Transcriptional analysis of the white spot syndrome virus major virion protein genes

Hendrik Marks, Melanie Mennens, Just M. Vlak and Mariëlle C. W. van Hulten

Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands

Correspondence
Just Vlak
just.vlak{at}wur.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
White spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5' and 3' ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5' RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5' ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
White spot syndrome virus is a large, circular, double stranded (ds) DNA virus that infects shrimps and other crustaceans (Wang et al., 1998; Wongteerasupaya et al., 1995). The virion particles, approximately 275x120 nm in size, are enveloped and have an ovoid-to-bacilliform shape with a tail-like appendage at one end (Durand et al., 1997; Nadala et al., 1998). Sequencing of three different WSSV isolates revealed that the dsDNA genome is about 300 kb in size (van Hulten et al., 2001a; Yang et al., 2001; GenBank acc. no. AF440570). Most of the 184 putative ORFs identified on the WSSV genome are unassigned, as they lack homology to known genes in public databases (van Hulten et al., 2001a). Based on its unique morphological and genetic features, WSSV has been accommodated in a new virus family, the Nimaviridae (genus Whispovirus).

The WSSV particle consists of five major and about 14 minor structural proteins (van Hulten et al., 2002; Huang et al., 2002). The five major proteins have been named according to their sizes in SDS-PAGE: viral protein (VP) 28, VP26, VP24, VP19 and VP15 (van Hulten et al., 2000a, b, 2002). VP26, VP24 and VP15 are present in nucleocapsid preparations, while VP28 and VP19 are found in envelope fractions of the virions. As an antiserum against VP28 was able to neutralize WSSV infection in the shrimp Penaeus monodon, this protein is most likely located on the surface of the virus particle and plays a key role in systemic WSSV infection in shrimp (van Hulten et al., 2001b). A putative function for VP15, a highly basic protein with no hydrophobic regions, is that of a histone-like, DNA-binding protein (van Hulten et al., 2002; Zhang et al., 2001).

Genes of most large dsDNA viruses infecting (in)vertebrates are expressed in a cascade fashion. Immediate early (IE) and early (E) genes are expressed before viral DNA replication, while expression of late (L) genes occurs after replication of the viral genome. For proper late gene expression, the motif that contains the transcription initiation site (TIS) often plays a prominent role in recognition by a virus-coded RNA polymerase (Davison & Moss, 1989; Garcia-Escudero & Viñuela, 2000; Kim et al., 2002; Morris & Miller, 1994; Weir, 2001). Information about pathogenicity, epidemiology, virion structure and the genomic sequence of WSSV is now available, but insight into gene regulation is limited. Transcriptional analysis has been performed on WSSV genes encoding the large and small subunit of ribonucleotide reductase (rr), protein kinase (pk), the chimeric thymidine kinase–thymidylate kinase (tk-tmk) and DNA polymerase (dnapol). These genes were identified in P. monodon 2 or 4 h post-WSSV infection (Liu et al., 2001; Tsai et al., 2000a, b; Chen et al., 2002), indicating that these genes are of the early type. The transcription of rr1, rr2, pk and dnapol is initiated 20–28 nt downstream of a TATA-box, suggesting a functional role for the TATA-box during early transcription. Both rr genes and dnapol share a consensus WCABT (W=a/t; B=c/g/t) sequence in which transcription is initiated (Chen et al., 2002). This somewhat degenerated consensus TIS motif could be an early promoter element of WSSV.

The present study focuses on the transcription and regulation of WSSV late genes. As no cell lines or primary crayfish or shrimp cell cultures are available, experiments were performed in vivo. Temporal gene expression of the major structural protein genes vp28, vp26, vp24, vp19 and vp15 (all putative late genes) was studied by transcriptional analysis using RT-PCR. 5' and 3' RACE analyses were used to map the TISs and polyadenylation sites of these major structural protein genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus infection.
The virus isolate used in this study originates from infected Penaeus monodon shrimp imported from Thailand in 1996 and was obtained as described before (van Hulten et al., 2000b). Crayfish (Orconectes limosus), equally susceptible to WSSV infection as P. monodon and used as a model, was injected intramuscularly with purified WSSV using a 26-gauge needle to initiate infection. At various time-points after injection, three crayfish were randomly selected, frozen in liquid nitrogen and stored at -80 °C. For infection of P. monodon, the same infection procedure was followed, using the same virus isolate.

