Department of Virology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands1
Author for correspondence: Eric Snijder. Fax +31 71 5266761. e-mail E.J.Snijder{at}LUMC.NL
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The order Nidovirales comprises two groups of animal, positive-stranded RNA viruses, arteriviruses and coronaviruses, which appear to have specialized in the use of polyprotein processing and sg mRNAs to regulate the expression of their polycistronic genome (de Vries et al., 1997 ; Lai & Cavanagh, 1997
; Snijder & Meulenberg, 1998
). The nidovirus replicase is expressed from the viral genome in the form of two polyprotein precursors, which are processed into at least 12 to 15 smaller functional subunits by internal viral proteinases (Ziebuhr et al., 2000
). The nidovirus structural proteins are derived from an array of separate genes. These are located in the 3'-terminal part of the genome and are translated from a set of six to eight sg mRNAs. A key feature of these sg transcripts is the fact that their 5'- and 3'-terminal sequences are identical to those of the viral genome. This 5' and 3' nested set structure is achieved by fusion of the genomic 5' leader sequence to specific body transcription-regulating sequences (TRSs) in the 3'-terminal one-third region of the genome. Almost all of the viral genes in the genomic 3'-terminal region are preceded by a TRS, which is termed intergenic sequence (IG) in the case of coronaviruses, and thereby they are positioned at the 5' end of the resulting sg mRNA. The 3' end of the genomic leader sequence also contains a TRS (leader TRS). Leader and body sequences are fused via an only partially understood mechanism of discontinuous transcription, which involves base-pairing between the genomic leader TRS and the body TRS complements in the viral minus-strand (van Marle et al., 1999
).
Equine arteritis virus (EAV) is the prototype member of the Arteriviridae family (Snijder & Meulenberg, 1998 ). The 12·7 kb EAV genomic RNA (den Boon et al., 1991
) is encapsidated by an isometric nucleocapsid containing a single nucleocapsid protein (N). The envelope which surrounds this core structure contains two major (M and GP5) and three or four minor (E, GP2b, GP3 and GP4) structural proteins (de Vries et al., 1992
; Snijder et al., 1999
; Snijder & Meulenberg, 1998
). As in all nidoviruses, the EAV replicase gene consists of two ORFs, 1a and 1b, which are both expressed from the genomic RNA, the latter by ribosomal frameshifting (den Boon et al., 1991
). The EAV ORF1a and ORF1ab polyproteins are processed by three viral proteinases, generating 12 nonstructural proteins (nsp112) and a large number of processing intermediates [for reviews, see Snijder & Meulenberg (1998)
and Ziebuhr et al. (2000)
]. A number of hydrophobic domains in the ORF1a polyprotein presumably anchor the EAV replication complex to intracellular membranes (van der Meer et al., 1998
; van Dinten et al., 1996
), resulting in their modification into characteristic double-membrane vesicles (Pedersen et al., 1999
). In addition to the two replicase ORFs and the seven structural genes in its 3'-terminal quarter, the EAV genome contains a 10th potential ORF, which is entirely located within the 211 nt genomic leader sequence (Kheyar et al., 1996
). This leader ORF (L-ORF; nt 14124) encodes a hypothetical 37 amino acid protein. Due to the nested structure of the EAV mRNAs, they all contain the L-ORF in their 5'-terminal region. However, it is unclear whether the EAV L-ORF is indeed expressed, in particular because translation initiation would have to occur very close to the 5' end of the RNA.
In addition to their function in genome encapsidation, RNA virus coat or N proteins can be involved in genome replication or mRNA transcription. Alfalfa mosaic virus coat protein associates with the viral replicase (Quadt et al., 1991 ) and plays a role in the initiation of infection and in asymmetric plus-strand accumulation (van der Kuyl et al., 1991
). The N proteins of the negative-stranded rhabdo-, paramyxo- and orthomyxoviruses are part of the helical ribonucleoprotein structure which is the template for viral RNA transcription and replication (Lamb & Kolakofski, 1996
; Lamb & Krug, 1996
; Wagner & Rose, 1996
). A role in viral RNA synthesis has also been postulated for the coronavirus N protein. Antibodies specific for mouse hepatitis coronavirus (MHV) N protein were able to almost completely inhibit viral RNA synthesis in an in vitro system (Compton et al., 1987
) and could specifically immunoprecipitate all leader-containing MHV mRNAs, as well as replicative intermediates (Baric et al., 1988
; Stohlman et al., 1988
). This finding led Baric et al. to propose that the coronavirus N protein might be involved in discontinuous subgenomic RNA synthesis. More recently, immunofluorescence and electron microscopy studies (van der Meer et al., 1999
) demonstrated that MHV N protein colocalizes with the viral replicase in membrane-associated complexes that are involved in RNA synthesis. MHV N protein was also shown to interact with the leader and IG sequences in in vitro binding assays (Nelson et al., 2000
). Furthermore, it was reported to interact with the cellular heterogeneous nuclear ribonucleoprotein A1 (Wang & Zhang, 1999
), which has been suggested to be involved in coronavirus sg RNA synthesis (Li et al., 1997
).
