Virus Research 86 (2002) 111– 122 www.elsevier.com/locate/virusres
Rescue of rubella virus replication-defective mutants using vaccinia virus recombinant expressing rubella virus nonstructural proteins Xiaojie Wang, Yuying Liang, Shirley Gillam * Department of Pathology and Laboratory Medicine, Uni6ersity of British Columbia, BC Research Institute for Children’s and Women’s Health, 950 West 28th A6enue, Vancou6er, BC, Canada V5Z 4H4 Received 26 October 2001; received in revised form 12 April 2002; accepted 12 April 2002
Abstract The genome of rubella virus (RV) is translated into a polyprotein precusor, p200, of the nonstructural proteins (NSPs). This is proteolytically processed by a viral-encoded protease into two mature products, p150 and p90. p150 contains sequence corresponding to the predicted methyltransferase and protease activities, while p90 has sequence for the proposed helicase and RNA-dependent RNA polymerase activities. Processing of p200 is essential for RV viral replication. RV NSPs are responsible for viral RNA replication, in which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, and that cleavage products p150/p90 are required for efficient production of positive-strand RNA. To determine whether p150 or p90 alone or together is involved in positive-strand RNA synthesis, vaccinia virus recombinants expressing individual NSPs were constructed and characterized. These were used in in vivo rescue experiments to complement replication-defective mutants in virus replication. A protease-inactive mutant was rescued by p200 or p150 provided in trans by using vaccinia virus recombinants. Thus this protease can function in trans. Rescue of cleavage-defective mutant by either p200 alone, or p150 plus p90 but not by p150 or p90 alone suggests that p150 and p90 function together as a replication complex in positive-strand RNA synthesis. © 2002 Published by Elsevier Science B.V. Keywords: Nonstructural proteins; RNA synthesis; Rubella virus
1. Introduction Rubella virus (RV) is an enveloped, positivestrand RNA virus in the Togaviridae family * Corresponding author. Tel.: +1-604-875-2474; fax: + 1604-875-3597. E-mail address:
[email protected] (S. Gillam).
(Francki et al., 1991). The RV genomic RNA is 9762-nt in length exclusive of 3%-terminal poly (A) tail and contains two large open reading frames (ORFs) (Dominguez et al., 1990). Two thirds of the 5% proximal ORF, from nt 41 to nt 6388, encodes nonstructural proteins (NSPs) that function mainly in viral RNA replication and the other one third of the 3% proximal ORF, from nt
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6512 to nt 9701, encodes the virion structural proteins (SPs) that function in virus assembly (Clarke et al., 1987; Gillam, 1994). In RV-infected cells, the NSP-ORF is translated from the genomic RNA whereas the SP-ORF is translated from subgenomic RNA co-linear with the 3% terminal region. The primary translation product, p200, of the NSP-ORF is cleaved by a viral protease into two mature products, p150 and p90 (Chen et al., 1996; Marr et al., 1994; Yao et al., 1998). p150 contains the predicted methyltransferase and papain-like cysteine protease sequences at its N- and C-terminal regions, respectively (Marr et al., 1994). p90 contains the proposed helicase and RNA dependent RNA polymerase (RdRp) sequences at its N- and C-termini, respectively. Experimental data for the protease (Chen et al., 1996; Liang et al., 2000) helicase (Gros and Wangler, 1996) and RdRp (Wang and Gillam, 2001) activities associated with the predicted motifs have been presented. The protease domains involved in trans- and cis-cleavage activities have been mapped by deletion mutagenesis and in vitro translation (Liang and Gillam, 2000). No evidence for RV methyltransferase has been reported so far. No complementation group has been assigned to RV NSPs due to lack of ts mutants and variants. Studies of the well-characterized members of the Alphavirus genus, such as Sindbis virus (SIN) and Semiliki Forest virus, show that the NSPORF functions mainly in RNA replication (Lemm and Rice, 1993a,b; Lemm et al., 1994, 1998) and in viral cytopathogenesis (Kuhn et al., 1992). NSPs of alphaviruses are translated as polyprotein p123 and/or p1234 and cleaved into final products nsP1, nsP2, nsP3, and nsP4 (Strauss and Strauss, 1994). The roles of alphavirus NSPs in RNA replication and transcription have been studied in detail using a vaccinia virus transient expression system (Lemm and Rice, 1993a,b; Lemm et al., 1994, 1998), complementation studies (Hahn et al., 1989a; Hahn et al., 1989b) and a reconstitution system based on vaccinia recombinants expressing NSPs (Li et al., 1991). The processing of RV NSPs is much simpler than that of SIN NSPs. There is only a single cleavage in the p200, although RV protease pos-
