Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus

Marieke A. Tijms1, Yvonne van der Meer1 and Eric J. Snijder1

Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, LUMC E4-P, 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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RNA synthesis (genome replication and subgenomic mRNA transcription) directed by equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) occurs on modified cytoplasmic membranes to which most viral replicase subunits localize. Remarkably, a fraction of non-structural protein 1 (nsp1), a protein essential for transcription but dispensable for genome replication, is present in the host cell nucleus, in particular during the earlier stages of infection. Expression of GFP-tagged fusion proteins revealed that nsp1 is actively imported into the nucleus. Although the signals responsible for nsp1 transport could not be identified, our studies revealed that another EAV protein with a partially nuclear localization, the nucleocapsid (N) protein, utilizes the CRM1-mediated nuclear export pathway. Inactivation of this pathway with the drug leptomycin B resulted in the unexpected and immediate nuclear retention of all N protein molecules, thus revealing that the protein shuttles between cytoplasm and nucleus before playing its role in cytoplasmic virus assembly.


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The replication of positive-stranded RNA (+RNA) viruses of eukaryotes depends on a unique process of cytoplasmic RNA-dependent RNA synthesis. A common feature is the involvement of host cell membranes, which are often modified to accommodate the +RNA virus replication complex (Carette et al., 2000 ; Chen & Ahlquist, 2000 ; Kujala et al., 2001 ; Mackenzie et al., 2001 ; Pedersen et al., 1999 ; Rust et al., 2001 ; Schaad et al., 1997 ; Schlegel et al., 1996 ; van der Meer et al., 1999 ; and references therein). The membrane association of +RNA viral non-structural (‘replicase’) proteins is often linked to their production from large polyprotein precursors by proteolytic processing. In nidoviruses (Arteriviridae and Coronaviridae), the replicase is translated from the genomic RNA in the form of two precursor polyproteins (for a review, see Snijder & Meulenberg, 1998 ). For the arterivirus prototype equine arteritis virus (EAV), the replicase ORF1a protein and the C-terminally extended ORF1ab ribosomal frameshift protein are 187 kDa (1727 aa) and 345 kDa (3175 aa), respectively. These polyproteins are autocatalytically processed into 12 non-structural proteins (nsps) by the proteases contained in nsp1, nsp2 and nsp4 (Snijder & Meulenberg, 1998 ). Seven additional genes, probably all encoding structural proteins (Molenkamp et al., 2000 ), reside in the 3'-terminal quarter of the genome. As in all nidoviruses, the structural protein genes are expressed from a 3'-coterminal nested set of subgenomic (sg) mRNAs, which contain the genomic leader sequence at their 5' end. The cotranscriptional fusion of the sg RNA leader and body segments most likely occurs during discontinuous minus-strand RNA synthesis, which is thought to yield the sg minus-strand templates for subsequent sg mRNA transcription (Sawicki & Sawicki, 1995 ; Sawicki et al., 2001 ; van Marle et al., 1999 ; Pasternak et al., 2001 ).

The EAV replication complex is associated with double membrane structures in the perinuclear region, which result from the modification of endoplasmic reticulum membranes (Pedersen et al., 1999 ; Snijder et al., 2001 ). Immunofluorescence microscopy revealed that most EAV nsps and a fraction of the nucleocapsid (N) protein localize to the same complex (Molenkamp et al., 2000 ; van der Meer et al., 1998 ; van Dinten et al., 1996 ). Using an anti-nsp1 antiserum (Snijder et al., 1994 ) in an immunofluorescence assay (IFA) (van der Meer et al., 1998 ), we have now established that the staining pattern for nsp1 is quite different and more complex (Fig. 1A). Nsp1 was observed both in the nucleus and in the cytoplasm, and staining patterns sometimes differed strikingly between cells in the same specimen (Fig. 1A). Nsp1 localization was analysed in more detail in a time-course experiment (Fig. 1B), which confirmed that the subcellular localization of nsp1 deviates from that of other EAV nsps. Especially during the earlier stages of infection, nsp1 was observed in the nucleus. However, later, the protein was also present in the cytoplasm, either in the perinuclear region to which all other nsps are confined, or dispersed throughout the cytoplasm (Fig. 1). The appearance of nsp1 in the cytoplasm was unrelated to the synthesis of structural proteins from sg mRNAs, which was monitored in double-labelling experiments using monoclonal antibodies against EAV structural proteins (data not shown). Similar observations were made in African green monkey (Vero) and rabbit kidney (RK-13) cells (data not shown). Subsequently, nsp1 was expressed from a transfected plasmid with a cytomegalovirus (CMV) immediate early promoter. Again, but now in the absence of EAV infection, substantial variation in nsp1 distribution and a partially nuclear localization were observed (data not shown), strongly suggesting that the variability in nsp1 distribution resulted from a property of the host cell, e.g. the stage of the cell cycle.



