Virology 284, 259–276 (2001) doi:10.1006/viro.2001.0908, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Recombinant Equine Arteritis as an Expression Vector

Antoine A. F. de Vries,1 Amy L. Glaser,2 Martin J. B. Raamsman,3 and Peter J. M. Rottier4

Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands Received November 20, 2000; returned to author for revision January 12, 2001; accepted March 14, 2001

Equine arteritis virus (EAV) is the prototypic member of the family Arteriviridae, which together with the Corona- and Toroviridae constitutes the order . A common trait of these positive-stranded RNA is the 3Ј-coterminal nested set of six to eight leader-containing subgenomic mRNAs which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are expressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral expression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3Ј end by EAV-specific transcription-regulating sequences was constructed. This genetic module was inserted into the recently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84–97)

between the genes for the M and the GL proteins. Confocal fluorescence microscopy of BHK-21 cells electroporated with capped RNA transcripts derived from the resulting plasmid (pBRNX1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfected cells, while the gradual spread of the infection through the cell monolayer showed that the recombinant virus was replication competent. The development of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precursor. Immunoprecipitation analyses of proteins from metabolically labeled BHK-21 cells infected with supernatant of the transfected cultures confirmed that the recombinant virus vector was viable and expressed viral genes as well as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the synthesis of nine instead of eight different EAV RNAs. These findings were corroborated by hybridization analyses. Mapping of the leader-to-body junctions of the ninth mRNA indicated that the 3Ј part of the GFP gene contains cryptic transcription signals which gave rise to at least five different RNA species ranging in size from 1277 to 1439 nt [without oligo(A) tract]. Furthermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant virus vector led to its gradual replacement by viral mutants carrying deletions in the GFP gene. The reduction in viral fitness associated with the insertion of the expression cassette into the EAV genome apparently caused genetic instability of the recombinant virus. © 2001 Academic Press Key Words: equine arteritis virus; Arteriviridae; Nidovirales; full-length cDNA clone; subgenomic mRNA; cryptic transcrip- tion signal; viral expression vector; green fluorescent protein; genetic instability.

INTRODUCTION productive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV). Although arteri- Equine arteritis virus (EAV) is an important equine viral viruses differ markedly from members of the family Coro- pathogen which causes reproductive and respiratory naviridae (genera Coronavirus and ) in biophys- disease in horses throughout the world (Doll et al., 1957; ical properties, virion size, genome length, and structural Timoney and McCollum, 1993; de Vries et al., 1996). EAV protein composition, their polymerase polyproteins are is a positive-stranded RNA virus which belongs to the phylogenetically related (for a review see de Vries et al., genus Arterivirus (family Arteriviridae) together with lac- 1997). The common ancestry of arteri-, corona-, and tate dehydrogenase-elevating virus of mice, porcine re- is further reflected by striking similarities in genome organization, replication mechanism, gene ex-

1 pression strategy, and (site of) virus assembly (reviewed Present address: Gene Therapy Section, Department of Molecular in Snijder and Meulenberg, 1998). Accordingly, the mo- Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, P.O. Box 9503, 2300 RA Leiden, The Netherlands. nogeneric family Arteriviridae and the bigeneric family 2 Present address: Department of Population Medicine and Diagnos- were recently grouped together in the tic Science, College of Veterinary Medicine, Cornell University, Ithaca, newly created virus order of the Nidovirales (Cavanagh, NY 14853. 1997). 3 Present address: Crucell N.V., Archimedesweg 4, P.O. Box 2048, 2301 CA Leiden, The Netherlands. EAV has a diameter of 50 to 70 nm and consists of a 4 To whom correspondence and reprint requests should be ad- putatively icosahedral core surrounded by an envelope dressed. Fax: ϩ31-30-2536723. E-mail: [email protected]. with small surface projections. The core particle is com-

0042-6822/01 $35.00 259 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 260 DE VRIES ET AL. posed of the unsegmented viral genomic RNA and a single phosphorylated nucleocapsid (N) protein of 14 kDa (Hyllseth, 1973; Zeegers et al., 1976). The viral en- velope contains at least five structural proteins which are embedded in a lipid membrane (Hyllseth, 1973; de Vries et al., 1992; Snijder et al., 1999). The large envelope glycoprotein (GL) of 30 to 42 kDa and the 16-kDa ungly- cosylated membrane (M) protein are major structural proteins; the 25-kDa small envelope glycoprotein (GS) and a poorly characterized third glycoprotein of 26/38 kDa are minor virion components. Finally, the 8-kDa unglycosylated envelope (E) protein (Snijder et al., 1999) is present in virus particles in intermediate amounts. The linear genomic EAV RNA contains a 5Ј cap struc- ture and a 3Ј poly(A) tail and has a length of 12,704 nt [exclusive of the oligo(A) tract] (den Boon et al., 1991; van Dinten et al., 1997; Glaser et al., 1999). Nine functional open reading frames (ORFs) have been identified in the viral genome (Fig. 1A; den Boon et al., 1991; Snijder et al., 1999). The two largest of these ORFs (1a and 1b) occupy the 5Ј three-quarters of the genomic EAV RNA and are translated directly from the viral genome (RNA 1 in Fig. 1A); expression of ORF 1b requires a Ϫ1 ribosomal frame shift (den Boon et al., 1991). As a result 2 multido- main polyproteins (1a and 1ab) are synthesized, which are proteolytically processed by three virus-encoded proteases into 8 and 12 nonstructural proteins, respec- tively (van Dinten et al., 1999, and references herein). The genes for the other viral proteins (ORFs 2a, 2b, and 3 through 7; Fig. 1A) are located in the 3Ј-terminal 3 kb of the EAV genome and are expressed from a 3Ј-coterminal nested set of six leader-containing subgenomic mRNAs (RNAs 2 through 7; Fig. 1A; de Vries et al., 1990). The Ј FIG. 1. (A) Structure of the EAV RNAs and expression of the viral 206-nt leader sequence is derived from the extreme 5 genes. RNA 1 corresponds to the EAV genomic RNA, RNAs 2 through end of the viral genome and is fused to the bodies of the 7 represent the viral subgenomic RNAs. The nine functional viral open subgenomic mRNAs at conserved hexanucleotide motifs reading frames (1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7) are indicated by boxes. [5Ј UCAAC(U/C) 3Ј] located upstream of every transcrip- The black triangles below RNA 1 represent the major leader-to-body Ј tion unit (Fig. 1A; de Vries et al., 1990; den Boon et al., fusion sites. The leader sequence with 5 cap structure is symbolized by a small white bar preceded by a tiny black triangle. The translation 1996). The generation of the EAV subgenomic mRNAs products of the EAV RNAs are indicated in the hatched boxes. The involves base pairing between the UCAACU box imme- glycoproteins encoded by EAV open reading frames 3 and 4 have been diately downstream of the leader sequence and comple- provisionally denominated GP3 and GP4, respectively. (A)n, poly(A) tract; mentary sequence motifs on the genomic minus strand nsp, nonstructural protein. (B) Structure of a part of the full-length EAV (van Marle et al., 1999). Leader-to-body fusion in the cDNA clones pBRNX1.38 (top), pBRNX1.38-5/6 (middle), and pBRNX1.38-5/6-gfp (bottom) and of the three smallest virus-specific other arteriviruses (Chen et al., 1993; Meulenberg et al., mRNAs specified by the viruses derived from these plasmids. The two 1993; Meng et al., 1996; Saito et al., 1996; Godeny et al., copies of the EAV RNA 7 leader-to-body fusion site are symbolized by 1998; Nelsen et al., 1999) and in coronaviruses (for re- black rectangles. The sizes of the subgenomic mRNAs are indicated in views see van der Most and Spaan, 1995; Lai and Ca- the boxes representing the leader sequence, and the binding sites for ϩ ϩ Ϫ Ϫ vanagh, 1997) is regulated by similar consensus se- primers A18 ( ), 750 ( ), 766 ( ), and 768 ( ) are also shown. On the left the anticipated length of the (RT-) PCR products is given for different quences. From the few studies on arterivirus mRNA primer combinations. nt, nucleotides. For an explanation of the other synthesis, it appeared that not all potential leader-to- symbols see above. body junction sites are used, which implies that addi- tional factors are involved in their selection. These fac- tors may include additional primary or secondary struc- at genomic sites that slightly deviated from the transcrip- ture elements in the viral genome, viral and/or cellular tion consensus sequence (see e.g., Chen et al., 1993; proteins, and other host cell components (van Marle et den Boon et al., 1996; de Vries et al., 2000). In most of al., 1999). Leader-to-body fusion has also been observed these cases, extra base-pairing possibilities outside the REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 261 conserved transcription motif seem to compensate for 1999). Arteriviruses are among the virus groups whose the mismatches occurring between leader and body se- usefulness as a viral (expression) vector has not yet quences at the junction site. Furthermore, fusion of been systematically investigated even though their leader and body segments can take place at different unique mode of virus replication and gene expression positions within the conserved transcription motifs, re- potentially allows expression of multiple heterologous sulting in subgenomic RNA sequence heterogeneity. genes. Furthermore, the ability of arteriviruses to cause There are even a few examples of subgenomic mRNAs persistent infections may offer specific advantages in in which the actual joining of leader and body sequences situations in which prolonged expression of (foreign) had occurred upstream of the transcription consensus genetic information is required, while their restricted sequence [e.g., RNAs 3, 6, and 7 of the Lelystad isolate host range may be a safeguard against unbridled spread of PRRSV (Meulenberg et al., 1993) and RNA 3 of EAV of virus recombinants into the environment. A final ad- (den Boon et al., 1996)]. vantage of using arteriviruses as viral expression vectors On the basis of extensive studies with coronaviruses, may be that they direct the synthesis of large amounts of several different models have been proposed to explain protein but have only a limited effect on host cell protein nidovirus transcription (van der Most and Spaan, 1995; synthesis (van Berlo et al., 1986). Lai and Cavanagh, 1997). In the classical leader-primed In this report, a recently developed full-length cDNA transcription model free leader RNAs transcribed from clone of EAV (Glaser et al., 1999; de Vries et al., 2000) has the 3Ј end of the anti-genome serve as primers for been used to assess the utility of arteriviruses for the subgenomic mRNA synthesis also from genomic nega- expression of heterologous genes. Although an infec- tive strands. According to another model the viral poly- tious cDNA clone has also been obtained for PRRSV merase is differentially halted at the transcription con- (Meulenberg et al., 1998), we preferred to use EAV for sensus sequences during negative-strand synthesis and this study since it is the only arterivirus that can be the nascent transcripts are relocated to the 5Ј end of the grown to high titers in vitro using a variety of established genome where they acquire an anti-leader sequence. cell lines. Our results showed that it is possible to con- The negative-stranded subgenomic RNAs that are pro- vert EAV into a replication-competent expression vector duced in this way are subsequently transcribed into by insertion of an extra transcription unit directing the plus-stranded subgenomic mRNAs. The subgenomic synthesis of a novel viral subgenomic mRNA in between mRNAs might also be generated by alternative splicing EAV ORFs 5 and 6. The recombinant virus vector was, of the viral genomic RNA or its negative-stranded com- however, greatly impaired compared to the parental virus plement. In the latter case, the negative-stranded sub- and was gradually outcompeted by viral mutants that had genomic RNAs serve as templates for subgenomic lost part of the expression cassette. Moreover, the intro- mRNA synthesis. duction of the expression module into the viral genome As a consequence of the peculiar transcription strat- triggered the formation of a collection of subgenomic egy, the EAV-specific intracellular RNAs are structurally mRNAs that had their leader-to-body fusion sites located polycistronic with the exception of the smallest sub- in the second half of the foreign gene. The appearance of genomic mRNA (RNA 7). However, it is generally ac- these aberrant transcripts, which may have contributed cepted that translation of each viral subgenomic mRNA to the poor growth characteristics of the vector virus, yields only one protein which is encoded by the first points toward a function of secondary structure elements gene downstream of the leader sequence. The viral sub- in arterivirus transcription. genomic mRNAs thus seem to be functionally monocis- tronic (Fig. 1A), e.g., translation of RNAs 5, 6, and 7 is RESULTS thought to result in the production of the G , M, and N L Construction of a replication-competent arterivirus proteins, respectively. The only exception to the rule vector expressing the GFP gene appears to be subgenomic mRNA 2, which directs the synthesis of the E protein encoded by ORF 2a and the GS Since each of the EAV gene products seems to be protein specified by ORF 2b (Snijder et al., 1999). required for the production of infectious virus particles In recent years many different groups of animal vi- (Molenkamp et al., 2000), the number of possibilities to ruses have been subjected to genetic manipulation ei- develop EAV into a replication-proficient expression vec- ther by homologous recombination or by direct mutagen- tor is limited. An obvious approach would be to insert esis of (cDNA copies of) their genomes. The availability one or more expression cassettes consisting of a heter- of reverse genetics systems for both DNA and RNA ologous gene flanked at its 3Ј end by a functional leader- viruses has created new perspectives for the use of to-body junction sequence into the viral genome in be- recombinant viruses as vaccines, expression vectors, tween existing EAV ORFs. Theoretically, this would give anti-tumor agents, gene therapy vectors, and drug deliv- rise to replication-competent viruses expressing the for- ery vehicles (Roth, 1994; Conzelmann and Meyers, 1996; eign genes from additional EAV-specific subgenomic Pastoret et al., 1997; Garcı´a-Sastre, 1998; Friedmann, mRNAs. Unfortunately, the overlapping arrangement of 262 DE VRIES ET AL. the EAV genes complicates the genetic manipulation of show obvious changes during the experiment. The cells the viral genome. To overcome this problem, we recently grown on coverslips were fixed at 12, 24, 36, and 48 h constructed infectious EAV cDNA clones in which ORFs posttransfection, detergent-permeabilized, and stained

