Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany1
Author for correspondence: Walter Fuchs. Fax +49 38351 7219. e-mail Walter.Fuchs{at}rie.bfav.de
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Abstract |
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Within the IRS and TRS sequences flanking the US region, two PrV genes encoding the homologues of the HSV-1 immediate-early proteins ICP4 (IE180) and ICP22 (RSp40) have been identified and latency-associated transcripts were detected in the antiparallel orientation to the IE180 mRNA (Cheung, 1989 , 1991
; Vlcek et al., 1990
; Zhang & Leader, 1990
). Between the two conserved genes, a short stretch of genomic DNA, located at the right end of the internal copy of the 7·7 kbp BamHI fragment 5 (Fig. 1b
), remained uncharacterized. Since earlier studies indicated the presence of an origin of viral DNA replication within the IRS and TRS sequences of PrV (Ben-Porat & Veach, 1980
), it was speculated that this regulatory element might reside within the uncharacterized DNA sequences.
In order to verify this, we cloned the 2·4 kbp BamHISalI fragment 5B and the overlapping KpnI fragments I and K (Fig. 1a) from virion DNA of PrV strain Ka (Kaplan & Vatter, 1959
) into plasmid pBS (-) (Stratagene). After digestion with suitable restriction enzymes, the inserts of the independently cloned plasmids pBS-BS5B-1 and -2 (Fig. 1c
) were shortened unidirectionally by treatment with exonuclease III and religated (nested-deletion kit, Pharmacia). Deletion plasmids were sequenced (Thermo Sequenase cycle-sequencing kit, USB) with the vector-specific M13 universal or reverse primers. Both strands of the DNA fragment investigated were analysed completely and proved to be complementary at all positions. To confirm the adjacent localization of the genomic BamHI fragments 5 and 10 or 5 and 12 (Fig. 1b
), the terminal part of pBS-KI (Fig. 1c
) was also sequenced. Finally, by sequencing of pBS-KK (Fig. 1c
) with PrV-specific custom primers (Gibco BRL), an overlap with characterized upstream sequences was generated. Data were assembled and analysed by using the GCG software package in UNIX version 10.0 (Devereux et al., 1984
).
The PrV DNA sequence obtained (GenBank accession no. AJ251976) is 2462 bp in length and exhibits a G+C content of 69·5%, which is slightly below the mean percentage of 73% (Ben-Porat & Kaplan, 1985 ). The 2396 bp inserts of the two plasmids analysed (pBS-BS5B-1 and -2) proved to be identical. At both ends, the novel sequence overlaps with previously described DNA sequences of the same PrV strain (Ka). Nucleotides 139 correspond to the reverse of the first bases of a sequence of the PrV IE180 gene region (Vlcek et al., 1990
; GenBank accession no. M34651) and nucleotides 24242462 are identical to bases 139 of BamHI fragment 10 (Zhang & Leader, 1990
; GenBank accession no. D00676).
The DNA fragment characterized contains several open reading frames (ORFs) of up to 315 codons (not shown). However, the deduced translation products share no homologies with known herpesvirus proteins. Furthermore, these ORFs are not preceded by TATA box-like elements and putative mRNA polyadenylation signals (AATAAA) are completely absent.
In order to investigate transcription of this part of the virus genome, porcine kidney (PSEK) cells were infected with PrV at an m.o.i. of 10 and incubated in the presence or absence of 100 µg/ml cycloheximide as an inhibitor of protein synthesis. Total RNA was prepared and Northern blot analyses were performed essentially as described previously (Fuchs & Mettenleiter, 1996 ). With 32P-labelled cRNA probes of pBS-BS5B (Fig. 1d
), a PrV-specific RNA of 1·8 kb was identified (Fig. 2a
), whereas no defined viral transcripts from the opposite strand were detectable (Fig. 2b
). The 1·8 kb RNA was shown to be polyadenylated, since it was bound by oligo(dT)-coupled magnetic beads (Dynal) (data not shown). Blot hybridization with labelled BamHI fragment 12 (probe a3, Fig. 1d
) as well as with pBS-BS5B subfragment a2 but not with a1 (Fig. 1d
) revealed that the transcript detected represents an mRNA of the RSp40 gene of PrV (data not shown). The RSp40 mRNA was described previously not to be expressed in the presence of cycloheximide (Zhang & Leader, 1990
). However, our results demonstrate expression of the 1·8 kb RNA under immediate-early conditions, although it becomes more abundant at early times after infection in the absence of drugs (Fig. 2a
). In contrast, the major immediate-early mRNA of IE180 (Fig. 2c
) accumulates only in the presence of cycloheximide, indicating that transcription of this gene is rapidly down-regulated after the onset of virus replication. Other transcripts of PrV, e.g. that of UL34 (Fig. 2d
) or UL23 (thymidine kinase, not shown), were not found in cycloheximide-treated cells, demonstrating that the inhibition of viral protein synthesis was sufficient in our experiments and that the discrepancy from earlier results might be due to the different cell line used or to a higher sensitivity of our hybridization probes. Thus, RSp40 of PrV, like the homologous ICP22 of HSV-1 and BHV-1 (Roizman & Sears, 1996
; Schwyzer et al., 1994
), can be expressed from an immediate-early RNA and is possibly required for efficient transcription of early and late viral genes.
