Gill-associated nidovirus of Penaeus monodon prawns transcribes 3'-coterminal subgenomic mRNAs that do not possess 5'-leader sequences

Jeff A. Cowley1, Christine M. Dimmock1 and Peter J. Walker1

Cooperative Research Centre for Aquaculture, CSIRO Livestock Industries, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly 4068, Australia1

Author for correspondence: Jeff Cowley. Fax +61 7 32142881. e-mail Jeff.Cowley{at}csiro.au


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Sequence analysis of the ~20 kb 5'-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a–1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining ~6·2 kb of the GAV genome downstream of ORF1a–1b to a 3'-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3'-coterminal subgenomic (sg) mRNAs of ~6 kb and ~5·5 kb. Primer extension and 5'-RACE analyses showed that the sgmRNAs initiate at the same 5'-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5'-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26·2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5'-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Gill-associated virus (GAV) is endemic in wild and farmed Penaeus monodon prawns in eastern Australia (Spann et al., 1995 ; Cowley et al., 2000a ; Walker et al., 2001 ) and has been associated with sporadic outbreaks of disease since 1996 (Spann et al., 1997 ). GAV causes similar pathology and is morphologically indistinguishable from yellow head virus (YHV) from southeast Asia, where it has caused significant production losses in farmed P. monodon since 1990 (Limsuwan, 1991 ; Boonyaratpalin et al., 1993 ; Chantanachookin et al., 1993 ). Sequence comparisons of ORF1b gene regions of GAV and YHV indicate that they represent closely related geographic topotypes (Cowley et al., 1999 ).

YHV contains a long (>22 kb) ssRNA genome (Wongteerasupaya et al., 1995 ; Nadala et al., 1997 ) and virions comprise three or four structural proteins of 170, 110–135, 62–67 and 20–22 kDa (Nadala et al., 1997 ; Wang & Chang, 2000 ). The 110–135 kDa protein is glycosylated (Nadala et al., 1997 ). The complete 26·2 kb (+)-ssRNA genome of GAV has recently been sequenced (Cowley et al., 2001 ). The 5'-terminal 20 kb gene of GAV encodes two long overlapping open reading frames (ORFs), the latter of which is translated via a -1 ribosomal frameshift (Cowley et al., 2000b ). Both GAV ORFs possess functional motifs, including an ‘SDD’ polymerase, indicating a distant relationship to the ORF1a–1b replicase polyproteins of vertebrate coronaviruses and arteriviruses. An unusual 3C-like proteinase and low levels of homology in the polymerase domain suggest that GAV, for which we have proposed the genus name Okavirus, may warrant establishment of a new family within the Nidovirales (Cowley et al., 2000b , 2001 ).

Other than the organization and relatedness of functional motifs in the long ORF1a–1b replicase polyprotein and its translation via a -1 ribosomal frameshift, the transcription of a nested set of four to eight 3'-coterminal subgenomic (sg) mRNAs of viruses in both the Coronaviridae and the Arteriviridae is a defining common feature of their replication strategy, contributing to the establishment of the Nidovirales (see review: de Vries et al., 1997 ). Moreover, the 5'-ends of each sgmRNA of coronaviruses (see review: Lai & Cavanagh, 1997 ) and arteriviruses (see review: Snijder & Meulenberg, 1998 ) characteristically possess a short 5'-genomic leader sequence. In both viruses, accumulated evidence now indicates that the sgmRNAs are derived by a process of discontinuous negative-strand synthesis (van Marle et al., 1999 ; Sawicki & Sawicki, 1998 ; Sawicki et al., 2001 ). In this process, a conserved transcription-regulating sequence (TRS) in each intergenic region attenuates synthesis of 5'-coterminal (-)-sgRNAs and then associates with the complementary TRS in the (+)-genomic 5'-leader RNA allowing synthesis of a 3'-antileader. Each nascent (-)-sgRNA strand then acts as a template for transcription of each 3'-coterminal (+)-sgmRNA. Berne virus, the prototype member of the genus Torovirus within the Coronaviridae, also transcribes a nested set of four 3'-coterminal sgmRNAs. However, rather than possessing common 5'-leader sequences, the 5'-termini of equine torovirus (ETV)-Berne sgmRNAs map directly to conserved sequences in each intergenic non-coding region (Snijder et al., 1990 ). Nested 3'-coterminal sgmRNAs are also synthesized by plant (+)-ssRNA plant viruses such as the closterovirus citrus tristeza virus (CTV) (Hilf et al., 1995 ; Karasev et al., 1997 ; Gowda et al., 2001 ), which possesses a 19·3 kb genome with some characteristics of nidoviruses (Karasev et al., 1995 ). Similarly to the Berne torovirus, the 5'-termini of the CTV sgmRNAs map directly to conserved intergenic ‘control element’ sequences which appear to act as terminators or promoters or both of (-)- and (+)-sgRNA synthesis (Gowda et al., 2001 ).