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Fig. 1. Subcellular localization of EAV nsp1 in EAV-infected BHK-21 cells. (A) Comparison of the staining for nsp1, nsp2 and nsp3 at 8 to 10 h p.i. IFAs were performed using previously described replicase antisera (Pedersen et al., 1999 ; Snijder et al., 1994 ) and a conventional Olympus fluorescence microscope. Note the variation (nucleus vs cytoplasm) in nsp1 staining between cells in the same specimen. (B) Time-course experiment. BHK-21 cells were infected with EAV or mock infected (M) and nsp1 IFAs were performed at 4, 6, 8, 10 and 12 h p.i. Typically, nsp1 was mainly detected in the nucleus during the earlier stages of infection, whereas the cytoplasmic, in particular perinuclear, localization became more apparent later in infection. All photographs in panel (B) were taken and printed with the same exposure times.

 
We have previously reported that EAV nsp1, the N-terminal cleavage product of the replicase, is essential for sg RNA transcription, but fully dispensable for genome replication (Tijms et al., 2001 ). Deletion of the nsp1-coding sequence from the viral genome completely blocked sg RNA synthesis, which was restored when nsp1 was provided in trans. To this end, nsp1 was expressed from an internal ribosomal entry site (IRES) which replaced a number of structural genes (replicon DITRAC; Tijms et al., 2001 ). We concluded that nsp1 connects two main levels at which genome expression is regulated: replicase translation/processing and sg mRNA transcription. Nsp1 (29 kDa; 260 aa) contains a papain-like cysteine protease (PCP{beta}) in its C-terminal half that rapidly cleaves the nsp1/2 site in nascent replicase polyproteins (Snijder et al., 1992 , 1994 ). Comparison of arterivirus sequences revealed remnants of an additional PCP domain, PCP{alpha}, that has lost one of its catalytic residues in EAV, but is functional in other arteriviruses, where it cleaves nsp1 internally (den Boon et al., 1995 ). Furthermore, the N-terminal region of nsp1 contains a putative zinc-finger (ZF) domain (Tijms et al., 2001 ). Mutagenesis studies indicated that the structural integrity of both nsp1 and its predicted ZF are essential for the transcription-specific function (Tijms et al., 2001 ), which remains to be characterized in detail.

The 29 kDa size of nsp1 is below the size exclusion limit of the nuclear pore complex. Although nucleo-cytoplasmic transport generally is a highly regulated process, even in the case of small proteins like histones, nsp1 might be able to diffuse through the nuclear pore complex (Gorlich, 1998 ; Nigg, 1997 ; Talcott & Moore, 1999 ). On the other hand, the relatively rapid change in nsp1 distribution, from mostly nuclear to cytoplasmic, suggested active transport across the nuclear envelope. Moreover, a much smaller C-terminally truncated 15 kDa deletion mutant of nsp1 (nsp1{Delta}1, lacking aa 157–260), which was expressed from a mutant DITRAC replicon (DITRAC{Delta}1; Tijms et al., 2001 ), was excluded from the nucleus (Fig. 2). This suggested the nuclear import of nsp1 to be an active process, which might depend on the C-terminal half of the protein.