Another common feature of the ICP22 mRNAs of HSV-1 and BHV-1 is the removal of introns from their 5'-untranslated sequences (Roizman & Sears, 1996 ; Schwyzer et al., 1994
; Wirth et al., 1991
). It appears likely that the 1·8 kb RSp40 mRNA of PrV is similarly spliced, since we localized its 5' end within BamHI fragment 5 but the predicted initiation codon is at positions 584586 and the polyadenylation signal is at positions 17821787 of the downstream BamHI fragment 10 or 12 (GenBank accession no. D00676; Zhang & Leader, 1990
). To test this hypothesis, we tried to characterize the 5' end of the detected mRNA by PCR amplification of oligo(dC)-tailed cDNA (5' RACE system, Gibco BRL), which was prepared from polyadenylated RNA obtained 3 h after PrV infection (m.o.i.=10) of PSEK cells. In repeated experiments with an oligo(dG) primer and the antisense primer PP22-R (reverse of nt 588610 of D00676; Fig. 1c
), we were not able to detect defined amplification products, indicating premature termination of cDNA synthesis at various positions. However, with primers PP22-F (nt 22602292 of GenBank accession no. AJ251976; Fig. 1c
) and PP22-R, a 498 bp cDNA fragment could be amplified reproducibly and plasmid-cloned (pBS-P22C, Fig. 1c
). Sequencing of pBS-P22C revealed the absence of two internal 121 and 155 bp fragments of genomic PrV DNA (nt 2410 of AJ251976 to nt 107 of D00676 and nt 161315 of D00676). The 5' and 3' ends of both introns and the surrounding nucleotides fit the consensus sequences of eukaryotic splice-donor and splice-acceptor sites (Breathnach & Chambon, 1981
). Although the precise 5' end of the RSp40 mRNA and possible further post-transcriptional modifications remain to be determined, we identified the next well-conserved TATA-box motif (TATATAT) at positions 20002006 of the novel DNA sequence. The functions of the extended mRNA and intron sequences upstream of the RSp40 coding region remain unclear.
Most alphaherpesvirus origins of DNA replication contain the conserved recognition sequence GTTCGCAC of the viral origin-binding protein (OBP) (Elias & Lehman, 1988 ; Koff & Tegtmeyer, 1988
). The DNA sequence of BamHISalI fragment 5B of the PrV genome contains three complementary pairs of this motif at positions 14071414 and 14571464, 16871694 and 17371744 and 19661973 and 20162023 (Fig. 1b
). The 42 nucleotides between the two predicted OBP-binding sites of each pair contain between 86 and 89% A and T residues, which corresponds to the base compositions in the central regions of other alphaherpesvirus origins located at related positions within the IRS and TRS sequences flanking the US genome regions of HSV-1 and -2, VZV, EHV-1 and BHV-1 (OriS) (Baumann et al., 1989
; Lockshon & Galloway, 1988
; Schwyzer et al., 1994
; Stow & McMonagle, 1983
; Stow & Davison, 1986
) or within the UL regions of HSV-1 and -2 and PrV (OriL) (Lockshon & Galloway, 1988
; Klupp et al., 1992
). In contrast to the situation in other alphaherpesvirus genomes, the AT-rich sequences of the predicted OriS of PrV are poorly palindromic. Whereas in the IRS and TRS sequences of HSV-1, EHV-1 and VZV, only single OriS elements were found, they are duplicated in HSV-2 and BHV-1 DNA. In the case of PrV, three directly repeated copies of the putative OriS domain were found close to each other. These are presumably the consequence of ancient duplication events, since nucleotides 13531630 share sequence identities of 88·5 and 86·2% with the following nucleotides 16311911 and 19122172, respectively.
In order to verify the function of PrV OriS in vitro, rabbit kidney (RK13) cells were co-transfected (lipofectamine plus reagent, Gibco BRL) with pBS-BS5B (Fig. 1c) and the control plasmid pUC-BS1D (Fig. 1a
), which contains a randomly chosen 2 kbp SalI fragment of the PrV genome cloned in pUC19 (New England Biolabs). One day after transfection, half of the cell sample was infected with PrV at an m.o.i. of 5 and harvested 8 h later. DNA of infected and uninfected cells was prepared as described previously (Fuchs & Mettenleiter, 1996
) and doubly digested with EcoRI and HindIII to release the cloning vectors pBS (-) (3·2 kbp) and pUC19 (2·7 kbp). Digestion with modification-specific restriction enzymes followed by electrophoretic separation and Southern blot hybridization with 32P-labelled pUC19 allowed discrimination between Dam-methylated input plasmids and newly synthesized DNA (Fig. 3
). After PrV infection, about a third of the pBS-BS5B DNA was resistant to cleavage by the methylation-dependent enzyme DpnI and, consequently, cleavable by DpnII, which is inhibited by Dam-methylation. In contrast, the control plasmid pUC-BS1D remained completely sensitive to DpnI and resistant to DpnII (Fig. 3
, left two lanes). Plasmid replication was not detectable in uninfected cells, indicating that viral proteins are required for initiation. Furthermore, it was confirmed (Fig. 3
, right part) that no unique elements of the vector pBS (-) are responsible for autonomous DNA replication of the cloned BamHISalI fragment 5B. The minimum sequence requirement of PrV OriS and the influence of the consensus sequence copy number on the efficiency of viral DNA replication remain to be tested.
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Footnotes |
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References |
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Received 7 January 2000;
accepted 8 March 2000.