sesses both cis- and trans-cleavage activity (Yao et al., 1998). It has been shown that uncleaved p200 functions in minus-strand RNA synthesis in cis, not in plus-strand RNA synthesis (Liang et al., 2000), whereas cleavage of p200 into p150 and p90 converts the complex to the capacity for efficient synthesis of positive-strand RNA both in cis and trans, but with higher efficiency in cis (Liang and Gillam, 2001). However, it is not known whether p150 or p90 alone can function in positive-strand RNA synthesis. To further define the role of RV NSPs in RNA synthesis, vaccinia virus recombinants expressing individual p200, p150 and p90 were generated and used in complementation studies. We found that the constructed vaccinia virus recombinants expressed authentic RV NSPs in high yields. A protease-inactive mutant (Liang and Gillam, 2000) could be rescued by p200 or p150 provided in trans, whereas a cleavage-defective mutant was rescued by p200 or by p150 plus p90 provided in trans. Rescue of protease-inactive mutant by p200 or p150 indicates that functional protease can be provided in trans to process defective p200. The requirement for both p150 and p90 to rescue a cleavage-defective mutant strongly supports the idea that a p150/p90 replication complex is involved in synthesis of positive-strand genomic and subgenomic RNAs.
2. Materials and methods
2.1. Plasmid constructs The recombinant vaccinia virus expression vector pTM3 (Moss et al., 1990) was used in this study. It is a pUC-derived plasmid that contains the T7 RNA polymerase promoter, the 5%-untranslated region (UTR) of encephalomyocarditis virus (EMCV), which facilitates cap-independent ribosome binding, a polylinker region for cloning foreign genes, TK region for homologous recombinant, E. coli guanine phosphoribosyltransferase (gpt) for selection of the positive recombinants, and a T7 transcription terminator. The AUG codon within the NcoI site in pTM3 is that at which cap-independent translation is initiated. In
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the RV M33 genome (Yao and Gillam, 1999) the initiation codon AUG starts at nt 41 within the NcoI site. RV cDNAs were positioned to the unique NcoI site in pTM3 to facilitate the authentic translation of RV genes. Cloning sites used in the construction of recombinant plasmids are shown in Fig. 1. To construct pTM3/p200, the NcoI-PstI fragment (nt 41 –2632) of pBRM33 was subcloned into the NcoI and PstI sites of pTM3, producing pTM3RV2632. A PstI-KpnI fragment (nt 2632– 6470) (the KpnI site was blunt-ended with T4 DNA polymerase) was then subcloned into the PstI-StuI sites of pTM3RV2632. The resulting construct was named pTM3/p200. pTM3/p150 was constructed by insertion of the PstI-EcoRV fragment (nt 2632– 4213) of pBR-150 (Liang et al., 2000) into pTM3RV2632 which had been digested with PstI and StuI. pBR-150 contains a stop codon at the cleavage site (nt 3945) in the NSP-ORF (Liang et al., 2000).
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To construct pTM3/p90, the NcoI-PstI fragment of pCMV5/p90 was subcloned into pTM3 that had been digested with NcoI and PstI restriction enzymes. pCMV5/p90 contains the RV 5%UTR (nt 1 – 40) and cDNA encoding p90 (nt 3945–4023). Since there are two NcoI sites (at nt 39 and 4023) within the cDNA, partial digestion with NcoI was carried out to isolate the fragment that retained the second NcoI site.
2.2. Generation of 6accinia 6irus recombinants Recombinant vaccinia viruses were generated by using human TK− 143 cells and identified by the gpt selection method (Falkner and Moss, 1988). Viruses were plaque purified three times under selective conditions (25 mg/ml of mycophenolic acid, 250 mg/ml of xanthine, and 15 mg/ml of hypoxanthine). Recombinants were characterized by protein analysis of infected cell lysates. The recombinant viruses expressing p200, p150 and
Fig. 1. Schematic diagram of RV NSP-ORF used in the construction of recombinant vaccinia viruses. RV NSP-ORF is shown as a dashed line. The 5%-UTR of EMCV, which facilitates cap-independent ribosome , is shown as a bold line. T7 RNA polymerase promoter (pT7) resides upstream of the EMCV UTR to direct the transcription from the foreign gene. The translation initiation site is located at the NcoI site. The locations of synthetic peptides used in the production of anti-peptide antibodies are indicated as ( — ). NS1 (residues 1 – 36); NS5 (residues 1598 –1637). Details of the plasmid construction are described in Section 2. The locations of restriction enzyme sites used in the plasmid construction are indicated. The cleavage site between p150 and p90 is shown by an arrowhead.
