriptase-template complex. The latter complex was inhibited in vitro by anti-NS antibodies, but not by anti-N (Harmon and Summers, 1982). However, the configuration of the replicase complex could be sufficiently different to now allow anti-N antibodies to inhibit replication on this enzyme-template complex. This explanation might certainly be true as well for anti-L serum which very effectively inhibited in vitro mRNA synthesis, but consistently stimulated in vitro RNA replication (see Fig. 7). If this were the case, it might suggest that one could use these specific antisera along with monoclonal antibodies to study the diverse functions (methylation, capping, polyadenylation, transcription, etc.) of the VSV enzymes subunits (L + NS) in in vitro transcription and replication systems. Perhaps pertinent to the observation that anti-NS inhibits in vitro RNA synthesis is the recent report of Keene et al. (1981) who, in their studies of methylation-protection in VSV nucleocapsids, showed that NS protein and not N or L proteins was able to bind to and protect a sequence of the middle of the VSV RNP leader sequence. They suggested that the NS protein was the “initiator protein,” at least for transcription. This finding certainly suggests a central role for the NS subunit of the enzyme in active RNA synthesis and our finding that anti-NS inhibited replication very effectively perhaps is in keeping with that report.

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Two reports (Blumberg et aZ., 1981; Blumberg and Kolakofsky, 1981) suggested that the nucleocapsid structural protein, N protein, plays a central role in controlling replication and transcription in the infected cell. Our findings, in part, support this theory in that anti-N antiserum caused a complete inhibition of replication, but had no effect on transcription in vitro. However, some of our observations in this report and other previous reports failed to support the model for the role of N protein as the major controlling element in transcription and replication. First, we had shown that although both I-S10 and U-S10 in vitro replication systems produced the same products in about the same ratios and with similar kinetics, there was a large disparity in the amount of N protein available to support replication. As reported previously the I-S10 had a sufficient pool of N protein to drive replication in the absence of protein synthesis for at least an hour (Hill et aZ., 1981). In the present report, the uninfected system had no such pool of available precursor N protein and was sensitive to inhibition by pactamycin. Nevertheless, in both cases, replication only accounted for a small percentage of total RNA synthesis, and even in the presence of a large pool of N (in the I-SlO) replication did not increase in amount. Furthermore, previous reports from this laboratory (Hill et al, 1979; Simonsen et aL, 197913)had shown that replicating templates seemed to behave very differently from transcribing templates in that the nascent messenger strands on transcriptional templates were unstable in Renografin gradients and were separated from the template. This was in opposition to replicating structures which remained intact in Renografin. This suggested that there is a basic difference in the enzyme-RNP-template complex involved in transcription as opposed to replication. A striking result was that the anti-L antiserum consistently caused stimulation of replication in vitro, despite the fact that this antiserum was shown to be an effective inhibitor of in vitro transcription. Perlman and Huang (1973) described experiments with a VSV temperature-sen-

SUMMERS

sitive mutant, ts 114, in which transcription was inhibited but replication was not at the nonpermissive temperature. There have been other reports of normal or increased transcription when replication had ceased (Wertz and Levine, 1973, Perlman and Huang, 1973; Martinet et cd. 1979). Other observations which suggest that there is a difference between the transcriptase and the replicase involved in VSV infections are those reports that have demonstrated that one can isolate a large number of host-range mutants of VSV and that these mutations often map in the L cistron. In each case the authors suggested that if the mutation did involve the large enzyme subunit and was dependent on the host-cell background, one can logically assume that there is some sort of host modification of the enzyme involved in the switch from transcription to replication (Levinson et al, 1978; Norwakowski et ah, 1973; Pringle, 1978; Simpson et al, 1974; Simpson et al, 1979). These observations and our results with the monospecific antisera suggest that there are very basic differences between the VSV transcriptase and the replicase which likely include host factors and possible structural differences. A similar in vitro replication system has been recently described by Davis and Wertz (1982) using rabbit reticulocyte lysates instead of a HeLa U-SlO. We have compared the reticulocyte and HeLa systems in our laboratory and have found the two systems to be similar in genomelength RNA synthesis. However, the recent report did not analyze the proteins contained in the RNP product, and thus other comparisons cannot be made. These studies of protein synthesis-dependent in vitro replication coupled with monospecific antisera now open up further possibilities to define the precise nature of the “replicase” involved in in vitro replication, and these studies are now underway. ACKNOWLEDGMENTS We thank Jerri Cohenour and Bobbie Bullock for typing the manuscript. This research was supported by NIH Grant 5ROl AI 12316-07 and NSF Grant PCM-81 10986.

VSV PROTEIN

REQUIREMENTS

REFERENCES

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