In this paper, we report that GAV transcribes 3'-coterminal sgmRNAs with 5'-termini that map to identical positions in two highly conserved intergenic sequences. The sgmRNAs do not contain genomic 5'-leader RNA sequences, as in coronaviruses and arteriviruses, suggesting GAV may employ a transcription mechanism similar to toroviruses. Like other nidoviruses, dsRNA equivalents of the GAV genomic and sgRNAs are produced in infected cells.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} RNA isolation and analysis.
Total RNA was isolated from pooled lymphoid organs or gills of GAV-infected P. monodon using TRIzol (Life Technologies), resuspended in DEPC-treated water and stored in aliquots at -70 °C. Total RNA was similarly isolated from lymphoid organs or gills of uninfected P. monodon and P. japonicus. Total RNA (10 µg per lane) was resolved in 0·6% neutral LMP agarose (Life Technologies)–TAE gels containing 0·5 µg/ml ethidium bromide (Sambrook et al., 1989 ). To identify dsRNA, lymphoid organ total RNA (10 µg) was digested in NTE pH 7·4 (10 mM Tris–HCl pH 7·4, 100 mM NaCl, 1 mM EDTA) containing 50 µg/ml RNase A at 37 °C for 30 min followed by incubation with 1U RQ1-DNase (Promega) at 37 °C for 15 min and resolved as above. Poly(A)+ RNA was selected from total RNA using the Oligotex mRNA midi kit (Qiagen).

{blacksquare} Probes and Northern blots.
Digoxigenin (DIG)-labelled DNA probes were generated by PCR using a dNTP mix containing DIG-dUTP (Roche) and the primer pairs shown in Table 1. Oligonucleotides were synthesized using a Beckman Oligo-1000 DNA synthesizer. Regions in GAV ORF1b, ORF2, ORF3 and a putative ORF4 between ORF3 and the 3'-genomic poly(A) tail of 317, 435, 285 and 188 nt, respectively, were amplified with Taq DNA polymerase (Promega) and 40 cycles of 95 °C/30 s, 55 °C/30 s and 72 °C/30 s. The relative positions of the probes in the GAV genome are shown in Fig. 1.


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Table 1. PCR primer pairs used to amplify DIG-labelled DNA probes and for 5'-RACE

 


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Fig. 1. GAV genome organization showing the positions of DIG-labelled DNA probes in ORF1b (1), ORF2 (2), ORF3 (3) and ORF4 (4) used to identify intracellular poly(A)+ genomic and subgenomic mRNAs. The two sgmRNAs initiate at conserved transcription start sequences in the intergenic regions upstream of ORF2 and ORF3.

