Expression of the two reovirus S1 gene products in transfected mammalian cells

VIROLOGY 178,223-23 1 (1990)

Expression of the Two Reovirus Sl Gene Products in Transfected Mammalian Cells

EDUARDO FAJARDO’ AND AARON J. SHATKIN’

Center for Advanced Biotechnology and Medicine, 679 Hoes lane, Piscataway, NewJersey 08854-5638

Received February 2 1, 1990; accepted May 8, 7 990

Reovirus genomic segment Sl, which has been implicated in the viral inhibition of cellular DNA synthesis, is tran- scribed into a single mRNA that encodes two proteins, the -49-kDa hemagglutinin, ~1, and the apparently nonstructural protein, ~14. These two polypeptides have been expressed in mammalian cells, together or independently, in order to assess their role in the shutdown of host DNA replication. Results obtained with transient and stable expression systems demonstrate that production of serotype 3 01 and p14 together or individually is not sufficient to change the kinetics of DNA replication in uninfected cells. However, inhibition of DNA synthesis by reovirus type 1 infection was enhanced in cells producing type 3 ~1 and p14 but not ~1 by itself. In addition, expression of ~14 alone led to increased cytopathic effects following infection by either type 1 or type 3 virus. The results suggest that interactions with other viral components are required to elicit the effects of the Sl-specified polypeptides on cellular DNA synthesis. 0 199oAcademic Press, Inc.

INTRODUCTION

Reovirus infection of mammalian tissue culture cells results in a general shutdown of host macromolecular synthesis, each of the three virus serotypes causing a different degree of inhibition (Sharpe and Fields, 1983). Inhibition of DNA replication in mouse L cells infected with reovirus type 3 is not due to cellular genome deg- radation (Ensminger and Tamm, 1969) alteration of precursor pools (Shaw and Cox, 1973), or to a decline in the activity of synthetic enzymes (Ensminger and Tamm, 1969; Shaw and Cox, 1973). Furthermore, the decrease in DNA synthesis appears not to be due to general cell damage because inhibition takes place before any gross cytopathic effect is evident (Hand et al., 197 1; Cox and Shaw, 1974; Ensminger and Tamm, 1970). Inhibition also precedes, and is probably not a secondary result of, the decline in cellular protein synthesis that accompanies infection (Shaw and Cox, 1973; Zweerink and Joklik, 1970; Hand and Tamm, 1973).

Reovirus particles inactivated by uv irradiation retain the ability to inhibit DNA synthesis, indicating that virus replication is not a prerequisite for inhibition (Hand and Tamm, 1973). However, empty particles (which are devoid of the 10 double-stranded RNA genome seg- ments) and noninfectious cores (lacking the outer capsid cell-attachment protein, al) are not inhibitory. Limited transcription takes place after infection by uv- inactivated virus (Shaw and Cox, 1973), and it is possi-

’ Present address: Department of Viral Oncology, Rockefeller Uni- versity, 1230 York Avenue, New York, NY 10021.

’ To whom requests for reprints should be addressed.

ble that this residual transcription results in the synthesis of a gene product responsible for inhibition.

Inhibition of DNA synthesis after Infection of L cells with serotype 1 reovirus is slow compared to the marked decrease caused by serotype 3, a phenotype assigned to the Sl genome segment (Sharpe and Fields, 1981). The Sl gene has been cloned from all three serotypes, and analysis of the cDNA sequences revealed the conserved presence of two open reading frames (ORF) (Cashdollar et a/., 1985). The first ORF spans the Sl gene almost from end to end and codes for polypeptide al while the second ORF begins at nu- cleotide 75, 66, or 71 (serotypes 1, 2, and 3, respectively) and results in a protein of ca. 14 kDa (Ernst and Shatkin, 1985). al which has been identified as the viral hemagglutinin responsible for virus attachment to cellular surface receptors (Weiner et al., 1980; Lee et al., 1981) plays a crucial role in the initial interaction of reovirus with the host and also carries serotype speci- ficity (Weiner and Fields, 1977).

