Department of Virology, Center of Infectious Diseases, Leiden University Medical Center, LUMC P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands1
Department of Biology, Division of Electron Microscopy, University of Oslo, Norway2
Author for correspondence: Eric Snijder. Fax +31 71 5266761. e-mail E.J.Snijder{at}LUMC.nl
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Abstract |
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Introduction |
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Equine arteritis virus (EAV) (Doll et al., 1957 ) is the prototype of the family Arteriviridae (Snijder & Meulenberg, 1998
), which has been placed in the order Nidovirales, together with the family Coronaviridae (Cavanagh, 1997
). The ancestral relationship between the two virus groups is most evident from the common features of their genome organization and expression. Arteri- and coronaviruses both (i) encode a similar array of functional domains in their replicase genes, (ii) use ribosomal frameshifting to express key replicative functions, (iii) employ extensive proteolytic processing of replicase precursor polyproteins (Ziebuhr et al., 2000
) and (iv) generate a nested set of subgenomic mRNAs to regulate the expression of their structural genes (Lai & Cavanagh, 1997
; Snijder & Meulenberg, 1998
).
EAV replicase gene expression results in the generation of two multidomain precursor proteins, the 1727 amino acid ORF1a protein and the 3175 amino acid ORF1ab protein (den Boon et al., 1991 ). These polyproteins are cleaved by three internal, ORF1a-encoded proteases (Snijder et al., 1992
, 1995
, 1996
). Our current understanding of EAV replicase processing is summarized in Fig. 1
. The ORF1a protein can be cleaved at seven sites (Snijder et al., 1994
, 1996
; Wassenaar et al., 1997
), yielding a number of processing intermediates and eight end-products, nsp1 to nsp8. The N-terminal cleavage products nsp1 and nsp2 are liberated rapidly by internal cysteine autoprotease activities (Snijder et al., 1994
). The remaining nsp38 intermediate (96 kDa) is processed by the nsp4 serine protease (SP), which also cleaves the ORF1b-encoded polyprotein three times (van Dinten et al., 1996
, 1999
). The latter part of the replicase contains a set of highly conserved functions that have been implicated in viral RNA synthesis (den Boon et al., 1991
). Among its processing products (nsp9 to nsp12) are the putative virus RNA-dependent RNA polymerase (RdRp, nsp9) and the recently characterized EAV helicase (Hel; nsp10) (Seybert et al., 2000
), which also contains a putative N-terminal metal-binding domain (van Dinten et al., 2000
).
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An interesting observation during analysis of the processing of the EAV ORF1a protein was the fact that two alternative pathways can be followed for processing of the C-terminal part (Fig. 1). Either the nsp4/5 site (major pathway) or the nsp5/6 and nsp6/7 sites (minor pathway) are processed (Wassenaar et al., 1997
). Cleavage of either of these sites is believed to render the alternative site(s) non-accessible. In an expression system, the presence of liberated nsp2 was found to determine whether the nsp4 SP could cleave the nsp4/5 site of the nsp38 precursor (Wassenaar et al., 1997
). It is possible that a specific folding or post-translational organization of the protein is required for this proteolytic event. In the nsp2/nsp38 complex, nsp2 is likely to have a strong interaction with nsp3, since the two proteins were previously found to co-immunoprecipitate, even under quite stringent conditions (Snijder et al., 1994
). Here, we show that nsp2 can be subject to an additional, internal cleavage, which is specific to Vero cells. Furthermore, both nsp2 and nsp3 were found to associate with membranes upon their individual expression by means of an alphavirus expression vector. However, only the expression of a self-cleaving nsp23 polyprotein induced the formation of the DMVs that are so typical of arterivirus infection.
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Methods |
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Labelling and immunoprecipitation of EAV nsps.
Cells were infected (m.o.i.=10) with an EAV stock grown and titrated in BHK-21 cells. To achieve the same m.o.i., Vero cells had to be infected with tenfold more virus than was used for either BHK-21 or RK-13 cells. All infected cell cultures were incubated at 39·5 °C. Radioactive labelling of intracellular proteins with [35S]methionine/[35S]cysteine (Expre35S35S protein labelling mix, DuPont NEN) was carried out between 5 and 8 h post-infection (p.i.) according to Snijder et al. (1994) . Pulsechase experiments were performed at 8 h p.i. by using a 15 min pulse and the labelling and chase protocol described previously (Snijder et al., 1994
). Cells were lysed in the buffer described by de Vries et al. (1992)
containing the protease inhibitors PMSF (400 µM), leupeptin (4 µM) and aprotinin (30 µM). Immunoprecipitations and SDSPAGE were carried out essentially as described by de Vries et al. (1992)
. For all antisera, the immunoprecipitation buffer contained 0·5 % SDS.