 
Lymphoid organ total RNA (2·5 µg) or poly(A)+ RNA (0·5 µg) from uninfected and GAV-infected P. monodon was denatured in formamide–formaldehyde at 70 °C for 10 min and resolved at 50 °C using ‘hot’ 1·2% agarose–TAE gels as described by Almeida et al. (2000) . RNA was transferred to Hybond-N+ nylon membranes (Amersham Pharmacia) using 50 mM NaOH and downward capillary transfer (Ingelbrecht et al., 1998 ). Membranes were washed briefly in 2xSSC (1xSSC: 0·15 M NaCl, 0·015 M trisodium citrate) and hybridized with denatured (100 °C for 10 min) DIG-labelled DNA probes (~10 ng/ml) by incubation at 42 °C for 16–18 h in UltraHyb solution (Ambion). Membranes were washed at 42 °C twice in 2x SSC, 0·1% SDS for 15 min and twice in 0·5x SSC, 0·1% SDS for 15 min. RNA hybridized to the DIG-DNA probes was detected by chemiluminescence using 1:2000 anti-DIG–alkaline phosphatase Fab fragments and 1:100 CDP-Star reagents (Roche) according to the manufacturer’s instructions.

{blacksquare} Primer extension analysis and 5'-RACE.
The putative 5'-termini of GAV sgmRNAs were determined by primer extension analysis and by an anchor-PCR method for the random amplification of cDNA ends (5'-RACE) (Dumas et al., 1991 ). Primer extension analyses utilized GAV-specific antisense primers downstream of intergenic regions spanning ORF1b–ORF2 (i.e. GAV48) and ORF2–ORF3 (i.e. GAV52) and total RNA isolated from the lymphoid organs of GAV-infected P. monodon. The primer sequences and their relative positions in the GAV genome are shown in Fig. 1. cDNA was synthesized using primer (50 ng) that had been 5'-phosphorylated with [{gamma}-32P]ATP (GeneWorks) and T4 polynucleotide kinase (Promega), 5 µg RNA and Superscript II reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. Plasmid DNA sequences were generated from a cDNA clone spanning these regions using either primer GAV48 or GAV52, [{alpha}-32P]dCTP (GeneWorks) and the SequenaseT7 DNA polymerase sequencing kit (United States Biochemical) adjusted to sequence close to the primer site. Primer extension and radiolabelled DNA sequencing products were resolved in 0·4 mm gels (Sambrook et al., 1989 ) prepared using 4% Long Ranger acrylamide mix (AT Biochem) and detected by autoradiography. The 5'-RACE also utilized lymphoid organ total RNA and the complementary anchor-PCR primers (156 and 2668) described previously (Walker et al., 1994 ). Briefly, cDNA was synthesized using 5 µg total RNA, 50 ng each primer and 200 U Superscript II reverse transcriptase by incubation at 42 °C for 1 h according to the manufacturer’s instructions. RNA was digested by incubation with 1·5 U RNase H at 37 °C for 1 h and the cDNA was purified through a Sephadex S400-HR column (Amersham Pharmacia). Primer 156 (0·2 µg), 5'-phosphorylated with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega), was ligated to cDNA 3'-ends with T4 RNA ligase (New England Biolabs) as described previously (Walker et al., 1994 ). Excess primer was removed using a Sephadex S400-HR column and the cDNA was amplified by PCR using the complementary primer 2668 in combination with either GAV-specific primer GAV48 or GAV52. PCRs were performed using Taq DNA polymerase (Promega) and 40 cycles of 95 °C/30 s, 60 °C/30 s and 72 °C/30 s followed by 72 °C/10 min. PCR products were purified using a QIAquick column (Qiagen), ligated into pGEM-T Vector (Promega) and transformed into competent E. coli DH5{alpha} cells; plasmids containing inserts were selected using standard methods (Sambrook et al., 1989 ).

