key: cord-1034124-332khr4a
authors: Mohammadi, Hakimeh; Sharif, Shayan; Rowland, Raymond R.; Yoo, Dongwan
title: The lactate dehydrogenase-elevating virus capsid protein is a nuclear–cytoplasmic protein
date: 2009-06-11
journal: Arch Virol
DOI: 10.1007/s00705-009-0410-0
sha: 495da6e82a1cb1a87f9b2a1a16959eefda207656
doc_id: 1034124
cord_uid: 332khr4a

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However, nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus, has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their normal cytoplasmic distribution. In the present study, the N protein of lactate dehydrogenase-elevating virus (LDV) of mice was examined for nuclear localization. The subcellular localization of LDV-N was determined by tagging N with enhanced green fluorescence protein (EGFP) at the N- and C-terminus. Both N-EGFP and EGFP-N fusion proteins localized to the nucleus and nucleolus of gene-transfected cells. Labeled N also accumulated in the perinuclear region, the site of virus replication. The LDV-N sequence contains a putative ‘pat4’-type nuclear localization signal (NLS) consisting of 38-KKKK. To determine its functional significance, a series of deletion constructs of N were generated and individually expressed in cells. The results showed that the ‘pat4’ NLS was essential for nuclear translocation. In addition, the LDV-N interacted with the importin-α and -β proteins, suggesting that the LDV-N nuclear localization may occur via the importin-mediated nuclear transport pathway. These results provide further evidence for the nuclear localization of N as a common feature within the arteriviruses.

Lactate dehydrogenase-elevating virus (LDV) is a murine virus causing lifelong persistent viremia in mice with no apparent clinical signs except an increased level of the plasma enzyme lactate dehydrogenase [4, 22, 24, 29] . LDV shows limited in vitro replication, which is restricted to specific murine primary macrophages and presents a low level of virus production in culture [2, 3, 20, 33] . LDV is an RNA virus with a diameter of 62-80 nm that belongs to the family Arteriviridae, a family of viruses whose members contain a single-stranded positive-sense RNA genome and which includes three other member viruses: porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV) [31] . The family Arteriviridae is grouped in the order Nidovirales along with the family Coronaviridae. Arteriviruses infect macrophages in the host animal and are responsible for a number of important diseases such as abortion and respiratory disorders in pigs by PRRSV, persistent viremia in mice by LDV, abortion and arteritis in horses by EAV, and hemorrhagic fever in monkeys by SHFV [15, 18, 32] .

The LDV genome is 14.1 kb in length, dominated by two large open reading frames, ORF1a and ORF1ab, in the 5 0 three-fourths of the genome, which code for two large polyproteins, PP1a and PP1ab. The polyproteins are predicted to be proteolytically cleaved into 12 cleavage products designated non-structural protein 1 (Nsp1) through Nsp12. Downstream from ORF1ab are ORF2 through ORF7, which encode at least seven structural proteins: GP2a, GP2b (E), GP3, GP4, GP5, matrix (M) and nucleocapsid (N) proteins [32] . The nucleocapsid protein of LDV is 115 amino acids in length and is the most abundant protein within the virion.

Nidoviruses replicate exclusively in the cytoplasm. However, the N protein of PRRSV, EAV and the coronaviruses avian infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV) localize to the nucleolus [25, 35, 40] . An interesting exception is the N protein of SARS-CoV. One study showed that SARS-CoV N protein is localized exclusively in the cytoplasm [28] , while other studies have reported that the SARS-CoV N protein is present in the both the cytoplasm and the nucleus/nucleolus [43, 44] . These findings suggest that localization of N to the nucleolus may not be a general property for all nidovirus N proteins. Once in the nucleus, the PRRSV and IBV N proteins interact with fibrillarin and nucleolin and may be involved in the cell cycle regulation [5, 40, 42] . For nuclear proteins, trafficking between the cytoplasm and the nucleus occurs through the nuclear pore complexes (NPC). While small molecules enter the nucleus by diffusion through NPC, large molecules enter the nucleus by an energy-dependent pathway using a nuclear localization signal (NLS) [8] . The NLS on the cargo protein interacts with importin proteins in the cytoplasm, which are then shuttled across the NPC and into the nucleus. Three types of NLSs have been identified. A ''pat4''-type NLS is a stretch of four basic amino acids such as 'KKKK'. A second type is ''pat7'', which is a string of amino acids starting with proline (P) followed by any other two amino acids, followed by four amino acids of which at least three are basic residues. The third type is a bipartite form. The bipartite NLS has two areas of basic residues separated by 10-12 amino acids. The first group of basic residues includes two basic amino acids, and the second group contains five amino acids, consisting of at least three basic residues [16, 26] . In this report, we examined the N protein nuclear localization of LDV and identified a functional 'pat-4' signal sequence essential for N protein translocation into the nucleus.

