Rinderpest Viruses Lacking the C and V Proteins Show Specific Defects in Growth and Transcription of Viral RNAs

Cells and virus.

Cells were maintained and virus stocks were grown and titered as described elsewhere (1). Rescue of rRPV from cDNA was performed as previously described (1) except that transfection was performed using FuGENE6 (Roche Biologicals) or Transfast (Promega), as described by the manufacturer, and using a ratio of 6 μl of reagent per μg of plasmid DNA for both reagents. For measuring viral growth rate, B95a cells (2 × 10⁶ per 35-mm-diameter well), Vero cells, or primary bovine skin fibroblasts (5 × 10⁵ per well) were infected with rRPV at a multiplicity of infection (MOI) of 0.005 to 0.04 (depending on the experiment) for 1 h. The inoculum was removed, the cells were washed twice with medium, and 2 ml of medium was added to each well. Samples were taken immediately and at various times thereafter and stored at −70°C. After thawing and clarifying at 2,500 rpm for 10 min, the 50% tissue culture infective dose (TCID₅₀) of released virus was quantitated by standard methods. All results were normalized to an initial infection with 10⁴ TCID₅₀ per well.

For observation of individual foci of infection, B95a cells were plated at 2 × 10⁶ cells per well 2 days before use. Virus at 10 to 100 TCID₅₀ per well was added and allowed to adsorb for 1 h, after which the cells were washed once with medium and then overlaid with Eagle's medium containing 4% fetal calf serum and 1% (wt/vol) carboxymethyl cellulose. After 5 to 6 days, most of the covering medium was removed and the cells were overlaid with undiluted Giemsa's stain (Merck) for 30 min at room temperature. The stain and the remaining carboxymethyl cellulose were washed off with water, and the stained foci were photographed.

Molecular biology and assays.

Except where indicated, all DNA manipulation was by standard methods. Plasmids were cloned and grown in Escherichia coli JM109 or DH5α and purified on CsCl gradients or using Qiagen columns. The change of the C open reading frame (ORF) initiation codon was introduced into pKSP (1) by using unique site elimination mutagenesis (20). The XmaI-XmaI fragment containing the mutation was swapped with the corresponding fragment from pMDBNP (1) to give pMDBNPCE, to which were added the M and F genes from pKSMF (1). The ClaI-SunI fragment containing the whole N, P, and M genes and most of the F gene was then swapped for the corresponding segment from the full genome cDNA clone in pMDBRPV. The change to editing site was performed by using the Quick-Change mutagenesis protocol (Stratagene) on pMDBNP; the sequenced ApaI-KpnI fragment was then used to replace the same section in pMDBNP (to give pMDBNPVE) or pMDBNPCE (to give pMDBNPVCE), and the single and double mutations were assembled into the full-length genome as described above. The introduction of stop codons in the C ORF was done by using two-stage overlap PCR (33), modified by using the proofreading DNA polymerase Pfu (Stratagene) instead of Taq DNA polymerase. The template was either pMDBNPCE or pMDBNPVE, and the XhoI-KpnI fragments containing the mutations were removed from these plasmids and inserted into pMDBNP-PacSbf, a version of pMDBNP where PacI and SbfI sites have been introduced at the ends of the N and P ORFs, respectively. The ClaI and SbfI sites at the beginning of the N gene and end of the P gene were then used to transfer the mutations into pMDBRPV2C, a version of pMDBRPV containing the same restriction sites (which have been found to have no effect on virus growth in vitro).

RNA from RPV-infected cells was prepared using Trizol (Life Technologies), and reverse transcription-PCR (RT-PCR) was performed as previously described (4). PCR products were sequenced directly using T7 DNA polymerase (Amersham Pharmacia Biotech) after treatment with shrimp alkaline phosphatase and exonuclease I (Amersham Pharmacia Biotech). Primer extension analysis was performed essentially as described previously (30), except that the primer used was 5′-GGTCGATTTCACGTCTGT-3′ and the stopping nucleotide was ddA rather than ddG.