Sindbis virus-based expression vectors.
The previously described Sindbis virus expression vector pSinEAV(2611677)His, a pSinRep5-derivative (Bredenbeek et al., 1993 ) expressing a C-terminally hexahistidine-tagged version of nsp27 (Pedersen et al., 1999
; Wassenaar et al., 1997
), was used to engineer similar vectors expressing nsp2, His-tagged nsp3 and His-tagged nsp23. These constructs were created by PCR deletion mutagenesis and were named pSRE-nsp2, pSRE-nsp3His and pSRE-nsp2+3His. pSRE-nsp2 was constructed by deleting the sequences encoding nsp3 to nsp7 and the His tag from pSinEAV(2611677)His. To obtain pSRE-nsp2+3His, the nsp4 to nsp7 coding sequences were deleted from pSinEAV(2611677)His and the His tag was fused in-frame to the nsp3 coding sequence. pSRE-nsp3His was obtained by deleting the nsp2 coding sequence from pSRE-nsp2+3His and fusing the engineered upstream ATG codon to the 5 end of the nsp3 coding sequence. A control Sindbis virus expression vector for the green fluorescent protein (pSinRep/GFP) was kindly provided by C. M. Rice (Washington University, St Louis, MO, USA). BHK-21 cells were transfected by electroporation with infectious RNA transcribed from pSinRep5-derived vectors (van Dinten et al., 1997
).
Immunofluorescence assays (IFAs) and electron microscopy.
For indirect IFAs, transfected cells were seeded on cover slips, fixed with paraformaldehyde at 6, 9 or 12 h after transfection and processed as described by van der Meer et al. (1998) . For EM, transfected cells were seeded in tissue culture dishes. Protocols for conventional Epon embedding and ultrathin sectioning and for cryoimmuno EM have been described by Pedersen et al. (1999)
. EM specimens were examined in a Philips CM100 transmission electron microscope.
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Results |
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The analysis of ORF1a protein processing in Vero cells (Fig. 2; lanes V) revealed a number of striking differences from the other two cell lines. The nsp2 antiserum precipitated a prominent additional band of approximately 18 kDa. At the same time, less nsp2 (61 kDa) was precipitated compared with the corresponding samples from the other cell lines. Furthermore, the nsp4 and nsp78 immunoprecipitations of the Vero cell sample revealed the appearance of a prominent additional band of about 44 kDa. The sizes of the two novel bands suggested strongly that they were derived from the cleavage of a proportion of the nsp2 at an internal site. The strong recognition of the 18 kDa protein by the anti-nsp2 antiserum, which was raised by using a 9 amino acid N-terminal peptide (Snijder et al., 1994
), suggested that this product contained the nsp2 N-terminal domain. Only a trace amount of the 44 kDa product was precipitated by the anti-nsp2 antiserum (see also below). The anti-nsp4 and anti-nsp78 antisera precipitated much larger amounts of the 44 kDa product. Taken together, these data suggested cleavage of nsp2 into an N-terminal 18 kDa product (nsp2N) and a C-terminal 44 kDa (nsp2C) product. The co-precipitation of the latter product, for which an antiserum is not available, upon use of the anti-nsp4 and anti-nsp78 antisera (Figs 2
and 3
) suggested that it is the C-terminal part of nsp2 that interacts with nsp3 and nsp3-containing processing intermediates.
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The incorporation of label into nsp2 was found to increase slightly during the first hour of the chase. This has been seen before (Snijder et al., 1994 ) and may be explained by completion of polyprotein synthesis after termination of the labelling and/or processing of nsp2 from short-lived precursors. The analysis made it clear that most of the nsp2N and nsp2C can be detected immediately after the pulse labelling. Furthermore, both cleavage products and their nsp2 precursor were found to be relatively stable proteins. A densitometric analysis of the nsp2 and nsp2N bands from the immunoprecipitation of the 1, 2 and 4 h chase samples with the anti-nsp2 serum (Fig. 3
) indicated that, in the long run, approximately 50% of the nsp2 molecules were cleaved. However, directly after the pulse labelling, there was clearly more nsp2N relative to nsp2 (Fig. 3
;
nsp2 panel, lane P), suggesting that the nsp2N/nsp2C cleavage occurred prior to cleavage of the nsp2/nsp3 site. For this estimation, the nsp2N C terminus was assumed arbitrarily to be residue 435 of the ORF1a polyprotein, which would give 18 kDa for the size of the nsp2N product, starting from the nsp2 N terminus at Gly-261 (Snijder et al., 1992
). The actual position of the nsp2N/nsp2C border is unlikely to differ from this position by more than 50 amino acids, a difference that would not affect the outcome of this analysis significantly. The densitometric data were corrected for the methionine/cysteine content of the two products.