4 and 5 and/or ORFs 5 and 6 were separated by newly by indirect immunofluorescence labeling for the EAV GL introduced restriction endonuclease cleavage sites (de protein using a Cy3-conjugated secondary antibody. A

Vries et al., 2000). A schematic drawing of the most red fluorescence indicative of the presence of the GL relevant part of construct pBRNX1.38-5/6 in which the protein was observed in cells electroporated with either overlap between ORFs 5 and 6 had been removed is of the three EAV transcripts but not in mock-transfected shown in the middle of Fig. 1B. The unique AflII site cells. Consistent with the progression of the cytopathic separating ORFs 5 and 6 was used for the insertion of an effect, the GL protein was detected first (i.e., at 12 h after expression cassette of 868 nt consisting of the green electroporation) in the pBRNX1.38 and pBRNX1.38-5/6 fluorescent protein (GFP) gene followed by a copy of the RNA-transfected cells and last (i.e., at 24 h posttransfec- RNA 7 leader-to-body junction region. The GFP gene was tion) in the cells electroporated with the pBRNX1.38-5/6- chosen because it yields an easily detectable product gfp transcripts. After irradiation with blue light some of which can be used as a marker for virus replication both the cells that had received pBRNX1.38-5/6-gfp RNA pro- in vitro and in vivo. The RNA 7-specific transcription duced a faint green fluorescence at 24 h after transfec- signal not only comprised the hexanucleotide consensus sequence at which leader-to-body fusion actually takes tion which was apparently associated with the expres- place, but also included some flanking sequences (65 nt sion of the GFP gene as it was not observed in the other at the 5Ј end and 50 nt at the 3Ј end) since it is unknown cell cultures. At later time points posttransfection more of to what extent the context of a given UCAAC(U/C) motif the pBRNX1.38-5/6-gfp RNA-transfected cells started to contributes to its functionality. Although we first consid- emit green light and the average intensity of the GFP ered duplicating the RNA 6 leader-to-body fusion region signal increased. A typical example of the fluorescence downstream of the GFP gene, we refrained from doing so patterns observed in each of the four cell populations at to minimize the risk of losing the foreign genetic infor- 36 h after electroporation is shown in Fig. 2. The jux- mation by homologous recombination (Lai, 1992). In- tanuclear staining of the GL protein is consistent with the stead, we provided the vector virus with two copies of the earlier observation that in EAV-infected cells this mem- RNA 7 leader-to-body junction sequence (Fig. 1B, bot- brane protein is predominantly localized in the Golgi tom) because genetic recombination between them complex (van der Meer et al., 1998). As a result of its would result in the elimination of the M gene and thus small size, which allows passive transport across the give rise to a replication-deficient virus. nuclear envelope (von Arnim et al., 1998), GFP was seen To test whether recombinant virus vector RNA was both in the cytoplasm and in the nucleus of the cell. The infectious and could direct the synthesis of GFP, BHK-21 relatively strong labeling of the latter compartment prob- cells were transfected with capped in vitro transcripts ably resulted from the efficient fixation of GFP to the derived from plasmid pBRNX1.38-5/6-gfp. In the same nuclear matrix. The microscopical examinations further experiment, we also performed electroporations with revealed that virtually all of the pBRNX1.38-5/6-gfp RNA- comparable amounts of RNA transcribed from the origi- transfected cells which stained positive for the GL protein nal full-length EAV cDNA clone pBRNX1.38 (Fig. 1B, top; also expressed the GFP gene, indicating that the expres- Glaser et al., 1999) and from plasmid pBRNX1.38-5/6, in sion of EAV ORFs and that of the GFP gene were not which ORFs 5 and 6 are separated. After transfection the mutually exclusive. cells were seeded onto glass coverslips and into culture To obtain definitive proof that the expression vector flasks. At regular intervals the cells were examined for was replication proficient, four parallel cultures of the development of cytopathic effect (cpe). The BHK-21 BHK-21 cells were transfected with DNA-free pBRNX1.38- cells that had received pBRNX1.38 transcripts showed extensive signs of infection already at 36 h posttransfec- 5/6-gfp RNA; the supernatants were harvested at 4 days tion, whereas in the monolayers of the pBRNX1.38-5/6- after electroporation and used to inoculate fresh BHK-21 gfp RNA-transfected cells infectious foci were first de- cells. In the same experiment, we also infected a batch tected at 48 h after electroporation. In the cells that had of BHK-21 cells with the pBRNX1.38-5/6 virus and per- received pBRNX1.38-5/6 RNA, cpe occurred at an inter- formed a mock infection. After 1 day at 37°C, the cells mediate pace, i.e., only few virus plaques were observed inoculated with the pBRNX1.38-5/6 virus all displayed at 36 h posttransfection. Moreover, at 72 h after electro- signs of infection, whereas only part of the pBRNX1.38- poration the pBRNX1.38 and pBRNX1.38-5/6 RNA-trans- 5/6-gfp virus-infected cells showed cytopathological fected cells had all detached from the plastic while the changes and the mock-infected cells were completely cells that had obtained pBRNX1.38-5/6-gfp RNA still ad- unaffected. Furthermore, many of the pBRNX1.38-5/6-gfp hered to the support despite their pronounced cytopa- virus-infected cells but none of the control cells pro- thology. As expected, the mock-transfected cells did not duced a green fluorescence. At 2 days after infection, a ELCTO-OPTN QIEATRTSVRSVECTOR VIRUS ARTERITIS EQUINE REPLICATION-COMPETENT

