The Sendai virus nonstructural C proteins specifically inhibit viral mRNA synthesis

VIROLOGY

189, 647-656

(1992)

The Sendai Virus Nonstructural JOSEPH CURRAN, Department

of Genetics

and Microbiology,

C Proteins JEAN-BAPTISTE

University Received

Specifically MARQ,

DANIEL

AND

of Geneva

School

of Medicine,

January

15, 1992;

accepted

Inhibit Viral mRNA Synthesis

CMU, May

KOLAKOFSKY’

9 Ave de Champed,

CH- 12 11 Geneva,

Switzerland

7, 1992

An in vitro transcription system for paramyxoviruses is described, in which polymerase-free templates are combined with cell extracts containing polymerase made in viva via transfected plasmids. Both P and L are required for polymerase activity, and both must be coexpressed for optimum activity. mRNA synthesis here was found to be inversely proportional to the level of C expression, whereas defective interfering genome replication was largely unaffected by the level of C in the extract. The inhibition of transcription appeared to be due to the C’ and C, but not the Y, and Y, proteins, and only occurred when C/C was coexpressed with P and L. C/C appears to intervene during polymerase formation, possibly by forming polymerase complexes which are inactive for transcription, but still competent for genome replication. @ 1992 Academic Press, Inc.

NDV, moreover, have no overlapping C ORF at all (Thomas et al., 1988). The possible function(s) of the C proteins has remained enigmatic. They are strongly basic proteins which are vastly underrepresented in virions relative to infected cells (relative to internal markers such as the P and NP proteins). However, there is a small amount of these proteins in virions, which appear to be bound to NCs (Yamada et a/., 1990; Lamb and Paterson, 1991; Kolakofsky et al., 1991), and C proteins made in vitro appear to bind to NCs present in infected cell extracts (Ryan and Kingsbury, 1988). Intracellularly, there appears to be little specificity in their localization; they are evenly distributed in the cytoplasm by immunofluorescence and are found in all cytoplasmic fractions by cell fractronation (Portner et al., 1986). However, in SEN-infected cells the C proteins are relatively abundant (at least as abundant as P). It is possible that there are specific binding sites for C on one or more of the NC proteins (NP, P, or L), but these sites are limited. This paper investigates the possibility that the C proteins are involved in mRNA synthesis.

The Sendai virus (SEN) C proteins were first described by Lamb et al. (1976) and Lamb and Choppin (1978) while cataloging the virus-induced polypeptides in infected cells. A pair of proteins with molecular weights in the 20-24 kDa range were noted, which were largely absent in virions, and named C’ and C as they had similar tryptic fingerprints. Since C proteins with slightly different electrophoretic mobilities were found when different viral strains were used to infect the same cells (Etkind eta/., 1980), it became clearthat these proteins were indeed virus coded. Later work then showed that the C proteins were coded for by the P gene mRNA (Dethlefsen and Kolakofsky, 1983; Dowling et a/., 1983), by an ORF frame which overlapped the N-terminus of the P ORF, and in the +l frame relative to that of P (Giorgi et a/., 1983; Shioda et a/., 1983). The various ORFs now known to be expressed by the SEN P gene mRNAs are schematized in Fig. 1. Remarkably, this gene expresses as many as eight primary translation products. These include all three reading frames and result from the use of five (and possibly six) different ribosomal start sites as well as “edited” mRNAs (reviewed in Kolakofsky et a/., 1991). In SEN, there are in fact four carboxy-coterminal C proteins, which start at four separate initiation sites, including an unusual ACG start codon at nt 81 (Curran and Kolakofsky, 1988; Gupta and Patwardhan, 1988). These are designated C’, C, Y, , and Y,. It is presently unclear why the C proteins are expressed in a nested set in some paramyxoviruses [eg, SEN and PlVl (Boeck et al., 1992)], but not in others (e.g., PIV3 and measles virus). The SV5/mumps group of viruses and

’ To whom

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requests

should

MATERIALS Cell cultures

AND

METHODS

and viruses

BHK and CVl cells were seeded on g-cm dishes and grown in MEM with 5% fetal calf serum. Cells were infected with Sendai virus (20-50 PFWcell) or a vaccinia virus recombinant expressing T7 RNA polymerase (vTF7-3, Fuerst et al., 1986: 2-3 PFU/cell). Infections were maintained in serum free medium at 33”. Construction

of mutants

and subclones

The constructions of pGEM-NP, pGEM-P/C, pGEML, pGEM-P, pGEM-C/Y 1 /Y2 (previously referred to as

be addressed. 647

0042-6822192

$5.00

Copyright Q 1992 by Academic Press. Inc. All r~ghis of reproduction I” any form reserved

