JOURNAL OF VIROLOGY, Nov. 1999, p. 9422–9432 0022-538X/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 11
Establishment and Characterization of Cytopathogenic and Noncytopathogenic Pestivirus Replicons N. TAUTZ,* T. HARADA, A. KAISER, G. RINCK, S.-E. BEHRENS,
AND
H.-J. THIEL
Institut fu ¨r Virologie (FB Veterina ¨rmedizin), Justus-Liebig-Universita ¨t Giessen, D-35392 Giessen, Germany Received 30 April 1999/Accepted 9 August 1999
Defective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease Npro in addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7insⴚ (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding -glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7insⴚ does not induce cell lysis. Accordingly, neither Npro nor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7insⴚ with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV. 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-Erns-E1-E2-p7-NS2; thus, in the polyprotein of DI9 the autoprotease Npro 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 Npro 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 Npro gene from
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: Npro–C– Erns–E1–E2–p7–NS2-3–NS4A–NS4B–NS5A–NS5B (9, 11, 17, 31, 45, 47, 55). Npro 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 Erns 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. * Corresponding author. Mailing address: Institut fu ¨r Virologie (FB Veterina¨rmedizin), Justus-Liebig-Universita ¨t Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany. Phone: 49-(641)-99 38375. Fax: 49-(641)-99 38359. E-mail:
[email protected]. 9422
VOL. 73, 1999
PESTIVIRUS RNA REPLICONS
this RNA interfered with its replication, the C-terminal part of Npro, 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-Cterminal hydrolases (22, 26, 44). Whether the residual part of Npro 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 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 DEAEdextran 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 F 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 g 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. For the comparative analysis mentioned in Discussion, transcripts of replicons HHDI9, FSubiNS3, Bi-NS2ins⫹, or Bi-NS3 were incubated with 5 U of DNase I (Boehringer GmbH, Mannheim, Germany) and 10 U of DpnI (New England Biolabs, Schwalbach, Germany) for 1 h at 37°C to ensure complete degradation of the template DNA. Subsequently the reaction mixture was extracted with phenol and chloroform followed by precipitation of the RNA with 0.9 M ammonium acetate–65% ethanol to avoid coprecipitation of free nucleotides. After the RNA was dried and resolved in H2O, its concentration was determined by measuring the optical density at 260 nm. The result was confirmed by agarose gel electrophoresis. From each transcript 2 g was used for 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 l containing 250 M 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 (MWGBiotech, 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. For determination of the GUS activities of transfected cell cultures, the culture media were collected, the volume was determined, and the cell layer was washed once with PBS and then resuspended in the lysis buffer supplied in the GUS-Light kit (Tropix, Bedford, Mass.), containing 0.2% Triton X-100. To the culture media 1/10 volume of 10⫻ lysis buffer was added. Measuring was carried out as recommended by the supplier of the kit 1 h after mixing of the samples
9423
with the substrate (Glucuron, supplied in the kit). For the detection an LB9501 luminometer (Berthold, Pforzheim, Germany) was used. 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. The origin of replication and the -lactamase gene of the plasmid were taken from plasmid Toto 57 (23). Except for the 21 bases at the 5⬘ end and 33 bases at the 3⬘ end, which were derived from BVDV strain Osloss (14, 39), all pestivirus sequences in the replicons used for the experiments described in this report were derived from BVDV CP7 (30, 48); the only exception is construct HHDI9, which encompasses a BVDV DI9-derived NcoI (including the start AUG)/HpaI (nucleotides 5434 to 5439) fragment (30) (see below). Upstream of the viral cDNA an SP6 RNA polymerase promoter was integrated in such a way that the start site of the RNA transcript is the first nucleotide of the BVDV cDNA. A single SmaI site allows linearization of the plasmids at the 3⬘ end of the cDNA, leading to molecules with three terminal C residues. The cDNA clones used for the assembly have already been described (30). Details of the cloning strategies are available on request. 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 DI9c⌬Nproubi (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, HHNpro*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 HHNpro*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. HHDI9⌬pol. 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. FSNS2insⴙ and FSNS2insⴚ. 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. FSNS2insⴙ⌬pol. The FSNS2ins⫹⌬pol construct was generated as described above for plasmid HHDI9⌬pol. 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-NS2insⴙ and Bi-NS2insⴚ. The NcoI (3⬘ end of FS element)/SacI (nucleotides 5855 to 5860) fragments from FSNS2ins⫹ and FSNS2ins⫺ were ligated
9424
TAUTZ ET AL.