FIG. 2. Confocal fluorescence microscopy of mock-infected BHK-21 cells and BHK-21 cells transfected with capped transcripts derived from plasmid pBRNX1.38 (clo) or from the mutant infectious EAV cDNA clones pBRNX1.38-5/6 (5/6) or pBRNX1.38-5/6-gfp (gfp). The cells were fixed in buffer containing 3% paraformaldehyde at 36 h after transfection, detergent-permeabilized, incubated with culture supernatant of the EAV GL protein-specific hybridoma 74 D(B), and stained by using Cy3-conjugated donkey anti-mouse IgG (HϩL) antibodies. In the top row, the intracellular distribution of the EAV GL protein is shown, whereas in the bottom row, the localization of GFP in the same cells has been recorded. 263 264 DE VRIES ET AL. pronounced cytopathic effect had also developed in the pBRNX1.38-5/6-gfp virus-infected cells, indicating active replication of the viral vector. To evaluate whether the pBRNX1.38-5/6-gfp viruses had retained the full-length GFP gene, total cytoplasmic RNA was extracted from each cell culture at 24 h p.i. and subjected to RT-PCR using an upstream primer [oligonu- cleotide 750 (ϩ)] localized in ORF 5 and a downstream primer binding to the 5Ј terminus of the ORF 6 gene [oligonucleotide 766 (Ϫ)] or of the GFP gene [oligonucle- otide 768 (Ϫ)]. For comparison, the same primer pairs were used to amplify the plasmids from which the viral genomes were transcribed. As is shown in the upper left of Fig. 3, amplification of both pBRNX1.38-5/6-gfp DNA and RNA obtained after a single passage of the corre- sponding virus yielded the expected PCR fragment of 1212 nt for oligonucleotides 750 (ϩ) and 766 (Ϫ). In addition, tiny amounts of smaller amplification products were obtained when the analysis was performed with RNA. As these small DNA fragments were not seen after PCR amplification of pBRNX1.38-5/6-gfp DNA and had different sizes and intensities depending on the specific pBRNX1.38-5/6-gfp virus-infected culture that was ana- lyzed, they were most likely not the result of false prim- ing. The experiments carried out with oligonucleotides 750 (ϩ) and 768 (Ϫ) corroborated the previous findings, i.e., RT-PCR amplification of RNA obtained from cells infected with either of the vector viruses yielded a single FIG. 3. RT-PCR analysis of total cytoplasmic RNA extracted from PCR product (Fig. 3, upper right). The apparent molecular mock-infected BHK-21 cells or from BHK-21 cells infected with mass of this DNA fragment matched with its anticipated pBRNX1.38-5/6 virus (5/6) or pBRNX1.38-5/6-gfp virus (gfp1 and gfp2). size of 344 nt, and a PCR product of the same length was The RT-PCR products (lanes marked RNA) shown in the upper two gels generated after PCR amplification of the pBRNX1.38-5/6- were obtained by using an upstream primer [oligonucleotide 750 (ϩ)] gfp plasmid. The other templates did not yield any spe- situated in ORF 5 and a downstream primer which was specific for either the EAV M gene [oligonucleotide 766 (Ϫ)] or the GFP gene cific amplification products. [oligonucleotide 768 (Ϫ)]. As a consequence, RNAs which originated from leader-to-body fusions downstream of the binding sites of oligo- Ϫ Ϫ The pBRNX1.38-5/6-gfp virus directs the synthesis of nucleotide 766 ( )or768( ) were not amplified. The arrangement of EAV-specific sequences in the resulting RT-PCR products thus corre- an additional subgenomic EAV mRNA sponded to that in the respective viral genomes (see also Fig. 1B). As The previous experiments did not reveal whether the a control, the same two primer pairs were also used for PCR amplifi- cation of the cDNA clones from which the recombinant viruses were GFP gene was expressed from a viral subgenomic derived (lanes marked DNA). The results in the lower left were obtained mRNA or translated directly from the viral genome. To with an ORF 6-specific antisense primer [766 (Ϫ)] and a leader-specific investigate if the pBRNX1.38-5/6-gfp virus directed the sense primer [A18 (ϩ)]. Using this primer pair subgenomic EAV mRNAs synthesis of an additional subgenomic EAV mRNA, RT- which had their leader-to-body fusion sites located immediately up- Ϫ PCR amplifications were performed with the leader-spe- stream of oligonucleotide 766 ( ) were preferentially amplified. In the ϩ experiment shown in the lower right the antisense primer was replaced cific oligonucleotide A18 ( ) and the ORF 6-specific by the GFP-specific oligonucleotide 768 (Ϫ), resulting in the selective oligonucleotide 766 (Ϫ). This should result in the prefer- amplification of mRNAs carrying the GFP gene. On the right sides the ential amplification of subgenomic EAV RNAs which positions and sizes of marker DNA fragments (obtained by HinfI diges- have their leader-to-body fusion sites located close to tion of pGEX-2T DNA) are indicated. The numbers on the left sides refer and upstream of the 5Ј end of ORF 6. Accordingly, for the to anticipated sizes of specific amplification products (see also Fig. 1B). pBRNX1.38-5/6 virus, major PCR products of 364 (ϳRNA 6) and 1206 nt (ϳRNA 5) were to be expected. Likewise, The lower left of Fig. 3 shows that the amplification RT-PCR amplification of the pBRNX1.38-5/6-gfp virus-spe- products of mRNAs 6 and 5 as well as those of RNAs 4, cific subgenomic mRNAs should primarily yield DNA 3, and 2 (de Vries et al., 2000) could be readily identified fragments of 379 and 1232 nt corresponding to the equiv- for the pBRNX1.38-5/6 virus. In contrast, RT-PCR analysis alent of wild-type RNA 6 (which was designated RNA 8Ј; of total cytoplasmic RNA extracted from pBRNX1.38-5/6- vide infra) and the conjectural GFP RNA, respectively. gfp virus-infected cells yielded the RNA 8Ј-specific DNA REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 265

TABLE 1 Nomenclature and Anticipated Sizes of the Virus-Encoded Subgenomic mRNAs

Virus

pBRNX1.38 pBRNX1.38-5/6 pBRNX1.38-5/6-gfp

RNA Size (in nt) RNA Size (in nt) RNAa Size (in nt)

1 12,704 1 12,721 1Ј 13,589 2 3,200 2 3,217 2Ј 4,085 3 2,715–2,640b 3 2,732–2,657 3Ј 3,600–3,525 4 2,234 4 2,251 4Ј 3,119 5 1,883–1,771c 5 1,900–1,788 5Ј 2,768–2,656 6 1,041 6 1,058 6Ј 1,926 7 659 7 659 8Ј 1,073 9Ј 659