{blacksquare} DNA sequencing and analysis.
Plasmid DNA was prepared using the Concert mini-DNA kit (Life Technologies). Automated sequencing of cDNA inserts utilized the Big Dye (Applied Biosystems Inc., ABI) dye-terminator reagent, universal pUC forward and reverse primers, and model 377 sequencing apparatus (ABI) at the Australian Genome Research Facility, Brisbane. SeqEd 1.0.3 (ABI) and MacVector 7 (Oxford Molecular) were used for chromatogram analyses and sequence alignments.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Conserved intergenic promoter sequences
We have recently determined the 3'-terminal sequence of the GAV genome downstream of the ORF1a–1b gene (Cowley et al., 2000b ) to a putative poly(A)-tail (GenBank accession no. AY039647). Downstream of the ORF1a–1b gene, the GAV genome contains a 93 nucleotide (nt) intergenic sequence proceeded by a 144 amino acid (aa) ORF2. The 57 nt intergenic sequence downstream of ORF2 is followed by a long (4·9 kb) gene encoding ORF3. The ~0·6 kb sequence from ORF3 to a putative 3'-genomic poly(A)-tail contains a putative short ORF4 of 83 aa positioned 256 nt downstream of ORF3. The GAV genome organization is shown in Fig. 1. The sequences of ORF2 (putative nucleocapsid protein), ORF3 (putative multiple transmembrane glycoproteins) and ORF4, will be described elsewhere.

Alignment of the GAV intergenic sequences upstream of ORF2 and ORF3 identified a remarkable degree of identity in a 32 nt region in which 29 nt were conserved (Fig. 2). In the longest continuous stretch, 21/22 nt were identical. Alignment to the putative non-coding sequence upstream of ORF4 identified a region with limited identity (14 nt with one point deletion). No significant homology was detectable between the conserved intergenic sequences and the 5'-genomic leader sequence upstream of the putative ORF1a start codon.



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Fig. 2. Conserved sequences in the 93 nt and 57 nt intergenic regions upstream of ORF2 and ORF3, respectively, and in the 256 nt region upstream of ORF4. The 5' ACAACC transcription start site identified for ORF2 and ORF3 sgmRNAs by primer extension analysis and sequencing of 5'-RACE clones is indicated.

 
Northern hybridization detection of sgmRNAs
Lymphoid organ total RNA from uninfected and GAV-infected P. monodon was resolved in denaturing ‘hot’ agarose–TAE gels, blotted onto nylon membranes and hybridized to DIG-labelled DNA probes targeted to regions in reading frames ORF1b, ORF2, ORF3 and ORF4 (see Fig. 1). As shown in Fig. 3, the ORF1b probe hybridized to a prominent smear of high molecular mass RNA significantly larger than the 6·6 kb RNA marker. The ORF2 probe hybridized to a discrete ~6 kb RNA. The ORF3 and ORF4 probes hybridized to the ~6 kb RNA in addition to a ~5·5 kb RNA. A minor diffuse ~5·0 kb RNA was also apparent in the hybridization using the ORF3 probe. Smeared RNA larger than the discrete ~6 kb RNA that initiated from the same relative position as the smeared RNA that hybridized to the ORF1b probe was also detected to lesser extents with the ORF2, ORF3 and ORF4 probes. Detection of the large smeared RNA with these probes was more obvious in longer exposures (data not shown).



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Fig. 3. Northern blot detection of GAV sgmRNAs. Total RNA (2·5 µg) from lymphoid organs of uninfected (-) and GAV-infected (+) P. monodon were resolved in a denaturing ‘hot’ 1·2% agarose–TAE gel, transferred to positively charged nylon membranes and hybridized to DIG-labelled DNA probes in ORF1b (1), ORF2 (2), ORF3 (3) and ORF4 (4) as shown in Fig. 1. RNAs hybridizing to the DNA probes were detected using anti-DIG–AP-conjugated antibody and chemiluminescence. The long (6 kb) gRNA smear and sgRNAs of ~6 and ~5·5 kb are indicated (), as is a diffuse ~5 kb band () detected by the ORF3 probe.

