INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, which together with the genera Flavivirus and Hepacivirus (proposed name) (hepatitis C virus), has been assigned to the family Flaviviridae (41, 50). The single-stranded pestivirus RNA genome is of positive polarity, usually has a length of about 12.3 kb, and encompasses one long open reading frame (ORF) which is flanked by untranslated regions (UTR) (3, 10, 15, 29, 35, 39, 42). In the 5' UTR a so-called internal ribosomal entry site (IRES) which promotes cap-independent initiation of translation has been identified (38, 43). The ORF of BVDV is translated into a polyprotein of about 4,000 amino acids which is co- and posttranslationally processed into at least 11 mature viral proteins as follows: N^pro-C-E^rns-E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B (9, 11, 17, 31, 45, 47, 55). N^pro is an autoprotease which generates its own C terminus. The cleavages leading to the release of the structural proteins are mostly catalyzed by host cell proteases (17, 45); for the structural glycoprotein E^rns an intrinsic RNase activity has been described (19, 20, 46, 53). Furthermore, a serine protease residing in the N-terminal region of NS3 is responsible for generation of most of the nonstructural (NS) proteins (54).

BVDV strains are classified as cytopathogenic (cp) or noncytopathogenic (noncp) according to their effects on tissue culture cells (27). Calves which are persistently infected with noncp BVDV as a consequence of an intrauterine infection may come down spontaneously with lethal mucosal disease (MD) (for a review, see reference 50). From those animals a cp BVDV can be isolated in addition to the persisting noncp virus; the appearance of the cp BVDV is considered to be essential for development of MD (6, 7, 13, 33, 37). The noncp and cp viruses from one animal with MD are called a virus pair. The molecular analysis of BVDV pairs revealed that cp BVDV strains evolve in vivo from noncp BVDV by mutation (32). The common thread for all cp BVDV strains is the expression of an 80-kDa protein, termed NS3. In cells infected with noncp BVDV, NS2-3, but not NS3, can be detected (16, 37). Expression of NS3 therefore is regarded as a marker specific for cp BVDV. NS3 is colinear to the C-terminal part of NS2-3 and is generated by a surprising variety of mechanisms which can often be deduced from the genome structures of the respective cp BVDV strains (32).

The genome alterations identified in cp BVDV genomes include insertions of cellular or viral RNA sequences, deletions of large genomic regions, or accumulated point mutations; in all cases the cp-virus-specific genome alterations concerned the NS2-3-coding region of the viral RNA (4, 24, 32). In the NS2 gene of BVDV strain CP7, a 27-nucleotide insertion has been identified and shown to be essential for NS2-3 cleavage and cytopathogenicity (30, 48). The cp BVDV isolate CP9 consists of a defective interfering particle (DI9) and a replication-competent noncp helper virus. In the genome of DI9 the deletion encompasses the genes for C-E^rns-E1-E2-p7-NS2; thus, in the polyprotein of DI9 the autoprotease N^pro is located directly upstream of NS3 and generates the N terminus of the latter protein. Biological characterization of this defective interfering particle revealed that it causes a cytopathic effect (CPE) in cells preinfected with a noncp helper virus (49). On the basis of the infectious cDNA of BVDV CP7, a subgenomic (sg) RNA with the genome structure of DI9 was generated. This was achieved by replacing in the CP7 cDNA the genomic region from the N^pro gene downstream to the 5' third of the NS3 gene with a corresponding cDNA fragment derived from DI9 (for details, see reference 30). The corresponding RNA exhibited the biological properties of DI9 with respect to interference and cytopathogenicity (30). More recently it was shown that this sg RNA is capable of replicating autonomously. Whereas a complete deletion of the N^pro gene from this RNA interfered with its replication, the C-terminal part of N^pro, encoded downstream of codon 42, could be substituted by ubiquitin (5); ubiquitin was used in this replicon to mediate generation of the N terminus of NS3 by cellular ubiquitin-C-terminal hydrolases (22, 26, 44). Whether the residual part of N^pro is important for replication remained an open question.

The major focus of the present study was the establishment of different pestivirus replicons in order to determine the sets of viral proteins essential for the cp and noncp phenotypes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells. MDBK cells were obtained from the American Type Culture Collection (Manassas, Va.). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS).