a RNAs 1Ј through 5Ј of the recombinant virus vector correspond to RNAs 1 through 5 of EAV Utr; RNAs 8Ј and 9Ј are equivalent to RNAs 6 and 7 and RNA 6Ј represents the transcript from which the GFP gene is expressed. b For RNA 3 of EAV Utr three leader-to-body junction sites were found by den Boon et al. (1996). c For RNA 5 of EAV Utr two leader-to-body junction sites were identified by de Vries et al. (2000). fragment of 379 nt and a number of larger PCR products sion site was thus indeed utilized for the synthesis of an of between 0.5 and 1.0 kb but not the 1232-nt DNA additional subgenomic mRNA which contains the GFP fragment predicted for the GFP RNA or any other long gene in a favorable position for translation to occur, i.e., amplification products. This result could be explained in immediately downstream of the EAV leader sequence. In three ways: (i) The newly introduced leader-to-body junc- addition, some larger amplification products, which most tion sequence was not functional or the GFP RNA was likely correspond to RNAs 2Ј through 5Ј, were obtained. transcribed in very low amounts. This possibility seemed hard to reconcile with the intense green fluorescence The pBRNX1.38-5/6-gfp virus directs the synthesis of observed in pBRNX1.38-5/6-gfp virus-infected cells. (ii) nine instead of eight EAV-specific RNAs The GFP gene contained sequence elements interfering with retrotranscription which resulted in the synthesis of The DNA fragments of 0.5 to 1.0 kb obtained after mainly short cDNA fragments. The failure to detect am- RT-PCR amplification of RNA extracted from pBRNX1.38- plification products of the equivalents of wild-type RNAs 5/6-gfp virus-infected cells with oligonucleotides A18 (ϩ) 2 through 5 (which were designated RNAs 2Ј through 5Ј; and 766 (Ϫ) suggested that cryptic leader-to-body fusion see below) in the RT-PCR samples derived from cells sites might be located within the GFP gene. To address infected with the pBRNX1.38-5/6-gfp virus would be com- this question, total cytoplasmic RNA extracted from patible with this hypothesis. (iii) The amplification of long mock-infected BHK-21 cells and from BHK-21 cells in- subgenomic mRNAs including the GFP transcript was fected with the first passage of the parental, pBRNX1.38, inhibited by the presence in the RNA preparation of a pBRNX1.38-5/6, or pBRNX1.38-5/6-gfp virus(es) was sep- large molar excess of smaller RT-PCR templates. The arated by agarose gel electrophoresis under denaturing fact that the RT-PCR samples derived from cells infected conditions and probed with the EAV-specific oligonucle- with the pBRNX1.38-5/6-gfp virus contained multiple otide 337 (Ϫ), which binds close to the extreme 3Ј ter- short DNA fragments in addition to the RNA 8Ј-specific minus of the viral genome and which should thus recog- amplification product was consistent with this idea. nize all EAV mRNAs. In a parallel experiment, oligonu- We therefore performed another RT-PCR in which the cleotide 768 (Ϫ) was used to specifically identify RNA downstream primer was replaced by the GFP gene- species carrying (the 5Ј part of) the GFP gene. Further- specific oligonucleotide 768 (Ϫ). Since this primer bound more, to facilitate interpretation of the results, the ex- close to the 5Ј end of the GFP gene the chance of pected sizes of the mRNAs without the poly(A) tail were missing the GFP RNA as a result of inefficient cDNA calculated for each of the viruses on the basis of the synthesis or preferential amplification of other templates leader-to-body junction sites identified earlier (de Vries was greatly reduced. With the new primer combination a et al., 1990, 2000; den Boon et al., 1996) (Table 1). As is DNA fragment of the anticipated length (344 nt) was evident from Fig. 4, both probes appeared to be highly amplified solely from total cytoplasmic RNA of cells that specific as neither of them bound to any of the RNA had been infected with the pBRNX1.38-5/6-gfp virus (Fig. species present in mock-infected cells while oligonucle- 3, lower right). The newly introduced leader-to-body fu- otide 768 (Ϫ) recognized only pBRNX1.38-5/6-gfp virus- 266 DE VRIES ET AL.

FIG. 4. In situ hybridization analysis of total cytoplasmic RNA purified from mock-infected BHK-21 cells or from BHK-21 cells infected with EAV Utr (par), pBRNX1.38 virus (clo), pBRNX1.38-5/6 virus (5/6), or pBRNX1.38-5/6-gfp virus (gfp1 and gfp2). Each of the recombinant virus stocks was prepared directly from RNA-transfected BHK-21 cells and the gfp1 and gfp2 viruses were derived from different transfection experiments. Total cytoplasmic RNA was extracted at 28 (par, clo), 40 (5/6), or 52 h p.i. (gfp1, gfp2) and separated in a denaturing agarose gel. After being washed and dried the gel was incubated with oligonucleotide 337 (Ϫ), which binds close to the extreme 3Ј end of the EAV RNAs, or with oligonucleotide 768 (Ϫ), which is specific for the 5Ј part of the GFP gene. The positions of the different EAV-specific RNA species and of the 28S and 18S ribosomal RNAs are indicated on the right sides. RNAs 1Ј through 5Ј of the pBRNX1.38-5/6-gfp virus are the functional homologs of RNAs 1 through 5 of the wild-type virus. Likewise, RNAs 8Ј and 9Ј of the vector virus correspond to RNAs 6 and 7 of the other viruses. The RNA expressing the GFP gene is denoted RNA 6Ј and the unforeseen ninth RNA species produced by the pBRNX1.38-5/6-gfp virus is designated RNA 7Ј. specific RNAs. After incubation with oligonucleotide 337 counterparts of RNAs 1 through 5. Indeed, the size dif- (Ϫ) (Fig. 4, left), the characteristic set of seven EAV- ference between the five largest RNAs from both sets specific intracellular mRNAs (RNAs 1 through 7; de Vries appeared to be approximately 0.9 kb, which is consistent et al., 1990) was clearly detected in the RNA preparation with the length of the expression cassette. The presence from cells infected with the parental virus. In addition, of RNA 7Ј was surprising as it could not be linked to any two bands were observed, which did not relate to any of the known viral mRNAs. The fact that this transcript particular viral mRNA but resulted from physical dis- hybridized both with oligo 337 (Ϫ) and with the leader- placement by the abundant 18S and 28S ribosomal specific oligonucleotide 441 (Ϫ) (data not shown) indi- RNAs. Very similar hybridization patterns were obtained cated that it did not represent a discrete degradation with the RNA samples extracted from cells infected with product of RNAs 1Ј through 6Ј. The hybridization exper- the parental virus, pBRNX1.38 virus, or pBRNX1.38-5/6 iment further showed that the pBRNX1.38-5/6-gfp virus- virus. In contrast, the pBRNX1.38-5/6-gfp virus directed infected BHK-21 cells contained relatively high amounts the synthesis of nine major mRNAs (RNAs 1Ј through 9Ј; of viral genomic RNA compared to BHK-21 cells infected Fig. 4, left). The second largest of these RNA species with the other viruses (Fig. 4, left). migrated only slightly faster than the 28S ribosomal RNA and may therefore be confused with displaced material. Ј Ј Multiple functional leader-to-body junction sites are RNAs 1 through 6 also hybridized to oligonucleotide located within the GFP gene 768 (Ϫ) (Fig. 4, right) and thus contained the 5Ј part of the GFP gene. On the basis of their electrophoretic mobili- The unexpected ninth RNA species (designated RNA ties and hybridization properties we concluded that 7Ј in Fig. 4) was recognized by oligonucleotide 337 (Ϫ) pBRNX1.38-5/6-gfp virus RNAs 9Ј and 8Ј corresponded to but not by oligonucleotide 768 (Ϫ), which implied that its RNAs 7 and 6 of the other viruses, respectively (Table 1). leader-to-body junction site was located within the GFP RNA 6Ј most likely represented the functional GFP mRNA gene. The size of RNA 7Ј further suggested that leader- as it roughly comigrated with RNA 5 of the parental virus to-body fusion had taken place in the 3Ј half of the GFP (Table 1) and was the shortest RNA recognized by oligo- gene. To confirm this hypothesis total cytoplasmic RNA nucleotide 768 (Ϫ). Their apparent molecular masses extracted from BHK-21 cells infected with pBRNX1.38-5/ suggested that RNAs 1Ј through 5Ј were the actual 6-gfp passage 1 virus was subjected to RT-PCR using the REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 267 leader-specific oligonucleotide A18 (ϩ) as the upstream pBRN1.38, pBRNX1.38-5/6, or pBRNX1.38-5/6-gfp virus- Ϫ primer and oligonucleotide 837 ( ), which binds to the (es). The m.o.i. used was approximately 2 TCID50 units extreme 3Ј end of the GFP gene, as the downstream per cell for the viruses lacking heterologous genes but primer. Analysis of the RT-PCR products by agarose gel was somewhat lower for the GFP-expressing viruses due electrophoresis revealed that five different DNA frag- to an initial overestimation of their infectious titers. Next, ments of between 0.3 and 0.5 kb had been generated as the cell cultures were metabolically labeled for 1 h with well as low amounts of some larger amplification prod- 35S-labeled amino acids at 24, 36, and 48 h p.i. and total ucts. After removal of the PCR primers by gel filtration, lysates of cells and media were prepared (de Vries et al., the DNA fragments were directly ligated into plasmid 1995). Aliquots of these lysates were then incubated with pNoTA/T7. Recombinant plasmids were selected by re- a polyclonal antiserum specific for GFP or with a cocktail striction enzyme analysis and 16 constructs carrying of antibodies directed against four of the EAV structural inserts of different sizes were subjected to nucleotide proteins. As is shown in Fig. 6 (right), a polypeptide of 29 sequence analysis. The nucleotide sequence of one of kDa was immunoprecipitated with the anti-GFP serum the inserts (No. 17) did not show significant identity with from the lysates of pBRNX1.38-5/6-gfp virus-infected any part of the EAV genome, suggesting that host cell cells. The molecular weight of this protein is in good RNA had served as the template for this RT-PCR frag- agreement with the predicted size of GFP (217 amino ment. Two plasmids (Nos. 02 and 09) contained only 5Ј acids; 26.9 kDa). Moreover, as a protein of the same size EAV-specific sequences probably as a result of errone- was not detected in the control samples we concluded ous attachment of the downstream primer (Fig. 5A). The that the 29-kDa polypeptide represented full-length GFP. other inserts comprised both EAV leader- and GFP-spe- In addition, trace amounts of the EAV N protein were cific sequences and thus represented subgenomic viral nonspecifically immunoprecipitated from the lysates of RNAs generated by discontinuous transcription. On the virus-infected cell cultures as has been documented basis of their leader-to-body junction sites six different before (see, e.g., de Vries et al., 2000). Importantly, no RNA species were identified. The largest of these tran- other proteins were observed in the lower part of the gel scripts (represented by plasmid 15) corresponded to the including the dye front, which indicated that translation of regular GFP RNA, as the fusion of leader and body RNA 7Ј did not yield N-terminally truncated GFP mole- sequences had occurred at the UCAACC box (located in cules unless these were not recognized by the GFP- the 3Ј end of ORF 5) that is normally used to produce EAV specific polyclonal antibodies. RNA 6 (Fig. 5A). The other mRNAs (designated RNAs 7.1Ј The results obtained with the antibody cocktail through 7.5Ј) were the result of leader-to-body joining at showed that each of the viruses that were analyzed five adjacent sites located within the 3Ј half of the GFP directed the synthesis of the N, M, GL, and GS proteins gene. The size difference between the largest and the (Fig. 6, left). For the parental and pBRNX1.38 virus, the smallest of these RNA molecules was only 163 nt, which highest protein synthesis levels were reached 24 h after explains why they were not resolved during gel electro- infection, whereas for the pBRNX1.38-5/6 virus structural phoresis (Fig. 4). As is shown in Fig. 5B, the fusion of protein production peaked at 36 h p.i. (data not shown) leader and body sequences did not occur at random and thus clearly lagged behind. In the pBRNX1.38-5/6-gfp positions within the GFP gene, but required the presence virus-infected cells the highest rate of structural protein of hexanucleotide sequences similar or identical to the synthesis was observed at around 48 h p.i. Moreover, the UCAAC(U/C) motif involved in the production of genuine pBRNX1.38-5/6-gfp virus appeared to produce relatively EAV mRNAs. small amounts of the EAV GL protein compared to the other viruses at each of the investigated time points (data not shown). It should be noted, however, that the immu- RNA 7Ј codes for an extended version of the EAV M noprecipitation analysis may not have been completely protein quantitative. Unexpectedly, a 22-kDa polypeptide was Translation of arteriviral mRNAs usually initiates at the detected in the lysates of pBRNX1.38-5/6-gfp virus-in- most 5Ј-proximal AUG codon downstream of the leader- fected cells but not in protein extracts obtained from to-body fusion site that is in a favorable context for cells infected with the other viruses. Additional immuno- translation initiation to occur. Accordingly, the unin- precipitation experiments demonstrated that this protein tended pBRNX1.38-5/6-gfp virus-specific transcripts col- species was recognized exclusively by the EAV M-spe- lectively denoted RNA 7Ј may give rise to N-terminally cific antibodies (data not shown). A close inspection of truncated GFP molecules (Fig. 5C). To investigate this the genomic region comprising the RNA 7Ј and 8Ј leader- possibility and to study whether the insertion of the GFP to-body fusion sites revealed the presence of an in-frame gene and/or the duplication of the EAV RNA 7 leader-to- AUG codon 165 nt upstream of the start codon of the EAV body junction sequence influenced normal viral protein M protein (Fig. 5C). Translation initiation at this AUG synthesis, BHK-21 cells were either mock infected or codon would result in the synthesis of a 217-residue infected with the parental virus or with passage 1 of the M-related polypeptide with a predicted molecular mass 268 DE VRIES ET AL. REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 269