In contrast to al, the information available about polypeptide pl4 is scarce. In all three serotypes, it has a relatively high content of basic amino acids and shares no significant homology with any sequences found in the data bank. The presence of pl4 has been demonstrated in the cytoplasm of reovirus type 3-infected cells (Ceruzzi and Shatkln, 1986), but it is unde- tectable or absent from purified virions (Ernst and Shat- kin, 1985) suggesting a regulatory role. To assess the functional properties of the reovirus Sl -specified polypeptides, we have analyzed the effects of ~1 and p14 expressed individually and together in mouse Cl27 cells.

223 0042-6822190 $3.00 Copynght @ 1900 by Acadcmlc Press. inc

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224 FAJARDO AND SHATKIN

MATERIALS AND METHODS

Enzymes and reagents

Restriction endonucleases were obtained from Bethesda Research Laboratories (BRL) and New En- gland Biolabs. MMLV reverse transcriptase, T7 and SP6 RNA polymerases, and T4 DNA ligase were purchased from BRL and calf intestine alkaline phosphatase from Boehringer-Mannheim. A nick translation kit was obtained from International Biotechnologies Inc. (IBI), and kits for T7 and SP6 RNA polymerase-depen- dent in vitro transcription and for rabbit reticulocyte lysate-based cell-free translation were from Promega and BRL, respectively. Assay conditions were according to suppliers’ recommendations. All tissue culture supplies were obtained from GIBCO Laboratories, and radioactive nucleotides and amino acids were Amer- sham products.

DNAs

Plasmids pBMTX (Pavlakis et al., 1987), pBCl2BI (Butnick et al., 1985), and pSV2neo (Southern and Berg, 1982) were kindly provided by B. Cullen; pSP6- S4 for in vitro synthesis of reovirus type 3 S4 RNA was from M. Giantini; and BPV-l/pML2d (Satver et al,, 1982) was purchased from IBI. Plasmid pT7-3 contains the entire type 3 reovirus Sl gene downstream of the bacteriophage T7 promoter in the polylinker region of pEMBL8(+) (Dente era/., 1983) and can be used to produce in vitro transcripts equivalent to authentic Sl mRNA (Fajardo, 1989). Single-stranded DNA for mutagenesis was obtained by infection of Escherichia co/i TGl cells carrying pT7-3 with phage Ml 3K07 (Vieira and Messing, 1987). Site-directed mutagenesis was performed using an Amersham kit according to the supplier’s recommendations. Construction of pCOS- TIS3 and pCOS-LTl has been described (Fajardo and Shatkin, 1990); pCOS-MTN2 is a pCOS-TIS3 derivative in which the Sl gene is intact except that the first three AUG codons of the p14 reading frame were mutated to ACG (positions 72, 174, and 177; Fajardo, 1989), third position changes without effect on the al sequence.

BPV-based vectors were used for the selection of stable cell lines expressing products specified by reovirus Sl sequences. pBMTX DNA (Fig. 1A) was linearized at the unique Xhol site downstream of the metallothionein promoter (mMT-l), and the ends were filled using reverse transcriptase. The &al-EcoRV Sl gene fragment (positions 42-497) that contains the pl4 coding sequence (Cashdollar et al., 1985), the HindIll frag- mentfrom pCOSTIS3 (Fajardo and Shatkin, 1990), and the Hindlll fragment from pCOS-MTN2 were blunt-

ended and ligated to the linearized, phosphatase- treated vector for the construction of BPXG, BPP2, and BPV-MTN2, respectively (Fig. 1 B). DNA manipulations were according to standard techniques (Ausubel eta/., 1988).

Ceils and virus

Mouse Cl 27 cells and monkey kidney COS7 cells were obtained from B. Cullen. Reovirus serotype 1 Lang strain, serotype 3 Dearing strain, and mouse L929 fibroblasts were purchased from the American Type Culture Collection. Cl27 and COS7 cells were grown in Dulbecco’s modified Eagle’s medium (high glucose, DMEM) supplemented with 10% fetal bovine serum (FBS). L929 cells were grown in minimum essential medium (MEM) supplemented with 5% FBS.

