RNP template of vesicular stomatitis virus regulates transcription and replication functions

Cell,Vol

35,175i&,November1983,Copyr1ghtQ

1983 by MIT

0092.8674/83/110175-11

$0200/O

RNP Template of Vesicular Stomatitis Virus Regulates Transcription and Replication Functions Jacques Perrault,* Gail M. Clinton,+ and Marcella A. McClure* *Department of Microbiology and Immunology Washington University Medical School St. LOUIS, Missouri 63110 ‘Department of Biochemistry Louisiana State University Medical Center New Orleans, Louisiana 70112

Summary Small leader RNAs, copied from the extreme 3’ ends of the minus and plus strands of the vesicular stomatitis virus (VSV) genome, are thought to play a central role in the regulation of viral transcription and replication. We describe here a novel class of VSV mutants, denoted pol R, in which termination at leader sites in vitro is specifically suppressed. We have assayed for the presence of leader RNAs and readthrough transcripts in reaction products from standard virion templates (plus leader) and defective interfering particle templates (minus leader). In both cases, mutant virions gave rise to a much higher proportion of readthrough transcripts than wild type (>80% vs -10%). Reconstitution experiments with separated ribonucleoprotein (RNP) templates and polymerase protein fractions revealed, surprisingly, that the N protein moiety of the RNP template was responsible for readthrough. This conclusion was further supported by protein analyses that showed a similar charge change in the N protein of two independently isolated pol R VSV mutants. These results lead us to propose that modification of the N protein may regulate termination at leader RNA sites. Introduction Gene expression in eucaryotic RNA viruses involves processes similar to those of their host cells, such as capping and polyadenylation, as well as features unique to viral RNA templates. Despite intensive investigations in several laboratories, many basic aspects of RNA synthesis directed by VSV, a model negative-strand virus, remain unclear. In particular, what controls the process of chain termination, crucial in regulating transcription versus replication functions, is unknown (for review see Ball and Wertz, 1981). A detailed study of this process is necessary for understanding mechanisms regulating virus replication in lytic and persistent infections. These studies may also shed light on the little understood mechanisms of transcriptional termination in eucaryotic cells. An important feature of RNA synthesis in negative-strand viruses is the apparently absolute requirement for an RNP template in both transcription and replication (Emerson and Wagner, 1972; Naito and Ishihama, 1976; Hill et al.,

1979). The VSV template consists of a helical nucleocapsid structure containing - 1500-2000 copies of the N protein (47 kd) tightly bound to the RNA genome (-1 I kb). The Inner core structure of the virion also contains -200 copies of the NS protein (25 kd) and - 100 copies of the L protein (190 kd) and can faithfully carry out the transcription process in vitro (Mellon and Emerson, 1978). VSV transcription begins with synthesis of a plus-strand leader RNA followed by the sequential appearance of the five monocrstronic VSV mRNAs in the order found on the genome, i.e., 3’.leader-N-NS-M-G-L-5’ (Ball and White, 1976; Abraham and Banerjee, 1976; Testa et al., 1980a; lverson and Rose, 1981). The transcription process IS polar in that each successive gene is copied less frequently as a function of the distance from the 3’ end of the template (lverson and Rose, 1981). Except for leader RNA which is dr- or tri-phosphorylated at its 5’ end, all five VSV mRNAs are released from the template as capped and polyadenylated transcripts that do not undergo splicing reactions. What signals termination for leader RNA is unknown but termination and polyadenylation of mRNAs is thought to occur when the polymerase reaches a stretch of seven undylate residues at the ends of the genes (Ball and Wertz, 1981). The single polymerase entry site model proposes that the transcriptase enzyme begins with synthesis of the plus strand leader RNA that is complementary to the first 47 residues at the 3’ end of the genome. A stop-start mechanism then presumably allows the template-bound enzyme complex to terminate and release completed transcripts before initiating synthesis at the next gene with reduced probability (Banerjee et al., 1977; Emerson, 1982). An alternative multiple entry site model has also been proposed whereby each gene is independently and simultaneously initiated, followed by elongation and completion of these short transcripts in a sequential manner (Testa et al., 1980a). An earlier model suggesting processing of mRNA precursors (Banerjee et al., 1977) although not entirely ruled out, lacks experimental support. What controls suppression of termination to allow replication of the negative-strand VSV genome is unclear. In VIVO, this process is coupled to viral protein synthesis (Wertz and Levine, 1973) and only recently have cell-free systems been developed that carry out this coupling of translation and replication in vitro (Hill et al., 1981; Davis and Wertz, 1982; Peluso and Moyer, 1983). The simplest hypothesis would suggest that once the polymerase complex begins synthesis at the 3’ end of the minus-strand template, a crucial regulatory decision is made at the leader-N gene junction. In the transcription mode, the complex would terminate synthesis and presumably reinittate at the beginning of the N gene, only four bases downstream. In the replrcatron mode, the same or perhaps a modified polymerase complex would readthrough this junction and continue synthesis uninterruptedly until the end cf the template. A previous study whose authors Included one of us