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p90 were named vTM3/200, vTM3/150 and vTM3/90, respectively.
2.3. Protein analysis of recombinant 6accinia 6irusinfected cells Monolayers of BHK cells in 35-mm tissue culture dishes were co-infected with vTF7-3 (Moss et al., 1990) and vaccinia recombinant viruses at a multiplicity of infection (MOI) of 2 pfu per cell. At 12-h post-infection (p.i.), the medum was removed and cells were washed and starved for 30 min in Dulbecco’s Modified Eagle Medium (D-MEM) lacking methionine and cysteine and containing 5% dialyzed fetal calf serum. [35S]methionine (20 mCi/ml) was added and incubated at 37 °C for 45 min. After labelling, the cells were lysed in lysis buffer (25 mM Tris –HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) and nuclei were removed by centrifugation. RV NSPs were immunoprecipitated with NS1 or NS5 antibodies. NS1 and NS5 are the rabbit sera produced against the N-terminal region of NSP-ORF (residues 1– 36) and the C-terminal region of NSP-ORF (residues 1598– 1637) respectively. After incubation at 4 °C for 1 h, 40 ml of 50% protein A-Sepharose beads (Pharmacia Biotech) was added and incubated for a further 1 h at room temperature with constant shaking. The beads were washed three times with 1 ml TNE buffer (50 mM Tris– HCl [pH 7.2], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), resuspended in SDS-gel loading buffer (50 mM Tris– HCl [pH 7.2], 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.1% bromophenol blue) and boiled for 5 min. After centrifugation, the immunoprecipitates were collected and then analyzed by sodium dodecylsulfate polyacrylamide gel (8%) electrophoresis (SDS-8% PAGE). Radiolabeled proteins were visualized by fluorescence autoradiography. In the pulse–chase experiments, at the end of labelling period, the medium containing the radiolabel was removed, and D-MEM containing the normal concentrations of methionine and cysteine was added. The cell monolayers were
scraped and suspended in phosphate-buffered saline (PBS) at various chase times and lysed in 200 ml of lysis buffer.
2.4. Western blot analysis BHK cells were co-infected with vTF7-3 and individual vaccinia virus recombinants at MOI of 2 pfu per cell. At 48-h p.i., cellular lysates were prepared, subjected to 8% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was washed briefly in TBS (0.15 M NaCl, 0.02 M Tris–HCl [pH 7.5]) containing 0.3% Tween 20 and blocked for nonspecific binding in TBS containing 4% powdered skimmed milk. The membrane was then incubated with rabbit anti-peptide sera (NS1 plus NS5 at 1:100 dilution) in the same solution for 2 h, washed with TBS/0.3% Tween 20, and then treated with goat anti-rabbit IgG conjugated to horseradish peroxidase for 2 h. The blot was washed as above and developed using ECL western blotting detection system (Amersham Life Science).
2.5. RNA transcription and transfection Recombinant plasmids were linearized at the HindIII site and RNA transcripts were synthesized with SP6 RNA polymerase (Promega) in the presence of m7Gpp(5%)G cap analog (1 mM) (Promega) in the reaction mixture (40 mM Tris– HCl [pH 7.9], 6 mM MgCl2, 10 mM DTT, 10 mM NaCl, 2 mM spermidine, 0.05% Tween-20, 0.5 mM NTP, and 1 U RNasin [ribonuclease inhibitor]) at 37 °C for 1–2 h. BHK cells were transfected by electroporation as described previously (Yao and Gillam, 1999). Briefly, BHK cells were trypsinized and washed with cold PBS (without Ca++ and Mg++) twice and resuspended at a concentration of 107 cells/ ml. RNA transcripts (10–20 mg) were mixed with 0.45 ml of cells, and the mixture was transferred into a 2-mm diameter cuvette. Electroporation was done with two consecutive 1.5 kV, 25 mF pulses with a Gene-Pulser (Bio-Rad). After electroporation, the cells were distributed into four culture dishes (35 mm).