648

CURRAN,

MARQ,

insertion site 1053 4 P 104

X 1523

-

\

1808 /

5’

+lG

t2G w 104 +

I

C’ 81

c

Yl

Y2

114

183

201

1061

FIG. 1. A schematic representation of the ORFs expressed from the P/C gene mRNAs. The mRNAs are shown as horizontal lines with the various ORFs as boxes. The P ORF is shown as a stipled box (0 frame), the C ORF as open boxes (+l frame), and the V ORF (-1 frame) as a black box. The mRNA at the top is an exact copy of the gene, and those below contain a 1 G or 2G inserted near nt 1053, as indicated. The numbers refer to nt positions of the first base of the various start and stop codons, relative to the 5’ end of the mRNA.

ACG8 1/ATG), and pGEM-C’/CN,N, (previously referred to as ATGl04/GCG) have all been described (Curran et a/., 1991; Curran and Kolakofsky, 1988, 1989). pGEM-C/Y,/Y, was prepared from a site-directed mutant, SP65 P/C BarnHI (the Sau3A site immediately following the P ATG/104 was changed to a BamHl site; ATGGATCA to ATGGATCC) by transfering a BamHIIXbaI fragment (nt 107-l 027 of the P/C gene) into pGEM3. pGEM-Y,N, was prepared from SP65 P/C by transfering a NarllXbal fragment (nt 156-l 027) into the SmallXbal sites of pGEM3.

AND

KO!-AKOFSKY

pared essentially as described by Carlsen et al, (1985). Briefly, the cell monolayers were washed once in icecold PBS before incubating on ice with 1 ml of buffer A (5% sucrose, 80 mM KCI, 35 mM HEPES, pH 7.4, 5 mM K,HPO,, pH 7.4, 5 mM MgCI,, 0.5 mM CaCI,) containing 250 pg/ml lysolecithin (Sigma) for 1 min. Buffer A was removed and the monolayer was scraped into 300 ~1 of transcription buffer (100 mM HEPES, pH 8.5, 150 mM NH&I, 4.5 mM MgAc, 1 mM DTT). This mixture was pipetted up and down 20 times to break the cell membrane before spinning out the nuclei and cell debris in an eppendorf centrifuge at 4”. RNP templates (NP:RNA) were prepared from infected BHK cells (Leppert et a/., 1977). They were banded twice on 20-40% CsCl gradients (38,000 rpm for 2 hr at 12” in a SW41 rotor) to remove the endogenous polymerase (P and L) before pelleting through 50% glycerol in 0.2% NP40, 30 mM NaCI, 10 mlVI HEPES, pH 7.4, and 1 mM EDTA onto a 1 00-~1 cushion of 68% sucrose (w/v) in D,O (43,000 rpm for 90 min at 18” in a SW60 rotor). NP:RNA templates isolated from one g-cm petri dish were resuspended in a final volume of 100 ~1. Both templates and extracts were freshly prepared for each experiment and were never frozen. In vitro transcription reactions contained 1 O-20 @Iof RNP template, 50-100 ~1 of lysolecithin cell extract, and 25 ~1 of a 10X energy mix (5 mlVI NTPs, 2 pglml actinomycin D, 400 U/ml creatine phosphokinase, 10 mM creatine phosphate) in a final volume of 250 ~1 (adjusted with transcription buffer). Reactions were incubated for 2 hr at 30”, and the RNA products were pelleted thru 20-40% CsCl gradients and analyzed by RNase protection (see below). Transcription/replication assays were performed under similar conditions except that the cold UTP present in the energy mix was replaced with 50 &i of 32P-UTP, and the templates were a mixture of nondefective and DI-H4 genomes (Leppert et al,, 1977). DI-H4 has recently been cloned and its structure determined; it is 1411 nt long and contains inverted terminal repeats of 1 10 nt (Calain et a/., submitted for publication). RNPs were then banded on CsCl gradients and analyzed on 1.5% agarose-formaldehyde gels. RNase protection