Immunofluorescence (IF) assay. The cultures were washed once with phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde-2% glutaraldehyde in PBS for 20 min at 4°C, and then washed again with PBS. Permeabilization of the cells was achieved by the addition of 1% N-octyl--D-glucopyranoside in PBS for 5 min at 4°C. After being washed with PBS, the cells were incubated with a 1:3 dilution of monoclonal antibody 8.12.7 (hybridoma supernatant), directed against BVDV NS3 (12), for 2 h. After a wash with PBS, a cyanogen 3-labeled secondary antibody directed against mouse immunoglobulin was added for at least 30 min.

Crystal violet staining of MDBK cells. Cells were washed with PBS and fixed for 10 min with 5% formaldehyde. After a wash with water, 1% (wt/vol) crystal violet in 50% ethanol was added for 5 min.

Transfection of RNA into MDBK cells. Transfections by the use of DEAE-dextran were carried out as described before (49).

RNA electroporation. MDBK cells were washed once with medium without FCS and once with PBS and then trypsinized. After the cells were pelleted by centrifugation (1,000 × g, 2 min) they were resuspended twice in PBS without Mg²⁺ and Ca²⁺. Confluent cells from one 10-cm-diameter dish were resuspended in 1.2 ml of PBS without Mg²⁺ and Ca²⁺, and 0.4 ml of the suspension was used for each electroporation. RNA and cells were mixed immediately before the pulse. For electroporation, a Gene Pulser II (Bio-Rad, Munich, Germany) was adjusted to 950 µF and 180 V. Each aliquot of electroporated cells was seeded into six 3-cm-diameter dishes; one-fourth and one-eighth of the cells were seeded and adjusted to 2 ml with medium including 10% FCS for analysis at 24 h posttransfection (p.t.) and at a later time point, respectively.

RNA transcription. For RNA transcription, SP6 RNA polymerase (NatuTec, Frankfurt, Germany) and 2 µg of linearized template DNA were used in a standard protocol. The amount of RNA was estimated by staining with ethidium bromide after agarose gel electrophoresis. One-third of the transcription reaction mixture was used for each electroporation.

PCR. For PCR a Genius thermocycler (Techne/thermo-DUX, Wertheim, Germany) was used. Standard conditions for DNA-based amplifications were used: 1 cycle of 2 min at 42°C and 2 min 96°C; 30 cycles of 96°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and one cycle of 3 min at 72°C. The reaction volume was 60 µl containing 250 µM deoxynucleoside triphosphates, 30 pmol of each primer, and 2 U of Biotherm DNA polymerase (NatuTec). The template DNAs and primers used are described for the respective constructs below.

Nucleotide sequencing. Sequencing was carried out with the Thermo Sequenase cycle sequencing kit (Amersham Buchler, Braunschweig, Germany). The primers used for sequencing were labeled with infrared dye IRD41 (MWG-Biotech, Ebersberg, Germany). Analysis of sequencing gels was carried out with an LI-COR 4000L automatic sequencer (MWG-Biotech).

Luminometric GUS release assay. The -glucuronidase (GUS) assay allows specific measurement of the lysis of cells which carry the bicistronic BVDV replicons. GUS was chosen as the marker enzyme for the cell lysis assay since it is known to be rather stable against proteolytic degradation and there has been no indication that this protein is sequestered from cells into the supernatant or that its expression is cytotoxic.

Construction of cDNA clones. Numbering of nucleotides throughout this work refers to the noncp BVDV SD-1 sequence (15), since no complete sequence of BVDV CP7 has been published so far. Moreover, the SD-1 genome is the only completely analyzed BVDV RNA without a strain-specific insertion. The use of the SD-1 numbering thus significantly facilitates comparisons between different cp BVDV genomes or polyproteins.

