Transcript mapping of the ‘early’ genes of Orf virus

Ann R. Wood and Colin J. McInnes

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK

Correspondence
Colin McInnes
mcinc{at}mri.sari.ac.uk


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The full complement of genes encoded by Orf virus (ORFV) is not yet known. A cDNA library was constructed using mRNA isolated 5 h post-infection from cells infected with ORFV in vitro and grown in the presence of cytosine arabinoside. Using 12 non-overlapping probes representing the entire genome of the Orf-11 strain of the virus, cDNA clones representing individual genes expressed early in infection were isolated. Thirty-eight early genes were identified, either via isolation of their cDNA from the library or via Northern blotting. Twenty-nine of the isolated cDNAs represented orthologues of other poxvirus genes or had been identified previously as genes of ORFV, whilst seven appeared unrelated to any known poxvirus gene or indeed to any known gene in the DNA databases. The sequences described in this paper constitute approximately 30 kb of the ORFV genome and contain the complete or partial sequence of 47 genes.


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Orf virus (ORFV) is the prototype species of the Parapoxvirus genus of the poxvirus family and is found throughout the world wherever sheep and goats are farmed. It causes a highly contagious pustular dermatitis in these animals which is self-limiting and rarely results in mortality, although secondary adventitious infections can cause severe complications (Haig & Mercer, 1998).

The ORFV genome is approximately 140 kb in length and has a G+C composition of about 63 % (Menna et al., 1979; Wittek et al., 1979). Although the complete genome sequence has yet to be published there are many reports describing individual ORFV genes and the general relationship between ORFV and the other poxviruses. The central region of the ORFV genome is collinear with that of Vaccinia virus (VACV), Variola virus (VARV) and Molluscum contagiosum virus (MOCV), both in the relative order and orientation of genes, and often also in the spacing between genes (Fleming et al., 1993; Mercer et al., 1995, 1996a). However, since the ORFV genome is considerably smaller than these other genomes not all of their genes will be found in ORFV. Determining the exact gene complement of the ORFV genome will be problematical due to its high G+C content compared with the majority of other poxviruses which have a lower G+C composition. Annotation of the sequence will rely on a number of different parameters including the presence of promoter-like sequences, transcription termination signals, theoretical translation comparisons and codon usage (Fleming et al., 1991, 1992; Mercer et al., 1995). As an aid to help identify ORFV genes we have isolated cDNA clones corresponding to viral genes expressed early in an ORFV infection.

RNA was isolated from foetal lamb muscle (FLM) cells 5 h after they were infected with the Orf-11 strain of ORFV (McInnes et al., 2001). Cells were infected at an m.o.i. of 20 TCID50 units and were grown in ‘199’ medium supplemented with 2 % (v/v) foetal bovine serum (Gibco) and 40 µg cytosine arabinoside (AraC) ml-1, a concentration that inhibits intermediate and late gene expression but not that of early viral genes (Cooper & Moss, 1979; Deane et al., 2000). TCID50 values were based upon the dilution of virus giving 50 % cytopathic effect in FLM cells.

Northern blotting was used to assess the approximate number of genes expressed by ORFV early in infection. Twelve {alpha}-32P-labelled double-stranded (ds)DNA probes, prepared from cloned fragments of the Orf-11 genome as illustrated in Fig. 1(A) were hybridized with the RNA as described previously (McInnes et al., 1993). The resulting blots are shown in Fig. 1(B). The number of bands appearing in each Northern is indicative rather than definitive in terms of the number of early genes being expressed from each region, although it is clear that early genes are scattered throughout the genome.



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Fig. 1. (A) Schematic representation of the Orf-11 genome showing the KpnI restriction enzyme sites. The restriction fragments are named alphabetically starting with the largest fragment. Fragments at the left-hand end that are identical to those found at the right-hand end are indicated by the superscript ('). The inverted terminal repeats are indicated by solid boxes. The regions covered by the 12 probes used for Northern blotting and to screen the cDNA library are indicated below the map. (B) Northern blots of total RNA isolated from FLM cells 5 h post-infection with Orf-11 (m.o.i.=20 TCID50) and grown in the presence of 40 µg AraC ml-1. The blots were probed with [{alpha}-32P]dCTP-labelled dsDNA fragments corresponding to the genomic regions indicated in (A). The probes used for each are indicated below the blots.