DNA transfections

For construction of stable transformants expressing the reovirus Sl gene products, mouse Cl 27 cells were seeded at 3 X lo5 per loo-mm plate 20 hr prior to transfection. Two micrograms of the appropriate BPV- derived chimeric plasmid mixed with 0.2 pug of pSV2- neo was transfected by coprecipitation of DNA with calcium phosphate (Howley et al., 1981). Selection was applied 48 hr later by adding fresh DMEM + 10% FBS containing 250 pg active G418 (Geneticin, GIBCO) per milliliter. Cells were subsequently fed twice a week; transformed foci could be seen 2 weeks later and were picked 4 weeks post-transfection. After the independent clones were grown to confluence in 25-cm2 flasks, cells were propagated in the absence of antibi- otic. Transfection of DNA into COS7 cells and analysis of transfected cells have been described previously (Fajardo and Shatkin, 1990).

Virus infections and DNA synthesis

DNA synthesis was measured in duplicate or tripli- cate in six-well tissue culture plates. Cells were plated at 5 X 1 O4 cells/well 1 day before infection. At the time of infection, monolayers were washed once with PBS before addition of 0.13 ml of virus inoculum. Virus was adsorbed at 4” for 2 hr at a multiplicity of 1 O-20 plaque forming units (PFU) per cell in PBS containing calcium and magnesium. Next was added 3 ml of warm DMEM supplemented with 5% FBS; this was considered zero time of infection. To measure DNA synthesis, medium was replaced, adding to each well 0.7 ml of DMEM + 5% FBS containing 20 &i/ml [methy/-3H]thymidine (25 Ci/mmol). After 30 min at 37’, the medium was as- pirated, and the monolayers were washed twice with cold PBS before addition of 1 ml cold 5% trichloroace- tic acid (TCA) to each well (Munoz et al., 1985). After

REOVIRUS Sl PROTEINS IN TRANSFECTED CELLS

aspirating the TCA, the monolayers were washed twice with ethanol, air-dried, and suspended in 0.3 ml of 0.1 M NaOH before removing 0.15 ml samples for liquid scintillation counting. Another 90-PI aliquot was neutralized with 10 ~1 of 1 M HCI and used to determine total DNA as follows.

DNA determinations

DNA samples containing an excess of RNA were as- sayed using the diaminobenzoic acid assay (Thomas and Farquhar, 1978). Briefly, 0.1 ml of 1 M perchloric acid was added to the 0.1 -ml samples followed by incu- bation at 75” for 1 hr. After cooling on ice and brief cen- trifugation, 0.2 ml of DABA reagent [l voi of freshly prepared 20% 3,5-diamiobenzoic acid dihydrochloride (Aldrich) mixed with 3 vol of 1 M NaOH containing 10 mM Na,CO, and incubated for 1 hr at room tempera- ture before use] was added to each sample. Mixtures were incubated for 1 hr at 37”, cooled on ice, and, after addition of 1 ml 1 M HCI, read in a Perkin-Elmer spec- trofluorometer with excitation set at 408 nm and emis- sion at 520 nm. Standards were made with sonicated calf thymus DNA (Pharmacia) in the range lOO- 5000 ng.

RESULTS

Construction and characterization of cell lines expressing reovirus products

Mouse Cl 27 cells were transfected with each of the BPV-derived plasmids shown in Fig. 1 B. Plasmid BPP2 encodes both reovirus Sl gene products, al and ~14, while plasmid BPX6 contains only the pl4 ORF. In plasmid BPV-MTN2 the initiation codon for p14 and the next two AUGs in the pi4 ORF were mutated to ACG (indicated in Fig. 1 B by x’s), changes that occur in the third codon position in the al ORF. Consequently, cells transfected with BPV-MTN2 can produce al but not ~14.