Cells, viruses, and bacterial strains MARC-145 cells [13] , a derivative of MA-104 cells, HeLa cells, and NIH-3T3 cells were used for this study. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) in a humidified incubator with 5% CO 2 at 37°C. PRRSV strain PA8 [38] was grown in MARC-145 cells. E. coli strains DH5a and XL-1 Blue (Stratagene, La Jolla, CA) were used for gene cloning and mutagenesis, and strain BL-21 (Stratagene) was used for protein expression.

The LDV-N gene was amplified by PCR from a plasmid containing the LDV genomic fragment, and using the BamHI sequences at the ends of both primers, cloned into pEGFP-N1 and pEGFP-C1 (Clontech, Mountain View, CA) upstream and downstream, respectively, of the enhanced green fluorescence protein (EGFP) gene. LDV-N was also subcloned using BamHI into pCITE-2C (Novagen) for in vitro transcription and translation.

Mutant construction for LDV-N Deletion constructs of LDV-N were made by PCR using the appropriate primer sets (Table 1 ) under the following Table 1 List of the primers used for EGFP fusion constructions for LDV-N a Primers were designed based on sequence information available for LDV b Lowercase letters indicate the BamHI recognition sequence conditions: preincubation for 5 min at 37°C, denaturation for 30 s at 95°C, annealing for 1 min at 56°C, and extension for 1 min at 72°C per cycle for 35 cycles, and final extension for 10 min at 72°C in a GeneAmp 2400 thermocycler (Perkin Elmer). PCR products were digested with BamH1 and subcloned into pEGFP-N1. Each construct was verified by restriction digestion and nucleotide sequencing of the junction between N and EGFP. Cloning and DNA manipulations were conducted according to standard protocols [30] . For mutagenesis, PCR-based site-directed mutagenesis was performed by overlapping extension using mismatch primers as described (Table 1 ) [39] . PCR was carried out using 15 ng of pLDV-N-EGFP plasmid DNA, 300 ng of each forward and reverse primer, 1 mM dNTPs, 19 buffer [10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO 4 , 0.1% Triton X-100], and 2.5 units of Pfu DNA polymerase (Stratagene) for 16 cycles as follows: denaturation at 94°C for 30 s, primer annealing at 56°C for 1 min, and primer extension at 68°C for 8 min. The PCR products were digested with DpnI and used to transform E. coli XL1-Blue. Transformants were randomly selected for plasmid DNA preparation using a QIAprep spin miniprep kit (QIAGEN, Santa Clarita, CA), and mutagenized sequences were verified by nucleotide sequencing in both directions.

Expression of N-GFP fusion proteins was determined in HeLa cells by transfection using Lipofectin (Invitrogen) according to the manufacturer's instruction. Cells were grown on microscope coverslips and transfected with GFP fusion constructs of LDV-N. PRRSV N gene and PRRSV N-GFP fusion constructs were included as positive controls [37] . At 24 h post-transfection, cells were fixed with methanol and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min, and fluorescence was observed using a fluorescence microscope (model AX70, Olympus). For PRRSV-infected cells, N protein was stained with MAb SDOW-17 [19] at a dilution of 1:400 in 1% BSA-PBS, followed by goat anti-mouse antibody conjugated with AlexaFluor 488 (Molecular Probes) at a dilution of 1:100. After five washes with PBS, cells were stained with DAPI (4 0 ,6-diamidino-2-phenylindole dihydrochloride, Sigma) for 5 min, washed again, and mounted on a slide using 40 ll of the mounting buffer (60% glycerol, 0.1% sodium azide in PBS), and fluorescence was examined by microscopy. fusion proteins and LDV-N were incubated overnight at 4°C with constant agitation. The Sepharose beads were washed five times with PBS, boiled for 5 min in SDS sample buffer [10 mM Tris-HCl (pH 6.8), 25% glycerol, 10% SDS, 10% b-mercaptoethanol, and 0.12% (wt/vol) bromophenol blue], and analyzed by 12% SDS-PAGE. Gels were dried and exposed for image analysis using a Phosphorimager (Molecular Dynamics).