J. VIROL.
FIG. 1. Schematic drawing of RNA replicons. The bar labeled “INSERTION” indicates the position of the CP7-specific insertion in NS2. ubi, ubiquitin gene. 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 Cterminally 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-NS2insⴙ⌬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-NS3⌬pol. Plasmid Bi-NS3⌬pol 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 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-virusspecific 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 char-
acteristics 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% antigenpositive cells (Fig. 2A). Importantly, a clear CPE appeared about 18 h p.t. (Fig. 2B). Transfection of a control replicon, termed HHDI9⌬pol, 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. For a replicon corresponding in its genome structure to HHDI9, the C-terminal part of Npro could be substituted by one ubiquitin monomer (5). Since a complete deletion of the Npro gene abrogated replication of this RNA, we next addressed the question of whether the N-terminal Npro fragment is required for replication and/or cytopathogenicity of sg BVDV RNAs. In replicon FSubiNS3 (Fig. 1), an almost
VOL. 73, 1999
PESTIVIRUS RNA REPLICONS
9425
FIG. 2. Analysis of MDBK cells transfected with HHDI9 or HHDI9⌬pol RNA. (A) IF analysis. MDBK cells were analyzed 24 h after transfection of HHDI9 or HHDI9⌬pol 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 HHDI9⌬pol 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.
complete deletion of Npro was achieved by truncation of the Npro-coding sequence; similar to the genome organization of the natural BVDV isolate DI13, only the 5⬘ 39 bases of Npro were included (25). In addition, we deleted base 10 of the ORF
and duplicated nucleotide 38. Accordingly, the reading frame of this construct shifts beyond nucleotide 9 and the original frame is recovered at base 40, the initial nucleotide of the ubiquitin-coding sequence. This 39-base sequence is referred
9426
TAUTZ ET AL.
J. VIROL.
FIG. 3. Analysis of MDBK cells transfected with FSubiNS3 or FSubiNS3⌬pol RNA. (A) IF analysis. FSubiNS3 or FSubiNS3⌬pol 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 FSubiNS3⌬pol 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).
to as the FS element. Replicon FSubiNS3 thus encodes the first 3 amino acids of Npro followed by 10 unrelated amino acids, ubiquitin, and the polyprotein of BVDV CP7 starting with glycine1590, the first amino acid of NS3. A derivative of this replicon, FSubiNS3⌬pol, served as a control. After transfec-
tion of FSubiNS3 RNA into MDBK cells, about 70% of the cells were positive for viral antigen in the IF assay whereas no positive cells could be detected after electroporation of FSubiNS3⌬pol RNA (24 h p.t. [Fig. 3A]). About 24 h after transfection of FSubiNS3 RNA, a clear CPE became apparent
VOL. 73, 1999
PESTIVIRUS RNA REPLICONS
9427
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.
(Fig. 3B). These results demonstrate that expression of Npro (except at least for the first three amino acids) is not essential for replication as well as for cytopathogenicity of BVDV replicons. 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).
In order to elucidate whether this replicon does not induce cell lysis or whether cell lysis caused by this RNA does not lead to a visible CPE, a more sensitive method for the detection of cell lysis was developed. Such an assay can be based on monitoring the release of an intracellular enzyme into the culture medium as an indicator of cell lysis. To reduce background signals arising from the lysis of untransfected cells, e.g., by the electroporation procedure, a marker enzyme was expressed by the BVDV replicon. GUS was chosen as a reporter because of its known high resistance to proteolytic degradation. In the first attempt a BVDV replicon with a GUS gene integrated between the FS element and the ubiquitin gene was established. The respective RNA replicated autonomously, but no GUS activity could be detected (data not shown); we assumed that the inactivation of GUS was caused by the fusion of ubiquitin to the C terminus of the enzyme. In the picornavirus field in particular, bicistronic viral RNAs have been established which enable two (poly)proteins from one RNA molecule to be independently expressed by using additional IRES elements, which mediate internal initiation of translation (2, 34). In our pestivirus system a corresponding bicistronic replicon would allow the expression of GUS without a C-terminal fusion. Accordingly, the IRES of EMCV (21, 36) was used to introduce a second ORF into the pestivirus replicons. In the resulting bicistronic replicons the FS element
9428
TAUTZ ET AL.