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(Leppert et al., 1979) proposed that readthrough of the leader-N gene junction in vivo is achieved by binding of soluble N protein to nascent plus strand leader RNA. This coupling of replication to RNP assembly is consistent with the presence of a binding site for N protein in plus strand leader RNA (Blumberg et al., 1983). The proposal also suggested that a minus strand leader RNA (46 nucleotides) performs an analogous function during synthesis of minus strands in vivo (see Figure 1 for sites of synthesis of leader RNAs). Some studies, however, have shown that protein synthesis is not required to achieve readthrough of leader RNA termination sites in vitro. For unknown reasons the analog AMP-PNP cannot replace ATP for initiation of transcription in vitro but will support chain elongation. Under these conditions, the VSV transcription process is inhibited and up to 50% of the products appear to be fulllength plus strand copies (Testa et al., 1980b). A low level readthrough of the leader-N gene junction has also been reported in the presence of various base analogs in vitro (Testa and Banerjee, 1978; Herman and Lazzarini, 1981; Chinchar et al., 1982). It is noteworthy that the requirement for a hydrolizable -r-/I ATP bond is also a characteristic of transcription initiation by eucaryotic RNA polymerase II in vitro (Bunick et al., 1982) and may reflect some common mechanism. Readthrough of the minus strand leader termination site can also be achieved in vitro in the absence of protein synthesis. VSV defective interfering particles (DI), whose 3’ end template sequence is identical to the 3’ end sequence of standard virus plus strands (see Figure I), normally synthesize only the minus strand leader RNA, but as with standard virus, replacement of ATP by AMP-PNP during the elongation process leads to synthesis of fullsize plus strand DI RNA copies (Chanda et al., 1980). We have recently reported the isolation of some novel VSV mutants, denoted pol R, that generate DI that appear to efficiently readthrough the minus strand leader termination site under standard in vitro conditions, and we have suggested that these viruses may be mutated in a function that regulates the switch from transcription to replication (Perrault et al., 1980). In the work presented here, we provide support for this hypothesis by showing that VSV pol R mutants also readthrough the plus strand leader-N gene junction very efficiently in vitro. Furthermore, we find that the responsible mutation maps in the template N protein. These findings lead us to propose a direct role for the N protein of the RNP template structure in termination of VSV RNA synthesis at leader sites, thereby regulating transcription versus replication pathways. Results Readthrough Synthesis of the Plus Strand Leader Termination Site by pot R VSV Mutants A detailed description of the isolation and in vivo growth properties of pol R mutants will be presented elsewhere

SYNTHESIS

OF

L

5”

VSV

, G C-b

LEADER

.M.NS.

RNAs

N

I b3

STANDARD TEMPLATES

3’.

L

, G (+)

.M.NS.

N

. -OS,

of the Sites of Syntheses

of VSV

*

Figure 1, Diagrammatic Leader RNAs

Representatron

Bold arrows indicate plus (47 nucleotides) and minus (46 nucleotrdes) leader RNA transcripts (not drawn to scale relative to the genome). Sequences rn open boxes are complementary to sequences in closed boxes. Thus complementarity consists of the first 14 out of 17 residues at the ends of the standard genome of the VSV Indiana strain. In DI templates, which contain only part of the L gene (L’), the extent of complementarity between the 5’ and 3’ ends varres in different isolates but extends to at least 45 nucleotrdes (Perrault, 1961). The 5’ ends of both plus and minus strand DI templates are homologous to the 5’ end of the genome minus strand, while the DI 3’ end are homologous to the 3’ end of the genome plus strand. Detergent-drsrupted standard virus therefore synthesize plus strand leader (as well as mRNAs) under normal in vrtro reaction conditions, whrle DI synthesrze only menus strand leader. In vrvo, termination events at leader sates are suppressed at least rn part to allow replication of the genome.

(Perrault et al., unpublished data). Let it suffice to say that the two mutants described were isolated independently after fifteen cycles of heat inactivation of virus lysates followed by growth of survivors. Pol RI and R2 virus stocks were derived from single plaque isolates and are stable upon passage. Their growth is restricted in vivo since yield of infectious or total mass of mutant virus per cell varies from 10% to 50% that of wild type at 37’C. In vitro transcription by mutant virus gives rise to a normal array of mRNAs except for longer poly (A) tails (Perrault et al., 1981). A sensitive assay for the specific quantitation of VSV plus strand leader RNA synthesis has been developed (Perrault and Semler, 1979; Leppert et al., 1979). In this assay, the VSV genome RNA is labeled specifically at the 3’ end with RNA ligase and 32pCp, annealed to complementary RNA products, and labeled duplexes protected from RNAase digestion are analyzed on polyacrylamide gels. When this assay is carried out with an excess of probe sequences (on a molar basis) one can easily obtain a measure of the number of transcripts that contain the exact complement of the genome 3’ end, as well as the position at which this complementary contiguity terminates on the genome template.

0

VSV Regulates 177

Transcrrptron

and Replrcatron

Functfons

Figure 2 illustrates the results of such an analysis with in vitro transcripts (freed of their RNP templates by CsCl centrifugation) from wild type and pol R VSV mutant viruses. The annealing reactions were carried out with varying concentrations of transcripts, ranging from probe excess (1 x and 1Ox) to product excess (100x). It should be noted that leader RNA represents -6% of the total mass of wild-type virus transcripts under our reaction conditions (not shown) in agreement with the reports from other laboratories (Carroll and Wagner, 1979; Herman and Lazzarini, 1981). The major labeled duplex obtained from wild-type reaction products (Figure 2, lanes b, c, and d) corresponds to the 47 bp long leader molecules as shown previously (Perrault and Semler, 1979; Leppert et al., 1979). In contrast, the same amounts of products from either pol RI (lanes e, f, and g) or pol R2 VSV (lanes h and i) contain fewer leader-size transcripts and show an array of larger size duplexes representing readthrough of the leader-N gene junction. The size range of these readthrough transcripts was determined more accurately by agarose gel analysis under totally denaturing conditions (Figure 3). The results indicate that the majority of pol RI mutant readthrough transcripts, under conditions of labeled probe

excess (which most accurately reflects the true size distribution of these products), are about 300 to 800 nucleotides long, with a distinct band slightly larger than the 1326 nucleotide long N mRNA marker (Figure 3A, lanes a and b). Under conditions of product excess, this distinct band is the major discrete species observed for both pol RI (Figure 3A, lane c) and pol R2 viruses (Figure 38, lane c). Note that wild-type products also contain small amounts of this large size readthrough transcript in addition to leader RNA (Figure 3A, lanes e and f). The results shown in Figures 2 and 3 were obtained in several independent experiments with different preparations of wild type and mutant viruses. We thus conclude that the mutant viruses, in contrast to wild type, frequently readthrough the leaderN gene junction under normal in vitro transcription conditions Most of these readthrough transcripts terminate -250 to -750 bases from the beginning of the N gene. The distinct readthrough product -50 nucleotides larger than the N mRNA could conceivably be the result of

abc nnl

def Rl

abc

wi Id-

L

N ,-leader

a b

hi

Figure 2. Detectron of Plus Strand Leader RNA and Readthrough scripts rn In Vitro Products from Standard Weld-Type and pol R VSV