The LDV nucleocapsid protein was expressed as an EGFP-N fusion protein in HeLa cells and examined for subcellular distribution. Cells were counterstained with DAPI for nuclear staining. EGFP alone was distributed throughout transfected cells (Fig. 1, panel b) . For the purpose of comparison, localization of the PRRSV N protein in virusinfected MARC-145 cells is shown (Fig. 1, panel c) . Detection of PRRSV N by N-specific MAb SDOW17 showed the accumulation of N in the nucleus and nucleolus in addition to its normal cytoplasmic distribution. In PRRSV N-and PRRSV N-GFP-transfected cells, N accumulated in the nucleus (Fig. 1 , panels e, f), and similarly, both LDV-N-EGFP and LDV-EGFP-N fusion proteins showed an accumulation of fluorescence in the nucleus (Fig. 1, panels g, h) , with a distribution pattern similar to that of PRRSV N, indicating that LDV-N localized both in the cytoplasm and the nucleolus.

Sequence analysis of the LDV-N protein identified the presence of a single basic region of KKK which resembled a 'pat-4' type NLS located between amino acid positions 38-41 (Fig. 2a) . The location of this lysine-rich region corresponded to the 'pat-7' motif of the PRRSV N protein.

To determine the functional significance of the 'pat-4' motif of LDV-N, a series of deletion mutants were made by progressively deleting from the C-terminus of N (Fig. 2b) . Cells were then transfected with the mutant constructs, and their fluorescence was examined. LDV-N-C40-EGFP, LDV-N-C65-EGFP, and LDV-N-C70-EGFP showed fluorescence in the nucleus and nucleolus (Fig. 3, panels b, c, d) .

In contrast, LDV-N-C82-EGFP, in which the putative motif was deleted, yielded cytoplasmic fluorescence but not nuclear fluorescence (panels e). These results indicate that the predicted 'pat-4' motif was indeed a functional NLS that translocates LDV-N to the nucleus and nucleolus of cells.

To further confirm the function of the predicted NLS and to examine amino acids important for this function, individual residues of '38-KKKK-41' were substituted with glycine (G) at single or multiple sites (Table 2) , and the individual mutants were expressed in HeLa cells (Fig. 4) . The data showed that two 'K's at positions 38 and 40 are important for nucleolar localization of LDV N, and the residues at positions 38, 40, and 41 play a crucial role for this function (Table 2; Fig. 4 ). The mutant in which all four 'K's were substituted with 'G's became exclusively cytoplasmic and did not localize to the nucleus or the nucleolus (Fig. 5a) . This confirms that the predicted 'pat-4' motif was indeed the functional NLS for nuclear localization of the LDV-N protein. Since LDV is a murine virus and HeLa cells were used for this study, the NLS function of LDV-N was further confirmed in mouse cells using the same constructs (Fig. 5b) . NIH-3T3 cells were transfected with either wild-type or NLS-null mutant LDV-N and their fluorescence patterns were examined. As expected, the PRRSV N protein was found in the nucleus and nucleolus in the murine cells. For LDV-N, the nucleolar localization was evident for the wild-type N protein, whereas the NLSnull mutant protein was cytoplasmic without distinct nuclear staining, indicating that the LDV-N nuclear localization is not cell-type-dependent but is rather a natural property of the N protein. 

To determine the basis for LDV-N nuclear localization, interactions between N and importin proteins were determined by GST pull-down assay. Importin-a and importin-b were individually expressed in E. coli as a GST-fusion proteins and coupled to glutathione-Sepharose beads. The radiolabelled LDV-N protein was then synthesized in vitro by transcription and translation, and importin-coupled GST beads were incubated with the radiolabelled N protein.