Several independent transformed foci were picked, grown, and analyzed by immunoprecipitation of [35S]- methionine-labeled cell lysates and polyacrylamide gel electrophoresis. Figure 2 shows the analysis of transformed clones treated with 1 PM CdCI, (Fajardo and Shatkin, 1990) in comparison with virus-infected cells (lane INF). Control cells were transformed with plasmid BPV-l/pML2d which lacks Sl-specific sequences (lane 1). A protein with an apparent molecular weight of ca. 49 kDa was specifically immunoprecipitated with ul-specific, F7 monoclonal antibody (Burstin et al., 1982). This protein, at the expected position for 01, was present in lysates from cells transformed with plasmids BPP2 (lane 2) and BPV-MTN2 (lane 4) but not with plasmid BPX6 (lane 3). lmmunoprecipitation of the

BPV-1

I I Sl GENE - p14

L I BPP2

--q-b _______ - __-_-------- BPXB

I I BPV-MTNZ x xx

FIG. 1, Vectors for stable expresston of reovrrus products tn transformed mouse Cl 27 cells. (A) Plasmrd pBMTX consrsts of the entire genome of bovrne papilloma vrrus 1 (BPV- 1). pBR322 sequences In- cludtng the ongrn of replrcatron (Orr) and amprcrllrn resrstance gene (Amp’), and a 3.8-kb fragment contatnrng the mouse metallothronern 1 gene (mMT-1) Reovrrus Sl gene sequences were Inserted Into the unrque Xhol sate downstream of the metallothronern promoter; the arrow tndtcates the dtrectron of transcription. (5) Schematic repre- sentatron of the sequences introduced Into pBMTX at the Xhol site to obtain the rndrcated vectors. The entire S 1 gene rncludrng the pl4 coding region (bracketed) IS present in BPP2. The dashed lines corre- spond to deleted sequences In BPXG, a construct that contarns the p14 ORF but lacks most of the 01 coding region. In BPVMTN2 the x’s show the relatrve posrtrons of mutations whtch change AUG codons to ACG rn the pl4 reading frame. The 5’.end sequences have been reported (Fafardo and Shatkln, 1990)

same cell lysates with rabbit antiserum against bacteri- ally expressed, gel-purified pl4 (which cross-reacts with crl , provided by M. Ceruzzi) revealed the presence of a 14-kDa protein in cells transformed with plasmid BPP2 (lane 2) or BPX6 (lane 3) but not in cells transformed with BPV-MTN2 (lane 4) or BPV-l/pML2d (lane 1). The same proteins were also expressed in the absence of Cd” induction due to baseline transcription of the metallothionein promoter (Fajardo, 1989; Fa- jardo and Shatkin, 1990). These results demonstrate the expressron of reovirus type 3 al and pl4 proteins, either together in the same cell line or independently in others. The stable cell lines were used to study the effects of these viral polypeptides on cell replication.

DNA synthesis in transfected cells DNA replication in stable transfectants expressing

the Sl products was measured at different times after

226 F/YARD0 AND SHATKIN

Anti-al Ab Anti-p1 4 Ab

14 -

FIG. 2. Expression of reovirus proteins in mouse Cl 27 cells transformed with plasmid BPV-l/pML2d (lanes l), BPP2 (lanes Z), BPX6 (lanes 3), or BPV-MTN2 (lanes 4). Cultures were treated with 1 PLM Cd& and labeled 4 hr later with [35S]methionine. Cell lysates were immunoprecipitated with monoclonal antibody F7 made against al (Burstin et al., 1982) or with rabbit antisera made against p14 expressed in Escherichia co/i(provided by M. Ceruzzi). Positions of molecular weight markers (in kilodaltons) are shown on the left. The anti- pl4 antibody cross-reacts with 01 (Fajardo, 1989), and the two -3O- kDa bands In lane 2 may be proteolytic cleavage products of ~1. The additional higher molecular weight bands in lysates of type 3-k-- fected mouse L cells (INF lanes) probably result from coprecipitation of virion structural components including the major protein ~1 c (-90 kDa).

induction with 1 PLM Cd&. From these measurements it was clear that expression of the Sl -specific proteins had little or no effect on DNA replication as compared to the almost complete shutoff observed in type 3-infected mouse L cells (data not shown). Independent isolates responded differentially to Cd&, but [3H]thymidine incorporation was generally correlated with the morphology of a particular cell line. It is well-docu- mented that BPV-containing cell lines can be isolated in the absence of morphological transformation (Mene- guzzi et al., 1984), and thymidine incorporation was greater in clones that acquired an elongated, fusiform appearance as compared to lines that retained the round and flat shape of the parental cells (data not shown).