J. VIROL.
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⫹.
is located upstream of the reporter GUS gene (ORF1); the EMCV IRES, which resides downstream of the stop codon of the GUS gene, initiates the translation of the pestivirus NS proteins encoded in ORF2 (Fig. 5). The bicistronic sg replicons termed Bi-NS2ins⫹ and BiNS2ins⫺ (Fig. 5) encode in ORF2 polyprotein fragments of either CP7 or CP7ins⫺; the pestivirus polyproteins start with p7*, analogous to the previously established monocistronic constructs (see above). After electroporation of these replicons, about 60% of the MDBK cells expressed viral antigen as judged by the IF assay (24 h p.t. [Fig. 6A]). No positive cells were detected after transfection of control replicon BiNS2ins⫹⌬pol (Fig. 6A). In order to evaluate the replication of the respective bicistronic RNAs, we next determined the GUS levels in the transfected cells. The GUS activities measured in the cells 24 h after transfection of similar amounts of RNAs Bi-NS2ins⫹ and BiNS2ins⫺ both were about 50- to 100-fold higher than that detected in control cells; the GUS activities further increased by a factor of 2 to 3 over the next 24 h. In line with the result obtained in the IF assay, the GUS activity in cells transfected with Bi-NS2⌬pol RNA was not significantly elevated over that measured for control cells (data not shown). MDBK cells electroporated in parallel experiments with similar amounts of replicons Bi-NS2ins⫹ and Bi-NS2ins⫺ were then assayed for cell lysis by determination of the GUS activities in the adherent cells and the culture media, respectively. Over a period of up to 10 days after transfection of Bi-NS2ins⫺ RNA, consistently only 1 to 3% of the total GUS activity of the cell culture dishes was detected in the culture media (Fig. 6B). These data show that replicon Bi-NS2ins⫺ does not induce lysis of its host cell. In contrast, after transfection of Bi-NS2ins⫹ RNA the part of the total GUS activity which was released from the adherent cells into the medium continuously rose until 96 h p.t.; at that
time up to 40% of the total GUS activity of the corresponding culture dishes was found in the culture media (Fig. 6C). These experiments demonstrated that replication of Bi-NS2ins⫹ RNA induces cell lysis, which can be quantitatively measured by the developed GUS release assay. Interestingly, even at 96 h p.t., when the GUS activity reached its highest level in the supernatant, no clear CPE could be detected by microscopy; this observation was in line with the results described above. In the GUS release assay described above, the samples used to determine the enzymatic activity of the culture media included detached cells. The removal of these cells from the GUS-containing culture media by centrifugation did not lead to a significant loss of GUS activity (data not shown). Accordingly, the GUS activity detected in the media of the transfected cell cultures reflects cell lysis rather than detachment of viable cells from the culture dish. Bicistronic replicons expressing BVDV proteins NS3 to NS5B. In the monocistronic replicons described above, the N terminus of NS3 was generated by a preceding Npro 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-NS3⌬pol 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). In order to monitor the replication of Bi-NS3 in a more quantitative way, the GUS activity was measured 24 h p.t. and
VOL. 73, 1999
PESTIVIRUS RNA REPLICONS
9429
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.
was found to be, on average, a factor of 40 above that of cells transfected with Bi-NS3⌬pol RNA, which showed only background levels of GUS activity (data not shown). The GUS activity in the cell cultures transfected with replicon Bi-NS3 further increased by about a factor of 10 between 24 and 48 h p.t. (data not shown). To monitor cell lysis after transfection with Bi-NS3 RNA, GUS activities in the cells and the culture media were mea-
sured. Similar to the results obtained previously with the bicistronic RNAs Bi-NS2ins⫹ and Bi-NS2ins⫺, about 2% of the total GUS activity was detected in the culture media at 24 h p.t. When the time period was extended, 12% (48 h) and 80% (72 h) of the total GUS activities were found in the supernatant of the transfected cell cultures (Fig. 7B). These data clearly demonstrated that neither Npro nor ubiquitin (expressed by the replicon) is required for the induction of cell lysis.
9430
TAUTZ ET AL.