 
Northern hybridizations using the four probes were also performed using poly(A)+ RNA selected from lymphoid organ total RNA of GAV-infected prawns. These hybridizations produced similar results to those obtained using total RNA, except that more smeared low molecular mass material was detected, presumably due to RNA degradation (data not shown). Nonetheless, the ORF1b probe hybridized to a smeared RNA >6 kb, the ORF2 probe to a ~6 kb RNA and both ORF3 and ORF4 probes to ~6 kb and ~5·5 kb RNAs. As for total RNA, the amount of smeared material >6 kb detected diminished with probes further from the 5'-end of the GAV genome. Interestingly, the minor diffuse ~5 kb RNA was also detected by the ORF3 probe using poly(A)+ RNA.

Primer extension and 5'-RACE to identify 5'-sgmRNA ends
To determine whether the conserved intergenic sequences represent transcription initiation sites for the ~6 kb and ~5·5 kb sgmRNAs, primer extension and 5'-RACE analyses were done using primers downstream of the ORF1b–ORF2 (i.e. GAV 48) and ORF2–ORF3 (i.e. GAV 52) intergenic regions, respectively. Primer extension analysis of cDNAs synthesized from lymphoid organ total RNA using 32P-labelled primers identified major products of ~55 nt with primer GAV48 and ~65 nt with GAV52 (Fig. 4a). A minor long cDNA product of ~600 nt, which was more obvious in longer gel exposures, was also synthesized with primer GAV52. Smeared bands between the 75–154 nt markers occurred with other primers (data not shown) and appear to be non-specific.



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Fig. 4. Primer extension analysis identifying the 5'-termini of GAV sgmRNAs initiated at intergenic sequences upstream of ORF2 and ORF3. cDNAs synthesized from total lymphoid organ RNA of GAV-infected P. monodon using 5'-[{gamma}-32P]ATP-labelled primers GAV48 or GAV52 were resolved in 4% polyacrylamide–TBE sequencing gels and detected by autoradiography. (a) Gel analysis of GAV52-primed and GAV48-primed cDNA and primers (P) alone. A 1 kb DNA ladder (Life Technologies) labelled with [32P]dCTP and Klenow DNA polymerase is shown to the left. (b) Primer extension cDNA products resolved alongside dideoxy-terminated DNA sequencing products generated using pG16, either primer GAV48 or GAV52, [32P]dCTP and a T7 Sequenase DNA sequencing kit. The nucleotide sequences generated with the primers GAV48 and GAV52 are indicated to either side and the sequence (UUACAA) encompassing both ORF2 and ORF3 sgmRNA 5'-ends is marked (-).

 
To identify the sgmRNA 5'-termini, primer extension products were resolved alongside DNA sequences generated from a cDNA clone using the same primers (Fig. 4b). Although the DNA sequence quality was compromised by the closeness of the primers, cDNAs generated using primers GAV48 and GAV52 aligned to positions 55 and 35 nt upstream of the AUG start codons of the ORF2 and ORF3 genes, respectively (Fig. 1). The data indicated that both RNAs were likely to initiate at the same position (i.e. 5'-AC) in the conserved intergenic sequences, although it was not clear whether the A or C residue represented the terminal nucleotide.

The 5'-A terminus of the GAV sgmRNAs was confirmed by sequencing multiple 5'-RACE clones generated using primers GAV48 and GAV52. Primer GAV52 generated two 5'-RACE PCR products equivalent in length to the short and long cDNAs detected by primer extension analysis (see Fig. 4a, data not shown). The terminal sequence of the short (~0·1 kb) 5'-RACE clones mapped to the same 5'-A position 35 nt upstream of ORF3 identified in the GAV52 primer extension analysis. The terminal sequence of the long (~0·7 kb) 5'-RACE clones mapped to the same 5'-A position 55 nt upstream ORF2 position identified in the GAV48 5'-RACE clones and primer extension analysis. As in similar analyses of the putative GAV 5'-genomic RNA terminus (Cowley et al., 2000b ), most 5'-RACE clones possessed one or two additional G nucleotides beyond the 5'-A position that did not match the GAV genome sequence.