In vitro RNA synthesis CVl monolayers on g-cm dishes were infected with vTF7-3. At 1 hpi, the infecting medium was replaced with a transfection mix composed of 2 ml of MEM and 20 ~1 of transfectACE (BRL) combined with the various plasmid DNAs as indicated. pGEM-P, pGEM-P/C, and the various forms of pGEM-C were always used at 5 pg/dish, pGEM-L at 1.5 pg/dish, and pGEM-NP at 2.5 pg/dish. At 18-20 hpi, cytoplasmic extracts were pre-

The SP65 leader/NP riboprobe used in these studies covered the entire leader region plus the first 87 nt of the NP gene, (Vidal and Kolakofsky, 1989). RNase protection using this riboprobe was performed essentially as described by Vidal and Kolakofsky (1989) except that no Tl nuclease was used for Figs. 5-8 and here the concentration of RNase A was increased to 80 pg/ ml. The protected products were analyzed on 6% polyacrylamide sequencing gels.

IN I/ITRO

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FOR

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649

blot analysis

Protein blots were performed as described in Curran and Kolakofsky (1988), except that the signal was visualized using the chemiluminescent substrate AMPPD (Boehringer). The polyclonal antiserum to the C proteins was raised in rabbits against a bacterial fusion protein. The NP and P monoclonal antibodies were a kind gift from Dr. Claes Oetvell, Stockholm.

RESULTS A novel in vitro system for mRNA synthesis We have recently described a system for SEN in which defective interfering (DI) genomes are replicated in viva without helper virus, but with viral proteins provided in trans from DNA plasmids. DI genome replication here is dependent on the NP, P, and L proteins, but none of the other P gene proteins, including any of the C proteins, appeared to be required (Curran et al., 199 1). As these DI genomes do not transcribe mRNAs (they contain the copy-back organization), we sought an analogous in viva system in which the protein requirements for mRNA synthesis could be examined. Our first approach was to transfect nondefective nucleocapsids (NCs) devoid of the majority of their P and L proteins (by banding in CsCl density gradients) into cells which expressed the P/C and L proteins (from pGEM-P/C and -L). We found that these NCs made relatively large amounts of NP mRNA, and that this activity was dependent on the presence of both plasmids. We then carried out many of the experiments previously described for genome replication (Curran et al., 1991), including the effect of abrogating C protein expression from the plasmid, and obtained very similar results (not shown). On reflection, however, we realized that the P and L proteins made from the plasmids were simply “priming the pump” of the nondefective NCs (which was intended), but that the levels of NP mRNAs that followed were mostly due to those of their templates, which were being amplified. Genome amplication, moreover, could not be prevented without interfering with the expression of the polymerase proteins from the plasmids. Since it was unclear how mRNA synthesis could be divorced from genome amplification in an in viva system, we investigated an in vitro system. As the CsCIbanded NCs were clearly active, they were combined with viral proteins prepared as extracts of cells transfected with the pGEM plasmids [the T7 polymerase was provided by coinfection with a vaccinia virus recombinant (vTF7-3, Fuerst et a/., 1986)]. After incubation, the RNA products which pelleted through a CsCl

55

+

FIG. 2. Demonstration of the in vitro system. (A) Cytoplasmrc extracts (100 ~1) of vTF7-3-infected CVl cells transfected with both pGEM-P/C and -L (lane (P/C + L), or a mixture of 100 ~1 each of extracts from cells individually transfected with pGEM-P/C or -L, were incubated with a constant amount of NP:RNA templates (20 ~1) (Materials and Methods). The reaction products were isolated as CsCl pellet RNA, and their levels were estimated by RNase protection. As negatrve controls, the templates were incubated without cell extracts (lane RNPs), or with extracts from cells infected only with vTF7-3 (lane vTF7-3). As a positrve control, the radiolabeled nboprobe (lane PROBE) was annealed wrth CsCl pellet RNA from SEN-infected cells (lane CTRL). The expected products (and their lengths) are indrcated on the left. The left-most lane shows an unrelated riboprobe of 55 nt, as a marker for the 55 nt leader RNA. (B) The relative amounts of P protein in the various cell extracts were determined by Western blotting, wrth a P-specific monoclonal antibody

gradient were followed by a RNase protection protocol (Materials and Methods). This method is demonstrated in Fig. 2. RNA synthesis here was carried out with unlabeled precursors, and a radioactive riboprobe containing nt l-142 as (-)RNA (plus additional vector sequences) was used to detect the products. When this probe is used with in