Tran-

Yrelds of 3H-UMP-labeled products per rg of vrrus template were equal to 6.0, 2.0, and 2.7 pg for weld type, pol Rl, and pol R2. respectively. They were purrfied free of their template RNP by CsCl centrifugation and varyrng amounts rangrng from 1.6 ng (lx) to 160 ng (100x) were hybndrzed to -450 ng (-30 k cpm) of 3’ end-labeled wild-type VSV RNA. RNAaseresrstant duplexes contarnrng “P-label were then analyzed on a 20% acrylamrde gel (see Expenmental Procedures). Lane a corresponds to a control labeled probe treated Identically but wrthout added products. XC rndrcates posrtron of xylene cyanol dye marker.

Figure 3. Srze Distributron of Plus Strand Leader Readthrough on Agarose Gels after Glyoxal-Denaturation

Transcripts

Hybndrzatron reactions were carned out as In the prevrous ftgure except that rn (A) -666 ng of labeled probe (-22 K cpm) was used wrth 100X products equalrng 360 ng. In (8) lane a IS the control probe not treated wrth RNAase, and lanes b and c are the same samples as In lanes e and h of Frgure 2. Marker positrons of unpolyadenylated N mRNA (1326 nucleotrdes) and polyadenylated L mRNA (-6200 nucleotrdes) were obtarned from fluorographrc analysrs of wild-type 3H-UMP labeled products run on a parallel lane

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termination near the end of this gene giving rise to a leader-N gene transcript. However, a consideration of the hybridization assay employed suggests a more likely explanation for the appearance of this labeled duplex. Both mutant and wild-type products contain similar large amounts of mRNA transcripts under these conditions (Perrault et al., 1981). It is reasonable to expect that formation of relatively abundant NS gene duplexes prevents annealing of small amounts of readthrough products in regions that extend beyond the N-NS gene junction. The presence of a branched duplex structure at this site would allow RNAase to cleave the genome at or very near the two base long intercistronic junction. This kind of competition is not a serious problem with the leader-N gene junction because readthrough transcripts spanning this site are obviously present in large amounts in the products from the mutant viruses (see below). The readthrough assay as described above also allows us to quantitate accurately the relative frequency of initiations at the 3’ end of the template as well as the relative frequency of readthrough for both wild type and mutant viruses. We have done this by carefully quantitating the amount of 32P RNA protected from ribonuclease digestion. Table 1 shows that, for the same amount of total products analyzed under conditions of probe excess, pol RI VSV initiates synthesis at the 3’ end of the template twice as frequently as wild-type virus (25 ng of pol Rl in vitro transcripts protect about twice as many cpm as 25 ng of wild-type products). The relative concentrations of leader RNA molecules and readthrough transcripts was obtained

Table 1. Quantitation of Leader and Readthrough In Vitro by Wild-Type and Pol RI VSV

Transcripts

Synthesized

Product Concentratron’

% Probe Protectior?

% in Leader”

% in Readthroughd

2.5 ng 25 ng 250 ng

2.3% 14% 60%

68% 69% 87%

12% 11% 13%

2.5 ng 25ng 250 ng

3.1% 29% 78%

16% 19% 17%

84% 81% 83%

Wild type

Pol Rl

a The indrcated amounts of 3H-UMP-labeled products were hybridized to -0.5 rg (-62 K cpm) of 3’ end-labeled wld-type VSV RNA as before (see Expenmental Procedures). b RNAase-resrstant cpm were determined on a portion of each sample after hybndrzatron and corrected for a background of 2.9% resistance for the probe treated identrcally but without added products. ’ RNAase dtgested samples were analyzed on a 15% acrylamrde gel as rn Ftgure 2 and a densitometric scan of the autoradiograph was used to quantitate the relative amounts of leader RNA In each lane. The band migrating slrghtly faster than leader was included in the calculatrons. d The proportion of readthrough transcripts (anything larger than leader) rn weld-type samples was calculated directly from the densttometric scans. Those rn the pal RI samples were calculated by subtractron assuming twrce as many total 3’ end initiations as wild type. Drrect quantrtation of the latter by densrtometry yielded slrghtly lower values presumably because the largest transcripts barely entered the gel.

directly by densitometric scanning of autoradiographs similar to Figure 2 since the end-labeling procedure insures equimolar representation. These results (Table 1) indicate that mutant pol Rl virus synthesize approximately 40% as much leader-size RNA molecules as wild type where this RNA species represents -88% of the total 32P label protected. This reduction in plus strand leader RNA synthesis by pol RI virus in vitro confirms our previous report where a-32P-GTP labeled leader-size RNA molecules were quantitated by densitometry (Perrault et al., 1981). In contrast to wild-type virus where the polymerase only occasionally fails to terminate at the leader-N gene junction in vitro, -83% of all pol R virus polymerase initiations at the 3’ end of the template yield readthrough transcripts (Table 1). The results shown in Figures 2 and 3 clearly indicate that 3’ end initiations by pol R2 virus are also quantitatively and qualitatively very similar to pol Rl. Additional experiments not shown here indicate that, under our transcriptase reaction conditions, neither wild type nor mutant viruses synthesize significant amounts of 3’ end transcripts that are smaller than the stable duplexes detected in our assay.