Taken together, these data indicated that the internal cleavage of nsp2 is relatively rapid and that the nsp2 molecules that are not cleaved within the first hour after their synthesis remain uncleaved. The pulsechase analysis presented in Fig. 3 also revealed that a minor, but increasing quantity of nsp2C was precipitated when the nsp2 antiserum was used. We interpret this to be co-precipitation of a proportion of the nsp2C molecules with a complex containing uncleaved nsp2 and an nsp3-containing intermediate (most likely nsp34). This could indicate that these complexes may contain multiple copies of nsp3, interacting with either nsp2 or nsp2C, or that a single nsp3 molecule can interact with multiple nsp2/nsp2C subunits.
Expression of nsp2 and nsp3 from alphavirus vectors
We have reported previously that the expression of EAV nsp27 from the 26S promoter of a Sindbis virus-based RNA vector (Bredenbeek et al., 1993 ) induced striking membrane re-arrangements in BHK-21 cells (Pedersen et al., 1999
). Double membranes and DMVs, strongly resembling those found in infected cells, were observed and were labelled for various replicase subunits in cryoimmuno EM. We employed this expression system to delineate the sequences required for DMV formation in more detail. The nsp27 expression vector was used as the basis for making deletion variants expressing nsp2, nsp23, nsp24, nsp3, nsp34, nsp38 and nsp57. With the exception of nsp57, each of these expression products is able to process itself due to the action of the nsp2 and/or nsp4 proteases (data not shown).
Previously, we found that nsp27 expression results in an IFA staining that is indistinguishable from the staining observed in infected cells (Pedersen et al., 1999 ). Thus, similar IFAs with our anti-replicase antisera were used for a first analysis of the novel set of expression vectors. This screening revealed that only co-expression of nsp2 and nsp3 in the form of nsp23 (construct SRE-nsp2+3His) or nsp24 (data not shown) produced the dense, perinuclear staining observed in EAV-infected cells (Fig. 4C
, D
). The presence of a His tag at the C terminus of nsp3 allowed us to demonstrate the exact co-localization of nsp2 and nsp3His in a double-labelling experiment (Fig. 4D
). Upon individual expression of nsp2 (construct SRE-nsp2) or nsp3 (construct SRE-nsp3His), both proteins seemed to associate with membranes (Fig. 4A
, B
), but did not produce the perinuclear staining observed upon co-expression. Remarkably, the expression of nsp2 strongly reduced the amount of labelling for the ER-resident protein PDI (Fig. 4A
).
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EAV nsp2 and nsp3 interact in the formation of DMVs
Our analysis of the morphological changes in cells transfected with SRE-nsp2, SRE-nsp3His and SRE-nsp2+3His was extended by using EM. Serial sections did not reveal the presence of double-membrane structures in Epon-embedded cells expressing nsp2 only (Fig. 5C), nsp3 only or GFP, which was used as negative control. In contrast, closely apposed ER membranes and DMVs were abundant in cells expressing the nsp2+3His protein (Fig. 5A
). Fig. 5(B)
shows that, as in EAV-infected cells (Pedersen et al., 1999
), the outer membrane of DMVs can be continuous with the ER membrane, creating a neck-like connecting structure. These results confirmed that, as suggested by the IFA results shown in Fig. 4
, the expression of EAV nsp2 and nsp3 is necessary and sufficient to induce DMV formation. The morphology of the DMVs induced upon nsp2+3His expression was somewhat more variable (both in size and shape) compared with the structures seen in EAV-infected cells.