J. VIROL.
FIG. 7. IF analysis of MDBK cells 24 h after electroporation of either Bi-NS3 or Bi-NS3⌬pol 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 Recently, an autonomous sg RNA replicon of BVDV which encodes the viral proteins Npro and NS3 to NS5B has been established (5). The same study also showed that the substitution of the C-terminal part of Npro by ubiquitin did not interfere with replication of this RNA; however, a complete deletion of the Npro-coding sequence from the replicon interfered with its viability. In the present study we addressed the question of whether the C-terminally truncated Npro molecule is essential for replication of sg BVDV RNAs. It could be shown that the first 39 nucleotides of the Npro gene are sufficient to promote replication. Due to an introduced FS the respective sg RNA encoded only the first 3 amino acids of Npro followed by 10 unrelated amino acids. While we did not determine the minimal Npro gene fragment sufficient to promote RNA replication, the results of our experiments exclude the possibility that the Npro protein, at least downstream of amino acid 3, is essential for RNA replication. One hint at a possible function of the Npro 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 Npro gene stimulates the efficiency of translation initiation (8). It is tempting to speculate that the 5⬘ region of the Npro 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 Npro 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
VOL. 73, 1999
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 Erns 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 Erns in the BVDV system since replicon Bi-NS2ins⫺, which does not encode Erns, fails to induce lysis of its host cell. In addition, our experiments show that neither any of the other structural proteins nor Npro 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
PESTIVIRUS RNA REPLICONS
9431
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. ACKNOWLEDGMENTS We thank Matthias Ko ¨nig for helpful assistance in photography. This study was supported by the SFB 535 “Invasionsmechanismen und Replikationsstrategien von Krankheitserregern” from the Deutsche Forschungsgemeinschaft. REFERENCES 1. Agapov, E. V., I. Frolov, B. D. Lindenbach, B. M. Pragai, S. Schlesinger, and C. M. Rice. 1998. Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proc. Natl. Acad. Sci. USA 95:12989–12994. 2. Alexander, L., H. H. Lu, and E. Wimmer. 1994. Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene. Proc. Natl. Acad. Sci. USA 91:1406–1410. 3. Becher, P., M. Orlich, and H.-J. Thiel. 1998. Complete genomic sequence of border disease virus, a pestivirus from sheep. J. Virol. 72:5165–5173. 4. Becher, P., M. Orlich, and H.-J. Thiel. 1998. Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus. J. Virol. 72:8697–8704. 5. Behrens, S.-E., C. W. Grassmann, H.-J. Thiel, G. Meyers, and N. Tautz. 1998. Characterization of an autonomous subgenomic pestivirus RNA replicon. J. Virol. 72:2364–2372. 6. Bolin, S. R., A. W. McClurkin, R. C. Cutlip, and M. F. Coria. 1985. Severe clinical disease induced in cattle persistently infected with noncytopathogenic bovine viral diarrhea virus by superinfection with cytopathogenic bovine viral diarrhea virus. Am. J. Vet. Res. 46:573–576. 7. Brownlie, J., M. C. Clarke, and C. J. Howard. 1984. Experimental production of fatal mucosal disease in cattle. Vet. Res. 114:535–536. 8. Chon, S. K., D. R. Perez, and R. O. Donis. 1998. Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus. Virology 251: 370–382. 9. Collett, M. S., R. Larson, S. Belzer, and E. Retzel. 1988. Proteins encoded by bovine viral diarrhea virus: the genome organization of a pestivirus. Virology 165:200–208. 10. Collett, M. S., R. Larson, C. Gold, D. Strick, D. K. Anderson, and A. F. Purchio. 1988. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology 165:191–199. 11. Collett, M. S., V. Moennig, and M. C. Horzinek. 1989. Recent advances in pestivirus research. J. Gen. Virol. 70:253–266. 12. Corapi, W. V., R. O. Donis, and E. J. Dubovi. 1990. Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus. Am. J. Vet. Res. 51:1388–1394. 13. Corapi, W. V., R. O. Donis, and E. J. Dubovi. 1988. Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea infections. J. Virol. 62:2823–2827. 14. de Moerlooze, L., C. Lecomte, S. Brown-Shimmer, D. Schmetz, C. Guiot, D. Vandenbergh, D. Allaer, M. Rossius, G. Chappuis, D. Dina, A. Renard, and J. A. Martial. 1993. Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5⬘ untranslated region. J. Gen. Virol. 74:1433–1438. 15. Deng, R., and K. V. Brock. 1992. Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathogenic bovine viral diarrhea virus strain SD-1. Virology 191:867–879. 16. Donis, R. O., and E. J. Dubovi. 1987. Differences in virus-induced polypeptides in cells infected by cytopathic and noncytopathic biotypes of bovine diarrhoea-mucosal disease virus. Virology 158:168–173. 17. Elbers, K., N. Tautz, P. Becher, D. Stoll, T. Ru ¨menapf, and H.-J. Thiel. 1996.