 
A cDNA library, constructed using poly(A)+ RNA from the same source as was used for the Northern blotting, was screened with the DNA probes indicated in Fig. 1(A). Clones were selected, the nucleotide sequence of each was determined in full and the sequence data were compared with the GenBank nucleotide database. Verification that the cDNAs corresponded to genes that were expressed early was obtained by using each as a probe in further Northern blots (results not shown). Since the cDNA library was constructed in the directional vector pCMV-Script XR (Stratagene) we were able to analyse the 3' end of each cDNA for the presence of a poly(A)+ tail, a transcription termination motif (T5NT) and a stop codon. Protein coding regions were predicted by translating each of the three frames and comparing the conceptual amino acid sequences with the SWISSPROT database. The majority of clones had one major open reading frame (ORF) running from the start of the clone through to a stop codon near to a T5NT motif and the poly(A)+ tail. Candidate initiation codons were present in some of the sequences. Nine of the clones corresponded to ORFV genes described previously. Nineteen of the clones represented orthologues of VACV genes, one an orthologue of a Leporipoxvirus gene and one an orthologue of an MOCV gene, based on the predicted translation products. Seven of the clones shared little or no sequence similarity to anything in either the nucleotide or protein databases. A summary of the sequence data is given in Table 1. A codon usage table was constructed using the genes encoding orthologues of VACV genes as well as those identified previously as functional ORFV genes (data not shown). The table contained data from approximately 5250 codons. There is a heavy bias towards a G or C residue in the third base of the codons. In fact for all the amino acids for which there is a choice of codon those with either a G or C in the third position are used between 78 % and 93 % of the time, with the exception of isoleucine where the AUC codon is used 69 % of the time. In addition codons which contain G or C residues at two positions are used approximately 71 % of the time. The codon usage table was used in conjunction with the program CODONPREFERENCE (GCG Version 10.0) to verify that the predicted ORF of those cDNAs which were apparently unrelated to anything in the databases matched the expected codon usage and also to check for frameshifts in those sequences that diverged abruptly from their VACV counterparts.


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Table 1. Summary of the sequence data obtained from the cDNA clones representing early ORFV genes

ORFV, Orf virus; MOCV, Molluscum contagiosum virus; SFV, Shope fibroma virus; VACV, Vaccinia virus; RPO, RNA polymerase subunit; VEGF, vascular endothelial growth factor; IL, interleukin; GM-CSF, granulocyte–macrophage colony-stimulating factor.

 
Some of the cDNAs contained two major ORFs, with the T5NT motif being found within or downstream of the second ORF. These are listed in Table 2 together with the identities of the complete or partial genes contained therein. It is not uncommon for there not to be a T5NT motif between the end of an early gene and the start of the next gene. Of the 56 early genes predicted to be present in the genome of Fowlpox virus (FPV) only 22 have a T5NT motif closely associated with their stop codon (Afonso et al., 2000).


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Table 2. cDNA clones containing sequence from more than one gene.

VACV, Vaccinia virus; RPO, RNA polymerase subunit; dsRNA, double-stranded RNA; IFN, interferon.

 
The majority of genes mapping to regions 10, 4, 9, 2, 5, 8 and 7 from Fig. 1(A) (corresponding to approximately 90 kb in the middle of the ORFV genome) are related to genes found in VACV and the other poxviruses (Table 1). Generally, they encode proteins involved in transcription and replication. In this region we found only three early genes that do not appear to have counterparts in the other poxvirus genera. Previously, we had identified one of these (clone 316) as a late gene encoding a protein (GIF) that binds and inhibits the ovine cytokines interleukin (IL)-2 and granulocyte–macrophage colony-stimulating factor (Deane et al., 2000). However, in this study we were able to detect a signal on a Northern blot of early RNA corresponding to this gene, suggesting that although the gene is undoubtedly expressed early it is done so either transiently or at very low levels (results not shown). Sequencing of clone 316 revealed a transcript terminating downstream of the T5NT motif situated 119 bases after the stop codon. The functions of the proteins encoded by the other two unknown genes from this region (clones 53 and 103) remain obscure as they do not appear to be related to anything in the protein databases.