650

CURRAN,

MARQ,

viva-made mRNA from H strain-infected cells, a single group of bands representing the beginning of the NP mRNA (nt 56-142) is detected (lane CTRL). In reactions where the templates were incubated either alone (lane RNPs) or with extracts from cells infected only with vTF7-3 (lane vTF7-3) no RNA products could be detected. When the cells were also transfected with both pGEM-P/C and pGEM-L, significant amounts of RNA products were seen (lane (P/C + L)). This included both NP mRNA and bands at ca. 142 nt which represent unencapsidated leader/NP readthrough RNAs. These latter bands are much more prevalent in Z strain infections (Vidal and Kolakofsky, 1989) the source of the templates used here. Similar to the in vivo situation, but in contrast to reactions with purified virions (Vidal and Kolakofsky, 1989) we found little or no evidence of the 55 nt leader RNA, which presumably turns over in the presence of the cell extracts. Extracts from cells transfected with pGEM-L alone never supported >l% of the transcription of extracts in which P and L were coexpressed, whereas extracts in which only P was expressed supported low but variable amounts of mRNA synthesis, which ranged from 2 to 10% (not shown here, but see Figs. 6,7, and 8). This latter variability presumably reflects small amounts of L which have not been removed from the templates. Additional activity could be restored by combining these extracts (lane (P/C) + (L), Fig. 2A; and lane(P) + (L), Fig. 8A). However, it was always considerably less than when the proteins were coexpressed, even though the amounts of P protein were at least the same as judged by immunoblotting (Figs. 2B and 8B). We were unable to estimate the L protein levels due to the lack of a suitable antibody (see Note added in proof). The effect of the C proteins on RNA synthesis In the above experiment, we used pGEM-P/C containing the wild-type gene. To determine what effect abrogation of C expression had, we replaced pGEM-P/ C with pGEM-P, which contains a stop codon in the C ORF just downstream of the Y, AUG, and which expresses P normally but not the C proteins (Curran eta/., 1991). When extracts from cells transfected with pGEM-L and either pGEM-P or -P/C were compared (Fig. 3), those without the ability to express the C proteins produced five times more products, even though both extracts contained equivalent amounts of P as judged by immunoblotting (not shown). Moreover, the increase in activity was roughly proportional to the fraction of pGEM-P extract in this mixing experiment. The apparent ability of the C proteins to inhibit transcription was unexpected, as they had little or no effect on DI genome replication in viva (Curran et a/., 1991). We therefore examined the effects of the C proteins on both transcription and replication in the same reaction.

AND

KOLAKOFSKY P P/C

455 FIG. 3. The inhibitory effect of the C proteins on mRNA synthesis. RNA synthesis was carried out with 200 @I of extract from either pGEM-L and -P/C (indicated as P/C, 20) or pGEM-L and -P transfected cells (indicated as P, 20) or a mixture of the two as indicated above the figure, and their levels were estimated as in Fig. 2. The numbers below show the relative amounts of the NP mRNA bands, determined by reexposing the gel in a Phosphorimager. The remaining lanes, including the left-most lane, are as described in Fig. 2.

This was carried out as above, except that (i) the templates were a mixture of nondefective and DI-H4 genomes from the H strain, (ii) pGEM-NP was also cotransfected to provide NP for genome replication, (iii) the in vitro reactions were directly radiolabeled and RNase protection of the CsCl pellet RNA was done with cold riboprobes, and (iv) the RNA from the NC band region of the CsCl gradient was electrophoresed on gels to directly estimate DI genome replication. Three sets of extracts were used. All contained pGEMNP and pGEM-L, and pGEM-P/C, pGEM-P, or a mixture of pGEM-P and pGEM-C’/C/Y,/Y,. This last plasmid, in which the P protein AUG/lO4 is now a noninitiator GCG, was used as a “natural” source of the C proteins, which are made in the same relative amounts as from the wild-type gene (see Figs. 5 and 6). As shown in Fig. 4A, elimination of the C proteins increased NP mRNA synthesis 5.7-fold, whereas rein-