quence within such a quasi-species is the product of the permanent competition between viruses with slightly dif- ferent genotypes. In contrast, genetically engineered vi- ral genomes are not shaped by natural selection and may thus give rise to viruses of relatively low fitness. As is evident from Figs. 4 and 6, the relative expression levels of the EAV-specific RNAs and virion proteins were clearly altered due to the incorporation of the GFP gene into the EAV genome. To investigate the consequences of these changes for the fitness of the recombinant viruses, the supernatants of two different cell cultures transfected with pBRNX1.38-5/6-gfp RNA were passaged three times in BHK-21 cells. After each round of virus amplification, total cytoplasmic RNA was extracted from FIG. 6. Immunoprecipitation analysis of EAV structural protein syn- thesis and of GFP gene expression in mock-infected BHK-21 cells or in these cells and subjected to RT-PCR using oligonucleo- BHK-21 cells infected with EAV UTR (par), pBRNX1.38 virus (clo), tides 750 (ϩ) and 766 (Ϫ). For comparison, RNA samples pBRNX1.38-5/6 virus (5/6), or pBRNX1.38-5/6-gfp virus (gfp1 and gfp2). obtained from BHK-21 cells infected with different pas- Each of the recombinant virus stocks was prepared directly from sages of the pBRNX1.38-5/6 virus were analyzed in par- RNA-transfected BHK-21 cells and the gfp1 and gfp2 viruses were allel (Fig. 7). RT-PCR amplification of viral RNA from cells derived from different transfection experiments. Mock- or virus-infected cells were pulse-labeled for 30 min with 35S-labeled amino acids at 24 infected with passage 1 of the GFP-expressing viruses (par, clo), 36 (mock, 5/6), or 48 (gfp1, gfp2) h p.i. Total lysates of cells and yielded a prominent DNA fragment of 1.2 kb. A PCR media were then prepared and mixed with a cocktail of monospecific product of identical size was obtained using pBRNX1.38- antibodies directed against the EAV M, N, GL, and GS proteins (left) or 5/6-gfp DNA as a template, which indicates the presence with a polyclonal rabbit antiserum recognizing GFP (right). The posi- of an intact GFP gene in virtually all the immediate tions of the N, M, GL, and GS proteins of EAV and of an N-terminally extended version of the EAV M protein (Mϩ) are indicated on the right progeny of the vector viruses. After further passage of side of the left panel. On the right, the protein band corresponding to the pBRNX1.38-5/6-gfp viruses, smaller RT-PCR products GFP is marked. The numbers on the left sides indicate positions and became apparent in addition to the 1.2-kb DNA fragment. 14 sizes (in kDa) of C-labeled marker proteins analyzed in the same gel. In contrast, a single DNA fragment of 0.3 kb was ob- tained for the pBRNX1.38-5/6 virus irrespective of the of 23.7 kDa. We therefore concluded that translation of viral passage number. It thus seems that the pBRNX1.38- RNA 7Ј yielded an N-terminally extended version of the 5/6 virus is stable, whereas the recombinant virus vector EAV M protein. Although RNA 7Ј might specify additional was gradually outgrown by GFP gene deletion mutants. polypeptides these were not detected with the M- and Consistently, the proportion of cells coexpressing EAV GFP-specific antisera. structural protein genes and the GFP gene rapidly de- clined after serial passage (data not shown). Interest- ingly, the sizes and relative amounts of the shorter am- The pBRNX1.38-5/6-gfp virus is genetically unstable plification products differed for both series of pBRNX1.38- As a result of the inaccuracy of viral RNA polymerases, 5/6-gfp virus passages. We therefore concluded that the genomes of RNA viruses consist of a collection of different genotypic changes can increase the fitness of closely related sequences. The prevailing genomic se- the vector viruses.

FIG. 5. (A) Overview of the pBRNX1.38-5/6-gfp virus-specific cDNA clones generated by RT-PCR with primers A18 (ϩ) and 837 (Ϫ). cDNA clones 02 and 09 are colinear with the 5Ј end of the viral genome; the other cDNA clones are the result of leader-to-body fusion events that occurred immediately upstream of or within the GFP gene. The small black triangles symbolize authentic EAV transcription-regulating sequences, the small white triangles correspond to leader-to-body fusion sites identified in the 3Ј half of the GFP gene. Leader sequences are shown in gray and functional open reading frames have been given a name (GFP) or a number (1a, 5, 6, and 7). (B) Nucleotide sequence of the leader-to-body junction regions of the RNA 7Ј subspecies. The cDNA clones used to determine the junction sequences of these EAV-specific RNAs were 13 for RNA 7.1Ј;06and19 for RNA 7.2Ј; 03, 20, and 21 for RNA 7.3Ј; 16 for RNA 7.4Ј; and 11, 12, 18, 23, and 24 for RNA 7.5Ј. The sequences are aligned with the most 5Ј-proximal UCAAC(U/C) box on the EAV genome, including 20 nt at its 5Ј and 3Ј end (5Ј Leader), and with the genomic sequences surrounding the transcription-regulating sequences (Genome). For comparison, the leader-to-body junction sequences of RNAs 6Ј and 8Ј are also included. In each case, nucleotides from the leader and genome sequences that unambiguously contribute to a specific sgRNA are shaded. The overall junction region consensus sequence is shown at the bottom with the most common nucleotides displayed in the middle line. (C) Sequence analysis of the 3Ј half of the GFP gene. cDNA sequences as well as deduced amino acid sequences are indicated and methionine residues are boxed. Please note that the EAV M protein (which starts with the sequence MGAID) is encoded in frame 1 and that GFP is encoded in frame 2. The amino acid sequence that is duplicated in the extended version of the M protein (which starts with residues MAWTS) is shaded and leader-to-body junction sequences located within and immediately downstream of the GFP gene are marked by single and double lines, respectively. 270 DE VRIES ET AL.