Unbound proteins were washed off and the bound proteins were resolved by SDS-PAGE and autoradiography. As with the PRRSV N protein, which was previously shown to interact with both importin-a and -b proteins [27] ; (Fig. 6,  lanes 7 and 9) , both importin-a and importin-b specifically bound LDV-N (lanes 8 and 10), while GST alone did not bind to PRRSV-N or LDV-N (lanes 5 and 6). This study demonstrates the specific interaction of LDV-N with both importin proteins and suggests that LDV-N nuclear localization may be importin-mediated. 

Nuclear localization of the capsid protein has been studied for several RNA viruses that restrict their replication to the cytoplasm, and capsid proteins of Semliki Forest virus, dengue virus, and hepatitis C virus have been shown to localize in the nucleus and nucleolus [7, 36, 41] . Nidoviruses also replicate exclusively in the cytoplasm of infected cells [1] , but the N protein has been shown to localize in the nucleus and nucleolus for PRRSV, EAV, IBV, MHV and TGEV [6, 10, 25, 35, 40, 45] . The N protein of PRRSV contains two putative NLS motifs, 'pat 4' and 'pat 7', and the ''pat 7'' located at positions [41] [42] [43] [44] [45] [46] [47] has been shown to be the active and primary NLS for N [27] . In the present study, we showed that the LDV-N protein localized to the nucleus and nucleolus in cells transfected with the N-EGFP gene, and using mutant constructs, we showed that the nuclear localization of N was dependent on a 'pat-4' motif located at positions 38-41 (Figs. 2a, 3) . The location of the 'pat-4' motif in LDV-N is similar to the region where the functional 'pat 7' motif is located in PRRSV N. We further identified two lysine residues at positions 38 and 40 in LDV-N that form core residues for nuclear transport of N. For PRRSV N, amino acids at positions 43 and 44 are critical residues for nuclear localization [14, 21, 27] . For a protein to localize in the nucleus, the cellular chaperone importin-a recognizes the NLS on the cargo protein, and subsequently, importin-b binds to the importin-a/cargo protein complex, transporting the complex in the presence of Ran-GDP to NPC on the nuclear membrane [8, 17] . We found that LDV-N binds to importin-a (Fig. 6, lane 8) , which is consistent with the utilization of the classical pathway for nuclear transport. It is interesting to note that LDV-N also binds to importin-b (Fig. 6, lane 10) . PRRSV N also binds to both importin-a and importin-b [27] , and several other viral proteins have been reported to interact with both importin-a and -b. The polyomavirus capsid protein requires interaction with both importin-a and -b to enter the nucleus [23] , and SV40 VP3 also interacts with both importin proteins for nuclear translocation [17] . The HIV Rev protein binds both importin-a and -b, but its binding to importin-a is NLS-independent, while importinb binding is NLS-dependent [9] . For PRRSV, two nucleolar proteins, nucleolin and B23, have been shown to bind to N (unpublished data), and thus a possible mechanism for LDV-N nuclear transport is the use of an importin-dependent pathway with a 'piggy-backing' mechanism, in which N binds to a nuclear-targeted cellular protein in addition to importin proteins.

Since N proteins of PRRSV, EAV, and LDV all localize to the nucleus and nucleolus, the nuclear role of N during infection is of major interest. The core protein of hepatitis C virus is believed to modify cellular function by preventing translocation of host cell proteins to the nucleus [12, 34] , and the N protein of TGEV has been suggested to play a role in the disruption of cell division [40] . For PRRSV N, disruption of NLS does not affect virus replication in cell culture, but in pigs, NLS-null viruses are attenuated in their virulence [14, 21] . The NLS motif of PRRSV N was associated with strong selective pressure for reacquisition of the nuclear function in vivo, suggesting the involvement of N nuclear localization in viral pathogenesis. N proteins of several member viruses in the order Nidovirales colocalize and interact with fibrillarin and nucleolin in the nucleolus, supporting the hypothesis that N modulates host-cell ribosomal biogenesis and cell cycle progression [5, 11] . In summary, we show that LDV-N contains a functional 'pat-4'-type NLS at positions 38-41 and localizes in the nucleolus of cell via the importin-mediated pathway. The nuclear localization of the capsid protein may be a common feature of members of the family Arteriviridae and may play a common role for replication and pathogenesis of this group of viruses. 

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