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2.6. RNase protection assay
3. Results
For RNase protection assay (RPA) experiments, a minus-polarity RNA probe (pb18) (Liang and Gillam, 2000) was used. pb18 was synthesized with SP6 RNA polymerase from EcoRI-digested plasmid pST18-pb. pST18-pb can protect 301-nt positive-strand genomic RNA and 188-nt subgenomic RNA against RNase digestion. The 35S-labeled pb18 probe was synthesized with SP6 polymerase in a 20 ml in vitro transcription reaction mixture incubated for 1–2 h at 37 °C. The reaction mixture contained transcription buffer (2 mM spermidine, 10 mM DTT, 6 mM MgCl2, 40 mM Tris – HCl, 0.05% Tween-20), 0.5 mM each of ATP, GTP, and UTP, 10 mM of CTP, 2.5 mCi/ml of [a-35S]CTP. After in vitro transcription DNase I (7500 U/ml) was added and incubated for 15 min at 37 °C. The probe was precipitated with ethanol after phenol/chloroform extraction and resuspended in 30 ml hybridization buffer. For positive-strand RNA assay, approximately 2 mg of total cytoplasmic RNA was hybridized with 5× 105 cpm of pb18 probe in 30 ml of hybridization buffer (40 mM PIPES [pH 6.4], 1 mM EDTA, 400 mM NaCl, 80% deionized formamide) overnight at 55 °C. RNase digestion was for 45 min at 30 °C in an RNase mixture (RNase A at 10 mg/ml, RNase T1 at 70 u/ml, 300 mM sodium acetate, 10 mM Tris– HCl [pH 7.5]). The samples were treated with SDS-protease K for 15 min at 37 °C, extracted with phenol– chloroform and ethanol precipitated with 5 mg of E. coli tRNA at −70 °C. Samples were denatured in boiling water and analyzed on a 5% polyacrylamide-7 M urea sequencing gel. The gel was fixed in 10% acetic acid infiltrated with enhancer (DuPont), dried and exposed to X-ray film overnight at − 70 °C.
3.1. Construction and expression of 6accinia 6irus recombinants containing RV NSPs
2.7. Image analysis Image analysis was employed to quantitate the relative amounts of protected RNAs. The program for image analysis was downloaded from the Scion Image program for Windows at http:// www.scioncorp.com.
To examine the involvement of different NSP species in RNA replication and transcription, a set of RV cDNAs encoding p200, p150 and p90 was constructed using plasmid pTM3 (Fig. 1). pTM3 contains the E. coli gpt gene which permits mycophenolic acid selection for recombinant vaccinia viruses (Falkner and Moss, 1988; Moss et al., 1990). The AUG within the NcoI site in pTM3 is that at which cap-independent translation is initiated (Moss et al., 1990). In the RV M33 genome, the initiation codon AUG starts at nt 41, within the NcoI site. RV cDNA encoding NSPs were positioned to the unique NcoI site in pTM3. Schematic diagrams of the constructed recombinant plasmids and cloning sites used in the plasmid construction are shown in Fig. 1. Vaccinia virus recombinants expressing individual NSPs were isolated by the gpt selection procedure (Falkner and Moss, 1988). To determine the functionality of vaccinia virus recombinants expressing RV NSPs, BHK cells co-infected with vTF7-3 (Moss et al., 1990) and various recombinant viruses were labelled with [35S]methionine at 12-h p.i. and cellular lysates were immunoprecipitated with antibodies specific to each NSP (Yao et al., 1998). The precipitated proteins were analyzed on SDS-8% PAGE. In vTM3/90-infected cells, a protein with a molecular weight of 90 kDa (p90) migrating slightly above the protein band in the wt vaccinia virus infected cells was only recognized by peptide antiserum NS5 (against the Cterminal NSP-ORF, residues 1598–1637) (Fig. 2, lanes 1 and 2). The host proteins in the region of p90 obscure the clarity of immunoprecipitated p90 in the recombinant vaccinia virus lanes. In vTM3/150-infected cells, a protein with an apparent molecular weight of 150 kDa (p150) was recognized by both peptide antisera NS1 (against the N-terminal NSP-ORF, residues 1–36) and NS5 (Fig. 2, lanes 3 and 8). The co-precipitation of p150 by antiserum NS5 was also observed in our previous studies in BHK cells transfected with infectious RV RNA transcripts (Yao et al., 1998).
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Fig. 2. Expression of RV NSPs in cells infected with recombinant vaccinia viruses. BHK cells were co-infected with vTF7-3 and individual vaccinia recombinant viruses. At 12-h p.i., the infected cells were pulse-labeled for 45 min. Cellular lysates were immunoprecipitated with antibodies specific to p90 (NS5) or p150 and p200 (NS1). The immunoprecipitates were analyzed by SDS-8% PAGE. The positions of the apparent molecular mass standards are indicated on the left (in kDa). Wt vv, wt vaccinia virus; v90, vaccinia virus recombinant containing p90 coding region; v150, vaccinia virus recombinant containing p150 coding region; v200, vaccinia virus recombinant containing RV-NSP-ORF.