Basic strategy. The backbone vector plasmid carrying nucleotides 22 to 335 of the BVDV CP7 cDNA directly followed by bases 11957 to 12274 of CP7 was assembled by standard cloning techniques. The terminal sequences and an SP6 RNA polymerase promoter upstream of the 5' end of the BVDV cDNA were inserted into this plasmid by pairs of complementary oligonucleotides (ol5'+ ol5' and o13'+ ol3'; see below). The terminal sequences of the BVDV cDNA in the replicons are 5'-GTA TAC GAG AAT TTG CCT AAC CTC GTA and CAA TGG TTG GAC TAG GGA AGA CCC TTA ACA GCC C-3'. Into this plasmid restricted with XhoI (nucleotides 224 to 229) and AatII (12268 to 12273), a corresponding XhoI/AatII fragment from plasmid pA/BVDV/D9 (30) was ligated, resulting in plasmid HHDI9. The XhoI (224 to 229)/SacI (5855 to 5860) fragment of this plasmid was replaced by the XhoI/SacI fragment of a derivative of DI9cN^proubi (5) in which the first codon of the NS3 gene (encoding glycine¹⁵⁹⁰) follows directly downstream of the last codon of the ubiquitin gene. The resulting plasmid, HHN^pro*ubiNS3, which was the basis for all further constructs, accordingly contained no BVDV DI9-derived sequences. The ubiquitin gene in these constructs mediates generation of the authentic N terminus of NS3 by cellular ubiquitin C-terminal hydrolases (26, 44).

FSubiNS3. After restriction of HHN^pro*ubiNS3 with XhoI (nucleotides 224 to 229) and NcoI (at the start AUG), the fragment was replaced by a PCR-derived fragment containing the two frameshifts (FSs) (see also Results). PCR: SP6 sequencing primer, olFS (see below), and CP7 cDNA as a template. The resulting clone, FSubiNS3, encompasses a unique NcoI site located at the first codon of the ubiquitin gene; the latter is located downstream of the FS element. The amino acid sequence encoded by the FS element is MELSQMNFYTKHT. This replicon thus contains the FS element followed by sequences corresponding to one monomer of ubiquitin and the polyprotein of BVDV CP7 from glycine¹⁵⁹⁰ (NS3) to the authentic stop codon.

HHDI9pol. In order to generate a replication-defective control, a ClaI/NgoMI fragment (nucleotides 11055 to 11339) of the NS5B gene, encompassing the coding sequence for the GDD motif in the active center of the RNA polymerase, was deleted from plasmid HHDI9. Religation of the vector was carried out after treatment with the Klenow fragment of DNA polymerase I.

FSNS2ins+ and FSNS2ins. By PCR an NcoI site was introduced into clones pC7.1 and pC7.1 INS (48) upstream of the codon for leucine¹⁰⁹⁹, which is located in the p7 gene; the two clones were identical except for the CP7-specific insertion in the NS2 gene missing in pC7.1 INS. In the resulting clones the NcoI site and two additional nucleotides precede the codon for leucine¹⁰⁹⁹ (nucleotides 3680 to 3682) and the downstream BVDV CP7 sequence. NcoI (PCR derived)/SacI (nucleotides 5855 to 5860) fragments of these clones were used to substitute a corresponding NcoI (5' end of the ubiquitin gene)/SacI (nucleotides 5855 to 5860) fragment in plasmid FSubiNS3, which led to the final constructs. The 3' part of the p7 gene was included in these constructs since it was assumed that the C terminus of p7 functions as a signal sequence which might be necessary for correct membrane insertion of NS2.

FSNS2ins+pol. The FSNS2ins+pol construct was generated as described above for plasmid HHDI9pol.

Construction of bicistronic replicons. In order to establish an assay which allows the quantitative measurement of the cp potential of an RNA replicon in MDBK cells, sg BVDV RNAs expressing a reporter gene were established. The Escherichia coli-derived GUS gene, obtained in plasmid pPCV812GUS (52), was fused in frame to the 3' end of the FS element in replicon FSubiNS3. Downstream of the stop codon of the GUS gene a PvuII/SacI fragment (nucleotides 3690 to 559) of pCITE2A (Novagen, Madison, Wis.) was integrated. This fragment encompasses the IRES of encephalomyocarditis virus (EMCV). The cDNA in the resulting plasmid, Bi1, which served as the basis for all further bicistronic constructs, encompassed the following downstream of the SP6 RNA polymerase promoter: the 5' UTR, FS element, GUS gene, EMCV IRES, and BVDV cDNA from nucleotide 5855 (SacI) to the 3' end.

Bi-NS2ins+ and Bi-NS2ins. The NcoI (3' end of FS element)/SacI (nucleotides 5855 to 5860) fragments from FSNS2ins+ and FSNS2ins were ligated into Bi1 restricted with the same enzymes. The final bicistronic constructs thus express from the 5' ORF the peptide encoded by the FS element with the C-terminally fused GUS; the 3' ORF located downstream of the EMCV IRES encompasses two vector-derived codons (those for methionine and alanine) followed by the sequence for BVDV CP7 (or CP7ins) polyprotein from the middle of p7 (beginning with leucine¹⁰⁹⁹) to NS5B.