RNA isolation.
After removal of the exoskeleton and the underlying epidermis of frozen crayfish, gill tissue was excised with a scalpel. For isolation of total RNA, 300 mg of frozen gill tissue was homogenized in 4 ml TRIzol Reagent (Invitrogen), using a glass-Teflon homogenizer, and subjected to chloroform extraction, isopropanol precipitation and ethanol washing according to the manufacturer's recommendations. The precipitated RNA was dissolved in 40 µl RNase-free water. The concentration of RNA was quantified by measuring the absorbance of the RNA solution at a wavelength of 260 nm. RNA from the gills of shrimp was isolated using the same method.

RT-PCR.
For the RT reaction, 20 µg of isolated RNA was treated with DNaseI (Gibco BRL), according to the manufacturer's protocol. To inactivate the DNaseI, the sample was heated to 65 °C for 10 min followed by a phenol/chloroform extraction and ethanol precipitation. The RNA was resuspended in 20 µl RNase-free water. Five µg of RNA was used in the RT reaction. This was performed using SuperScript RNase H- reverse transcriptase according to the manufacturer's (Invitrogen) protocol. PCR on 1 µl of the cDNA obtained was performed using Taq DNA polymerase (Promega) with specific primer sets for the different genes, listed in Table 1(a). For vp28, vp26, vp24, vp19 and vp15 25 cycles were run; for actin and rr1 33 cycles were used. The PCR machine used was the GeneAmp PCR System 2400 (Applied Biosystems). Twenty µl of each PCR product was analysed by agarose gel electrophoresis.


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Table 1. Primers used for RT-PCR (a) and for the 5' and 3' RACE (b)

Primer coordinates are according to the WSSV sequence deposited as GenBank acc. no. AF369029 (van Hulten et al., 2001a).

(a) RT-PCR

 

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(b) 5'/3' RACE

 
5' and 3' RACE.
Both 5' and 3' RACE were carried out using a commercial 5'/3' RACE kit (Roche) following the manufacturer's instructions. Total RNA was isolated from the gills of O. limosus 6 days post-infection (p.i.) as described above. In the case of the 3' RACE, first-strand cDNA was synthesized using the oligo(dT) anchor primer. The resulting cDNA was amplified using one specific forward primer (see Table 1b; RACE-F1 primers) and the anchor primer. For 5' RACE, the primers used for synthesis of the cDNA are described in Table 1(b) (RACE-R1 primers). This cDNA was purified using the High Pure PCR product purification kit (Roche) and a homopolymeric 3' d(A)-tail was added to the cDNA in a mixture with a total volume of 20 µl, using terminal transferase and dATPs included in the kit. Five µl of this mixture was used in a first-round PCR performed with an oligo(dT) anchor primer and a nested primer, shown in Table 1(b) (RACE-R2 primers). For vp28 and vp24, the protocol was slightly modified. Instead of poly(A)-tailing, the cDNA was 3'-tailed with dTTPs and the first PCR cycle was performed with an oligo(dA) anchor primer instead of the oligo(dT) anchor primer. For vp24 and vp15, a second-round nested PCR was performed using the anchor primer and a second nested primer, shown in Table 1(b) (RACE-R3 primers). The final products of the 5' and 3' RACE were cloned into the pGEM-T easy vector (Promega) and sequenced.

DNA sequencing and computer analysis.
Plasmid clones carrying RACE products were sequenced at the Laboratory of Molecular Biology (Wageningen University, the Netherlands), using universal M13 forward and reverse primers. Sequence data were analysed using the software package DNASTAR 4.2 and the data were edited in GeneDoc, version 2.6.000 (Nicholas et al., 1997).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
All five WSSV major structural virion protein genes are present as single copies in the WSSV genome (van Hulten et al., 2001a). Polyadenylation motifs are present downstream of, or overlapping with, the translational stop codon of the ORFs, suggesting that the transcripts are polyadenylated. The regions upstream of the translational start sites of the major structural protein genes all contain stretches of A/T-rich sequences. Only vp15 contains a consensus TATA-box sequence, 87 nt upstream of the translational start codon. The nucleotides surrounding the methionine start codons of the five major structural protein genes are consistent with the Kozak rule for efficient eukaryotic translation initiation (Kozak, 1989).