In vTM3/200-infected cells, three protein species with molecular weights of 200 kDa (p200), 150 kDa (p150) and 90 kDa (p90) were recognized by peptide-antiserum NS5 (Fig. 2, lane 4), while two protein species with molecular weights of 200 and 150 kDa were recognized by peptide-antiserum NS1 (Fig. 2, lane 9). The co-precipitated protein species with molecular weights of 140, 130 and 70 kDa are likely host proteins or degraded RV NSPs (Fig. 2). At present we do not know if these proteins are involved in RV replication. Due to the interference of host 90 kDa protein in the region of p90, we carried out western blot analysis to demonstrate the synthesis of p90 in vaccinia recombinant viruses. At 2 days p.i., cellular lysates were prepared and separated on 8% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes and tested for their immuno-reactivities to both NS1 and NS5 peptide-antisera. Vaccinia virus recombi-
nant expressing p200 carrying a mutation at its cleavage site (v1301) or protease active site (v1052) was used as a control for p200. We found that p90 was clearly visible in vaccinia virus recombinants containing p90 and p200 (Fig. 3, lanes 2 and 4), and p200 was hardly detectable after 2 days p.i., due to the efficient processing of p200 into p150 and p90 (Fig. 3, lane 4). No cleavage products were observed in vaccinia virus recombinants containing a mutation at the cleavage site or protease active site (Fig. 3, lanes 5 and 6). Our results indicate that RV NSPs synthesized in cells infected with recombinant viruses were expressed efficiently and underwent post-translational modification similar to that observed in RV-infected cells. The kinetics of RV NSP polyprotein-processing were examined by pulse– chase analysis of vTM3/ 200-infected cells. At 24-h p.i., vTM3/200-infected cells were starved for 30 min in DMEM lacking
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methionine and cysteine, labeled for 45 min with [35S]methionine and then chased with normal medium for periods up to 5 h. As shown in Fig. 4, the intensity of the p200 band decreased substantially throughout the chase period (Fig. 4, lanes 3 – 5). No residual fraction of p200 could be detected after 3 h chase (Fig. 4, lane 5), whereas the intensity of p90 and p150 bands increased slightly or remained constant throughout (Fig. 4, lanes 3 –5). p150 and p90 bands were observed even after 45 min of pulse labeling (Fig. 4, lane 2), indicating that the cleavage of p200 is very efficient, consistent with the finding of Yao et al. (1998) in BHK cells transfected with a full-length RV RNA transcript (pBRM33).
3.2. Replication of replication-defecti6e rebulla 6irus mutant with 6accinia 6irus recombinants as helpers Since RV and alphavirus share similar strategies in viral RNA replication (Liang and Gillam,
Fig. 3. Immunoblot analysis of cellular lysates from BHK cells infected with vaccinia recombinant viruses. Lysates from BHK cells infected with vaccinia virus recombinants were subjected to SDS-PAGE, transferred to nitrocellulose membrane and probed with rabbit anti-peptide sera, followed by goat antirabbit IgG conjugated to horseradish peroxidase. wtvv, wt vaccinia virus; v90, vaccinia virus recombinant containing p90 coding region; v150, vaccinia virus recombinant containing p150 coding region; v200, vaccinia virus recombinant containing RV-NSP-ORF p200; v1301, vaccinia virus recombinant containing p200 coding region carrying a mutation at its cleavage site (G1301S); v1152, vaccinia virus recombinant containing p200 coding region carrying a mutation at the protease active site (C1152S). The positions of the apparent molecular mass standards are indicated on the left (kDa).
Fig. 4. Kinetics of RV nonstructural polyprotein processing. BHK cells were co-infected with vTF7-3 and vTM3/200 as described in Section 2 (lanes 2 – 5). Cells were pulse-labeled with [35S]methionine for 45 min, and chased for 0, 1, 3, and 5 h after 24-h p.i. Cell lysates were immunoprecipitated with a mixture of antibodies specific to RV p150 (NS1) and p90 (NS5). Protein samples were analyzed by SDS-8% PAGE. RV-specific proteins are indicated on the right, and molecular mass standards are indicated on the left. Lane 1: wild-type vaccinia virus infected BHK cells.