of ORF 2b to achieve its expression from a modified subgenomic mRNA 2. As a result of the concomitant disruption of ORFs 2a and 2b the expression vector did not direct the synthesis of infectious progeny virus. In the second construct, the CAT gene was inserted immedi- ately upstream of ORF 7 to accomplish its expression from subgenomic mRNA 7. Using this approach, ORF 6 was disrupted while the N gene was silenced, which again precluded the formation of infectious virus parti- cles. We therefore chose an alternative vector design in which the heterologous gene was expressed from an additional subgenomic mRNA without inactivating any of the EAV genes (Fig. 1B, bottom). Consequently, the FIG. 7. RT-PCR analysis of total cytoplasmic RNA extracted from BHK-21 cells infected with pBRNX1.38-5/6 virus (5/6) or pBRNX1.38-5/ pBRNX1.38-5/6-gfp virus described in this report is the 6-gfp virus (gfp1 and gfp2) of different passage number (lanes 1, 2, and first example of a replication-proficient arterivirus vector. 3) using an ORF 5-specific sense primer [oligonucleotide 750 (ϩ)] and The genome of EAV contains 12 UCAAC(U/C) motifs, 6 an ORF 6-specific antisense primer [oligonucleotide 766 (Ϫ)]. In lanes of which have been unambiguously shown to direct the 1, RNA extracted from BHK-21 cells that had been inoculated with virus synthesis of viral subgenomic mRNAs (de Vries et al., obtained from transfected cells was analyzed. To create internal stan- dards, the same primer pair was used for the PCR amplification of 1990; den Boon et al., 1996). The remaining UCAAC(U/C) plasmids pBRNX1.38-5/6 and pBRNX1.38-5/6-gfp (DNA lanes). The num- sequences are either nonfunctional or give rise to unde- bers on the right refer to anticipated sizes of specific amplification tectably low amounts of EAV transcripts and there are no products (see also Fig. 1B). On the left the positions and sizes of indications for the use of transcription signals that devi- marker DNA fragments (obtained by HinfI digestion of pGEX-2T DNA) ate from the hexanucleotide consensus sequence apart are indicated. from those involved in the synthesis of minor species of EAV RNAs 3 and 5 (den Boon et al., 1996; de Vries et al., 2000). It thus seems surprising that the 3Ј half of the GFP DISCUSSION gene, which contains only 1 perfect UCAAC(U/C) motif, In this paper we have documented the construction of directs the synthesis of at least 5 different subgenomic a replication-competent arterivirus expressing the GFP mRNAs. A similar phenomenon has been described by gene via an extra subgenomic mRNA. Although the re- Fischer et al. (1997) after the replacement of gene 4 of combinant virus vector appeared to be severely impaired MHV by a copy of the GFP gene. In addition to the compared to wild-type EAV, it directed the synthesis of authentic GFP transcript in which the joining of leader significant amounts of GFP as evidenced by light micros- and body sequences had occurred at the intergenic copy and immunoprecipitation analysis. An unexpected sequence in front of gene 4, 11 RNA species that had consequence of the insertion of the GFP expression their leader-to-body fusion sites located in the 3Ј half of cassette into the EAV genome was the synthesis of a the GFP gene were found. For EAV the junction of leader series of subgenomic mRNAs which had their leader-to- and body sequences was observed solely at positions in body fusion sites located in the 3Ј half of the foreign the GFP gene containing at least four of six nucleotides gene. Translation of at least some of these transcripts of the consensus transcription signal (Fig. 5B) while for yielded an extended version of the EAV M protein. We MHV the formation of the aberrant GFP-related tran- further showed that repeated passage of the vector virus scripts did not require a substantial similarity between resulted in the gradual loss of foreign gene expression the genomic site at which leader-to-body fusion took due to the appearance of viral deletion mutants. place and the intergenic consensus sequence. Since the Recently two other studies reported the use of nidovi- anomalous leader-to-body fusion events occurred in a ruses for the expression of foreign genes. In one study, region of the MHV genome where the mRNA 5 consen- the nonessential gene 4 of mouse hepatitis virus (MHV; sus transcription signal would normally be located, genus Coronavirus) was replaced by the GFP gene using Fischer et al. (1997) proposed that discontinuous tran- homologous recombination (Fischer et al., 1997). The scription could sometimes depend solely on the struc- recombinant virus generated in this manner directed the ture of the viral genome which in turn would be deter- synthesis of an MHV mRNA 4 equivalent specifying GFP mined by long-range RNA–RNA and/or RNA–protein in- instead of the ns4 protein. The GFP gene was, however, teractions. Alignment of the genomes of the wild-type poorly expressed and its product could be detected only and the pBRNX1.38-5/6-gfp virus from either their 3Ј or by Western blotting. In another study, van Dinten et al. their 5Ј end (Fig. 5A) indicated that a similar situation (1997) inserted the gene for chloramphenicol acetyltrans- does not apply to EAV. We therefore favor the hypothesis ferase (CAT) at two different positions in the EAV ge- that the GFP gene contains sequence elements that nome. In the first construct, the CAT gene replaced part promote leader-to-body fusion. Because the poly- REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 271 merases of the distantly related arteri- and coronavi- initiation codons to the translation machinery (Fig. 5C). ruses are unlikely to recognize the same primary RNA Several of these AUG codons are in frame with ORF 6 sequences, the signal inducing the formation of the ab- and are in a reasonably favorable context for translation errant transcripts probably resides in a secondary RNA initiation to occur. The most 3Ј of the in-frame AUGs structure. The difficulty in obtaining RNA 1Ј- through would give rise to a 217-aa protein with a predicted 6Ј-specific RT-PCR fragments when using oligonucleo- molecular weight of 23.7 kDa, which is close to the tide 766 (Ϫ) as the primer for cDNA synthesis may estimated size (22 kDa) of the extended version of the M indeed indicate that the 3Ј half of the GFP gene is highly protein displayed in Fig. 6. In view of the high abundance structured. It is thus conceivable that the anomalous of RNA 7Ј in pBRNX1.38-5/6-gfp virus-infected cells, it formation of subgenomic mRNAs is not a general con- may seem surprising that the M-related fusion protein is sequence of the insertion of foreign genetic information synthesized in such low amounts. However, as is shown into the EAV genome, but relates to a specific property of in Fig. 5C, the ribosomes have to read through two to four the GFP gene. Further evidence for this hypothesis might termination signals (depending on the RNA 7Ј subspe- be obtained by analysis of the transcripts produced by cies) before reaching the proper start codon. Thus, un- recombinant viruses in which other heterologous genes less the M-related fusion protein is the unlikely result of have been incorporated or in which different parts of the internal translation initiation, its presence in pBRNX1.38- GFP ORF have been deleted. Interestingly, a recent study 5/6-gfp virus-infected cells implies that (some of) these of the effects of flanking sequences on the utilization of stop codons are poor translation terminators. a specific leader-to-body junction sequence in MHV also The synthesis in pBRNX1.38-5/6-gfp virus-infected pointed to an important role of RNA secondary structures cells of an N-terminally extended version of the EAV M in the regulation of Nidovirales transcription (An and protein may provide an explanation for the poor growth Makino, 1998). Finally, the nucleotide sequence analysis characteristics of the recombinant virus vector. Since the of the RT-PCR products of RNAs 7.1Ј and 7.5Ј (Fig. 5B) M-related fusion protein contains two copies of the ORF demonstrated that these unforeseen transcripts had ob- 6-specific amino acids 97 through 136 (Figs. 1B, bottom, tained their entire leader-to-body junction sequence from and 5C) it might inhibit virion assembly or interfere with the GFP gene. These results are most consistent with a virus infectivity through nonproductive interactions with model for discontinuous RNA synthesis in which the other EAV components. However, given the low relative hexanucleotide consensus sequences in the 3Ј part of abundance of the M-related fusion protein such effects the viral genome serve as transcription terminators and cannot be explained by direct competition between func- the nascent subgenomic negative-stranded RNA mole- tional (i.e., wildtype) and nonfunctional (i.e., extended) M cules are translocated to the 5Ј end of the viral genome molecules. Alternatively, genomic size constraints im- where they interact with the UCAACU box upstream of posed by the nucleocapsid structure may account for the the polymerase gene thereby acquiring an anti-leader low (genetic) stability of the recombinant virus. However, sequence (van Marle et al., 1999). However, we cannot this possibility seems unlikely since insertion of the ex- exclude the possibility that the presence of GFP se- pression cassette added no more than 885 nt to the EAV quences at the leader-to-body fusion sites of EAV RNAs genome, while SHFV can accommodate 3 kb of addi- 7.1Ј,7.4Ј, and 7.5Ј is the result of trimming of leader tional sequence information compared to EAV by using a transcripts attached to the genomic minus strand by a nucleocapsid protein which contains only one extra 3Ј 3 5Ј nuclease activity associated with the viral tran- amino acid (Godeny et al., 1995). Moreover, the SHFV scriptase (van der Most et al., 1994). In this context, it is genome contains three additional ORFs, in comparison interesting to note that Mizutani et al. (2000) recently to the genomes of the other arteriviruses, which most obtained strong evidence for a transcription mechanism likely have been acquired during a single recombination of coronaviruses in which subgenomic mRNAs are syn- event that did not involve any changes to the nucleocap- thesized by discontinuous plus-strand synthesis involv- sid protein gene (Godeny et al., 1998). Another reason for ing binding of positive-stranded leader transcripts to the the reduced fitness of the recombinant virus vector could various leader-to-body junction regions on the full-length be that the insertion of the GFP module influenced over- anti-genome. all transcription and/or replication rates. The presence of Our immunoprecipitation analyses revealed that relatively high amounts of viral genomic RNA in pBRNX1.38-5/6-gfp virus-infected cells contained an ex- pBRNX1.38-5/6-gfp virus-infected cells compared to cells tended version of the EAV M protein. The presence of infected with the other viruses (Fig. 4) would be consis- these unusual molecules most likely is a consequence of tent with the idea that the insertion of the expression the formation of the aberrant GFP-related mRNAs. cassette has an effect on the ratio between genomic and Whereas RNA 6 is generated by a leader-to-body fusion subgenomic RNA synthesis. Finally, the introduction of event that positions the start codon of the M gene im- the GFP gene and a second copy of the RNA 7 leader- mediately downstream of the leader sequence, upstream to-body fusion site into the viral genome could have joining of leader and body segments exposes additional altered the relative expression levels of the EAV sub- 272 DE VRIES ET AL. genomic mRNAs. As this would result in suboptimal the successful expression of a MHV M protein-specific molar ratios of the EAV structural proteins it may have a nonapeptide as an integral part of the ectodomain of the profound effect on viral fitness. Indeed, Fig. 6 suggests EAV M protein (de Vries et al., 2000). Another possibility that in pBRNX1.38-5/6-gfp virus-infected cells less GL would be to replace EAV (structural protein) genes by protein is present relative to the N, M, or GS protein than foreign genetic information and to provide the missing in cells infected with wild-type EAV. Interestingly, from the viral polypeptides in trans by using either packaging cell same experiment it appeared that the changes to the 5Ј lines or (defective) helper constructs. noncoding region of RNA 6 did not have a clear effect on the relative expression level of the EAV M protein. MATERIALS AND METHODS As comparatively little is known about the regulation of arterivirus transcription and replication, the precise rea- Antisera, cells, and viruses son for the observed changes in the viral RNA profile Cells were cultured in a humidified air–5% CO atmo- caused by the insertion of the expression cassette is 2 sphere. The Utrecht variant (de Vries et al., 1990) of the unclear. From studies on coronaviruses it appeared that Bucyrus strain (Doll et al., 1957) of EAV (EAV Utr) and the primary sequence of the leader-to-body junction mo- engineered mutants thereof were propagated in baby tifs is not the only factor involved in transcriptional reg- hamster kidney [BHK-21 (C-13)] cells (American Type ulation. Other factors that have been shown to influence Culture Collection) maintained in Glasgow minimum es- their activity include: (i) the specific position in the viral sential medium (GMEM; Gibco BRL Life Technologies) genome (Hiscox et al., 1995; van Marle et al., 1995), (ii) containing 10% fetal bovine serum (FBS), 100 U/ml pen- the number of downstream leader-to-body fusion sites icillin, and 100 ␮g/ml streptomycin (GMEM–10% FBS). (van Marle et al., 1995; Krishnan et al., 1996), (iii) the Infectious titers of EAV stocks were determined by distance between functional leader-to-body junction se- TCID assays according to Hyllseth (1969) using rabbit quences (Joo and Makino, 1995; van Marle et al., 1995), 50 kidney (RK-13) cells grown in Dulbecco’s modified Ea- and (iv) the nucleotides flanking the leader-to-body fu- gle’s medium (DMEM; Gibco BRL Life Technologies) with sion sites (Makino and Joo, 1993; Hiscox et al., 1995; 10% FBS and the aforementioned antibiotics. Jeong et al., 1996; An and Makino, 1998). In addition, The production and characterization of rabbit anti- sequence elements located at the 5Ј and 3Ј terminus of peptide sera directed against the C-termini of the EAV M the coronavirus genome appear to influence transcrip- and G proteins have been documented previously (de tion levels while coronavirus replication seems to be S Vries et al., 1992). The EAV G -specific murine monoclo- primarily controlled by 5Ј and 3Ј end genomic sequences L nal antibodies (MAbs) 74D(B) and 93B were generated by (reviewed by Lai and Cavanagh, 1997). Since in arterivi- Glaser et al. (1995). The preparation of a rabbit antiserum ruses replication and transcription efficiencies may be directed against an EAV N-specific bacterial fusion pro- determined by a similarly complex interplay of signals tein has been described elsewhere (Weiland et al., 2000). located at different positions on the viral genome, it will An affinity-purified rabbit polyclonal antiserum raised be hard to predict the effects of any genomic alteration against a red-shifted variant of GFP was purchased from on these processes. Clontech. If expression of heterologous ORFs from extra sub- genomic mRNAs in the context of a complete genome RNA purification would turn out to be troublesome irrespective of the genomic locus used for insertion, the number of dupli- Total cytoplasmic RNA was extracted from 20-cm2 cated EAV-specific nucleotides, or the identity of the (sub)confluent monolayers of mock- or EAV-infected foreign gene, other strategies could be pursued to con- BHK-21 cells. To this end, the cells were washed once vert EAV into a viral expression vector. One approach with ice-cold phosphate-buffered saline (PBS) and lysed would be to incorporate heterologous genes and flank- in 500 ␮l of 20 mM Tris–HCl (pH 7.4)–150 mM NaCl–1 ing sequences encoding cleavage sites for one of the mM EDTA containing 0.5% Triton X-100 and 0.5% 1,5- viral proteases at suitable positions in EAV ORF 1. This naphthalenedisulfonic acid (disodium salt) at 0°C. The would result in the expression of the foreign genetic lysate was centrifuged for 10 min at 2000 g and 4°C and information as part of the ORF replicase polyproteins and the postnuclear supernatant was mixed with 500 ␮lof20 the release of the heterologous gene product(s) during mM Tris–HCl (pH 7.4)–150 mM NaCl–1 mM EDTA con- proteolytic processing of the polyprotein precursors. Al- taining 7 M urea and 2% sodium dodecyl sulfate (SDS). ternatively, internal ribosomal entry segments could be The mixture was extracted once with an equal volume of utilized to generate multicistronic mRNAs that direct the buffer-saturated phenol, three times with phenol:chloro- synthesis of a viral protein and one or more foreign gene form:isoamyl alcohol (25:24:1; Sambrook et al., 1989), and products. Furthermore, heterologous sequences could once with water-saturated ether at room temperature. be expressed as an integral part of one or more of the The RNA was precipitated by the addition of 100 ␮lof3 viral (structural) proteins. Indeed, we recently reported M sodium acetate (pH 5.2) and 2.5 ml of ice-cold 96% REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 273