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Fig. 2. Transport of nsp1 to the nucleus is an active process. The nsp1–nGFP fusion protein (see text) was expressed in BHK-21 cells from a transfected pNT-TOPO expression plasmid or from the EAV-derived RNA replicon DITRAC (Tijms et al., 2001 ). The protein was observed in the nucleus in both cases. In contrast, the nsp1–cGFP fusion protein was retained in the cytoplasm when expressed from pCT-TOPO or DITRAC. Cells were fixed at 24 h post-transfection and images were obtained using the GFP autofluorescence signal. Additional IFAs with the anti-nsp1 rabbit antiserum were performed and yielded essentially similar results (data not shown). The two rightmost panels show an nsp1 IFA for the wild-type DITRAC replicon (upper panel; partially nuclear localization) and C-terminally truncated nsp1{Delta}1 (expressed from replicon DITRAC{Delta}1; lower panel), which was retained in the cytoplasm.

 
To confirm the active nuclear import of nsp1, the protein was fused to GFP to increase its size above the 50–60 kDa size exclusion limit of the nuclear pore complex (Nigg, 1997 ; Pemberton, 1998 ). Both an N-terminal (nsp1–nGFP) and a C-terminal (nsp1–cGFP) fusion were generated with sizes of 59 and 58 kDa, respectively. Although there was no consensus PCP{beta} cleavage site at the nsp1–GFP border, PCP{beta} proteolytic activity was prevented by replacing the active site Cys-164 with Ser (Snijder et al., 1992 ). The expression of fusion proteins of the correct size was confirmed by Western blot and immunoprecipitation analyses (data not shown).

The nsp1–GFP fusion proteins were expressed from the CMV promoter in expression vector pNT/CT-TOPO (Invitrogen) and in the more natural setting of the DITRAC replicon. The nsp1–nGFP protein partially localized to the nucleus in both expression systems (Fig. 2), suggesting that the nuclear import of nsp1 is an active process. Interestingly, the nsp1–cGFP fusion protein of the same size did not localize to the nucleus (Fig. 2). This might have been due to steric hindrance by the GFP tag, since the results obtained with the nsp1{Delta}1 protein (see above and Fig. 2) suggested a role for the C-terminal nsp1 domain in nuclear import. Furthermore, the nsp1–nGFP and nsp1–cGFP proteins expressed from the DITRAC replicon were both unable to trans-complement sg RNA synthesis (data not shown; see also Tijms et al., 2001 ), indicating that in this respect also the GFP tag interfered with normal nsp1 function.

EAV nsp1 sequence analysis did not reveal the presence of known nuclear localization (NLS), exclusion (NES) or shuttling (NS) signals. This indicated that nsp1 transport might be mediated through an interaction with a cellular protein containing such signals. Numerous nuclear import pathways and a few nuclear export mechanisms have been described. Of the latter, the best studied example depends on the CRM1 transporter (exportin 1; for a review, see Pemberton, 1998 ). CRM1 can be inactivated by the drug leptomycin B (LMB) (Nishi et al., 1994 ), which blocks its interaction with NES-containing proteins (Kudo et al., 1999 ). To determine whether CRM1 is involved in intracellular transport of nsp1, EAV-infected BHK-21 cells were given 10 ng/ml LMB at 6 h post-infection (p.i.) and were fixed 2–6 h later. Double labelling experiments were performed to detect nsp1 and the N protein, the only other EAV protein known to have a partially nuclear localization (Molenkamp et al., 2000 ). Nsp1 localization was unaltered, indicating that its intracellular distribution does not depend on CRM1 function (Fig. 3). A major surprise, however, was the observation that upon LMB treatment all of the EAV N protein accumulated in the nucleus (Fig. 3), which normally contains only trace amounts of the protein. Subsequently, we analysed the effect of a complete block of nuclear export throughout the EAV life-cycle by administering LMB 2 h prior to infection and keeping the drug present throughout the experiment. The N protein was not detected in the cytoplasm at any time point, suggesting its immediate post-translational targeting to the nucleus.



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Fig. 3. Inactivation of the CRM1-dependent nuclear export pathway with leptomycin B (LMB). EAV-infected BHK-21 cells (upper panels) were given 10 ng/ml LMB at 6 h p.i. (lower panels) and were fixed at various time-points. Cells fixed at 8 h p.i. are shown after a double IFA with an anti-nsp1 rabbit antiserum (Snijder et al., 1994 ) and a mouse monoclonal antibody (3E2; MacLachlan et al., 1998 ) directed against the EAV N protein. Images were recorded with a Zeiss confocal laser scanning microscope. LMB treatment did not change the subcellular distribution of EAV nsp1. However, upon treatment with the drug, the N protein accumulated in the nucleus, in particular in foci that most likely are nucleoli in view of the data previously obtained with PRRSV (Rowland et al., 1999 ). This suggests that all N protein molecules shuttle between cytoplasm and nucleus (see text).