2001), we used vaccinia virus recombinants expressing RV NSPs to analyze the roles of NSPs in the synthesis of distinct RV RNAs in complementation experiments. To carry out complementation experiments, two components are required in the assay. The first is the trans-acting protein and the other is the appropriate RNA template. In our experiments, the trans-acting proteins of RV NSPs were expressed via recombinant vaccinia viruses, while the RNA template used was either a protease-inactive (pBRM33/C1152S) or a cleavage-defective (pBRM33/G1301S) mutant construct (Liang and Gillam, 2000). To determine the conditions for complementation using a vaccinia virus recombinant and the RNA template, BHK cells were infected with wt vaccinia virus or recombinant viruses at MOI of 2 pfu/cell. At 4-h p.i., infected BHK cells were isolated by trypsinization and then electroporated with a full-length RNA transcript from pBRM33 (Yao and Gillam, 1999). Synthesis of positivestrand RNAs was analyzed by RPA. We observed that RV replication was greatly reduced in the
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vaccinia virus-infected cells (data not shown). However, when BHK cells were first electroporated with RNA transcript, and then infected with wt vaccinia virus or recombinant vaccinia viruses 4 h after electroporation, no inhibition of positivestrand RNA synthesis was detected (data not shown). Thus, in our complementation studies, BHK cells were electroporated with replication-defective RNA template 4 h prior to virus infection. We have shown previously that a full-length protease-inactive mutant construct (pBRM33/ C1152S) can synthesize minus-strand RNA but not plus-strand genomic or subgenomic RNA (Liang and Gillam, 2000). To determine whether C1152S lethal mutation can be rescued to synthesize plusstrand RNAs by providing functional NSPs in trans, BHK cells were infected with recombinant vaccinia viruses 4 h after electroporation of RNA transcript derived from a full-length protease mutant pBRM33/C1152S. Total cytoplasmic RNAs were extracted at 0, 8, 24 and 48 h after electroporation and subjected to RPA. Protected RNA samples were denatured and analyzed by elec-
trophoresis on a 5% polyacrylamide-7 M urea gel. To attempt to equalize the intensity of RNA bands in the mutants, only one tenth of the RNA sample from the wt (pBRM33) was loaded into lanes 3–6 of the gel. As illustrated in Fig. 5, large quantities of protected genomic RNA (301-nt) representing the input genomic RNA were observed at 0 h post-electroporation (Fig. 5, lanes 3, 7 and 11). By 8 h post-electroporation, the input genomic RNA was mostly degraded (Fig. 5, lanes 4, 8 and 12). Both protected bands, 301-nt (representing plusstrand genomic RNA) and 188-nt (representing subgenomic RNA), were clearly observed at 24 h post-electroporation (Fig. 5, lanes 5, 9 and 13), indicating that RNA replication occurred in rescued C1152S mutant, and that functional protease can be provided by p200 or p150 in trans to process p200 in the C1152S mutant by using vaccinia virus recombinant expressing p200 or p150. The smaller protected fragment (148 nt) observed in vaccinia virus recombinant (v200) was due to the 3%-end sequence of p200 that overlapped with probe pb18 (nt 6323–6623).
Fig. 5. Rescue of protease-inactive and cleavage-defective mutants with recombinant vaccinia viruses. BHK cells were transfected with full-length RNA transcripts of protease-inactive mutant pBRM33/C1152S by electroporation (lanes 7 – 14). Four hours after transfection, the cells were co-infected with vTF7-3 and either vTM3/200 (lanes 7 to 10) or vTM3/150 (lanes 11 to 14). In other experiments, BHK cells were transfected with full-length RNA transcript of cleavage-defective mutant pBRM33/G1301S, and then co-infected with vTF7-3 and either vTM3/200 (lanes 15 –18), or vTM3/150+vTM3/90 (lanes 19 – 22) 4 h after electroporation. As a positive control, BHK cells were transfected with wt RV full-length RNA transcript from pBRM33 (lanes 3 – 6, only one tenth of the RNA sample was loaded). As a negative control, BHK cells were co-infected with vTF7-3 and either vTM3/200 (lanes 23 – 26), vTM3/150 (lanes 27 – 30) or vTM3/90 (lanes 31 –34) in the absence of RNA template. Total cytoplasmic RNAs were extracted with TRIzol reagent and subjected to RPA at the indicated times. Lane 1, 2 ×103 cpm of pb18; Lane 2, BHK cells were electroporated in the absence of RNA.