Semiquantitative RT-PCR was performed essentially like ordinary RT-PCR except that specific primers, rather than random hexamers, were used for the RT step. Total RNA (1.2 μg) was transcribed with 10 pmol of RPV primer or 100 μg of oligo(dT)₁₅ (Promega) in a final volume of 20 μl; all samples from one set of time courses (two of each type of virus, four virus types, and four time points) were transcribed at the same time with aliquots of the same master mix of RT enzyme, deoxynucleotide triphosphates, and buffer. For strand-specific priming of the RT reaction, the primers used were 5′-GTAGGCTGGTGAGTAATCT-3′ (primes on genome positions 309 to 291) and 5′-TGATTCCCCGG-ATAGCC-3′ (primes on antigenome positions 201 to 185). For PCRs, 5 μl of RT reaction product was used in a reaction volume of 50 μl. The program was 95°C for 1.5 min followed by the indicated number of cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 20 s. Genome-specific PCR used primers 5′-GTAATCTCAAGTCTGGATACC-3′ (primes on genome positions 297 to 277) and 5′-AGGATCGGGAAGCAGACA-3′ (corresponds to genome positions 83 to 100). Antigenome-specific PCR primers were 5′-ACCAGACAAAGCTGGGTAAGGA-3′ (corresponds to antigenome positions 1 to 22) and 5′-GCGCCTCGAGCCTGTGCCCTAAGCCT-3′ (primes on antigenome positions 181 to 165). P-gene-specific primers were 5′-CCCAGTGTGATCCGTTC-3′ (corresponds to antigenome positions 3187 to 3203) and 5′-CCTGCAGGAGATCAGCTATGTTGT (primes on antigenome positions 3338 to 3356), and H-gene-specific primers were 5′-CCCGTGGGACCGCAAACT-3′ (corresponds to antigenome positions 8971 to 8988) and 5′-GGCCCTGGTTTATAA-3′ (primes on antigenome positions 9112 to 9126). The actin control primers were BA1 and BA2 (23). Five-microliter samples from each PCR were analyzed in 1.3% agarose gels using 1× Tris-borate-EDTA buffer; gel and buffer contained 0.05 μg of ethidium bromide per ml. The fluorescence of the ethidium bromide-stained DNA was recorded using a Bio-Rad Chemi Doc system, and the peak area for each band (average pixel density per row of pixels multiplied by the number of rows), determined using the QuantityOne software (Bio-Rad), was taken as a measure of the relative amount of PCR product. Where no DNA was visible, the pixel density in the equivalent region was determined. The Chemi Doc system consists of a charge-coupled device camera coupled to a digitizing board, the limited spatial resolution of which accounts for the grainy quality of the resulting images.

Antibodies and immunoprecipitation.

Rabbit antisera recognizing the RPV C protein were raised against bacterially expressed glutathione S-transferase fusion proteins, using the pGEX vector. The rabbit anti-V antiserum was the kind gift of D. Briedis, Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada. Mouse monoclonal antibody 2-1 recognizing the RPV P protein was the kind gift of M. Sugiyama, Department of Veterinary Public Health, Faculty of Agriculture, Gifu University, Gifu, Japan. For immunoprecipitation studies, cells in six-well plates were infected with rRPV for 2 days, incubated for 1 h in Eagle's medium without methionine or cysteine, and then incubated for 2 h in 0.5 ml of the same medium containing 5 to 10 μCi of PROMIX (Amersham Pharmacia Biotech), a mixture of amino acids containing [³⁵S]methionine and [³⁵S]cysteine. The medium was removed, and the cells were lysed in 0.5 ml of lysis buffer (1% [vol/vol], Nonidet P-40, 50 mM Tris-Cl [pH 7.5], 0.5 M NaCl, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 mM iodoacetamide, 100 ng each of leupeptin, pepstatin, antipain, and chymostatin per ml). The lysate was incubated first with nonimmune rabbit serum and protein A-Sepharose (Amersham-Pharmacia) for 1 to 2 h at 4°C, centrifuged to remove the protein A-Sepharose, and then incubated with the specific antiserum and protein A-Sepharose overnight at 4°C. Immunoprecipitation of P protein was routinely from one-fifth the amount of lysate used from which to immunoprecipitate V or C. Where the primary antibody was a mouse monoclonal antibody, 0.5 μl of rabbit anti-mouse immunoglobulin G (Dakopatts) was added in addition to bind the mouse antibody more efficiently to the protein A. Sepharose pellets were washed, and samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described elsewhere (3). SDS-PAGE was performed using the buffer system of Laemmli (40); gels were fluorographed using sodium salicylate (12).