In a different approach to the problem, plasmid replication was measured in a transient expression assay. Monkey kidney COS7 cells (Gluzman, 1981) were transfected with plasmid pCOS-TIS3, pCOS-LTl I or pBC12BI, resulting in the production of 01 and ~14, p14 only, or, as control, rat preproinsulin, respectively (Fajardo and Shatkin, 1990). Plasmid pt3lNV, which contains the type 3 reovirus S4 gene in the reverse ori- entation with respect to the LTR-directed transcription unit (Giantini and Shatkin, 1989), was cotransfected at

a 1: 1 molar ratio and used as a reporter plasmid for measuring DNA replication. Total cellular DNA was

prepared 48 hr post-transfection and analyzed by Southern blot hybridization to a nick-translated S4-specific probe. The extent of reporter plasmid replication was similar in all of the transfectants, as judged by sus- ceptibility of the isolated DNA to methylation-sensitive EcoRll digestion (Fig. 3). The results demonstrate that plasmid DNA synthesis is unaffected by Sl gene expression in COS cells.

It has been suggested that al exerts its inhibitory effect on DNA synthesis at the membrane level after binding to cellular receptors (Sharpe and Fields, 1981). Therefore, DNA replication was measured in cells treated with in vitro synthesized al or with 03 as a control. Reovirus serotype 3 Sl and S4 capped mRNAs were prepared using plasmid pT7-3 and T7 RNA polymerase or plasmid pSP6-S4 and SP6 RNA polymerase, respectively. The RNAs were translated in rabbit reticulocyte lysate, and the yields of al and 03 were determined (Fajardo, 1989). DNA synthesis in L cells was not altered by treatment with aliquots of lysate incuba- tion mixture containing 01 equivalent to 10 PFU/cell (data not shown).

These combined results suggested that the Sl-encoded proteins under these conditions were not capa- ble of inhibiting DNA synthesis and that a strong inhibitory effect could only be detected in the context of an infection where other viral proteins are available for po- tential interactions. To explore this possibility, DNA replication was measured in stable transformants in-

1234 ---I

FIG. 3. Plasmid replication in COS cells transiently expressing reovirus Sl gene products. Reporter plasmid pt3lNV was transfected into COS7 cells by itself (lane 4) or (in duplicate) with plasmid pCOS- TIS3 (lanes l), pCOS-LTl (lanes 2), or pBCl2BI (lanes 3). Plasmid replication was assessed by Southern blot analysis of total cellular DNA hybridized with a reovirus type 3 nick-translated gene S4 probe. Before loading the sample onto a 1.1% agarose gel, the DNA was incubated with EcoRll which cleaves the replicated molecules but does not cut the methylated form of the parental plasmid DNA used for transfection.

REOVIRUS Sl PROTEINS IN TRANSFECTED CELLS 2‘!7

10 20 30 40 50 60

Hours Dost-infection

0.6

0.6

0.2 Type 1

10 20 30 40 50 60 IO 20 30 40 50 60

Hours post-infection Hours post-lnfectlon

06

0 t.‘-““l”““““.j IO 20 30 40 50 60

0.6 -

0.6 -

0.2 ; Type 3

Ot.. 10 20 30 40 50 60

06

06

Hours post-infection Hours post-infection Hours posl~infection

FIG. 4. DNA syntheses in Infected cells expressing reovrrus Sl gene-specified proteins. Mouse C 127 cells were transformed with (A) plasmrd BPP2 and producrng both Sl products [ran p14(+)]; (B) BPV-MTN2 synthesizing only 01 [cl(+)]; and (C) BPX6 yielding p14 only [p14(+)]. BPV-I/ pML2d-transformed cells were included in all experiments as controls without Sl sequences (-) to take into account any variabrltty. Bars Indicate ranges of values for duplrcates (A,B) and triplicates (C except 13.hr points in upper panel are duplicate samples). Cells were Infected wrth reovrrus type 1 (upper panel) or type 3 (lower panel) at 10 PFU/cell. Graphs show ratio of cpm of [3H]thymrdine incorporated into DNA In Infected vs mock-infected cultures