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Since the cleavage site mutant (pBRM33/ G1301S) that produces uncleaved p200 can only synthesize minus-strand RNA but not plus-strand genomic or subgenomic RNA (Liang and Gillam, 2000), we addressed the question whether both p150 and p90 are required in plus-strand RNA synthesis. We have demonstrated that p150 and p90 can be co-expressed in BHK cells without interfering with the expression of each other using vaccinia virus recombinants (Fig. 2, lanes 5 and 10). Thus these recombinant vaccinia viruses were used in complementation experiments. BHK cells were infected with either vTM3/200, vTM3/150, vTM3/90, or co-infected with vTM3/150 and vTM3/90 4 h after electroporation of BHK cells with RNA transcripts from the full-length cleavage site mutant (pBRM33/G1301S). We found that the mutant was rescued by p200 (Fig. 5, lanes 17 and 18) as well as by p150 plus p90 (Fig. 5, lanes 21 and 22) provided in trans, but not by p150 or p90 alone (Fig. 5, lanes 35– 42). No protected 301-nt or 188-nt fragment was detected in BHK cells infected with either vTM3/200, vTM3/150 or vTM3/90 vaccinia virus in the absence of RNA template (Fig. 5, lanes 23– 34). Again, the protected 148 nt observed in vaccinia virus recombinants (v200 and v90) infected cells was due to the 3-end sequence of p200 and p90 that overlapped with pb18. Our results indicate that p150/p90 together form the principal positive-strand replicase complex for the synthesis of both genomic and subgenomic RNAs, and p90 alone cannot serve as a replicase for the synthesis of positive-strand RNAs.
3.3. Comparison of the relati6e amounts of RNAs produced by wt and rescued mutant 6iruses Replication and transcription of RV RNA is dependent on the viral NSPs that are the components of the viral replicase– transcriptase complex responsible for the synthesis of three viral RNAs: the genomic minus-strand RNA, the plus-strand genomic RNA and a subgenomic RNA. The replication efficiency of rescued mutants can be assessed from the accumulated RNA in infected cells. To quantitate the amount of positive-strand genomic and subgenomic RNAs, intensities of the
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Table 1 Comparison of the relative amounts of positive-strand RNAs produced by the wt and rescued mutants Samples
pBRM33
Relative amounts of RNA accumulationa
SG/Gb
Subgenomic
Genomic
Molar ratio
1.0
1.0
2.6 9 0.2
0.4 9 0.1 0.4 90.1 0.3 90.1 0.4 90.1
0.7 90.2 0.7 9 0.2 0.6 9 0.1 0.7 90.2
Vaccinia 6irus recombinants 1152+v200 0.1 9 0.0 1152+v150 0.1 90.0 1301+v200 0.1 90.0 1301+(v150 0.1 9 0.0 + v90)
The values shown are the results of at least two independent experiments. a The positive-strand RNA, either genomic or subgenomic, produced at 24 h post-electroporation was assessed and normalized against that of the wt which is set a value of 1.0. b The SG/G molar ratio is the calculated molar ratio of subgenomic to genomic RNA 1152: protease-inactive mutant (C1152); 1301: cleavage-site mutant (G1301S). v200: vaccinia recombinant virus vTM3/200; v150: vaccinia recombinant virus vTM3/150; v90: vaccinia recombinant virus vTM3/90.
protected RNA bands at 24 h post-electroporation were determined by image analysis of autoradiographs. Results for the rescued viruses were normalized against those of the wt. The molar ratio of subgenomic RNA to genomic RNA was also calculated. We found that the rescued mutants produced about 30–40% of the level of wt genomic RNA and 10% of the subgenomic RNA produced by the wt. The molar ratio of subgenomic/genomic RNA ranged from 0.6 to 0.7 for the rescued mutants, about 4-fold lower than that of the wt (Table 1), indicating that NSPs provided in trans are not as efficient as in cis in RNA replication.