RT-PCR
Temporal WSSV gene expression of the major structural virion protein genes was studied in crayfish using RT-PCR. To compare WSSV transcription in crayfish (O. limosus) with shrimp (P. monodon), both species were infected with WSSV using injection to synchronize infection. The gills, which are well infected by WSSV early in infection, were used for the isolation of RNA. RNA was isolated from crayfish from 2 h p.i. until 7 days p.i., and from shrimp at 1 and 2 days p.i. RT-PCR was performed on DNase I-treated RNA for the WSSV genes vp28, vp26, vp24, vp19, vp15 and rr1 (Fig. 1). Actin mRNA of the host was included in this analysis as an internal control for RNA extraction and was detected in every sample (Fig. 1). Purified WSSV DNA was taken as a positive control for the PCR and no template as a negative control (Fig. 1, C+ and C- respectively). PCR was also performed on RNA that was not reverse transcribed. These PCRs were all negative, confirming that no detectable amount of viral genomic DNA was present in the RNA samples used. Rr1 gene transcripts were detected from 6 h p.i. until 7 days p.i. (Fig. 1), which is comparable with data obtained by Tsai et al. (2000a). Compared to the rr1 gene, the vp genes are all transcribed late in infection. Transcripts of vp15 were clearly detected from 16 h p.i., while transcripts of vp28, vp26, vp24 and vp19 were detected from 1 day p.i. onwards. The RT-PCR suggests that expression of vp24 mRNA was the lowest (very weak band at 1 day p.i.) of the major structural protein genes. This correlates with the low amount of VP24 protein in WSSV virions (van Hulten et al., 2002). The RT-PCRs at 1 and 2 days p.i. showed that there is little if any difference in transcription of the WSSV major structural protein genes between O. limosus and P. monodon.



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Fig. 1. RT-PCR time-course for the WSSV vp28, vp19, vp26, vp24, vp15 and rr1 mRNAs and the host messenger actin mRNA in crayfish (O. limosus) and shrimp (P. monodon). Lane headings show time-points hours (h) or days (d) post-infection. M represents a 100 bp DNA marker, C+ and C- are, respectively, the positive and negative control for the PCR.

 
Transcription initiation sites
To determine the TISs of the five major structural protein genes, 5' RACE was performed, using RNA isolated from gills of the crayfish O. limosus. A time-point of 6 days p.i. was chosen for analysis of the mRNA, as abundant mRNAs are present for all analysed genes (Fig. 1). The 5' RACE protocol was slightly modified for vp28 and vp24 as long stretches of thymidines are present in the 5' upstream sequences, which could result in aspecific binding of the oligo(dT) anchor primer to the cDNA. Therefore, the cDNA of these genes was 3'-tailed with dTTPs instead of poly(A)-tailing and an oligo(dA) anchor primer was used for the PCR. The 5' RACE PCR products were cloned and for each gene at least three clones were sequenced. The results of the 5' RACE experiments are shown in Fig. 2.



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Fig. 2. Sequences 100 nt upstream and downstream of the major structural protein genes of WSSV showing the major transcription initiation and polyadenylation sites of each gene, indicated by arrows (TIS by arrow above sequence, polyadenylation by arrow below sequence). The 5' and 3' termini of the different clones sequenced for each gene are underlined (each underlined nucleotide represents a clone). The number beneath the underlining shows the number of similar clones. The start codon of each gene is shaded black. The TATA-box for vp15 is double underlined. The stop codons are shaded dark grey and the poly(A) signals light grey.

