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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ABSTRACT |
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MAIN TEXT |
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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 -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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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 56·5 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.
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ACKNOWLEDGEMENTS |
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Received 24 June 2003;
accepted 4 August 2003.
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