Digital image processing.

Original gel pictures (autoradiographs or fluorographs) or photomicroscopy transparencies were photographed using a Kodak DCS420 digital camera or scanned with a Linotype-Hell Saphir, transferred to a PowerMac 7500, converted to grey scale and clipped in Adobe Photoshop, laid out and labeled in QuarkXPress, and printed on a Kodak XLS 8600 printer. Gel images from the Chemi Doc system were exported as TIFF files, cropped to the appropriate region, and imported to ClarisDraw for labeling and printing as described above.

Introduction of mutations to abolish the expression of C or V protein.

The three P gene ORFs that are utilized by the virus are illustrated in Fig. 1a. The AUG initiator codon for the P and V proteins has a suboptimal context (C at −3, rather than the preferred A or G [38]); the C ORF starts at the next AUG, 20 bases downstream (Fig. 1b). To abolish C expression, we first changed this latter AUG codon to ACG, this being the only change to that codon that would be silent in the P protein (Fig. 1b). Although the rescued viruses containing this mutation [RPV(CE) and RPV(VCE)] did not express C protein (see below), we were concerned that a reversion could occur through a single point mutation, and RNA viruses have a relatively high mutation rate, making such a reversion possible. In addition, ACG is used as the C′ protein start codon in SeV (15, 29), and so it was possible that some C protein might be expressed from this ACG, even though the next base after the ACG codon in our mutant (C) is the least conducive to such usage (28). We therefore introduced two stop codons into the C ORF (again, with no change to the P protein), in addition to the change in the initiation codon (Fig. 1b), giving rise to recombinant viruses RPV C− and RPV VC−. In fact, no difference was seen in the behavior of viruses with the single change or with both changes to the C ORF, and data on their growth rates in tissue culture were pooled.

Expression of the RPV V protein requires the nontemplated insertion of an extra G base in the sequence UUAAAAAGGGCACAGA, extending the run of G's from three to four. Although the minimal sequence required for editing has been mapped to only a 24-base section of SeV (49), it is generally assumed that the run of purines followed by the three G's is essential. Four changes were made simultaneously to this motif (Fig. 1c) to ensure that no editing should occur, that no V protein should be made, and that no single mutation could restore V expression. All of the changes were silent in the P ORF.

All of the mutations were made first in a cDNA clone of the P gene, and the C− and V− mutations were combined to make the VC− P gene. The different versions of the P gene were then incorporated into the full-length clone of the RPV genome for rescue into live virus (see Materials and Methods for details). All mutant viruses were rescued without major difficulty, although the double (VCE or VC−) mutants could not be rescued until we had improved the efficiency of our transfection system by changing the transfection reagent from Lipofectin/LipofectACE (1) to FuGENE6 or Transfast (see Materials and Methods), which gave a 12- or 20-fold increase in transfection efficiency. RT-PCR was used to amplify viral RNA from specific regions of the genomes of mutant viruses, and the PCR products were sequenced to confirm the presence of the appropriate mutation. All of the studies reported here were performed on two separately isolated clones of the virus type in question. In addition, although the B95a lymphoblastoid cell line that we used for most of the studies reported here seems to be the best line so far discovered for growing RPV (37), allowing direct culture of wild viruses without adaptation by blind passage, we have noticed that repeated passage of viruses in B95as does increase the rate of replication of RPV in these cells (normally noticeable by about passage 6), and therefore we performed all studies on virus stocks of passage 2 or 3.