A Translation Product of pol R VSV Causes Readthrough Synthesis of the Minus Strand Leader Termination Site We reported previously that DI derived from the pol Rl VSV mutant also appear to readthrough the minus strand leader termination site (see Figure 1) in vitro at a frequency exceeding 80% (Perrault et al., 1980). This tentative conclusion was based on the detection of large transcripts, including a small proportion very near the size of the DI template (wild-type DI synthesize only the 46 nucleotide long minus strand leader). We have since established that these large transcripts do initiate at the 3’ end of the DI templates and not internally as expected for molecules that readthrough the minus leader RNA termination site (Perrault, unpublished data). The mutated function(s) in pol R virus therefore appears to affect termination at both leader RNA sites in a similar fashion. A priori the pol R mutation(s) responsible for the readthrough phenotype could involve RNA sequence changes at the leader termination sites resulting in weaker signals. Alternatively, mutations in protein components involved in the termination step might be involved. To choose between these alternatives, we have previously constructed DI particles whose proteins are entirely derived from the pol R mutant but contain a wild-type DI RNA template and viceversa (Perrault et al., 1980). These heterologously encapsidated DI RNA templates were obtained by coinfecting cells with standard mutant virus and wild-type DI or standard wild-type virus and mutant DI (see Experimental Procedures). The DI RNA templates, which do not encode any translation products (Perrault, 1981) were faithfully replicated and assembled with standard helper virus-coded proteins as expected (not shown). We reported previously that the heterologous DI contain-

VSV Regulates 179

Transcriptron

and Replrcatron

Functions

ing pol Rl proteins and wild-type DI RNA templates incorporate about lo-fold more 3H-UTP in vitro than DI containing wrld-type proteins and wild-type RNA, suggesting that the readthrough activity might be a property of pol Rl proteins (Perrault et al., 1980). In order to establish that this is indeed the case, we have examined the size of products from heterologously encapsidated DI to determine readthrough activity directly. As shown in Figure 4, large amounts of readthrough transcripts (>46 nucleotides) were synthesized by DI containing wild-type template RNA and pol Rl proteins (lane a). In contrast, the great majority of transcripts from DI containing pol RI template RNA and wild-type proteins correspond to the 46 nucleotide long menus leader RNA (lane b). The autoradiograph shown in this figure was purposefully overdeveloped to demonstrate that, as in the case of plus strand leader readthrough, wild-type proteins are also capable of synthesizing a small amount (~10%) of readthrough transcripts (Figure 3A, lanes e and f and Figure 4, lane b). The results shown in Figure 4, lanes a and b, are very similar to those obtained with pure pol RI DI and pure wild-type DI, respectively (Perrault et al., 1980) except for the size of the largest transcripts which in each case, as expected, correlates with the size of the template. Similar results were also obtained with a different source of wild-type template (not shown). We conclude from these experiments that the readthrough activity of pol R mutants is caused by a

ab

c

loader

Frgure 4. Readthrough ologously Encapsidated

of Mrnus Strand Leader Termrnatron Weld Type and pol RI DI

Site by Heter-

In vrtro 3H-UMP-labeled products were analyzed on agarose gels after glyoxal denaturation (see Experimental Procedures). DI containrng wild-type RNA and pot RI proteins (lane a) synthesized -2.3 pg products per *g of template. DI containrng pol Rl DNA and wrtd-type proterns (lane b) synthesrzed 0.28 pg/pg template. Products from the same amount of template RNAs were loaded in each lane. Arrows Indicate the positions of 3H-urrdrnelabeled HeLa ribosomal and transfer RNA markers run in a parallel lane Note that the srze of pol RI DI RNA template (represented by the largest product rn lane b) IS slightly smaller than that of wild type (-2300 vs. -2400 nucleottdes: Perrault et al , 1980).

mutation(s) in a protein component(s) that affects termination of RNA synthesis at the ends of VSV leader RNA transcripts.

Reconstitution Experiments Identify the VSV Template N Protein as the Determinant of Readthrough Activity at Both Leader RNA Termination Sites The template-polymerase complex of VSV can be separated into a soluble polymerase protein fraction, containing L and NS, and a template fraction containing only N protein assembled with the RNA genome. When both of these inactive fractions are combined, polymerase activity is restored (Emerson and Wagner, 1972). To identify more precisely the altered protein component(s) responsible for readthrough in pol R VSV, we carried out such reconstitution experiments using various combinations of mutant and wild-type soluble protein and RNP template fractions, and assayed the products of readthrough of plus and minus leader RNA termination sites as before. To our surprise, readthrough of either plus strand leader (Figure 5) or minus strand leader (Figure 6) was obtained with all combinations which included pol Rl RNP templates regardless of the source of solubilized polymerase proteins (Figure 5, lanes d and g; Figure 6, lanes e and f). Similarly, no significant readthrough was obtained in all combinations that contained wild-type standard RNP (Figure 5, lanes c and f) or wild-type DI RNP templates (Figure 6, lanes c and d) regardless of the source of polymerase proteins. The small amount of readthrough observed with wild-type standard RNP plus pol Rl solubilized proteins (Figure 5, lane f) was most likely because of small amounts of pol Rl RNP contaminating the soluble fraction (see legend to Figure 5). We conclude then from these experiments that a protein component(s) in the RNP template fraction of pol Rl VSV is responsible for readthrough. Both standard and DI RNP fractions employed in our reconstitution experiments were extensively purified through renografin and CsCl gradients to ensure complete removal of all virus proteins except N (see Experimental Procedures). The SDS-polyacrylamide gel analysis of DI RNPs shown in Figure 7 attests to their purity (similar results were obtained with standard virus RNPs). The only protein component detected in these RNPs was the N protein. In concert with the results obtained with heterologously assembled Dl (Figure 4) these data indicate that the N protein is responsible for readthrough. Note also in Figure 7 that the pol RI VSV N protein migrates slightly faster than its wild-type counterpart. Closer examination of readthrough transcripts for both plus strand leader (Figure 5) and minus strand leader (Figure 6) reveals an additional unexpected result. The banding pattern reflecting the heterogeneity in termination sites of readthrough transcripts on both standard and DI mutant templates is essentially identical when comparing control (unfractionated samples) and reconstituted samples (compare Figure 5, lanes d, e, and g and Figure 6,