4. Discussion Replication and transcription of RV RNA is dependent on the viral NSPs that are the components of the viral replicase–transcriptase complex responsible for the synthesis of genomic negativestrand RNA, genomic positive-strand RNA and a
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subgenomic positive-strand RNA. We have shown that synthesis of these three RV RNA species is regulated by NSP cleavage (Liang and Gillam, 2000). Uncleaved p200 forms the replication complex for negative-strand RNA synthesis and cleavage of p200 into p150 and p90 converts the complex into one with the capacity for efficient positive-strand RNA synthesis (Liang and Gillam, 2000). Although these experiments suggest that p150 and p90 are involved in positivestrand RNA synthesis, it does not address the question whether p150 or p90 alone can carry out this function. In this report, we demonstrated by complementation experiments using vaccinia virus recombinants expressing individual RV NSPs, that p150 and p90 are both required as components of the active replication complex in the synthesis of positive-strand genomic and subgenomic RNAs. In complementation experiments, a protease-inactive mutant (pBRM33/C1152) was rescued by a vaccinia virus recombinant expressing p200 or p150 in trans (Fig. 5). Successful rescue of a protease-inactive mutant by trans-supplied p200 or p150 indicates that functional protease can be provided in trans to process p200 in the pBRM33/ C1152S mutant. The products, p150 and p90, are produced and positive-strand genomic and subgenomic RNAs are synthesized in replication-defective mutant pBRM33/C1152S. The requirement for both p150 and p90 for rescue of cleavage-defective mutant (pBRM33/G1301S) in RNA replication strongly supports the idea that a p150/p90 replication complex is involved in positive-strand RNA synthesis. p150 or p90 alone is incapable of functioning as an active replication complex for synthesizing positive-strand RNAs (Fig. 5). We demonstrated that vaccinia virus recombinants expressing RV NSPs can successfully rescue the RNA replication of pBRM33/C1152S and pBRM33/G1301S, which contain a mutation at the protease active site and cleavage site respectively. Replication and transcription of pBRM33/ C1152S and pBRM33/G1301S by vaccinia virus recombinants were detectable, but at a lower efficiency. It is unlikely that the rescued virus replication is due to RNA recombination in infected cells. We have shown previously that replication-
defective constructs with an in-frame deletion within the helicase or protease region in NSP used as RNA template were not rescued by providing p200, p150 or p90 in trans (Liang and Gillam, 2001; Wang and Gillam, 2001). RV replicates slowly with a latent period lasting more than 12 h. Viral RNA synthesis is not detectable until at least 10-h p.i. and the peak rate of synthesis occurs as late as 56-h p.i. (Hemphill et al., 1988). In our complementation experiments, we detected the accumulated genomic and subgenomic RNAs at 24 h post-electroporation. In the absence of helper viruses we did not detect any positivestrand RNAs even at 48 h post-electroporation (Fig. 5). Therefore, the rescue of pBRM33/ C1152S and pBRM33/G1301S mutants by wildtype NSPs could not be due to the generation of recombinant genomes. In addition, RV RNA recombinants have been detected only after multiple passages at high MOI and homologous recombination occurred mostly in RV SP-ORF (Pugachev et al., 2000). The low complementation efficiency obtained in the rescue experiments (Fig. 5) as well as the lower molar ratio of subgenomic/genomic RNA in the rescued mutant viruses (Table 1) may be due to the cis-preferential replication of RV (Liang and Gillam, 2001). For a number of positive-strand RNA viruses, RNA replication requires the translation of part or all of the NSP coding region in cis. The existence of cis-limited replication in positive-strand RNA virus has been reported. For example, in poliovirus, the co-replication of deletion variant RNAs depends on the presence of a translatable nonstructural cistron. Mutations in the 2C NSP are poorly complementable in trans (Telerina et al., 1995). The replication machinery of turnip yellow mosaic virus displays a cis-preference, such that the essential replication proteins made from a given genomic mRNA molecule assemble most efficiently into a replication complex on that same RNA molecule, facilitating the switch in the role of the RNA from mRNA to replicational template (Weiland and Dreher, 1993). In RV the advantage of cis-translation appears to lie in the preferential cis action of p200 in synthesizing RV negative-strand RNA and the cis and trans action
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of the p150/p90 complex in synthesizing positivestrand RNA, with higher efficiency in cis. The vaccinia virus recombinants expressing individual RV NSPs are useful tools for studying RV replication. The expression of functional RV NSPs via vaccinia virus recombinants opens many possibilities for further investigation of these proteins. Vaccinia virus recombinants expressing RV NSPs provide a convenient, heterologous system for the characterization of RV NSPs that is free of replication competent RV-specific RNAs and structural proteins. RV NSPs prepared from recombinant vaccinia virus-infected cells may be useful for in vitro biochemical analyses of RNA binding or enzymatic activities associated with the NSPs, such as methyltransferase, RNA helicase and RNA-dependent RNA polymerase. Another application of RV NSP vaccinia virus recombinants is to use as a helper system to supply wild-type NSPs in trans for complementing RV mutants with lethal lesions in the NSPs.
Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. Y.L. is supported by a studentship from the British Columbia Children’s Hospital Foundation. S.G. is an investigator of the British Columbia Children’s Hospital Foundation.
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