Subgenomic dsRNAs
Lymphoid organ total RNA from GAV-infected P. monodon was digested with RNase A and DNase I and resolved in non-denaturing agarose–TAE gels to identify intracellular double-stranded (ds)RNA forms of the GAV sgmRNAs. As shown in Fig. 5, three discrete dsRNAs of approximately 22, 5·8 and 5·2 kb were detected. Any dsRNAs <1 kb in length, however, could not be detected due to the smear to degraded ssRNA.



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Fig. 5. Intracellular dsRNA forms of the GAV genomic and sgRNAs. Lymphoid organ total RNA from GAV-infected P. monodon was digested with RNase A and RQ1-DNase and resolved in a native 0·6% agarose–TAE gel. A 1 kb DNA ladder (left) and HindIII-cut {lambda} DNA (right) were used as size markers.

 

   Discussion
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Introduction
Methods
Results
Discussion
References
 
In this study, we report evidence that GAV of penaeid shrimp, which contains a 20 kb ORF1ab gene indicating that it is a crustacean nidovirus (Cowley et al., 2000b ), also synthesizes 3'-coterminal sgmRNAs. The transcription of a nested set of sgmRNAs with common 3'-termini is a defining feature of the replicational strategy employed by nidoviruses (Lai et al., 1984 ; see reviews Spaan, 1988 ; Lai, 1990 ; van der Most & Spaan, 1995 ; Snijder & Horzinek, 1995 ; de Vries et al., 1997 ; Lai & Cavanagh, 1997 ; Snijder & Meulenberg, 1998 ; Sawicki & Sawicki, 1998 ). In coronaviruses, differing models have been proposed for the mechanism by which sgmRNAs are transcribed and acquire a genomic leader RNA at their 5'-end and the role in this process of a conserved transcription-regulating sequence (TRS) that occurs at the leader-body junction of each sgmRNA and in the leader and each genomic intergenic sequence. Recent data on the synthesis of (-)-sgRNAs and the accumulation of subgenomic replicative intermediate (RI) and replicative form (RF) dsRNAs favours a discontinuous (-)-RNA synthesis model (Sawicki & Sawicki, 1990 , 1998 ; Baric & Yount, 2000 ; Sawicki et al., 2001 ). In this model, the full-length (+)-genomic RNA is used as a template for the synthesis of 5'-coterminal (-)-sgRNAs. The synthesis of (-)-sgRNAs is attenuated at each intergenic TRS, which then associates in trans with the TRS in the (+)-5'-leader RNA to prime the synthesis of a 3'-antileader sequence (Sawicki & Sawicki, 1998 ). Each (-)-sgRNA then acts independently as a template for transcription of a complementary 5'-leader-containing sgmRNA. Arteriviruses also synthesize 3'-coterminal sgmRNAs containing genomic 5'-leader sequences (de Vries et al., 1990 ; den Boon et al., 1996 ; Snijder & Meulenberg, 1998 ). Mutagenesis of the leader and intergenic body TRS elements of infectious cDNA clones has revealed that arteriviruses employ a similar discontinuous (-)-RNA synthesis process to coronaviruses involving direct base-pairing of the TRS element in the nascent subgenomic (-)-RNA to the (+)-5'-leader RNA TRS to prime addition of a 3'-antileader (van Marle et al., 1999 ).