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+ Ior

Idal -lea Idw

xc

xc

Frgure 5. Analysis of Plus Strand Leader Readthrough Activity Followrng In Vitro Reconstitutron of Solubilrzed Polymerase Proteins with Purified Standard Virus RNP Template Fractions Hybridrzation of products (25 ng of each) to an excess of probe (-590 ng, -82 K cpm), and detection of RNAase-resistant duplexes (In thus case on a 15% acrylamrde gel) were camed out as in Figure 2. The yield of total 3H-UMP-labeled products (2.5 hr reactrons) per pg of template m the vanous samples was as follows: unfractionated wt std virus, 7.9 pg; wt RNP + wt sup, 430 ng, pol Rl RNP + wt sup, 220 ng; unfractionated pol Rl std vrus, 2.0 pg; wt RNP + pol Rl sup, 28 ng; pol Rl RNP + pol Rl sup, 70 ng. Reactrons containrng RNPs only or supernatants only ytelded ~5 ng. A sample of the probe treated identically but without added products is shown rn lane a, without RNAase treatment, and in lane h, with RNAase drgestion.

lanes e, f, and g). These remarkable results imply that the solubilized VSV polymerase complexes, following dissociation and rebinding to RNP templates that have been exposed to harsh conditions during purification, nevertheless recognize the same array of termination sites as before dissociation. This phenomenon bears important implications regarding the mechanisms controlling termination of RNA synthesis in VSV (see Discussion).

Evidence That a Charged Amino Acid Substitution in the N Protein of VSV pol R Mutants Is Responsible for Readthrough The polymerase reconstitution cate the N protein in readthrough sites in the mutant viruses. We biochemical evidence that pol one alteration in the amino acid

experiments clearly impliof leader RNA termination provide here corroborating R VSV mutants do contain sequence of their N protein

Figure 6. Analysts of Minus Strand Leader Readthrough Activrty Followrng In Vrtro Reconstitutron of Solubrlized Polymerase Proteins with Purified DI RNP Template Fractrons Total reaction products from wrid type and pol Rl virus fractions were labeled wrth CY~*P-GTP and analyzed in a denaturing 20% acrylamrde gel containing 7M urea. Products (3 hr reactions) synthesized from equrvalent amounts of templates were loaded on the gel in all cases except for unfractronated DI whrch represent one tenth as much template. Product yrelds per ag of template were equal to 0.35 Pg for wild-type DI and I .9 pg for pal Rl DI. Reactions containrng RNPs only yielded no detectable products on gels (not shown).

which is most likely the mutational change responsible for the readthrough property. The two VSV mutants, pol Rl and pol R2, were isolated independently but exhibit very similar properties (Figures 2 and 3; unpublished data). We therefore reasoned that they might contain a similar or related nonlethal N protein mutation. In order to detect such alterations, we compared 35S-methionine-labeled virion proteins of wild type and pol R mutants by two-dimensional gel electrophoresis using the procedure of O’Farrell (1975). The RNP template protein N and the glycoprotein G were the major proteins detected by this method in agreement with earlier reports (Raghow et al., 1978; Levinson et al., 1978; Hsu and Kingsbury, 1982a). The VSV M protein is too basic to focus

VSV Regulates 181

Transcnptron

wildtype

and Replicatron

Functrons

pal R1

Frgure 7. Protern Gel Analysrs of 01 RNP Cores Employed tutron Expenment of Figure 6

rn the Reconstt-

Whole vtrus (- 15 Mg protean) and RNPs (- 7 pg) were electrophoresed on a 12% acrylamrde resolving gel (3004, acrylamtde:bis-acrylamrde) and starned wrth Coomasste blue as described In Expenmental Procedures From left to rrght weld-type vrrus. wild-type RNP, pol RI RNP, pol Rl virus.

In the stable pH range of the ampholines we have employed, and as rn other studies, the minor L and NS proteins were not clearly visualized using 35S-methionine label. Inspection of the patterns of migration (Figures 8a, 8b, 8c) suggested that the G protein from both mutants was not greatly altered. This suggestion was verified when mixtures of wild type plus pol Rl, and wild type plus pol R2 VSV, were analyzed for co-migration in the two-dimensional gels (Figures 8d and 8e). Each of the eight species of G protein co-migrated, demonstrating that no charge or size alteration had occurred in the G proteins of the mutants. The basis for the G protein heterogeneity over a pl range of 6 to 7 has been attributed to differential oligosaccharide sulfation (Hsu and Kingsbury, 1982b). The nucleocapsid N protein was resolved into four differently charged species, Nl through N4, ranging in pl from -6.2 to -6.7 (Figures 8a, 8b, 8~). Others have previously reported two specres with pls of about 5 (Raghow et al., 1978) or three species of N protein with pls from 6.8 to 7.2 (Hsu and Kingsbury, 1982a). The cause for charge heterogeneity in the N protein has not been determined, but could very well be the result of posttranslational modifications since only one mRNA species is translated into the N protein. The wild type and both mutant viruses all contained four N protein charged species. Inspection of their posrtions in the gels relative to the G protein indicates that all four species of N protein from both pol Rl and pol R2 viruses were similarly shifted from 0.02 to 0.09 pH units toward the more acidic region of the isoelectric focusing gel as compared to wild type. This charge shift is most clearly seen in the mixed samples of