Expression of C and V proteins by mutant viruses.

The effectiveness of the introduced mutations in preventing the expression of the C or V protein was assessed by immunoprecipitation of ³⁵S-labeled proteins from lysates of labeled infected cells. As can be seen in Fig. 2, all three P-gene-derived proteins were made in cells infected with ordinary RPV. No C protein was found in cells infected with RPV CE, VCE, C−, or VC−, and no V protein was found in cells infected with RPV V−, VCE, or VC−. To determine if the mutations that we had introduced into the editing site of the P gene had abolished nontemplated base insertion completely, we used a modification of the primer extension technique that we had used previously to examine P gene editing in different strains of the virus (30), in which a labeled primer is extended on isolated mRNA, or PCR products derived from mRNA, until the first A residue is incorporated. As the only source of adenine is ddATP, this terminates extension after only 10 to 12 bases. This technique should show one band for the normal unmodified P gene transcript, with an additional band for transcripts containing an additional base (Fig. 3a). Further bands will be seen for transcripts containing two or more nontemplated bases: such editing events are normally seen, to various extents, in morbilliviruses (5, 10, 30). Due to the mutations introduced into the P gene in order to abolish editing, the predicted size of the primer extension product from the unedited P(V−) gene transcript is one base longer than that from the P gene of the unmutated virus (Fig. 3b). Although editing of transcripts from the P gene of normal RPV could easily be seen (including +2 and +3 bases), there was clearly no editing in viruses containing the V− mutation (Fig. 3c), the single primer extension product being 29 bases long, the same length as the product from the V mRNA cDNA clone.

Growth of mutant viruses in tissue culture cells.

We examined the replication rates of all four virus types in tissue culture using B95a cells, the monkey kidney-derived Vero cell line, or primary bovine skin fibroblasts. Although unmodified RPV could be grown to titers of 10⁶ to 10⁷ TCID₅₀/ml after only a few passages, we found that none of the mutants could be grown in large-scale culture to greater than 10⁵ TCID₅₀/ml, and the double mutant could barely be grown to 10⁴ TCID₅₀/ml. All growth curves therefore had to be multistep, with an initial infection at an MOI of approximately 0.01 to enable all viruses to be used at the same (or nearly the same) infectious dose. In B95as, we found that the initial growth rate of the virus was unaffected by the absence of the V protein, but there was a decrease in the final titer reached (Fig. 4a). In contrast, the initial growth rate of viruses lacking the C protein (RPV C− or RPV VC−) was significantly lower (Fig. 4a). Since the final titer reached is dependent on the relative rates of viral replication and decay (RPV is not particularly stable at 37°C, and newly made virus particles lose infectivity during subsequent culture of the infected cells), it is not surprising that the more slowly growing RPV C− mutants also grew to a lower final titer. Identical results were seen in Vero cells (not shown). In bovine skin fibroblasts, the main difference observed (Fig. 4b) was that the double mutant grew more slowly than that lacking only the C protein, while the RPV V− mutant continued to grow almost identically to the normal RPV. In these cells, it appears that the absence of the V protein is somehow more deleterious to viral replication in the absence of the C protein than in its presence.

Although the RBOK vaccine strain of RPV is not particularly lytic in cell culture and does not form good plaques, we have found that we can observe the morphology of individual foci of infection by overlaying infected cells with carboxymethyl cellulose and fixing and staining the cells after 5 to 6 days using Giemsa's stain (see Materials and Methods). Examples of the viral foci observed for the four types of virus are shown in Fig. 5: clear differences were seen due to the presence or absence of the individual V or C proteins. Normal RPV shows large areas mostly cleared of cells, with several small syncytia in each infected focus. RPV V− developed similar-size plaques but with many more and larger syncytia, while RPV C− developed plaques similar to those formed by RPV only much smaller, as might be expected from its lower growth rate. The foci of growth of RPV VC− showed both changes, being smaller than normal RPV and having the increased syncytium-forming tendency of RPV V−. The effects of lack of the individual V or C proteins are thus clearly distinct and additive, as both phenotypes are combined when the mutations are combined.