fected with either type 1 or type 3 reovirus to determine if type 1 infection of cells expressing type 3 S 1 products resulted in increased inhibition characteristic of type 3 infections. As shown in Fig. 4A, constitutive expression of ~rl and p14 enhanced the inhibition of DNA replication in cells infected with type 1 reovirus (upper panel) but did not affect serotype 3 infections (lower panel). By contrast, infected cells that expressed al in the absence of pl4 showed no significant difference in inhibition compared to control, infected cells (Fig. 4B), suggesting that ~14 is involved in the faster inhibition observed in type 1 infection of cells expressing “endogenous” type 3 01 and ~14. Expression of type 3 pl4 alone was associated with a greater inhibition of DNA replication in cells infected by either serotype (Fig. 4C); however, this may not be a specific effect on DNA synthesis but rather a generalized alteration of cell me- tabolism (see below).

Effect of p14 during virus infection

The enhanced inhibition of thymidine incorporation by cells expressing pl4 and infected with either type 1 or type 3 reovirus occurred concomitantly with the rapid appearance of a marked cytopathic effect, result-

ing in many trypan blue stainable, nonviable cells that detached from the monolayer. To quantitate the loss of cells, the amount of DNA in infected cultures was determined at different times after infection in comparison to mock-infected cultures. As shown in Fig. 5A, the DNA content of ~14 expressing, type S-infected cultures (upper panel) was three- to four-fold less than in infected, nonexpressing cells at 48 hr (lower panel) and six- to eight-fold less compared to mock-infected cultures. In addition, there was a net loss of DNA in infected cultures expressing ~14 (Fig. 5A, upper) as compared to an overall increase in nonexpressing cultures during the same period (Fig. 5A, lower). Although the loss of cells cannot account completely for the enhanced inhibition of thymidine incorporation in pl4-expressing cells, the inhibition probably reflects the generalized cytopathogenesis rather than a specific effect on DNA replication. The enhanced cytopathic effect accompanying ~14 synthesis during type 3 infection also occurred but to a lesser extent in type l-infected cells (Fig. 5B). Other differences, e.g., viral antigen ac- cumulation measured by Western blot and immuno- fluorescence, were not evident in type 1 or type 3 infected cultures that were expressing, as compared to not expressing, “endogenous” pl4 (data not shown).

228 FAJARDO AND SHATKIN

A5000 .I,I.I,I'I'I'I'I'(' B 1600

- p14(+) 1400

4000 - z E 1200

e 3000

1000 -

?

g

8 E 600 2000 - 600

0 j’l’)‘I’I’I’I”‘(“’ 0 0 10 20 30 40 50 10 20 30 40 50 60

Hours post-infection Hours post-infection

4000 z

1600

g

g 3000 E m E E

1200

8 8 5 2000 2 600 0

1000 400

0 0 0 10 20 30 40 50 10 20 30 40 50 60

Hours post-infection Hours past-infection

FIG. 5. DNA content of pl4-expressing cultures infected with reovirus. DNA levels were measured in samples of TCA-precipitated lysates of Cl 27 cells transformed with BPX6 [p14(+)] or BPV-l/pML2d (-) and mock-infected or infected with 10 PFUlcell of (A) reovirus type 3 or(B) type 1. Bars indicate range for triplrcate samples.