mutant and wild-type viruses (Figures 8d and 8e). Although we could detect a small increase in the mobility of both mutant N proteins relative to wild type in a single dimension SDS gel (Figure 7 and data not shown) no differences in relative migration were detected in the second dimension of the two-dimensional gels, indicating that the size of these proteins was not greatly altered. The magnitude of the pl shift in the mutant N proteins is consistent with a single charge change (King and Newman, 1980). These results therefore strongly argue that a similar amino acid substitution has occurred in the N protein of both pol R viruses. A number of additional comparisons between wild type and mutant viruses revealed only two other protein alterations: an acidic charge shift in the two NS protein species (two-dimensional analysis of 32P-labeled proteins) of pol R2 but not pol RI, and a very small but reproducible increase in the migration of pol Rl M protein, but not pol R2, in high resolution SDS gels (not shown). Since these changes were not common to both mutants, they cannot be responsible for the readthrough phenotype but they do confirm the independent origin of pol RI and pol R2. It seems likely that the mutants contain only few undetected protein alterations as compared to wild type because no differences were seen by tryptic peptide analyses of 35Smethionine-labeled N and M proteins; SDS gel analysis of unlabeled N protein partially digested with V8 protease; and Tl oligonucleotide fingerprint analysis of 32P-labeled genome RNAs (not shown). The observed charge alteration in the mutant N proteins is therefore most likely responsible for the readthrough phenotype. Discussion We have shown here that the VSV pol R mutants, in contrast to wild-type virus, frequently readthrough leader RNA termination sites in vitro. A number of important features of this readthrough phenotype need to be emphasized. First, both plus and minus strand leader termination sites are affected about equally (~80% readthrough) even though the template RNA sequence at these sites bears little or no homology (Keene et al., 1980). Second, the approximately -/-fold increase in readthrough relative to wild-type virus IS specific for termination at leader sites since no such increase was detected for mutant mRNA termination sites in vitro (Perrault et al., 1981). Third, the change in pol R mutants represents a major change in termination activity for the virus-bound polymerase because Initiation at the 3’ end of the template is far more frequent than productive initiation of mRNAs (attenuation phenomenon). Fourth, initiation of RNA synthesis in vitro by pol R VSV mutants is 5 to lo-fold more resistant to replacement of ATP by the analog AMP-PNP than wildtype virus (Perrault and McClear, unpublished data). It should be recalled that the presence of this analog following initiation of RNA synthesis by wild-type virus in vitro leads to readthrough of leader RNA sites (Testa et al.,

Cell

182

Figure 8. Two-Dimensronal Gel Electrophoretlc Analysts of %methronine-Labeled Wild Type and pal R VSV Proteins Solubilized in SDS The first dimension Isoelectric focusing gel ran from left (acidic) to right (basic) while the second dimension SDS gel ran from top to bottom. (a), (b), and (c): wild type; pal Rl ; pol R2 VSV. (d) and (e): wild type plus pol Rl; wild type plus pol R2. (f): a diagrammatic representation of the various species of G and N proteins showing the charge shift in the N proteins of the pol R mutants (dotted

lines) versus wild type (solid lines). The arrows indicate the position of the major G5 protein spe cles as a reference

1980b; Chanda et al., 1980). All these considerations argue strongly that an important controlling mechanism has been altered in the mutants. It is therefore not surprising that these nonconditional mutants are somewhat restricted in their growth in vivo (yields are -lo%-50% of wild type). Interestingly, we have also detected a relative increase in nonencapsidated readthrough transcripts in pol R VSVinfected cells (Perrault, unpublished data). The in vitro reconstitution experiments we have presented leave little doubt that the readthrough property of pol R mutants is mediated by its RNP template fraction. The source of solubilized polymerase proteins (L and NS) does not affect this property and this is true for both standard and DI RNP templates. On the other hand, the properties of DI constructed in vivo with wild-type template RNA and pol R proteins, or mutant template RNA and wildtype proteins, establish that it is not a template sequence change at the site of leader termination that is responsible. The fact that both leader sites are affected in pol R mutants also reinforces this conclusion. The RNPs employed in our reconstitution experiments were extensively purified to eliminate significant contamination with proteins other than N. The inescapable conclusion from all this is that a mutated N protein mediates readthrough of leaders. It is therefore gratifying to find a qualitatively and quantitatively similar charge shift alteration in this protein for both independently isolated pol R viruses. Other genome sequence changes have undoubtedly accumulated in these mutant viruses relative to wild type (we detected at least one in each) but, on the basis of our comparative protein analyses, these appear to be few and in all probability unrelated to the readthrough phenotypic change. The replication model of Leppert et al. (1979) proposes that binding of soluble N protein to nascent leader RNA chains is necessary for readthrough activity. Our results with the pol R mutants, however, indicate that readthrough can easily occur without concurrent assembly and solely as a result of an alteration in the N protein (exchange of N protein subunits from template to products is unlikely

point.

because the readthrough transcripts were purified away from RNPs by CsCl centrifugation). These considerations lead us to propose an alternative model where the already assembled RNP template structures (and not assembly of nascent leaders) determine whether readthrough of leader termination site, a required but not necessarily sufficient step for replication, can occur. We suggest that wild-type standard virus or DI package RNPs whose structure promotes termination at leader RNA sites (and transcription of mRNAs in the case of standard virus) under the usual in vitro reaction conditions. In vivo, these “transcription RNPs” would be modified to “replication RNPs” whose structure promotes readthrough of the plus and minus strand leaders. The substrate for this modtiication, we suggest, is the N protein of the assembled RNP. The mutation in the pol R N protein can then be viewed either as promoting the change from a “transcription” to a “replication RNP” structure and/or allowing the packaging of “replication RNP” structures normally excluded from wildtype particles. Additional observations lend support to our proposal. First, N protein appears to be a substrate for posttranslational modification since at least four differently charged species are found in standard virions (Figure 8). Whether these various species correlate with terminatiqn functions, however, is unknown. Second, conformational differences between virion and intracellular RNPs, due solely to N-N or N-RNA interactions have been reported previously (Naeve et al., 1980). These differences could be modulated by protein modification. Third, our reconstitution experiments indicate that the heterogeneous pattern of readthrough transcript termination sites from pol R viruses (and the much more limited heterogeneity in wild type) is essentially identical to that of the unfractionated virus that served as the source of RNPs (Figures 5 and 6). We are forced to conclude from this that once RNPs have been assembled in standard or 01 virions, termination sites for leader RNA and readthrough transcripts are already predetermined.