Of the 14 early genes found in the flanking regions of the genome, (regions 3, 11, 6, 1 and 12 in Fig. 1A), seven have been described previously in ORFV (Fraser et al., 1990; Lyttle et al., 1994; Sullivan et al., 1995a; Fleming et al., 1997; McInnes et al., 2001; Rziha et al., 2003), two are orthologues of VACV genes and the remaining five appear unrelated to anything in the nucleotide or protein databases. Only two of the ORFV genes have had a function ascribed to them: those encoding the viral vascular-endothelial growth factor (VEGF) and viral IL-10 (Meyer et al., 1999; Wise et al., 1999; Fleming et al., 2000; Savory et al., 2000; Imlach et al., 2002). The remaining five, together with the five unknown genes reported here, have not been functionally characterized. Analysis of the predicted amino acid sequences for membrane-spanning regions, signal peptides, DNA-binding regions and functional motifs and fingerprints yielded little clue to their function (results not shown). However two (ANK-2 and ANK-3) contain a number of ankyrin-like repeat elements (Rziha et al., 2003).

Most of the genes identified in this study have G+C contents of between 58 % and 73 %, but there are a few exceptions. These include the genes encoding the viral IL-10 (47 %) and the putative chemokine-binding protein (51 %), leading to the suggestion that their G+C content is a reflection of their recent acquisition from the host (Fleming et al., 2000). If this is true then the same could be said for the orthologue of VACV A33R, clone 11 (44 %), and the equivalent of ORFV NZ-2 B3L, clone 377 (51 %). However, the proteins encoded by these genes are not related to any known mammalian proteins. Since orthologues of the VACV A33R are found across the poxvirus genera it is likely that it was present in an ancestral virus. Thus an alternative explanation for the low G+C content of some ORFV genes would be the requirement to maintain the structural integrity of the proteins encoded by them.

We did not isolate cDNA clones corresponding to all the signals obtained on the Northern blots and in particular for those representing the larger mRNAs found in regions 4, 9 and 8 (see Fig. 1B). However, using plasmid subclones of the ORFV genome corresponding to internal regions of the putative ORFV RNA polymerase (RPO) 147 kDa and 132 kDa subunit genes (orthologues of the VACV J6R and A24R respectively) as probes, we were able to confirm that these genes were expressed early and indeed corresponded to the larger mRNAs from regions 4, 9 and 8 (results not shown).

The sequences reported in this paper together constitute approximately 30 kb of the ORFV genome and contain the complete or partial sequences of 47 genes, at least 36 of which are expressed early in infection. We also found that the ORFV orthologues of the VACV J6R and A24R genes were expressed early. Orthologues of VAVC E9L, E3L and F12L and the ORFV genes provisionally named G1L, E3L and E2L (not present in the Orf-11 genome) have all been reported previously to be expressed early (Mercer et al., 1989, 1996b; Fleming et al., 1995; Sullivan et al., 1995b; Cottone et al., 1998, 2002; McInnes et al., 1998). Taken together this would suggest that there are at least 44 early genes within the ORFV genome. This compares to 30 predicted to be present in the similarly sized genome of Swinepox virus (SWPV) and 56 in the FPV genome, which is approximately twice the size (Afonso et al., 2000, 2002). In both these viruses prediction of whether or not a gene was expressed early was based on the presence of early promoter-like sequences upstream of the predicted initiation codon. Twelve of the SWPV early genes are found in the flanking regions of the genome and may not be present in ORFV. Thirteen corresponded to genes found in this study whist the remaining five were not detected. However, of the early ORFV genes found in this study that have orthologues in the SWPV genome there were eight where no prediction was made for the SWPV genes and a further two which were predicted to be expressed late in infection. One of these, however (the orthologue of VACV A5R), was predicted to be expressed early by FPV.

Whether or not all the ORFV early genes have been identified is unknown. Early viral proteins are produced in cells treated with AraC, in comparison to those treated with cycloheximide, and may have had an effect on the transcription pattern observed in this study. For example, the VACV E9L gene is expressed during the early phase of infection. In cells grown in the presence of AraC transcripts reach a peak about 2·5 h post infection and are barely detectable by 5–6·5 h, whereas in the presence of cycloheximide they continue to accumulate (McDonald et al., 1992). Early expression of the ORFV orthologue of VACV E9L has been detected in cells grown in the presence of cycloheximide (Mercer et al., 1996b), whereas we did not isolate the corresponding cDNA nor did we detect an mRNA on a Northern blot of early RNA using an appropriate probe (results not shown). Nevertheless, studying the promoter regions of the genes isolated in this study may provide us with a better understanding of the ‘core’ elements of ORFV early promoters, and once the ORFV genome sequence becomes available should help to identify any early genes that were not identified here.


   ACKNOWLEDGEMENTS
 
This work was funded by the Scottish Executive Environment and Rural Affairs Department.


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Received 24 June 2003; accepted 4 August 2003.



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