In contrast to virion proteins, polypeptide p14 was relatively unstable in infected cells. Mouse L cells infected with reovirus type 3 at 10 PFWcell were labeled with [35S]methionine for 1 hr at 14 hr after infection, followed by addition of medium containing excess methionine. Cell lysates prepared at different times after the chase were analyzed by immunoprecipitation and gel electrophoresis. As shown in Fig. 6, pl4 decayed with a half life of less than 4 hr, while virion structural proteins, including the cross-reacting al and the 90 kDa plc which cosedimented in the immunoprecipi- tate, decreased relatively little during the chase period. Similar results were obtained with uninfected Cl27 transformants stably expressing pl4 and with either infected or uninfected COS7 cells expressing the same protein transiently (data not shown). These findings are consistent with a regulatory role for p14 in the virus replication cycle. In infected cells ~14 expression in- creases concomitantly with structural proteins, without obvious temporal regulation (Fajardo, 1989; and data not shown). Intracellular pl4 levels apparently are determined by a combination of high protein turnover and suboptimal translation initiation due to the presence of the upstream crl initiator codon in the Sl mRNA (Fajardo and Shatkin, 1990).

DISCUSSION Polypeptide al, a quantitatively minor but function-

ally important component of the reovirion outer capsid,

has been well-characterized as the hemagglutinin responsible for cell attachment. By contrast, polypeptide pl4 is apparently nonstructural and of unknown function. To study these proteins further, mouse C 127 cell lines were obtained that express the Sl -specified serotype 3 products from a BPV-based vector containing

Hours post-chase A?

M iI 2 4 6 ?i --mm---

FIG. 6. Stability of pl4 in infected cells. Monolayers of mouse L929 fibroblasts were infected with reovirus type 3 and labeled with [%- methionine as described under Materials and Methods. Cell lysates were prepared at the indicated times after addition of chase medium, and incorporation of radiolabel into TCA-insoluble material was determined by liquid scintillation counting. The same amounts of radio- labeled proteins were immunoprecipitated with antibody made against gel-purified ~14, and precipitates were analyzed by electrophoresis in a 17.5% SDS-polyacrylamide gel followed by fluorogra- phy. Controls included immunoprecipitates of mock-infected L cells (lane 1) and COS7 cells transfected with plasmid pCOS-LTl (lane Pl4).

REOVIRUS Sl PROTEINS IN TRANSFECTED CELLS 229

the mMT-1 promoter. Sl expression could be induced by Cd&, 1 r.lfl/l resulting in the highest amounts of viral protein (Fajardo and Shatkin, 1990). Because Cd+* toxicity was evident (30% reduction in DNA replication at 1 PM even in the absence of Sl sequences) and independent clones had different Cd+* sensitivities, heavy metal-induced cultures were not used to evaluate the effects of the expressed proteins on DNA replication. However, constitutive function of the mMT-I promoter (Scholer et al., 1986; Braam-Markson et a/., 1985) yielded basal levels of al and ~14 in uninduced cells that were within three-fold of the amounts produced in L cells infected with type 3 reovirus at 10 PFU/cell (Fajardo, 1989). Nevertheless, Sl -expressing cell lines continued to divide (at different rates among independent isolates), indicating that the Sl gene products by themselves do not inhibit DNA replication. However, isolation of the cell lines may have selected for events that permit the continuous presence of a potentially inhibitory product. It should be noted in this context that the Sl gene IS often mutated during the establishment of persistent infections (Ahmed and Fields, 1982).

Although a lower number of transformants per mi- crogram of DNA would be expected if the transforming plasmid resulted in a toxic product, the number obtained was not significantly different for any of the plasmids shown in Fig. 1. In addition, there were no differences in the extent of reporter plasmid replication in COS cells transiently expressing al or p14 as compared to control cells containing the original COS vector which encodes rat preproinsulin II, i.e., the viral proteins by themselves again were not sufficient to inhibit DNA replication. However, plasmid amplification may not accurately reflect chromosomal replication, espe- crally in this system where the plasmid is reproduced rapidly and independently of cell cycle control.