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SYSTEM

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PARAMYXOVIRUSES

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t.ui 2

FIG. 4. The C proteins strongly inhibit transcription, but not genome replication. RNA synthesis was carned out with 3ZP-UTP and extracts from cells transfected with pGEM-NP, -L. and -P/C, -P, or both -P and C, as indicated above. As a negative control, extracts from cells infected only with vTF7-3 were also used (lane none, the same as lane vTF7-3 in Figs. 2 and 3). NP mRNA levels In 10% of the resulting CsCl pellet RNA were determined with an excess of nonradiolabeled riboprobe l-1 42 (A). These levels, determined In a Phosphorimager, are indicated below. The film shown here was exposed overnight. All of the RNAs from the NC band region of the CsCl gradient were drrectly separated on a 1.5% agarose-HCHO gel (B). The film shown was exposed for 5 days, but the DI RNA levels (shown below) were determined in a Phosphorimager after a 2-day exposure [the intensity of the band in lane NP + P/C + L was nine times that of the background levels (lane none)]. (C) Shows the estimations of P protein levels In the extracts by Western blotting.

troduction of the C proteins via pGEM-C’/C/Y,/Y, decreased synthesis 2.7-fold (both values are relative to the wild-type construct). DI genome replication in this system was found to be much less efficient than mRNA synthesis, and it was difficult to produce a clear autoradiograph of the results even with a 5-day exposure of the film (Fig. 4B). However, clear results could be obtained by exposing the gel in a Phosphorimager for 2 days, and here the intensity of the DI band in lane NP + P/C + L was nine times that of the negative control (lane none). The Phosphorimager is considerably more sensitive and accurate than film for determining low amounts of radioactivity. When the relative amounts of DI genome replication were compared, it was found to be increased only slightly by elimination of the C proteins and decreased only slightly by their reintroduction (numbers at the bottom of Fig. 4B). Again, these effects were not due to changes in the levels of P proteins, which were equivalent in the three extracts (Fig. 4C). The C proteins therefore appear to have a much stronger inhibitory effect on transcription than on replication. Characterization transcription

of C protein

inhibition

of

The ability of the P and L proteins to efficiently transcribe the NC template in this system depends on their having been coexpressed. We therefore examined

whether the ability of the C protein(s) to inhibit this reaction was similarly dependent on its coexpression with P and L. Transfected cell extracts were prepared in which pGEM-C’/CIY,N2 was either coexpressed with pGEM-P and -L or expressed separately and then combined with extracts containing P and L.. To eliminate possible differences due to the amount of cell extract used, a similar amount of extract from cells infected only with vTF7-3 was added to those reactions which did not receive a separate C protein extract, and all extracts were monitored for protein levels. As shown in Fig. 5, the ability of the C protein(s) to inhibit transcription was also highly dependent on their coexpression with P and L. The C protein(s) made in the absence of P and L had lost all their inhibitory activity. This loss could not be accounted for by changes in either the C protein(s) or P protein levels in the extracts, which remained constant (Fig. 5B). Also included in this immunoblot is an extract from cells transfected with pGEMs P/C and L, which shows the ratios of C and P proteins made from the wild-type construct. In this experiment, two to three times more C (relative to P) is made from pGEM-C’/C/Y,/Y, than from the wild-type construct and presumably explains the even stronger inhibition by C here. As expected, RNA synthesis in this system is dependent on adding NTPs to the reactions (lane (P + L) - NTPs). Similar to natural virus infections, the C protein (AUG/114) is by far the predominant of these proteins

652

CURRAN,

MARQ,

A

~

leader NP (142)

+

NP(87)

AND KOLAKOFSKY

mRNA synthesis (Fig. 6A). We found that expression of pGEM-CN,IY, strongly inhibited transcription like that of pGEM-C’/CN,IY,, and pGEM-C’/Y,N, inhibited even more strongly. In this latter case, however, P levels are reduced, which may also account for the decreased mRNA synthesis. pGEM-Y,/Y*, in contrast, inhibited only slightly (<2-fold). In this experiment, slightly less Y,N, is made relative to C’ or C from the other constructs, but in other experiments where Y,/Y, levels from pGEM-Y,/Y, were equivalent, there was still only minimal inhibition of polymerase activity (not shown). It appears that one region of the C proteins which is critical for this inhibition lies near the NH, -terminus of the C protein chain. Independent

expression

of the viral L protein

In this system, the ability of the P, L, and C proteins to act, either positively or negatively, depends on their