 
The TIS of vp28 could be located within the nucleotide sequence AAC, 33 nt upstream of the ATG. A single 5' RACE clone was identified with a 5' mRNA terminus 5 nt upstream of the translational start codon and is most probably the result of RNA breakdown, although the possibility of a second TIS cannot be excluded. The TIS of vp26 is located on GC, 71 nt upstream of the ATG. For vp24 a TIS was identified on CA, 27 nt upstream of the ATG and for vp19 the TIS was identified within 64 to 71 nt of the translational start codon. A minor TIS (one clone) for vp19 was found 151 nt upstream of the translational start codon (not shown in Fig. 2). Transcription from the initiation sites identified for vp19 is not regulated by the TATA-box, which is located 254 nt upstream of the translational start codon. For vp15 the TIS was found 24 nt downstream of the TATA-box. Transcription starts 57 nt upstream of the translational start codon of vp15.

Analysis of WSSV putative promoter motifs
The TISs of the WSSV major structural virion protein genes were aligned to identify putative consensus motifs in the surrounding regions (Fig. 3). Transcription of vp24 and vp15 is initiated at exactly the same sequence, TCATGAC. The conserved TCATGAC motif of vp15 and vp24 occurs 37 times in the total genome sequence of which three are in a putative promoter region. Except for vp24 and vp15, the sequence is found in the 100 bp sequence upstream of the putative transcriptional start site of ORF158, which encodes a protein with no homology to proteins present in GenBank. This motif, however, is not present in the 5' upstream sequences of the other vps. Transcription of all vps starts 25 nt downstream of an A/T-rich sequence, which includes the TATA-box present in the vp15 5' upstream sequence (Fig. 3). No other conserved motifs could be detected in the sequences surrounding the transcriptional start sites of the five major structural proteins.



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Fig. 3. Alignment of the TISs in the 5' upstream region of the major structural virion protein genes. Shading is used to indicate the occurrence of identical nucleotides (black, 100 %; dark grey, 80 %; light grey, 60 %). The TISs and A/T-rich region are indicated. The translational start codon of each gene is included in the sequence.

 
Polyadenylation
To determine the polyadenylation sites of the major structural protein genes, a 3' RACE was performed for all five genes on RNA isolated 6 days p.i. A clear 3' RACE PCR product was obtained for each of the genes. This product was cloned and at least two clones for each gene were sequenced. For each of the genes, the polyadenylation starts 15/16 nt downstream of the poly(A) signal, as shown in Fig. 2. Two internal 3' RACE clones (one for vp28 and one for vp19; not shown in Fig. 2) were found, probably caused by aspecific binding of the oligo(dT) anchor primer to long stretches of adenosines in the 3' untranslated region.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
WSSV has a broad host range, infecting shrimp, crayfish and other crustaceans. Its dsDNA genome of around 300 kb (van Hulten et al., 2001a; Yang et al., 2001; AF440570) is among the largest viral genomes, but little is known about gene regulation of this virus. To obtain insight into this process, transcription of five putative late WSSV genes was studied using crayfish as host. The five major structural protein genes were selected, as we assumed that their expression is coregulated to secure correct assembly of the virions. The WSSV virus isolate derived from Thailand was used in this study (van Hulten et al., 2001a). The three complete WSSV genome sequences available so far are highly similar (sequence identity of over 99 %) and the major differences occur in repeat regions and in a 13 kb region absent from the Thailand isolate (position 31135). Putative promoter sequences analysed in this study are identical in all three isolates.

The transcription of the WSSV genes was studied using RT-PCR, as this technique allows the most sensitive detection of transcripts. Compared to the expression of rr1 (Tsai et al., 2000a), which is presumed early as it should precede DNA replication to provide dNTPs, the WSSV major structural proteins genes are late (Fig. 1). This is in accordance with many structural virion protein genes from other large DNA viruses such as baculoviruses, herpesviruses and African swine fever virus (ASFV). All six WSSV mRNAs studied in the RT-PCR could be detected very late in infection (2 to 7 days p.i., Fig. 1), which can be explained by the multiple rounds of WSSV infection which occur in gill tissue. In contrast to the expression of the other major structural protein genes, vp15 is clearly expressed at 16 h p.i. preceding the other vps, which suggests an earlier role of VP15 in the virus replication and assembly process as a DNA binding protein. Vp15 is the only major structural protein gene with a consensus TATA-box (Fig. 2). The earlier onset of transcription of vp15 (Fig. 1) could be regulated by this TATA-box, as in other WSSV genes which have been shown or are likely to be expressed early in infection (rr1, rr2, pk and dnapol) (Tsai et al., 2000a; Liu et al., 2001; Chen et al., 2002).