Since

termination

at such

a large

number

of sites

VSV Regulates 183

Transcriptron

and Replication

Functrons

for readthrough transcripts is unlikely to reflect RNA sequence signals, it is most easily explained by variable modification of some N protein subunits. Future work in progress will hopefully shed light on the role of template protein modifications in regulating termination of RNA synthesis in RNA viruses and possibly their host cells. Experimental

Procedures

Growth, Source, and Purification of Virus Detarls of the rsolation procedure of Rl and pot R2 VSV mutants will be detailed elsewhere. Sources of wild-type standard VSV Indiana and DI from either weld type (DI 0.22) or pol Rl (DI 0.21) were described previously (Perrault et al., 1980). Growth and rnfectron of BHKZI cells were carried out as before (Perrault and Leavrtt. 1977). To obtain pol RI mutant DI RNA templates assembled wrth wild-type proterns, or vice versa, cells were cornfected wrth standard vrrus (free of DI) at an mar of 20 and a previously trtrated amount of purified heterologous DI (free of standard virus) under condrtrons srmilar to homologous DI infections. All DI rnoculae consisted of particles banded in sucrose gradients (see below), then stored wrth an equal volume of glycerol at -20°C. These were trtered for optrmum yreld per cell by cornfectron with standard vrrus. 35S-methronine-labeled virus was prepared by replacing the growth medrum wrth MEM containing -25 &i/ml ?S-methionine (New England Nuclear) (1237 Cr/mmole) and 2 pg/ml acttnomycrn D 2 hr after Infectron. followed by a chase with regular MEM -6 hr later Vrrus purifrcatton was carned out as previously described (Perrault and Leavrtt, 1977) wrth manor modrfrcatrons. Sucrose velocrty gradients, 5% to 40% (w/v) rn TE buffer (10 mM Tns-HCI (pH 7.7). 1 mM EDTA) plus 0 1 M NaCI, were centrrfuged for 35 mm at 35 K rpm, rn the Beckmann SW41 rotor. Followrng tartrate gradrent centrrfugation and dialysis, vrrus was concentrated by centnfugation for l-2 hr in the SW50.1 Beckmann rotor at 48 K rpm, 4°C. It was then resuspended by sonrcatton in a small volume of TE buffer and stored at -20°C after adding an equal volume of glycerol Standard vrrus and DI concentrations were determined spectrophotometrn tally (5.7 and 4.0 OD&mg vrrus, respectively) In Vitro Polymerase Assay of Standard Virus and DI and Purification of RNAs All polymerase reactrons contained the following unless otherwise stated: 50 mM Tns-acetate (pH 8.2). 8 mM Mg-acetate. 0.3 M K-acetate, 1 mM ATP. GTP, CTP. 0.1 mM UTP, 2 mM dithiothrertol and 0.1% Nonidet-P40. DI-templated assays contatned 200 rg/ml DI and 40 &r/ml ‘H-UTP (ICN) (38 Cr/mmole). Standard vrrus assays contarned purrfied transcrrbrng cores (obtarned by low salt drsruptron) equrvalent to 1 mg/ml of vrrus and 100 @J/ml 3H-UTP. The use of 0.3 M K-acetate rather than the more common 0 1 M NaCI, as well as rsolation of transcrrbrng cores from standard virus, were described by Brerndl and Holland (1976). lncubatrons were carned out at 30°C and rncorporation measured as before (Perrault et al., 1980). Purrftcatron of standard vrrus RNA transcrrpts free of templates and unrncorporated label was accomplrshed by CsCl centnfugatron. followed by proternase K drgestron, and phenol-chloroform extractron. as descrrbed prevrously (Perrault and Leavrtt. 1977; Perrault and Semler, 1979). DI products were extracted together with therr template by omrttrng the CsCl step. Unrncorporated precursors were then removed by centrrfugatron - 1,000 xg, 2 mn) through a small column containing an 8 ml bed volume of G-50 Sephadex Vrrus park& RNAs were srmrlarly extracted, omrttrng the last step End-labeling and Nuclease Protection Assays End~labelrng of genome RNA wrth cytidine 3’, 5’.brs3’P-phosphate and RNA lrgase was descrrbed before (Perrault and Semler, 1979) For quantrtatron of end label protected from RNAase. genome RNA was frrst purrfred over a sucrose gradrent before labelrng Annealrng reactron of labeled probe plus products were carned out by frrst denaturrng rn TE buffer at 100°C for I-2 mm. adlustIng to 0.3 M NaCI, and heatrng to 70°C for 30 mm. DIgestron wrth RNAases A, Tl at 10 fig/ml and 5 U/ml, respectrvely, proceeded for 20 mm at 37C, rn the same buffer salt solutron This was followed by

proternase K drgestion and erther direct preciprtatron phenol-chloroform extractron before loading on gels.