Also in arteriviruses, the N protein is encoded by the most 3' structural gene and is abundantly expressed from the smallest sg mRNA (mRNA7). To assess a possible role of EAV N in viral RNA synthesis, we first investigated its subcellular localization by confocal immunofluorescence microscopy. Three different cell lines [baby hamster kidney (BHK-21), rabbit kidney (RK-13) and African green monkey kidney (Vero)] were infected with EAV as described previously (van der Meer et al., 1998 ). N expression was monitored during time-course experiments and visualized by using monoclonal antibody 51A (Glaser, 1995
). N expression was first detected around 6 h post-infection in BHK-21 and RK-13 cells (data not shown) and about 2 h later in Vero cells (Fig. 1
), in which EAV replication is somewhat slower. In all cell lines, the replication cycle was rather asynchronous, since replicase-positive, but N-negative cells could be observed up to 3 h later. The early N signal was characterized by staining of the nucleoli of the infected cell, an observation previously also made for the porcine arterivirus (Rowland et al., 1999
), and a perinuclear, cytoplasmic staining that overlapped almost completely with that for the EAV replicase (Pedersen et al., 1999
; van der Meer et al., 1998
). The partial colocalization with the replicase remained visible throughout infection, also when N accumulated and staining of the entire cytoplasm of the infected cell was observed (Fig. 1
).
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Our results with the gene knockouts also indicated that none of the structural proteins (the products of ORFs 2a7) was required for RNA synthesis. However, the ORF5 and ORF6 mutants that we engineered could still express N-terminal fragments of the GP5 and M proteins (25 and 16 aa, respectively). In addition, we could not rule out that knockout mutants produced small amounts of protein or truncated protein, e.g. due to aberrant or internal initiation of translation or reversion of mutated AUG codons. To circumvent these potential problems, we constructed a set of deletion mutants (Fig. 2A) which lacked various parts of the structural protein coding region of the EAV genome. Each of these deletions completely ruled out the possibility of expression of one or more of the structural genes. Furthermore, they enabled us to assess which RNA sequences in the 3' end of the EAV genome are required for replication. The nomenclature of the deletion mutants reflected the size (in nt) of the deletion, for example 030-1615 lacked a 1615 nt sequence. Since the RNA2 body TRS of EAV is located within the replicase gene, all mutants were (in principle) able to produce (a truncated) mRNA2. Some of the mutants had also retained the RNA6 and/or RNA7 body TRSs. The largest deletion (2a-2594) extended from nt 9756 to 12350. This mutant lacked the complete sequence of ORFs 2a6 and lacked the 5'-terminal 40 nt of ORF7.
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The largest deletion that did not significantly affect genome replication was that in construct 2a-2594. However, this mutant did show a severe reduction of mRNA2 synthesis compared to the wt EAV030 (Fig. 2B). Mutant 030-2319, which contained the same 3'-terminal sequences but retained an intact ORF2a, produced wt amounts of mRNA2 (data not shown). Since we have previously shown that the ORF2a product (the E protein) is dispensable for genome replication and transcription (Snijder et al., 1999
), the mRNA2 transcription defect of mutant 2a-2594 is most likely due to an effect of the deletion on the cis-acting RNA sequences required for the synthesis of sg mRNA2. An RNA secondary structure prediction of the RNA2 body TRS region in 2a-2594 showed considerable differences compared to that of the same region in wild-type EAV030 (data not shown). Alternatively, the reduction of 2a-2594 mRNA2 levels might reflect differences in stability of this transcript, which contains an AUG to AUA mutation of the ORF2a translation initiation codon and is probably untranslated.
By deletion analysis of an EAV defective interfering (DI) RNA, we have previously mapped the boundary of the EAV 3' replication signals to the region between nt 354 and 1066 upstream of the genomic 3' end (Molenkamp et al., 2000 ). A construct containing a deletion leaving only 354 nt of the 3' end failed to replicate (EDIC2-3457; Molenkamp et al., 2000
). Mutant 030-2319, however, contains a similar deletion and apparently is able to replicate normally. This clearly shows that the sequence requirements for EAV DI RNA replication might be similar, but are not necessarily identical, to those of full-length genome replication. Similar findings have been reported for the alphavirus Sindbis virus (Niesters & Strauss, 1990
).
The experiments in this paper suggest that expression of the EAV replicase gene only is required for genome replication and sg mRNA transcription. Furthermore, the products of EAV ORFs 2a7 were all found to be essential for the production of infectious virus particles, whereas the functionality of the L-ORF has become highly doubtful. Formally, we cannot rule out the possibility that the L-ORF protein or one of the EAV structural proteins exerts a modest effect on viral RNA synthesis, but it is clear that their presence is not essential for the two main processes of RNA synthesis in the EAV life-cycle. The transcriptional involvement of the N protein in particular is unlikely, since the inactivation of its expression in different ways produced consistent results. In a previous study (van Marle et al., 1999 ), inactivation of the RNA7 body TRS did not affect genome replication or transcription of the other sg mRNAs. For a number of these TRS mutants, mRNA7 transcription could not be detected even by sensitive RTPCR methods, suggesting the complete absence of N protein expression. Here, both mutagenesis of the ORF7 AUG codon and deletion of the first 40 nt of ORF7 did not affect replication or transcription. Also mutant 030-2511, containing only the 3'-terminal 80 nt of the N gene, showed a low level of genome replication.
Despite the fact that it apparently is dispensable for RNA synthesis, part of the EAV N protein clearly colocalized with the replication complex (Fig. 1). In our opinion, the most obvious explanation for this colocalization is that it points towards the site of EAV genome encapsidation. Although genome replication and virus budding occur at distinct sites in arterivirus-infected cells, little is known about the intermediate stage of nucleocapsid formation. Newly made genomes may be encapsidated almost immediately, resulting in a preformed nucleocapsid structure that subsequently migrates to the site of virion budding. Possibly, the colocalization of the coronavirus replicase and N protein should be explained in a similar manner, although the pronounced differences between arterivirus and coronavirus N proteins also leave room to speculate on auxiliary functions of the latter, e.g. in viral RNA or protein synthesis. To address this issue, the recent generation of infectious cDNA clones for coronaviruses (Almazán et al., 2000
; V. Thiel and others, personal communication) will prove to be instrumental.
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References |
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Received 19 May 2000;
accepted 30 June 2000.