It has been suggested that ~1 causes inhibition of DNA replication after binding to receptors by triggering a signal at the cell membrane (Sharpe and Fields, 198 1). However, L cells exposed to in vitro-synthesized ~1 under the same conditions used for virus infection showed no difference in the extent of DNA replication as compared to control cells treated with PBS or viral polypeptide 03. Similarly, al purified from transformed E. co/i retained hemagglutinin and cell attachment ac- tivities but did not inhibit L cell DNA replication (P. Lee, personal communication). These results should be considered in light of the finding that 01 anti-idiotypic antibodies can mimic reovirus-mediated inhibition of DNA synthesis (Gaulton and Greene, 1989). It is possi- ble that ~1 made in vitro does not form multimers, as does the virion-associated protein (Bassel-Duby el al., 1985, 1987). Although not demonstrated for the in vitro synthesized product used in our studies, ul purified

from virions and from Sl -transformed bacteria does spontaneously associate into dimers and tetramers (Yeung et al., 1987; P. Lee, personal communication). Multimerformation of al would be necessary for cross- linking of the receptors, apparently a prerequisite for inhibition of DNA replication (Gaulton and Greene, 1989). It is worth mentioning in this regard that the re- ports in the literature fail to explain the lack of DNA synthesis inhibition by empty particles (Hand and Tamm, 1973) which contain crl in a conformation that is presumably similar to that in virions.

It is of interest that (~1 and ~14 produced by stable transfectants enhanced the course of type 1 viral infections, presumably by association of “endogenous” proteins with other viral products. DNA synthesis decreased in cell lines producing al and pl4 earlier than in control, BPV-l/pML2d transformed cells (Fig. 4A, upper). In contrast, the kinetics of DNA inhibition following infection by type 3 reovirus was unaffected by the presence of the “endogenous” proteins (Fig. 4A, lower), perhaps because in this situation the net result is a small increase in the total amount of ~1 and pl4 of the same serotype. Although DNA synthesis inhibition in type 1 infections was accelerated, it did not reach the level caused by type 3 virus, possibly because the type 3 S 1 proteins are effectively out-competed by their type 1 counterparts for interaction with other viral components, i.e., interaction(s) between polypeptides of the same viral serotype may be favored (Chen et al., 1989; Moody and Joklik, 1989). Another interesting possibility is that the stronger Inhibition is brought about by progeny pseudotypes containing type 3 al in the outer capsid available for binding to receptors on other cells. Progeny virions, or virus-specific macromolecular assembly complexes, could also attach to receptors intracellularly, before they reach the membrane, and trigger events similar to those resulting from external binding. An analogous situation has been observed in the intracellular activation of the krnase associated with the platelet-derived growth factor receptor by the v-sis gene product (Huang ef a/., 1984).

The lack of a measurable effect in infected cells expressing al in the absence of ~14 (Fig. 4B) suggests that p14 may influence ul activity. The presence of both protein coding sequences in the same genome segment, an arrangement that precludes reassortment of the two Sl genotypes, is consrstent with “homolo- gous, ” serotype-specific interaction(s) between these two polypeptides. This is supported by the observation that type 1 pl4 encoded by the infecting vrrus does not allow the putative interactions of “endogenous” type 3 ~1 in BPV-MTN2-transfected cells that result in faster inhibition of DNA replication. Interestingly, the products of bicistronic mRNAs of other animal RNA viruses,

230 FAIARDO AND SHATKIN

notably influenza (Shaw el a/., 1983) and paramyxovi- ruses (Giorgi et a/., 1983), include a structural protein and small, nonstructural polypeptide(s). In influenza 6 virus, genome segment 6 contains two overlapping ORFs that encode the neuraminidase, a protein inter- acting with the cell at the membrane surface, and nonstructural polypeptide NB. It has been suggested that the small nonstructural product plays a role in influenza virus toxicity (Shaw et al., 1983), a situation perhaps analogous to the reovirus Sl gene.

ACKNOWLEDGMENTS

We thank Drs. Bryan Cullen, Marion Ceruzzi, Bernard Fields, and Michael Giantini for essential materials, Alba LaFiandra for invaluable assistance, and M.G. for critically reading the manuscript.

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Expression of the two reovirus S1 gene products in transfected mammalian cells