B

xvt Q\~ Qx

cl x QxvG

A .I& tP

C C Yl +

Y2

must be coexpressed with P and L for their FIG. 5. The C proteins inhibitory activity. (A) RNA synthesis was carried out with a total of 200 ~1 of cell extracts as follows: the reaction in lane (P + L) received 100 PI from cells cotransfected with pGEM-P and -L and 100 PI of extract from cells infected only with vTF7-3; lane (P + L) + (C) with 100 ~1 of pGEM-P plus -L extract and 100 ~1 of pGEM-C’/CN,N, extract; lane (P + L + C) with 100 ~1 of pGEM-P plus -L plus -C’/CN,I Y, extract and 100 pl of vTF7-3 extract; and lane RNPs with 200 pl of vTF7-3 extract. Lane CTRL is the positive control using infected cell CsCl pellet RNA to protect the probe, and lane (P t L)-NTPs shows another control in which the nucleoside triphosphates were omitted from the reaction. The numbers below show the levels of the NP mRNA band determined in a Phosphorimager. (6) An equal amount of the various cell extracts indicated above (10 ~1) were monitored for P and C protein levels, with a mixture of P and C specific antibodies.

CP C’

expressed from pGEM-P/C and pGEM-C’/CN,N, (Figs. 5 and 6). To determine whether each of the C proteins could equally inhibit transcription, other plasmids were constructed in which either or both C’and C were not expressed and designated pGEMs C’/Y,/Y,, CN,IY,, and Y,N,. These are shown in Fig. 6B (C’, C, Y, , and Y, are 215, 204, 191, and 185 aa long, respectively). Each of these plasmids was then coexpressed with pGEM-P and -L, and the resulting cell extracts were assayed for their relative abilities to carry out

C Yl +

Y2

FIG. 6. C’ and C, but not Y, and Y,, inhibit mRNA synthesis. RNA synthesis was carried out with extracts of cells cotransfected or singly transfected with the plasmids indicated above, and the RNA products were estimated as before (A). The levels of the NP rnRNA band, determined in a Phosphorimager, are indicated below each lane. The positive (CTRL) and negative (RNPs) controls are as described before. An equal amount of each cell extract was monitored for P and C protein levels (B).

IN VlTRO

TRANSCRIPTION

SYSTEM

coexpression. We have followed the levels of P and C in the extracts, and as they are generally independent of their coexpression with other viral proteins, their inability to act when expressed independently cannot be due to their instability. Unfortunately, we have been unable to follow L levels directly. The only antisera we have is weak and can only detect amounts of L which are already strongly inhibitory for RNA synthesis. It then remains possible that L is unstable when expressed without P, or is somehow lost during extract preparation (see Note added in proof). We therefore sought other evidence of whether L was present in the extract when expressed independently. We first added either L or P expressed independently to extracts in which P and L were coexpressed at optimum levels and found that they had little or no effect (Fig. 7). However, when P or L expressed independently were added to extracts in which P, L, and C were coexpressed, the P extract reproduceably increased mRNA synthesis, whereas L further decreased polymerase activity (Fig. 7). Since the extent of the effect is the same in both cases, namely 2-fold, this suggests that L, like P, is not lost when expressed independently. Figure 7B shows the estimations of the P and C protein levels directly in this experiment (by immunoblotting) and again compares their levels relative to those obtained from the wild-type construct (pGEM-P/C).

Possible

interactions

of the C and P proteins

Optimal polymerase activity in this system requires that P and L be coexpressed, but some activity is found by expressing each protein alone and then combining the extracts (Fig. 2). Since the inhibitory effect of the C protein(s) also depends on their coexpression with P and L (Fig. 5) it was of interest to determine the effect of coexpressing C with P and L individually on the activity obtained upon combining the extracts. As shown in Fig. 8, in this experiment extracts in which only L was expressed showed little or no activity [lane(L)], but those containing only P [lane(P)] showed about 10% of maximal activity (i.e., relative to extracts in which P and L were coexpressed [lane (P + L)], and combining these extracts increased the activity to about 18%. When C was coexpressed with Land then this extract was combined with that containing only P [lane (P) + (L + C)], there was only a slight inhibition due to C expression (to 15%) even though polymerase activity here is completely dependent on L expression. The lack of a strong effect here, it should be noted, also provides evidence that C inhibition of polymerase activity is unlikely to be due to the loss of L from the extract