A smeared RNA originating from an RNA estimated to be >20 kb in length and two RNAs of 6·0 and 5·5 kb were detected in Northern blots using DNA probes to ORF1a, ORF2, ORF3 and ORF4 and either total or poly(A)+ RNA from GAV-infected prawn lymphoid organ. The >20 kb smeared RNA was detected only by the ORF1a probe, this and the 6 kb RNA by the ORF2 probe and these in addition to a 5·5 kb RNA were detected by the ORF3 and ORF4 probes. This is consistent with these RNAs representing degraded full-length genomic RNA and nested 3'-coterminal sgmRNAs initiating in the intergenic regions upstream of ORF2 and ORF3. However, no obvious small RNA of a size (0·4–0·6 kb) predicted for an ORF4 gene sgmRNA was detected by the ORF4 probe. A minor ~5·0 kb RNA was consistently detected by the ORF3 but not the ORF4 probe. The origin of this RNA is unknown. However, the data suggest it may be equivalent to the 5·5 kb RNA but truncated at the 3'-end upstream of the ORF4 probe site. It may be noteworthy that a 63 nt highly A/T-rich (46A/15T) sequence resides in the intergenic sequence just downstream of ORF3 at a position that would result in a 3'-truncated ORF3 sgmRNA of this size (J. A. Cowley, unpublished).

In contrast to coronaviruses and arteriviruses in which each sgmRNA contains a 5'-leader, primer extension analysis and sequencing of 5'-RACE clones identified that each GAV sgmRNA initiated directly at a common 5'-AC position central to highly conserved sequences in the intergenic regions upstream of ORF2 and ORF3. Alignment of these regions (29/32 nt conserved) identified a continuous stretch of 20/21 identical nucleotides. The absence of a genomic leader RNA at the 5'-termini of GAV sgmRNAs has also been observed with the four 3'-coterminal sgmRNAs of ETV-Berne, the type member of the genus Torovirus within the Coronaviridae (Snijder et al., 1990 , 1991 ). Interestingly, the 5'-termini of the ETV-Berne sgmRNAs also map directly to 5'-A(C/A) residues upstream of conserved intergenic sequences (Snijder et al., 1990 ). There is no obvious conservation in the intergenic sequences downstream of the sgmRNA start sites in GAV and ETV-Berne. However, like GAV, two of the four ETV-Berne sequences display significant conservation in the region immediately upstream of the sgmRNA start sites. It has been suggested that these upstream sequences may have a role in regulating the synthesis of each ETV-Berne sgmRNA (Snijder et al., 1990 ). As the two GAV sgmRNAs identified are the primary candidates for translation of the virion structural proteins, they are likely to be required in high levels during replication. Although not directly quantified, the relative amounts of the two GAV sgmRNAs co-detected in Northern blots using either the ORF3 or ORF4 probe were similar (see Fig. 3). The availability of cell culture and reverse genetics systems will greatly assist in examining the mechanism by which the GAV sgmRNAs are transcribed and the role of the conserved intergenic sequences in facilitating this process.

As for the two sgmRNAs, 5'-RACE analysis has previously identified a 5'-AC at the putative 5'-terminus of the GAV (+)-genomic RNA (Cowley et al., 2000b ). However, no significant homology exists between the downstream regions of the genomic and the ORF2 and ORF3 sgmRNAs. This poses a perplexing question regarding the nature of the complementary sequences in the (-)-genomic RNA recognized by the GAV RNA polymerase to facilitate transcription of the full-length (+)-genomic RNA. In truncated defective-interfering (DI) RNAs of ETV-Berne, a sequence comparable to each intergenic ‘core promoter’ has been identified immediately downstream of the 5'-terminus of the (+)-DI RNA (Snijder et al., 1990 , 1991 ). Similarities in the nature of sgmRNAs of GAV and ETV-Berne suggest they are likely to share similarities in their transcription/replication processes. With regard to this mechanism, similarities in genome organization and the synthesis of 3'-coterminal sgRNAs have been noted between nidoviruses and (+)-ssRNA plant closteroviruses such as CTV (Hilf et al., 1995 ; Karasev et al., 1997 ; Gowda et al., 2001 ). Of particular relevance and similarly to GAV and ETV-Berne, CTV also synthesizes nested 3'-coterminal sgmRNAs with 5'-termini that map directly to intergenic sequences. In CTV, recent evidence suggests that sequences in each intergenic region act as terminators and initiators or both for the transcription of (-)- and (+)-sgRNAs (Gowda et al., 2001 ). Strand-specific probes were not used to look specifically for (-)-sgRNAs in GAV-infected cells. However, identification of dsRNAs equivalent in size to the genomic and two sgmRNAs suggests that (-)-sense sgRNAs are formed as part of the GAV replication process. Whether the conserved GAV intergenic sequences facilitate both termination of (-)-sgRNA synthesis and initiation of transcription of 3'-coterminal sgmRNAs similarly to CTV remains to be determined.