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Discussion |
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In view of these crucial functions of nsp2, it is quite remarkable that about 50% of this protein was now found to be internally cleaved upon EAV replication in Vero cells. The internal cleavage site was estimated to be close to residue 435 of the ORF1a protein. However, this estimation was based solely on the migration of nsp2N (18 kDa) and nsp2C (44 kDa) upon SDSPAGE, which cannot be considered a very reliable method. In principle, one of the three EAV proteases may be involved in the nsp2 cleavage, although potential cleavage sites for these enzymes appear to be lacking from the amino acid sequence of the region that should contain the nsp2N/nsp2C junction (residues 385485 of the ORF1a protein). Instead, this part of nsp2 stands out for its high content of basic residues (13%) and prolines (19%). Together with the fact that the internal nsp2 cleavage is completely lacking in both BHK-21 and RK-13 cells, these observations suggests strongly the involvement of a host cell-specific protease. Such an enzyme might be located in the cytoplasm, but it might also reside in the lumen of intracellular compartments like the ER and act on a luminal domain of a partially translocated nsp2 protein. The latter mechanism is employed during the processing of polyproteins generated by members of the family Flaviviridae (see Ryan et al., 1998 ; and references therein).
EAV nsp2 contains two somewhat hydrophobic regions around residues 450 and 490 (ORF1a polyprotein numbering). If one of these spans the membrane, a luminal cleavage downstream of this domain would generate two products with sizes that are relatively close to those estimated from SDSPAGE gels for nsp2N and nsp2C. The main hydrophobic domain of nsp2 is found between residues 520 and 640 and is large enough to span the lipid bilayer several times. Although it is clear that, at least at some point, the nsp2 N- and C-terminal domains (containing the nsp2 protease and the nsp2/3 cleavage site) must be on the same side of the membrane, the exact topology of this unusual non-structural protein remains to be elucidated.
The functional implications of the internal nsp2 cleavage for EAV replication in Vero cells are unclear. Although the EAV replication cycle is somewhat delayed in Vero cells compared with BHK-21 or RK-13 cells (15 h versus 12 h), it is premature to attribute this difference to the internal processing of nsp2. Previous IFA studies have not revealed any major differences between the nsp2 labelling patterns in the three different cell lines (van der Meer et al., 1998 ), suggesting that the nsp2N cleavage product remains associated with membranes. This observation could be explained by assuming that nsp2N indeed contains one of the hydrophobic domains of nsp2 and/or becomes part of a stable, membrane-associated complex before its partial internal cleavage. Thus, at the moment of nsp2N/nsp2C cleavage, both parts of the protein may be anchored within the replication complex by interactions with other partners or interactions between the N- and C-terminal domains of nsp2 itself. For nsp2C, an obvious partner is nsp3, on the basis of the observed co-immunoprecipitation with nsp3-containing intermediates (Figs 2
and 3
). Furthermore, both nsp2N and nsp2C contain clusters of conserved Cys residues (Snijder et al., 1994
, 1995
) with unknown functions.
Despite the fact that detailed information on the biochemical properties of nsp2 and the mechanism of its membrane association/translocation is lacking, our IFA studies show that the protein by itself can associate with membranes, probably those of the ER. Remarkably, the expression of nsp2 seems to reduce the amount of PDI in the cell dramatically, an observation that has occasionally also been made in EAV-infected cells, late in infection (van der Meer et al., 1998 ). Although EM studies did not reveal any morphological changes of the ER in these cells, this phenomenon may still signify biochemical changes resulting from the interaction of nsp2 with the ER. The membrane association of individually expressed nsp3 (Fig. 4B
) is not very surprising, since computer analysis predicts the hydrophobic nsp3 N terminus to be a quite reasonable signal sequence. Some of the expression products with nsp3 at their N terminus (e.g. nsp34 and nsp38) were even partially transported to the Golgi complex (data not shown). Since staining of the Golgi complex with anti-replicase antisera has never been observed in EAV-infected cells (van der Meer et al., 1998
), one consequence of the nsp2nsp3 interaction appears to be the retention of nsp3-containing proteins in the ER membrane.
The IFA (Fig. 4) and EM (Figs 5
and 6
) studies presented in this paper have clearly shown that co-expression of nsp2 and nsp3 leads to the formation of paired membranes and DMVs (Fig. 5B
) that are labelled for the two proteins (Fig. 6
). Apparently, the third major hydrophobic domain in the ORF1a protein, that in nsp5, is dispensable for the formation of these structures. Using the EAV infectious cDNA clone (van Dinten et al., 1997
) and Sindbis virus expression vectors, we should now be able to dissect the interaction between nsp2 and nsp3 in more detail, e.g. by using site-directed and deletion mutagenesis. These future studies can be expected to shed light on the co-ordination of replicase polyprotein processing and membrane association, the mechanism of DMV formation (Pedersen et al., 1999
; Schlegel et al., 1996
) and the role of DMVs in viral RNA synthesis.
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Acknowledgments |
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Received 26 October 2000;
accepted 26 January 2001.