Bi-NS2ins+pol. In order to generate a replication-defective control a ClaI/NgoMI fragment (nucleotides 11055 to 11339) of the NS5B gene was deleted from plasmid Bi-NS2ins+. Religation of the vector was carried out after treatment with the Klenow fragment of DNA polymerase I.

Bi-NS3. By standard PCR techniques an NcoI site was added to the 5' end of the NS3 gene; an NcoI/SacI (nucleotides 5855 to 5860) fragment of this cDNA was ligated into Bi1, from which an NcoI/SacI fragment had been deleted by the same enzymes. In the resulting construct the ORF downstream of the EMCV IRES encompasses the methionine start codon followed by the sequence for the CP7 polyprotein starting with glycine¹⁵⁹⁰.

Bi-NS3pol. Plasmid Bi-NS3pol was generated as described for Bi-NS2ins+pol.

Oligonucleotide primer sequences. Sequences of oligonucleotide primers were as follows: ol5'+, 5'-CCG CTA GCA TTT AGG TGA CAC TAT AGT ATA CGA GAA TTT GCC TAA CCT CGT A-3'; ol5', 5'-TAC GAG GTT AGG CAA ATT CTC GTA TAC TAT AGT GTC ACC TAA ATG CTA GCG GGT AC-3' (the underlined sequences represent the BVDV-derived cDNA sequence); ol3'+, 5'-CAA TGG TTG GAC TAG GGA AGA CCC TTA ACA GCC C-3'; ol3', 5'-GGG CTG TTA AGG GTC TTC CCT AGT CCA ACC ATT GAC GT-3'; and olFS, 5'-GGG CCA TGG TAT GTT TTG TAT AAA AGT TCA TTT GTG ACA ACT C-3'.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

One remarkable feature of the BVDV system is the natural occurrence of cp and noncp virus strains. Infectious cDNA copies of two cp BVDV strains have recently been established (28, 30, 51). In both cDNAs the deletion of the cp-virus-specific insertion from the NS2 gene resulted in isogenic noncp BVDV clones and the abolishment of NS2-3 cleavage (28, 30, 48). Unfortunately, these cDNA clones are difficult to handle; for example, they have to be maintained in plasmids with very low copy numbers because of stability problems. In comparison, the recently established sg BVDV replicons have the striking advantage of easy cloning in medium-copy-number plasmids. Furthermore, mutants with altered replication characteristics which occasionally appear will not spread in cell culture, and the absence of RNA packaging further facilitates investigations on the replication of these RNAs. Because of these advantages we intended to establish cp and noncp sg BVDV RNA replicons for further studies on the molecular basis of pestivirus cytopathogenicity.

Cytopathogenicity of sg pestivirus RNA. It has recently been shown that BVDV replicon DI9, which corresponds in its genomic structure to BVDV DI9, replicates in the absence of a helper virus (5). The starting point of our project was the question of whether such an sg RNA alone induces a CPE. Apart from differences in the 5'- and 3'-terminal sequences, the sg RNA used for this approach, termed HHDI9 (Fig. 1), is identical to replicon DI9c (see Materials and Methods). The investigation, however, was hampered by the fact that the DEAE method which had been used so far for the transfection of RNA into MDBK cells yielded less than 1% antigen-positive cells when tested 24 h p.t. by an IF assay (data not shown). In order to achieve a higher transfection efficiency, we set up an electroporation protocol for MDBK cells. Electroporation of HHDI9 RNA into MDBK cells led to about 70% antigen-positive cells (Fig. 2A). Importantly, a clear CPE appeared about 18 h p.t. (Fig. 2B). Transfection of a control replicon, termed HHDI9pol, which carries a deletion in the NS5B polymerase gene (see Materials and Methods), led neither to a positive signal in the IF assay (Fig. 2A) nor to the development of a CPE (Fig. 2B). These results demonstrated that HHDI9 RNA induces a CPE in MDBK cells in the absence of a helper virus and that replication of the sg RNA is essential for this process.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Schematic drawing of RNA replicons. The bar labeled "INSERTION" indicates the position of the CP7-specific insertion in NS2. ubi, ubiquitin gene.