TABLE 2 Oligonucleotides

Namea Sequenceb Positionc Use

A18 (ϩ) GCTCGAAGTGTGTATGGTGC 1–20 Analytical PCR; PCR to amplify RNA 7Ј subspecies 337 (Ϫ) CTTCAACATGACGCCACACAGGAG 12658–12681 Hybridization analysis 441 (Ϫ) CCGAGAGGGGCCCACAAG 63–80 Hybridization analysis 724 (ϩ) AGGGTACCATCGCCAACTGGTGG 12187–12204 PCR to amplify RNA 7 leader-to-body junction sequence 725 (Ϫ) AGGGTACCTTAAGCTTGTTACCAACTGCG 12290–12308 RT-PCR to amplify RNA 7 leader-to-body junction sequence 750 (ϩ) CGGGATCCTCCGCCAATTACTGTGGTT 11684–11705 Analytical PCR 766 (Ϫ) CCAGATGCTACATACCTAGTA 11990–12010 Analytical RT-PCR 768 (Ϫ) ccctctccgctgacagaaa * Analytical RT-PCR; hybridization analysis 837 (Ϫ) CGGAATTcacttgtacagctcgtcca * RT-PCR to amplify RNA 7Ј subspecies

a Positive-sense primers are marked (ϩ); negative-sense primers are symbolized by (Ϫ). b Nucleotides not specific for the EAV genome or the GFP gene are singly underlined, GFP-specific nucleotides are shown in lowercase. c The positions of the virus-specific sequences encoded by individual primers are based on the complete genomic sequence of EAV Utr (den Boon et al., 1991; van Dinten et al., 1997; Glaser et al., 1999).