The transcriptional start and stop sites of the vps were determined by 5' and 3' RACE. Splicing events can be excluded for these mRNAs, as has been demonstrated by the cloning and sequencing of cDNAs (Yang et al., 2001) and the expression of the VPs in insect cells (van Hulten et al., 2002). The polyadenylation sites of the five major structural protein genes, as determined by 3' RACE, were all between 15 or 16 nt downstream of the poly(A) signal (Fig. 2). The polyadenylation sites of vp26 and vp15 are in agreement with the 3' ends of these genes determined from RNA isolated from WSSV-infected P. japonicus (Zhang et al., 2001, 2002), in these papers referred to as p22 and vp6.8 respectively. However, the TISs for vp26 and vp15 described by these authors, using sequencing of a single cDNA clone, do not match the TIS found in the present study. Probably part of the 5' end of the mRNAs is missing in these studies (Zhang et al., 2001, 2002), because intact 5' ends of mRNA are rarely recovered from cDNA libraries. Polyadenylation of the WSSV genes rr1, rr2, pk and dnapol is, respectively, 12, 13, 16 and 17 nt downstream of the polyadenylation signal (Liu et al., 2001; Tsai et al., 2000a; Chen et al., 2002). All identified WSSV polyadenylation sites are within the range of regular polyadenylation in eukaryotic mRNAs, which is typically located 15 to 25 nt downstream of the sequence AAUAAA (Fitzgerald & Shenk, 1981).

For all vps, except for vp19, only one TIS has been identified. However, the presence of other TISs cannot be excluded as less abundant mRNAs at defined time-points are difficult to detect in vivo, where no absolute synchronized infection can be obtained. For proper late gene expression of large dsDNA viruses, the motif that contains the TIS is often an important viral promoter element. Mutations in the late motif TAAG of baculoviruses (Morris & Miller, 1994), the late TAAAT motif of poxviruses (Davison & Moss, 1989), the TATA initiator of ASFV (Garcia-Escudero & Viñuela, 2000) and the more diverged initiator YYANWYY (N=any nucleotide; W=t/a) of herpesviruses (Kim et al., 2002; Weir, 2001) often lead to a (dramatic) downregulation of gene expression. The 5' RACE analyses indicate that transcription of the WSSV genes vp28, vp26, vp24, vp19 and vp15 did not start at a consensus sequence for all five genes (Fig. 3). However, a conserved TIS motif (TCATGAC) was identified for vp15 and vp24. This motif might be involved in WSSV gene regulation, but not for a large group of genes, as it only appears in three WSSV putative promoter regions. Interestingly, all vps contained an A/T-rich sequence 25 nt upstream of the TIS (Fig. 3) and this sequence could have a function as a promoter element, as has been shown for baculoviruses, poxviruses and ASFV late genes. For baculoviruses, these A/T tracts serve as a protein binding site (Burma et al., 1994), while for the other viruses, the A/T sequences are thought to play a role in facilitating the unwinding of the double-stranded DNA and consequently, in the initiation of transcription (Davison & Moss, 1989; Garcia-Escudero & Viñuela, 2000). Further studies are needed to elucidate the role of the vp15 and vp24 TCATGAC motif and the A/T tracts in WSSV gene expression. However, the lack of a suitable cell culture system, either a cell line or primary cell cultures, hampers WSSV promoter as well as WSSV transcription studies.

Except for the features mentioned, no other conserved motifs could be detected in the sequences surrounding the transcriptional start sites of the five WSSV major structural virion protein genes. The absence of such a motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.


   ACKNOWLEDGEMENTS
 
This work was supported by Intervet International BV, Boxmeer, The Netherlands. We thank Professor Dr Rob Goldbach for continuous interest and advice.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 28 November 2002; accepted 4 February 2003.