9432
18. 19. 20.
21. 22. 23.
24. 25. 26. 27.
28.
29. 30. 31. 32. 33. 34. 35.
36. 37.
TAUTZ ET AL.
Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7. J. Virol. 70:4131–4135. Hoff, H. S., and R. O. Donis. 1997. Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection. Virus Res. 49:101–113. Hulst, M. M., G. Himes, E. Newbigin, and R. J. M. Moormann. 1994. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology 200:558–565. Hulst, M. M., F. E. Panoto, A. Hoekman, H. G. P. van Gennip, and R. J. M. Moormann. 1998. Inactivation of the RNase activity of glycoprotein Erns of classical swine fever virus results in a cytopathogenic virus. J. Virol. 72: 151–157. Jang, S. K., M. V. Davies, R. J. Kaufman, and E. Wimmer. 1989. Initiation of protein synthesis by internal entry of ribosomes into the 5⬘ nontranslated region of encephalomyocarditis virus RNA in vivo. J. Virol. 63:1651–1660. Jonnalagadda, S., T. R. Butt, B. P. Monia, C. K. Mirabelli, L. Gotlib, D. J. Ecker, and S. T. Crooke. 1989. Multiple (a-NH2-ubiquitin)protein endoproteases in cells. J. Biol. Chem. 264:10637–10642. Kuhn, R. J., H. G. Niesters, Z. Hong, and J. H. Strauss. 1991. Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. Virology 182: 430–441. Ku ¨mmerer, B. M., D. Stoll, and G. Meyers. 1998. Bovine viral diarrhea virus strain Oregon: a novel mechanism for processing of NS2-3 based on point mutations. J. Virol. 72:4127–4138. Kupfermann, H., H.-J. Thiel, E. J. Dubovi, and G. Meyers. 1996. Bovine viral diarrhea virus: characterization of a cytopathogenic defective interfering particle with two internal deletions. J. Virol. 70:8175–8181. Mayer, A. N., and K. D. Wilkinson. 1989. Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus. Biochemistry 28:166–172. McClurkin, A. W., M. F. Coria, and S. R. Bolin. 1985. Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from the spleen of cattle acutely and chronically affected with bovine viral diarrhea. J. Am. Vet. Med. Assoc. 186:568–569. Mendez, E., N. Ruggli, M. S. Collett, and C. M. Rice. 1998. Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity. J. Virol. 72: 4737–4745. Meyers, G., T. Ru ¨menapf, and H.-J. Thiel. 1989. Molecular cloning and nucleotide sequence of the genome of hog cholera virus. Virology 171:555– 567. Meyers, G., N. Tautz, P. Becher, H.-J. Thiel, and B. Ku ¨mmerer. 1996. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J. Virol. 70:8606–8613. Meyers, G., N. Tautz, R. Stark, J. Brownlie, E. J. Dubovi, M. S. Collett, and H.-J. Thiel. 1992. Rearrangement of viral sequences in cytopathogenic pestiviruses. Virology 191:368–386. Meyers, G., and H.-J. Thiel. 1996. Molecular characterization of pestiviruses. Adv. Virus Res. 47:53–118. Moennig, V., H.-R. Frey, E. Liebler, P. Polenz, and B. Liess. 1990. Reproduction of mucosal disease with cytopathogenic bovine viral diarrhoea virus selected in vitro. Vet. Rec. 127:200–203. Molla, A., S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer. 1992. Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus. Nature 356:255–257. Moormann, R. J. M., P. A. M. Warmerdam, B. Van der Meer, W. M. M. Schaaper, G. Wensvoort, and M. M. Hulst. 1990. Molecular cloning and nucleotide sequence of hog cholera virus strain brescia and mapping of the genomic region encoding envelope glycoprotein E1. Virology 177:184–198. Pelletier, J., G. Kaplan, V. R. Racaniello, and N. Sonenberg. 1988. Capindependent translation of poliovirus mRNA is conferred by sequence elements within the 5⬘ noncoding region. Mol. Cell. Biol. 8:1103–1112. Pocock, D. H., C. J. Howard, M. C. Clarke, and J. Brownlie. 1987. Variation
J. VIROL.
38. 39. 40. 41. 42. 43.