View larger version (82K): [in this window] [in a new window]   FIG. 2.   Analysis of MDBK cells transfected with HHDI9 or HHDI9pol RNA. (A) IF analysis. MDBK cells were analyzed 24 h after transfection of HHDI9 or HHDI9pol RNA. After fixation of the cells, a monoclonal antibody directed against NS3 and a secondary cyanogen 3-labeled antibody directed against mouse immunoglobulin were used to detect viral antigen. Magnification, ×100. (B) Cell morphology. Cells were fixed at 24 h after transfection of HHDI9 or HHDI9pol RNA and visualized by microscopy (magnification, ×100); for the upper panels phase-contrast microscopy was used. Whereas cells in the upper panels were not stained, the cultures shown in the lower panels were incubated with crystal violet after fixation to contrast remaining cells (30). Only electroporation of HHDI9 RNA induced a CPE.

View larger version (91K): [in this window] [in a new window]   FIG. 3.   Analysis of MDBK cells transfected with FSubiNS3 or FSubiNS3pol RNA. (A) IF analysis. FSubiNS3 or FSubiNS3pol RNA was transfected into MDBK cells; 24 h after transfection the cells were assayed for viral antigen (for antibodies see the legend to Fig. 2A). Magnification, ×100. (B) Cell morphology. Cells were fixed 24 h after transfection of FSubiNS3 or FSubiNS3pol RNA and visualized by microscopy (magnification, ×100); for the upper panels phase-contrast microscopy was used. The cell cultures shown in the lower panels were stained with crystal violet (see the legend to Fig. 2B).

Generation of a pair of BVDV replicons. After the successful establishment of cp BVDV replicons we aimed at generating a pair of comparable cp and noncp replicons. We decided to establish such a replicon pair on the basis of the infectious clones of BVDV CP7 and its isogenic noncp counterpart, CP7ins; the latter is missing the cp-virus-specific insertion in the NS2 gene (30). Accordingly, we had to construct replicons expressing the viral polyproteins from NS2 to NS5B. Previous studies had suggested that the C-terminal part of p7 functions as a signal sequence required for correct processing and membrane translocation of NS2 (reference 17 and unpublished results); this region (designated p7*) was therefore included in the ORF of the replicons. The p7* region and the sequences corresponding to the polyproteins of CP7 and CP7ins were cloned downstream of the FS element, leading to replicons FSNS2ins+ and FSNS2ins, respectively (Fig. 1). Transfection of the corresponding RNAs into MDBK cells resulted in about 70% antigen-positive cells (24 h p.t. [Fig. 4]), which demonstrated that both RNAs are capable of autonomous replication. However, to our surprise no obvious CPE could be detected after transfection of the cp BVDV-derived replicon FSNS2ins+ (data not shown).

View larger version (63K): [in this window] [in a new window]   FIG. 4.   IF analysis of MDBK cells transfected with FSNS2ins, FSNS2ins+, or FSNS2ins+pol RNA. Cells were fixed 24 h p.t. and tested for expression of NS2-3 or NS3 (for antibodies see the legend to Fig. 2A). No viral antigen was detected after transfection of the control, FSNS2ins+pol RNA. Magnification, ×100.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Schematic drawing of bicistronic RNA replicons. ORF1 encompasses the GUS gene, which is inserted in frame downstream of the FS element. The pestivirus NS proteins are encoded in ORF2 downstream of the EMCV IRES. The bar labeled "INSERTION" indicates the position of the CP7-specific insertion in NS2 of Bi-NS2ins+.