ethanol, washed with 70% ethanol, and dissolved in 100 cDNA synthesis and PCR amplification ␮ lofH2O. Total cytoplasmic RNA (10.5 ␮l) from mock- or EAV- infected BHK-21 cells was mixed with 1 ␮lof50␮M RNA gel electrophoresis and in situ hybridization reverse transcription primer, incubated for 10 min at analysis 65°C, and immediately cooled on ice. Next, 4 ␮lof250 Gel electrophoresis of RNA under denaturing condi- mM Tris–HCl (pH 8.3 at 25°C)–200 mM KCl–25 mM ␮ ␮ tions was carried out in a 2.2 M formaldehyde–1.5% MgCl2–2.5% Tween 20, 2 l of 100 mM DTT, 0.8 lof25 agarose gel using 10 mM 3-(N-morpholino)propanesul- mM each dNTP, 0.7 ␮l of RNAguard (33.4 U/␮l; Amer- fonic acid (sodium salt)–5 mM sodium acetate–1 mM sham Pharmacia Biotech), and 1 ␮l of Expand reverse EDTA as electrode buffer. Before application to the gel, 6 transcriptase (50 U/␮l; Boehringer Mannheim) were ␮l of each RNA sample was mixed with 14 ␮l of dena- added and the sample was incubated for1hat42°C. turation buffer (10.3% formaldehyde, 69.4% formamide, The reaction was then stopped on ice or the template 1.4ϫ electrode buffer), incubated at 70°C for 10 min, and RNA was first degraded by RNase H treatment (de supplemented with 4 ␮l of loading buffer (15% Ficoll 400, Vries et al., 2000) and the sample was frozen at 0.3% Orange G). The gel was run for 16 h at 50 V and Ϫ20°C. room temperature while the electrode buffer was recir- PCR amplifications were routinely performed in 50-␮l culated beginning 30 min after the start of the electro- volumes using thin-walled 0.5-ml vials. A typical reaction phoresis. Next, the gel was dried on Whatman 3MM filter mixture contained 1 ng of (linearized) plasmid DNA or 0.2 paper, divided in two equal halves, detached from the to 2 ␮l of cDNA, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 2 ␮ paper support, and prehybridized for 30 min at room mM MgCl2, 200 to 400 M each dNTP, 500 mM both temperature in binding buffer [5ϫ SSPE, 5ϫ Denhardt, forward and reverse primers, and 1.25 to 2.5 U of Ther- 0.05% SDS, 0.01% alkali-degraded yeast RNA (free acid) mus aquaticus DNA polymerase (Gibco BRL Life Tech- (homomix I; Jay et al., 1974)]. One piece of the gel was nologies). The samples were prepared on ice, overlaid then incubated overnight at 55°C with 100 ng of oligo- with 25 ␮l of mineral oil, and placed in a preheated nucleotide 337 (Ϫ) (Table 2) in binding buffer. The other thermocycler (Perkin–Elmer) at 94°C for 2 min. The target half of the gel was probed under the same conditions sequences were amplified in 25 cycles. Each round of with 100 ng of oligonucleotide 768 (Ϫ) (Table 2). Before amplification consisted of a denaturation step at 94°C for addition to the hybridization solution, the oligonucleo- 30 s, followed by an annealing period of 10 to 15 s at 50 tides were radiolabeled at their 5Ј ends using bacterio- to 55°C, depending on the specific primer pair used, and phage T4 polynucleotide kinase and [␥-32P]ATP (Ϯ3000 an elongation step at 72°C for 1 min with an extension Ci/mmol; Amersham Pharmacia Biotech). The gel pieces period of between 0 and 3 s per cycle. The resulting DNA were subsequently washed three times for 15 min at the fragments were analyzed in 1 to 2% agarose gels con- hybridization temperature with 5ϫ SSPE–0.05% SDS, taining 1 ␮g/ml ethidium bromide in the gel solution and covered with plastic foil, and exposed at Ϫ70°C to 100 ng/␮l intercalator in the electrode buffer. In one case, BioMax MS film (Eastman Kodak) sandwiched between a the PCR products were cloned into the pNoTA/T7 vector BioMax TranScreen from the same company. using the Prime PCR Cloner kit (5 Prime 3 3 Prime). 274 DE VRIES ET AL.

Construction of plasmid pBRNX1.38-5/6-gfp RNA transcription Recombinant DNA procedures were either adopted In vitro transcription reactions were performed essen- from Ausubel et al. (1987) or Sambrook et al. (1989) or tially as described by Olkkonen et al. (1994) except that performed according to the instructions supplied with G(5Ј)ppp(5Ј)G (Amersham Pharmacia Biotech) instead of specific reagents. Escherichia coli strain PC2495 m7G(5Ј)ppp(5Ј)G served as cap analogue while XhoI was (Phabagen) was used for all cloning steps and DNA was used to linearize the DNA templates. The reactions were transfected into these bacteria by high-voltage treatment stopped on ice, the integrity and yield of the RNA prod- using a Gene Pulser II (Bio-Rad) electroporation appara- ucts was determined by agarose gel electrophoresis tus and electroporation cuvettes with a 2-mm electrode under native conditions, and the samples were tran- Ϫ gap (BTX). The generation of the full-length EAV cDNA siently stored at 20°C. When indicated the plasmid clone pBRNX1.38 (Fig. 1B, top) and its derivative DNA was degraded after completion of the RNA synthe- pBRNX1.38-5/6 in which ORFs 5 and 6 are separated by sis by a treatment for 30 min at 37°C with1UofRQ1 a unique AflII restriction enzyme recognition site (Fig. 1B, RNase-free DNase (Promega) and the nuclease was middle) has been described elsewhere (de Vries et al., inactivated by a 10-min incubation at 65°C. 2000). To construct the EAV-based expression vector pBRNX1.38-5/6-gfp, the “humanized” gene for a GFP vari- Infection and transfection experiments ant with enhanced specific fluorescence was excised BHK-21 cells were transfected with EAV-specific RNA from plasmid pGreen Lantern-1 (Gibco BRL Life Technol- (and cDNA) by electroporation (Olkkonen et al., 1994). ogies) by using NotI and ligated into pUCBM20 (Boehr- Briefly, 80-cm2 monolayers of newly confluent cells were inger Mannheim). For this purpose, the cloning vector trypsinized, suspended in 10 ml of GMEM–10% FBS, and had been digested with the same restriction endonucle- pelleted at 400 g for 5 min. The cells were washed in 10 ase and dephosphorylated with shrimp alkaline phos- ml PBS without CaCl2 and MgCl2, repelleted, and resus- phatase (Amersham Pharmacia Biotech). Recombinant pended in 800 ␮l PBS. One-fifth to two-fifths of the tran- plasmids in which the GFP gene was flanked at its 5Ј scription reaction was then added to the cell suspension end by the AflII site of the vector were identified by and the mixture was transferred to electroporation cu- restriction enzyme analysis. One of these constructs vettes with a 4-mm gap size (BTX). The cells were pulsed (pAVI-gfp) was incubated with KpnI and dephosphory- twice at 800 V, 25 ␮F and, with the pulse controller set at lated using shrimp alkaline phosphatase. Subsequently, infinite, resuspended in 20 ml of GMEM–10% FBS and a 127-nt EAV-specific PCR product containing the RNA 7 seeded into 185-cm2 flasks and onto glass coverslips. leader-to-body junction sequence in the middle was li- After transfection, the cells were transferred to a CO2 gated into the KpnI-digested plasmid. The latter frag- incubator of 37°C. ment, which corresponds to nucleotide positions 12187 to 12308 of the genome of EAV Utr, was generated using Confocal laser scanning microscopy oligonucleotides 724 (ϩ) and 725 (Ϫ) as primers (Table Mock-transfected BHK-21 cells and BHK-21 cells trans- 2) and EAV cDNA clone PB106 (de Vries et al., 1990) as fected with pBRNX1.38, pBRNX1.38-5/6, or pBRNX1.38- template. Before insertion into pAVI-gfp, the amplification 5/6-gfp RNA were seeded on glass coverslips and fixed product was purified from gel with the aid of the QIA- at different time points after electroporation with 3% quick gel extraction kit (Qiagen) and cut with KpnI. A paraformaldehyde in PBS for 20 min at ambient temper- number of plasmid DNA clones were subjected to nu- ature. Before addition of the fixative, the cells were briefly cleotide sequence analysis using a T7 DNA polymerase washed twice with PBS at room temperature. The para- ␣ 33 Ն sequencing kit and [ - P]dATP ( 2500 Ci/mmol; Amer- formaldehyde was inactivated by three 5-min washes sham Pharmacia Biotech), and one of the constructs with PBS containing 10 mM glycine (PBS–Gly) and the containing an intact and properly oriented copy of the cells were permeabilized for 5 min with 0.5% Triton X-100 EAV RNA 7 leader-to-body fusion site was incubated with in PBS–Gly containing 5% FBS (PBS–Gly–FBS). Following BfrI. This yielded an 868-nt fragment which was ligated three 5-min washes with PBS–Gly–FBS, the cells were into the AflII site of plasmid pAVIs5/6 (de Vries et al., incubated for 30 min at ambient temperature with the 2000) to produce pAVIs5/6-gfp. In the latter vector, the EAV GL-specific MAb 74D(B) in hybridoma culture super- GFP gene is preceded at its 5Ј end by the 3Ј terminus of natant. The cells were then stained with a Cy3-conju- EAV ORF 5 and is followed at its 3Ј end by the copy of the gated donkey anti-mouse IgG (HϩL) antibody (Jackson transcription-regulating sequence that directs EAV RNA ImmunoResearch Laboratories), diluted 1:200 in PBS– 7 synthesis. Finally, pBRNX1.38-5/6-gfp was constructed Gly–FBS. After a wash, the coverslips were briefly rinsed by insertion of the 1.2-kb SfiI–XbaI fragment from pAVIs5/ in H2O and mounted on glass slides using a Mowiol 6-gfp into SfiI- and XbaI-digested pBRNX1.38 (Fig. 1B, 40-88 (Aldrich)-based mountant (Heimer and Taylor, bottom). 1974) containing 2.5% 1,4-diazobicyclo[2,2,2]octane as an REPLICATION-COMPETENT EQUINE ARTERITIS VIRUS VECTOR 275 antibleaching agent. The samples were analyzed by la- ACKNOWLEDGMENTS ser scanning confocal light microscopy using a Leica We thank the people within our unit for stimulating discussions, Paul TCS 4D system equipped with an appropriate set of Masters for supplying us with plasmid pGreen Lantern-1, Guido van filters. Images were processed in Adobe PhotoShop ver- Marle for assistance with denaturing RNA agarose gel electrophoresis, sion 5.0 and printed on a Hewlett–Packard DeskJet 720C and Henrik Garoff for his support during the preparation of the manu- printer. script. These investigations were supported by the Council for Chem- ical Sciences of the Netherlands Organization for Scientific Research (CW-NWO) with financial aid from the Netherlands Technology Foun- Metabolic labeling, immunoprecipitation, and dation (STW). fluorography BHK-21 cells in 4-cm2 culture dishes were either mock REFERENCES infected or infected with the parental or one of the re- An, S., and Makino, S. (1998). 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