FOR

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FIG. 7. The effect of P or L expressed Independently. RNA synthesis was carried out with 100 ~1 of extract from cells cotransfected with erther pGEM-P and -L or pGEM-P, -L, and -C’/C/Y,/Y, (shortened to simply C here), and 100 IJI of vTF7-3 extract (lanes P + L, and P + L + C), 100 pl of P extract (lanes 2P + L, and 2P + L + C), or 100 ~1 of L extract (lanes P + 2L, and P -t 2L + C), and the levels or NP mRNA made were determined (In a Phosphorimager) as before. Two expenments were carrred out and normalrzed by setting the P + L reaction in each to a value of 100. The results are shown In (A) where the bars show the range of the two determinatrons. Lane none IS a reaction which recerved 200 ~1 of vTF7-3 extract alone and is the same as the vTF7-3 lane In other figures. (B) The estimations of the P and C proteins In 10 ~1 of the various extracts by Western blotting with a mixture of P and C specrfrc antrbodtes.

when C and L are coexpressed. When C was coexpressed with P, on the other hand, and then combined with an extract containing only L [lane (P + C) + (L)], a much stronger inhibition was found (to 6%). As before, all reactions received the same amount of extract (the difference being made up with extracts from cells infected with vTF7-3 alone), and we have estimated the levels of P and the C proteins present in these extracts by immunoblotting (Fig. 8B). As their levels are basically unchanged whether they were expressed individually or in combination with the other proteins, it appears that C may be interacting with P to inhibit mRNA synthesis.

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FIG. 8. The effect of coexpressing C with either P or L expressed individually. RNA synthesis was carried out with 60 ~1 of extracts from cells which were either cotransfected and to which 60 ~1 of extract from cells infected only with vTF7-3 was also added (indicated by including the plasmid names within one set of brackets), or with 60 ~1 each of cells individually transfected with plasmids (indicated by the two sets of brackets), as indicated. pGEM-C’/CN,N, is listed as simply C here. The results of the RNase protection of radiolabeled riboprobe, as quantitated in a Phosphorimager, are shown as a bar graph in (A). The activity of coexpressed P and L was set as 100, and a background of vTF7-3 alone, which represented 2.4% of the (P + L) activity, was subtracted from each value. (B) The estimations of the P and C proteins in 10 ~1 of the various extracts by Western blotting with a mixture of P and C specific antibodies. Protein levels from the wild-type plasmid (P/C + L) are shown for comparison.

AND

KOLAKOFSKY

to the NH,-terminal half of P (Curran et a/., 1991). We presume that P and L must be coexpressed for maximal activity because they only efficiently form an active complex during or shortly after their synthesis. In similar experiments, which focus on the protein requirements for DI genome replication, Moyer and colleagues have found that P and L must also be coexpressed to provide the polymerase for replicase activity (Horikami et al., 1992). By comparing mRNA synthesis in the presence and absence of the C proteins, we found that (i) the C proteins inhibit transcription, whereas they have little effect on genome replication, and that this inhibition takes place in the presence as well as in the absence of added NP; (ii) this inhibition is predominantly due to C’ and C; and (iii) the ability of C/C to inhibit also depends on their coexpression with P and L. Unlike virion polymerase reactions or those based on infected cell extracts, there are no prepositioned polymerases on the template here. The entry of the P-L complex onto the NP:RNA template and chain initiation are then likely to be the rate-determining steps in RNA synthesis. This system then measures the number of active polymerases formed. Our data argue that C interacts with the polymerase components during polymerase formation, as adding C after P and L have been coexpressed has no effect. For example, C could be interacting with P and L to form a C-P-L complex (as indicated in Fig. 9) but more direct biochemical studies are required to

NP

P

C

L

DISCUSSION The in vitro system described here is a marked improvement over virion polymerase reactions. It is more active (data not shown), and more importantly, it provides a relatively simple way of varying the protein composition of the polymerase complex. Both P and L are required here for SEN mRNA synthesis, confirming earlier work with Newcastle disease virus (Hamaguchi et a/., 1983) and they must also be coexpressed for optimal activity. There is evidence that P and L form a stable complex both on and off the template (Hamaguchi et a/., 1983; Pot-trier et al., 1988), and we have recently mapped the site(s) on P which interacts with L

replication

transcription

FIG. 9. A hypothetical flow diagram of polymerase formation for transcription and replication. For transcription, P and L first form a complex (P-L), which can then engage the NP:RNA template. For replication, an NP-P complex is also required. C is hypothesized to form a complex with P and L (“C-P-L”) which is inactive for transcription. C-P-L, however, can be recruited for replication, either as such, or because it can be converted to P-L by interacting with the NP-P (or NP alone) required for replication.