Effects of defects in V or C expression on synthesis of viral RNAs.

Although the V− mutation had no detectable effect on viral growth rate in tissue culture, it was clear from the above results that this mutation did change the way the virus replicated, inasmuch as the balance between virus-induced cytopathic effect and viral protein-induced cell-cell fusion was altered in viruses carrying this mutation. As we were unable to grow virus to high titer and hence unable to infect at high MOI, we were unable to generate sufficiently strong signals in Northern blots to study the synthesis of viral RNAs at early time points (0 to 24 h postinfection [hpi]) when viral replication is still effectively in single-step mode (growth curves show that new virus is released from cells only at 20 to 24 hpi). We therefore used semiquantitative RT-PCR (35) to compare the four virus types with each other. Initial experiments showed that the amount of PCR product was proportional to the input RNA over a range of about 50-fold, though the optimum number of cycles depended on the maximum and minimum of the range of input RNA concentrations. RNA was isolated from cells infected at an MOI of approximately 0.005 at various times postinfection, and RT reactions were performed with primers specific for mRNA [oligo(dT)], genome sense RNA, or antigenome sense RNA. Primer pairs for PCR amplification of antigenome sequences included one primer from a promoter (non-mRNA) region. The results of these studies are shown in Fig. 6. We used the amplification of actin mRNA as an internal control to show that RNA from the same number of cells was included in each reaction mixture. The actin signal was in the linear range after 15 or 16 cycles, similar to the results found for cells infected with SeV at high MOI (35). To detect the RPV genome, however, 20 to 22 cycles were required, while antigenome amplification required about 28 cycles (Fig. 6). We consistently found less antigenome than genome in infected cells (compare the amounts of PCR product found for genome and antigenome at 22 cycles in Fig. 6a). The number of cycles required for detecting viral mRNAs depended on the gene, since RPV, like all members of the order Mononegavirales, shows a transcription gradient along its genome, with most transcription taking place from the N gene (the nearest to the genome promoter), followed in decreasing amounts by the P, M, F, H, and L genes, in order along the genome (3′ to 5′) (11). This arises, it is thought, because primary interaction of the viral polymerase with the genome can occur only at the genome promoter, and the polymerase has only a finite probability of initiating transcription from any one gene after it has finished transcribing and polyadenylating the previous one. Transcription from either the P gene or the H gene was clearly depressed in the absence of the C protein (compare P or H gene mRNA from RPV with RPV C− and RPV V− with RPV VC in Fig. 6), suggesting that the initiation or extension of viral mRNA transcripts was decreased in these mutants and that the C protein of RPV has a role in these processes.

In contrast, levels of genome and antigenome in RPV and RPV C− were indistinguishable (Fig. 6), while mutants lacking the V protein (RPV V− and RPV VC−) clearly showed higher levels of such transcripts at 24 hpi, suggesting either an earlier onset or more rapid rate of replicative transcription in the absence of the V protein. Given these higher amounts of genome RNA, the template for transcription of viral mRNAs, one would expect the V− and VC− mutants to show a correspondingly greater amount of viral mRNAs as well, which is what is observed. However, the increase in mRNA levels seen in the mutants lacking the V protein is less than the corresponding increase in genome RNA levels. The V protein may therefore cause an increase in the general efficiency of mRNA transcription from the viral genome, although it is not clear whether it does so by inhibiting replicative transcription or promoting that of message.

We also observed that most of the time zero samples contained detectable amounts of both genome and antigenome RNAs, presumably derived from the initial inoculum; the time zero samples from RPV VC−-infected cells always contained much more genome and antigenome than any of the others. Given that all experimental wells were infected with the same dose of infectious virus (as measured by TCID₅₀), this suggests that preparations of RPV VC− had far higher amounts of viral RNA, either as intact virus or unenveloped nucleocapsid, per infectious unit than was the case for the other virus types, suggesting in turn a greatly decreased efficiency of incorporation of viral genomes into infectious particles.