 
The nuclear (and nucleolar) localization of the arterivirus N protein was first described for the porcine arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) by Rowland et al. (1999) , who also identified putative (bipartite) nuclear and nucleolar localization signals. The EAV N protein also localized to nuclear foci, most likely nucleoli in view of the data obtained with PRRSV (Rowland et al., 1999 ). Interestingly, these foci seemed to be the only regions of the nucleus from which nsp1 was excluded (Fig. 3). Despite the presence of many basic residues, a canonical NLS could not be identified in the EAV N protein sequence. However, a Leu-rich region between residues 54 and 70 displays some similarity with known NESs (Leu-XXX-Leu-XX-Leu-X-Leu; Elfgang et al., 1999 ; Nigg, 1997 ).

The results obtained with LMB (Fig. 3) revealed that, although only small quantities of the N protein are normally present in the nucleus, all N protein molecules are initially transported to the nucleus, since they can be trapped in this organelle upon inactivation of CRM1-mediated export. The EAV N protein is dispensable for viral RNA synthesis (Molenkamp et al., 2000 ), which was corroborated by the fact that genome replication and mRNA synthesis could even continue with the N protein trapped in the nucleus throughout the replication cycle. However, both nucleocapsid formation and budding of arteriviruses are assumed to be strictly cytoplasmic events (for a review, see Snijder & Meulenberg, 1998 ). Consequently, the N protein has to be shuttled back to the cytoplasm to fulfil its role in virion biogenesis. At present, we can only speculate on the reasons for the nucleo-cytoplasmic shuttling of the arterivirus N protein. Nuclear shuttling frequently involves protein phosphorylation and the EAV N protein is a phosphoprotein (Zeegers et al., 1976 ). Possibly, this pathway is used to achieve an essential post-translational modification, like phosphorylation. A more trivial explanation is that the N protein might be transported to the nucleus ‘accidentally’, e.g. due to its phosphorylation, its binding to a cellular factor or the presence of clusters of basic residues, which are often found in NLSs (Nigg, 1997 ). In this scenario, the virus may have adopted the use of the CRM1 pathway to return the N protein to the cytoplasm. Finally, the nuclear/nucleolar localization of the N protein may be part of a strategy to modulate host cell functions. In addition to being the site of rRNA synthesis and modification of snRNAs and snRNPs, nucleoli have been implicated in a variety of host cell processes (Olson et al., 2000 ). In this context, it is remarkable that recent studies on members of the second nidovirus family, the Coronaviridae, have also revealed nuclear and nucleolar import of the N protein (Hiscox et al., 2001 ; Wurm et al., 2001 ), a process that was postulated to disrupt host cell division (Wurm et al., 2001 ). Although the coronavirus and arterivirus N proteins are unlikely to share common ancestry, these observations suggest that the nuclear import of the N protein is important for a mechanism common to nidoviruses.

Similar considerations apply to the nuclear localization of EAV nsp1. We have not obtained nsp1 mutants that are both retained in the cytoplasm (like nsp1{Delta}1; Fig. 2) and still capable of fulfilling the transcription-specific function of nsp1. Thus, the nuclear localization (or shuttling) of nsp1 may be required for a specific modification of nsp1 or the recruitment of a cellular factor to the viral transcription complex. On the other hand, the nuclear localization of nsp1 may be irrelevant for sg RNA synthesis and the protein may be used to influence a host cell-specific function.


   Acknowledgments
 
We thank Udeni Balasuriya and James MacLachlan (University of California, Davis) for monoclonal antibodies and Dr F. Prins (LUMC Department of Pathology) for assistance with confocal microscopy. We are grateful to Sietske Rensen and Jessika Dobbe for technical assistance, and to Fred Wassenaar, Leonie van Dinten, Richard Molenkamp, Alexander Pasternak and Willy Spaan for helpful comments. M.A.T was supported by Grant 348-003 from the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW) to E.J.S.


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Received 16 November 2001; accepted 14 December 2001.