It is noteworthy that 5'-RACE clones of most ORF2 and ORF3 sgmRNAs possessed one or two additional G nucleotides beyond the 5'-A position mapping to the GAV genome sequence. One or two G nucleotides have also been detected in 5'-RACE clones of the putative 5'-AC terminus of the GAV genomic RNA (Cowley et al., 2000b ). We currently do not know whether these originate from the GAV transcription process. However, it may be significant that additional non-templated nucleotides have also been detected at the 3'-termini of the (-)-genomic and -sgRNAs of some (+)-ssRNA plant viruses including CTV (Karasev et al., 1997 ; Yang et al., 1997 ) and the carmovirus turnip crinkle virus (Guan & Simon, 2000 ). In these viruses, the addition of these non-templated nucleotides appears to be mediated by the viral RNA polymerase, although their role in the transcription process is not yet well understood.

A region with only limited homology to the highly conserved intergenic sequence upstream of the ORF2 and ORF3 genes that encompasses the 5'-sgmRNA start sites was identified in the untranslated region upstream of the putative ORF4 gene of GAV. Moreover, a G rather than an A nucleotide occurs at the position of the 5'-A terminus determined for the ORF2 and ORF3 sgmRNAs and no discrete ORF4 sgmRNA was obvious in Northern hybridizations. It appears, therefore, that this sequence is either inactive or results in very low abundance transcription of a sgmRNA for translation of the 83 aa ORF4 polypeptide. It may be relevant that there is no evidence of a virion structural protein of this size in the closely related YHV (Nadala et al., 1997 ; Wang & Chang, 2000 ). In the coronavirus mouse hepatitis virus (MHV), an internal ribosome entry site (IRES) has been identified in RNA5 which permits translation of a second internal reading frame in this sgmRNA (Thiel & Siddell, 1995 ; Jendrach et al., 1999 ). The GAV intergenic region between ORF3 and ORF4 is quite long (256 nt) and sequences in this region may facilitate ORF4 translation by internal initiation on the ORF2 or ORF3 sgmRNAs. We are currently preparing antiserum to recombinant ORF4 polypeptides to determine whether this protein is expressed in GAV-infected cells.

We have recently proposed, based on the presence of an ORF1a–1b polyprotein homologue translated by a -1 ribosomal frameshift, that GAV of penaeid shrimp represents the first nidovirus to be isolated from an invertebrate (Cowley et al., 2000b ). The identification of two 3'-coterminal sgmRNAs in infected cells further strengthens the argument for placement of GAV, for which we have proposed the taxon Okavirus, within the order Nidovirales. The use of an sgmRNA transcription mechanism not reliant upon a 5'-genomic leader RNA, however, clearly distinguishes GAV and the torovirus ETV-Berne from coronaviruses and arteriviruses.


   Acknowledgments
 
We thank Tony Vuocolo for supplying phosphorylated primer 156.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Almeida, T. A., Pérez, J. A. & Pinto, F. M. (2000). Size-fractionation of RNA by hot-agarose electrophoresis. Biotechniques 28, 414-416.[Medline]

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Received 4 July 2001; accepted 23 November 2001.