View larger version (57K): [in this window] [in a new window]   FIG. 6.   (A) IF analysis 24 h after transfection of MDBK cells with Bi-NS2ins, Bi-NS2ins+, and Bi-NS2ins+pol RNA (control). No viral antigen was detected in cells transfected with Bi-NS2ins+pol RNA. For antibodies see the legend to Fig. 2A. Magnification, ×400. (B and C) GUS release assay with Bi-NS2ins and Bi-NS2ins+. MDBK cells transfected with replicon RNA were monitored for GUS activity up to 10 days p.t. The GUS activities present in the culture media and adherent cells were determined separately. The total GUS activity of each dish is the sum of the GUS activities detected in the adherent cells and the culture medium. The fraction of the total GUS activity which was detected in the culture medium of a specific dish at a given time point is depicted in the graph as a percentage of the total GUS activity. Values obtained from cultures which originated from the same RNA transfection are indicated by bars with identical patterns. (B) Cell cultures derived from five parallel transfections of Bi-NS2ins RNA were monitored for up to 10 days at different time points. While at 24 h p.t. one culture of each of the transfections was used for the determination of the GUS activity, three cultures from three independent electroporations were analyzed at 48, 72, and 144 h p.t. At 168 and 240 h p.t., the GUS activities of two cultures originating from two independent electroporations were measured. (C) Three transfections of Bi-NS2ins+ RNA were carried out in parallel, and the cells were monitored for 4 days. Every 24 h one culture of each of the three electroporations was analyzed.

Bicistronic replicons expressing BVDV proteins NS3 to NS5B. In the monocistronic replicons described above, the N terminus of NS3 was generated by a preceding N^pro or ubiquitin. To rule out the possibility that these proteins play an essential role in the cytopathogenicity of the sg BVDV RNAs, we decided to construct a bicistronic replicon expressing only BVDV proteins NS3 to NS5B downstream of the EMCV IRES. In this replicon, termed Bi-NS3, the ORF downstream of the EMCV IRES encompasses the methionine start codon and the sequence corresponding to the polyprotein of BVDV CP7 starting with glycine¹⁵⁹⁰, the first amino acid of NS3 (Fig. 5). Replicon Bi-NS3pol served as a control. Replication of the corresponding RNAs was demonstrated by the IF assay, which detected about 30% antigen-positive cells 24 h after transfection of replicon Bi-NS3 (Fig. 7A). No CPE appeared after transfection of Bi-NS3 RNA, similar to the observations made for replicon Bi-NS2ins+ (data not shown).

View larger version (67K): [in this window] [in a new window]   FIG. 7.   IF analysis of MDBK cells 24 h after electroporation of either Bi-NS3 or Bi-NS3pol RNA (magnification, ×400); for antibodies see the legend to Fig. 2A. (B) GUS release assay with Bi-NS3. MDBK cells transfected in three parallel experiments with Bi-NS3 RNA were analyzed for GUS activity at different times p.t. The transfected cell cultures were monitored for a period of 3 days; every 24 h one dish derived of each of the transfections was analyzed. (For further details see the legend to Fig. 6B and C.) Values obtained from cultures which originated from the same RNA transfection are indicated by bars with identical patterns.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Recently, an autonomous sg RNA replicon of BVDV which encodes the viral proteins N^pro and NS3 to NS5B has been established (5). The same study also showed that the substitution of the C-terminal part of N^pro by ubiquitin did not interfere with replication of this RNA; however, a complete deletion of the N^pro-coding sequence from the replicon interfered with its viability. In the present study we addressed the question of whether the C-terminally truncated N^pro molecule is essential for replication of sg BVDV RNAs. It could be shown that the first 39 nucleotides of the N^pro gene are sufficient to promote replication. Due to an introduced FS the respective sg RNA encoded only the first 3 amino acids of N^pro followed by 10 unrelated amino acids. While we did not determine the minimal N^pro gene fragment sufficient to promote RNA replication, the results of our experiments exclude the possibility that the N^pro protein, at least downstream of amino acid 3, is essential for RNA replication. One hint at a possible function of the N^pro sequence comes from a recent study in which the IRES of BVDV strain NADL was analyzed in vitro. It was shown that the presence of the complete N^pro gene stimulates the efficiency of translation initiation (8). It is tempting to speculate that the 5' region of the N^pro gene represents part of the BVDV IRES, a situation reminiscent of the one described for hepatitis C virus (40, 57).

After we had further defined the set of genes necessary for establishing an autonomous replicon, the main aspect of this study was to determine the sets of viral proteins essential for cp and noncp BVDV replicons. We could show that the replication of an sg RNA (FSubiNS3) which encodes, of the BVDV proteins, only the first three amino acids of N^pro in addition to NS3-NS4A-NS4B-NS5A-NS5B induces a CPE in MDBK cell cultures. This result demonstrates for the first time that the replication of an RNA encoding this set of viral proteins is sufficient for the induction of a CPE. Accordingly, there is not essential role for helper virus proteins or replication of the helper virus in this process.