44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
54. 55.
56. 57.
in the intracellular polypeptide profiles from different isolates of bovine viral diarrhea virus. Arch. Virol. 94:43–53. Poole, T. L., C. Wang, R. A. Popp, L. N. D. Potgieter, A. Siddiqui, and M. S. Collett. 1995. Pestivirus translation occurs by internal ribosome entry. Virology 206:750–754. Renard, A., D. Dino, and J. Martial. 1987. Vaccines and diagnostics derived from bovine diarrhea virus. European patent application 86870095.6. Publication 02.08672. Reynolds, J. E., A. Kaminski, H. J. Kettinen, K. Grace, B. E. Clarke, A. R. Carroll, D. J. Rowlands, and R. J. Jackson. 1995. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14:6010–6020. Rice, C. M. 1996. Flaviviridae: the viruses and their replication, p. 931–959. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed., vol. 1. Lippincott-Raven Publishers, Philadelphia, Pa. Ridpath, J. F., and S. R. Bolin. 1995. The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a noncytopathic BVDV. Virology 212:39–46. Rijnbrand, R., T. van der Straaten, P. A. van Rijn, W. J. M. Spaan, and P. J. Bredenbeek. 1997. Internal entry of ribosomes is directed by the 5⬘ noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon. J. Virol. 71:451–457. Rose, I. A. (ed.). 1988. Role of ubiquitin-protein isopeptidase action in protein breakdown. Plenum Press, New York, N.Y. Ru ¨menapf, T., G. Unger, J. H. Strauss, and H.-J. Thiel. 1993. Processing of the envelope glycoproteins of pestiviruses. J. Virol. 67:3288–3295. Schneider, R., G. Unger, R. Stark, E. Schneider-Scherzer, and H.-J. Thiel. 1993. Identification of a structural glycoprotein of an RNA virus as a ribonuclease. Science 261:1169–1171. Tautz, N., K. Elbers, D. Stoll, G. Meyers, and H.-J. Thiel. 1997. Serine protease of pestiviruses: determination of cleavage sites. J. Virol. 71:5415– 5422. Tautz, N., G. Meyers, R. Stark, E. J. Dubovi, and H.-J. Thiel. 1996. Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion. J. Virol. 70:7851–7858. Tautz, N., H.-J. Thiel, E. J. Dubovi, and G. Meyers. 1994. Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion. J. Virol. 68:3289–3297. Thiel, H.-J., P. G. W. Plagemann, and V. Moennig. 1996. Pestiviruses, p. 1059–1073. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed., vol. 1. Lippincott-Raven Publishers, Philadelphia, Pa. Vassilev, V. B., M. S. Collett, and R. O. Donis. 1997. Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts. J. Virol. 71:471–478. Walden, R., C. Koncz, and J. Schell. 1990. The use of gene vectors in plant molecular biology. Methods Mol. Cell. Biol. 1:175–194. Windisch, J. M., R. Schneider, R. Stark, E. Weiland, G. Meyers, and H.-J. Thiel. 1996. RNase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J. Virol. 70: 352–358. Wiskerchen, M., and M. S. Collett. 1991. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology 184:341–350. Xu, J., E. Mendez, P. R. Caron, C. Lin, M. A. Murcko, M. S. Collett, and C. M. Rice. 1997. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication. J. Virol. 71:5312–5322. Zhang, G., S. Aldridge, M. C. Clarke, and J. W. McCauley. 1996. Cell death induced by cytopathic bovine diarrhoea virus is mediated by apoptosis. J. Gen. Virol. 77:1677–1681. Zhao, W. D., E. Wimmer, and F. C. Lahser. 1999. Poliovirus/hepatitis C virus (internal ribosomal entry site-core) chimeric viruses: improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides. J. Virol. 73: 1546–1554.