with ethanol or prior

Purification of Standard and DI RNPs and In Vitro Reconstitution Both standard virus and DI (100-200 pg/ml) were drsrupted for 3 hr at O”C, rn IO mM Tris-HCI (pH 7.7), 1.0 M NaCI, 2% Trrton X-100, 10% glycerol, 1 mM drthiothreitol, banded twice rn 15%-76% renografrn step gradients, and once In a 20%-40% (w/w) CsCl gradient by centnfugation at - 160,ooO X g for 18 hr, at 4°C. Purified RNPs, recovered by side puncture, were dialyzed exhaustively against 10 mM Tris-acetate (pH 7.9) 0 1% Nonrdet-P40. 20% glycerol, 0.2 mM mercaptoethanol before berng stored at 0°C or -20°C. Concentration of cores (equivalent to -4 mg/ml of standard vrrus and -3.5 mg/ml of DI) was estimated from Coomassre blue stained protern gels. Reconstitution with standard RNPs was carned out as follows. Solubrlrzed polymerase proterns were prepared from weld type and pol RI vrrus by disrupting 2.5 mg of standard virions in -0.5 ml of the disruption solutron described above for the preparatron of RNPs. The suspenston was then overlaid with 10 mM Tns-HCI (pH 7.7) and centrifuged for 90 min at 49 K rpm, 4”C, in the SW50.1 Beckman rotor. The polymerase contarnrng supernatants rn the glycerol layer were carefully withdrawn wrth a prpette and 160 ~1 of this solution were added to 100 /II of weld type or pol Rl purrfred RNPs. The mrxture was then diluted to 1 ml wrth IO mM Trrs-acetate (pH 8 2), 15% glycerol to lower the NaCl concentration to 0.16 M, and rebrndrng of the L and NS proteins to RNPs was allowed to proceed for 90 mm on Ice (Mellon and Emerson, 1978) The reconstrtuted enzymatrcally actrve cores were then centrifuged through a glycerol step gradtent as rn the rsolatton of transcnbrng cores by the method of Breindl and Holland (1976). The pellets were resuspended in a small volume of IO mM Trrsacetate 8.2 and polymerase assay components, Including 100 pCr/ml 3HUTP. were added to give a final reactron volume of 200 ~1, No attempts were made to quantrtate the extent of rebrndrng of solubrlrzed L and NS to the RNP template. Unfractionated wild type and pol RI standard vrrus (200 rg/ml) were assayed in parallel. Reconstrtutron wrth DI RNPs was as follows Solubrlrzed polymerase proterns were prepared by disrupting 1 mg of standard vrrions rn 0.5 ml and centnfugrng as above. The supernatants were then desalted by centrrfugrng twice (-1000 xg. 2 mm) through an 8 ml bed volume of G-25 Sephadex equrlrbrated wrth IO mM Trrs acetate (pH 8.2) 0.1% nonrdet-P40, 10% glycerol and recovered rn a volume of -700 pl. 100 pl of this enzyme fractron (recoveries of L and NS were not monrtored) were then combrned wrth 70 AI of punfred DI RNPs and polymerase reactron components adjusted as before (except for substrtutrng 0.1 mM GTP, 750 &r/ml o-~‘PGTP, and 1 mM UTP) to a frnal volume of 400 pl. Unfractronated weld type and pol RI DI (200 lg/ml) were assayed rn parallel RNA Gel Analyses Agarose gel analysts of glyoxal-denatured RNAs by fluorography (3H samples) or drrect autoradrography (“P samples) was carrred out as before (Perrault and Semler, 1979) except for addtng a frnal concentratron of 3.3% acrylamrde (30:1, acrylamrde:brs-acrylamrde) lust before pourrng the bonzontal gels. No precautrons were taken to insure complete polymenratron of the acrylamrde. but thus modrfrcatron Improved resolution of small moles cules and the mechanrcal strength of the gels. Analysrs of duplex molecules (no urea) and transcripts (In the presence of 7 M urea) rn hrgh percentage polyacrylamrde gels was described prevrously (Perrault and Semler, 1979, Perrault et al., 1981). Quantrtatron of 32P-labeled specres in autoradrographs was carrred out by densrtometry (EC910 Transmrssron Densitometer) and rntegratton of peak areas wtth a Numonrcs drgrtrzer. Protein Gel Analyses Srngle drmensron SDS gel analysrs was performed as described by Laemmlr (1970).Two-drmensronal gel analysis followed the procedure of O’Farrell (1975) SDS drsruption of vrral proterns was carned out by solubrlrzatron of centrrfuged vrrus rn the sample buffer of Laemmlr (1970) and rncubatron rn a borlrng water bath for 2 mm Vrral proterns were then precrpitated wrth 10% TCA and washed twice wrth 10% TCA and twrce with ice-cold acetone Vrral proterns, collected by centnfugatron. were suspended rn the frrst drmensron lysrs buffer contarnrng 9.5 M urea (Schwartz/Mann, ultrapure

Cell 184

grade, 2% Nonldet-P40 (Particle data), 2% ampholines composed of 1.6% (pH 5 to 8) ampholines and 0.4% (pH 3.5-10) ampholines (LKB), and 5% @mercaptoethanol. Following Isoelectric focusing, the pH !n 1 cm slices of a blank sister gel that did not contain a protein sample was measured by eluting the ampholines from the slices Into distilled, deionized and degassed water. The pH gradlent in this blank gel was used to determine the isoelectric pH of protein species In the sister gel. The isoelectric focusing gels were embedded in agarose at the top of a 12% polyacrylamide slab gel and electrophoresed for 1040 volt-hours. The gels were dried and exposed to x-ray film for autoradiography. Acknowledgments We thank Joanne Shuttleworth and Aileen Torres for excellent technical assistance and Drs. John Lenard and David Apirion for critical reading of the manuscript. This work was supported by a research grant (Al14365) and a Research Career Development Award from the National Institutes of Health to J. P. and a National Science Foundation grant (PCM-8208155) to G. M. C. The costs of publication of this article were defrayed in parl by the payment of page charges. This article must therefore be hereby marked “advetiisement” in accordance with 18 U.S.C. Sectlon 1734 solely to indicate this fact. Received

of vesicular stomatitis virus replicating gradtents. Virology 99, 75-83.

complexes

isolated

In renografin

HIII, V. M., Marnell, L., and Summers, D. F. (1981). In vitro replication and assembly of vesicular stomatitls virus nucleocapsids. Virology 7 13, 109118. Hsu, C.-H., and KIngsbury, D. W. (1982a). NS phosphoproteln of vesicular stomatitis virus: subspecies separated .by electrophoresis and isoelectric focusing. J. Virol. 42, 342-345. Hsu, C.-H., and Kingsbury, D. W. (1982b). Contribution of oligosaccharide sulfation to the charge heterogeneity of a viral glycoprotein. J. Biol. Chem. 257, 9035-9038. Iverson, L. E., and Rose, J. K. (1981). Localized attenuation uous synthesis during vesicular stomatitis virus transcription. 484.

and discontinCell 23, 477-

Keene. J. D.. Schubert. M., and Lazzannl, R. A. (1980). lntervenlng sequence between the leader region and the nucleocapsid gene of vesicular stomatitls virus RNA. J. Vlrol. 33, 789-794. King, A. M. Q., and Newman, J. W. I. (1980). Temperature sensitive mutants of foot-and-mouth disease virus with altered structural polypeptides. I. ldentificatlon by electrofocusing. J. Vlrol. 34, 59-66. Laemmli. U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Leppert, M., Rlttenhouse. L., Perrault. J., Summers, D. F., and Kolakofsky, D. (1979). Plus and minus strand leader RNAs in negative-strand virusinfected cells. Cell 18, 735-748.

July 6, 1983

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