i/V WTRO

TRANSCRIPTION

SYSTEM

elucidate the nature of the complexes formed. Contrary to our previous report that C interacts with CsCl banded NCs which contain only the NP protein (Kolakofsky et a/., 1991), more carefully controlled experiments now find little or no evidence for this (data not shown). As C binds to holo-NCs containing P and L (Ryan and Kingsbury, 1988), it must then be binding to P or L. The data in Fig. 8 would indicate that it is binding mostly to P. C, however, has little effect on genome replication (Curran et al., 1991, and Fig. 4). This could mean that the polymerases which initiate transcription and replication are formed in different pathways, and that the latter pathway is insensitive to the presence of C. Alternatively, the hypothetical C-P-L complex might be competent for replication as such, together with a source of unassembled NP (e.g., NP-P), or because NP or NP-P could displace C from the C-P-L complex. NP and P are known to form a complex for genome replication in VSV (Peluso and Moyer, 1988; Howard and Wertz, 1989) and to associate in measles virus (Huber eta/., 1991). Most recently, Horikami et al. (1992) have found that genome replication can be reconstituted with two extracts containing P and L and NP and P, respectively. We also know that, at least for genome replication, the ratio of L to P is important. Excess L to P ratios inhibit both VSV and SEN RNA synthesis (Meier et al., 1987; Gotoh et a/., 1989; Curran e2 a/., 1991; Horikami et al., 1992). If C specifically binds to P, C could also be inhibitory simply by increasing the L to P ratios at the time of complex formation. It is then possible to explain why excess L exacerbates C inhibition of P-L activity under conditions where it has little effect in the absence of C (Fig. 7). Further understanding of this area, however, will require the determination of the precise nature of these complexes, including the stoichiometries of their components. Given that the C proteins are coded for by the P gene, and that they are not associated with viral or host membranes, it seemed reasonable that they would somehow participate in RNA synthesis. It had long been speculated that C was not required for transcription because there was so little C in virions, but could be involved in genome replication. C expression, however, has little effect on genome replication, and the present finding that C expression inhibits mRNA synthesis, although unexpected, does provide a coherent explanation for one of its possible functions. As there is so little C in virions, mRNA synthesis at the onset of infection would occur in an unimpeded fashion. During infection, however, the C levels would increase with time and preferentially inhibit transcription, thereby favoring genome replication. In this view, the switch from transcription to replication would still be mainly oper-

FOR

655

PARAMYXOVIRUSES

ated by the intracellular levels of unassembled NP (Vidal and Kolakofsky, 1989), but would be aided by that of the C proteins as well. Of the various proteins expressed from the P gene, only P is essential for RNA synthesis. All the others which have so far been shown to affect RNA synthesis do so negatively, e.g., V (and W) inhibits genome replication (Curran et al., 1991). Moreover, not all paramyxoviruses express all of these other P gene products. The SV5/mumps group of viruses and NDV do not contain a C ORF, and PIVl does not contain a V ORF (Matsuoka et a/., 1991). Nevertheless, (i) these other ORFs are conserved at least as well as that of P in those viruses that do express them, (ii) no evidence has been found that either the C or V ORFs can be dispensed with in long-term persistent measles virus infections of human brain (Cattaneo et al., 1989; M. Billeter, personal communication), and (iii) for PlVl , C’ expression is conserved even though it is initiated from a different non-AUG codon than that used in SEN (Boeck et a/., 1992). These examples of conservation argue that C and V must be important for those viruses which have them, even though some viruses can apparently do without them. However, whether C and V are truely essential to some viruses, or simply provide “luxury” functions, can best be studied by creating viruses such as SEN in which these ORFs have been closed.

ACKNOWLEDGMENTS We thank Sue Moyer (FlorIda) and Laurent Roux (Geneva) for dlscussions and comments on the manuscript, and Claes Orvell (Stockholm) for provldlng monoclonal antlbodies to the NP and P proteins. This work was supported by a grant from the SWISS National Science Fund. Nofe addedinproof. In the Interval, we have obtained an L protelnspecific monoclonal antibody which can estimate L levels expressed from the transfection of 1.5 pg of pGEM-L per dish, i.e., levels at which polymerase activity is still optimal. Using this antibody, we see no differences in L levels whether or not L has been coexpressed with P.

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