Investigations concerning the cytopathogenicity of BVDV are hampered by the observation that the CPE caused by cp BVDV strains in general is not very prominent and moreover depends strongly on various parameters, in particular the multiplicity of infection and the density of the cells at the time of infection. Thus, the absence of a CPE following electroporation of replicons Bi-NS2ins+ and Bi-NS3 was not totally unexpected.

To establish a reliable basis for further investigations, we decided to develop a reproducible and highly sensitive system for the quantitative determination of BVDV-induced cell lysis. The GUS release assay in combination with the established bicistronic BVDV replicons represents such a detection system. This approach enabled us to demonstrate that replicons Bi-NS2ins+ and Bi-NS3 induce the lysis of transfected cells. However, no CPE was observed (see below). Interestingly, for each of the cp replicons cell damage became apparent at a defined time p.t. This aspect was verified by a comparative experiment in which similar amounts of replicons HHDI9, FSubiNS3, Bi-NS2ins+, and Bi-NS3 were transfected in parallel into MDBK cells (data not shown). Replicon HHDI9 induced a clear CPE at about 18 h p.t., while FSubiNS3 RNA led to comparable cell damage at 24 h p.t. For the bicistronic RNAs, host cell lysis had to be determined by the GUS release assay since no visible CPE appeared. Bi-NS3 RNA induced cell lysis between 48 and 72 h p.t. and thus about 24 h earlier than Bi-NS2ins+ RNA. When different amounts of the latter RNA were used for transfection, no changes in the time course of cell lysis were observed (data not shown). These experiments show that the kinetics of cell lysis differ among the replicons and are an intrinsic property of each sg RNA. The bicistronic replicons in particular appear to be delayed with respect to the induction of cell death. The slow process of cell lysis in the cultures may enable untransfected neighboring cells to mask the CPE by cell division.

Taken together, these results indicate that cell lysis is triggered by each cp replicon with a given efficiency. Whether these variations correlate with differences in replication among the respective RNAs is currently under investigation. In this context it is intriguing that the amount of intracellular viral RNA is elevated in cells infected with the cp BVDV strain ACNR/NADL in comparison with the isogenic noncp strain ACNR/cIns-NADL (28).

Recent studies revealed that the CPE induced by cp BVDV is caused by apoptosis of the host cells (18, 56). The prime candidate for the induction of the cell death is NS3. It is still unknown, however, whether the apoptosis is induced by a direct interaction of NS3 with host cell factors, e.g., activation of a caspase(s) by the NS3 protease, or in a more indirect way by NS3-driven deregulation of viral replication, which then induces the death pathway.

For classical swine fever virus, another member of the genus Pestivirus, it has been demonstrated that inactivation of the virus-encoded RNase which resides in the glycoprotein E^rns changes the biotype of the virus from noncp to cp. The authors proposed that RNase activity is required for maintenance of the noncp phenotype of classical swine fever virus (20). Our data exclude such a function of E^rns in the BVDV system since replicon Bi-NS2ins, which does not encode E^rns, fails to induce lysis of its host cell. In addition, our experiments show that neither any of the other structural proteins nor N^pro is needed for an active maintenance of the noncp phenotype.

In general, bicistronic replicons are well suited for the expression of foreign genes. The noncp replicon Bi-NS2ins is also a promising candidate for long-term protein expression. To enable stable long-time propagation of the replicon in cell culture, the expression of a selectable marker by the sg RNA could be applied. Recently, noncp Sindbis virus vectors have been established. These replicons, however, are restricted in noncp replication to BHK cells (1). The noncp BVDV replicon enables this approach to be extended to other cells and may provide an attractive tool to deliver defined antigens in vivo e.g., for immunization purposes.

Cytopathogenicity of BVDV and BVDV-derived replicons is always correlated with the expression of NS3. The noncp phenotype of Bi-NS2ins obviously depends on the absence of NS2-3 cleavage. The NS2 domain in NS2-3 can therefore be considered a cis-acting negative regulator of BVDV-induced cell lysis. The consequences of NS2-3 cleavage for polyprotein processing and replication of BVDV replicons are promising subjects for further studies aimed at the elucidation of the mechanism of pestivirus cytopathogenicity. The established pair of bicistronic replicons, which mimic two complete isogenic cp and noncp BVDVs with regard to their effect on tissue culture cells, will significantly facilitate these studies.