key: cord-0701735-tg52rgpr
authors: Takahashi-Omoe, H.; Omoe, K.; Sakaguchi, M.; Kameoka, Y.; Matsushita, S.; Inada, T.
title: Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins
date: 2003-08-23
journal: Comp Immunol Microbiol Infect Dis
DOI: 10.1016/s0147-9571(03)00053-5
sha: 657602c5e4a96575433b73793bbf7e33110262e7
doc_id: 701735
cord_uid: tg52rgpr

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells.

The Arteriviridae are enveloped positive-stranded RNA viruses that comprise a variety of animal pathogens, including lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), and porcine reproductive and respiratory syndrome virus (PRRSV) [1] . The ability to replicate in a variety of cell lines is characteristic of EAV, SHFV, and PRRSV, but not of LDV, which has a strict host cell specificity. In mice, it is known that only a subpopulation of peritoneal macrophages and other macrophages support LDV replication [2] . To date, no cell lines in which LDV can replicate or LDV receptors responsible for cell tropism have been identified; therefore, the mechanism underlying LDV susceptibility restriction remains unclear.

Because of an essential role in attachment to the plasma membrane of receptive host cells, it is important for studying the biological features of arterivirus envelope proteins. Arterivirus acquire their envelope by budding into the lumen of smooth membranes of the exocytic pathway, probably including those of the Golgi complex [3 -7] . The specific roles of the various envelope proteins in arterivirus assembly and infectivity have not yet been reported, however, the recent development of infectious cDNA clones for arteriviruses [8 -10] has opened the possibility of studying arterivirus assembly by modifying the expression of envelope proteins.

LDV has two major envelope proteins. The smaller of the two is an 18 -19-kilodalton (kD) nonglycosylated protein M/VP-2, encoded by open reading frame (ORF) 6. It is close to the N-terminal end of three potential adjacent transmembrane segments that mimic sequences in the corona virus M protein [11] . The larger protein is the envelope glycoprotein VP-3, encoded by ORF 5. It is generally heterogenous in size (25 -40 kD) due to varying amounts of N-glycosylation [12] . Li et al. [13] have postulated that LDV VP-3 may be the virus attachment protein. Their studies have shown that the neutralization epitope is located in the short envelope glycoprotein ectodomain and is associated with polylactosaminoglycan chains, which may affect neutralizing antibody binding to LDV virions.

LDV M/VP-2 and VP-3 are present in virions as heterodimers that are covalently linked by disulfide bonds, probably between single cysteine residues in the protein ectodomains [14] . Because disulfide bond breakage causes viral infectivity loss, linkage between M/VP-2 and VP-3 appears to be required for host cell entry and is perhaps achieved by generating the virion receptor attachment site. Further analysis of the interaction between LDV VP-3-M/VP-2 heterodimer envelope proteins and host cells will require an expression system that includes glycosylation and cell surface localization. Using transient mammalian expression systems based on Sindbis and vaccinia virus-based expression vectors, the non-structural proteins 2 and 3 (nsp2 and 3) of EAV and ORF 2, 4, 5, 6, and 7 products of PRRSV have been reported [15 -17] . Although in vitro translation of LDV ORF 5 and ORF 6 transcripts has been reported in a rabbit reticulocyte lysate system [18] , a mammalian cell system expressing LDV proteins remains unestablished. While experiments with the transient expression systems described above are convenient for studying cytotoxicity [17] , cellular immune responses [15] , and viral processing [16] , there is considerable variability between individual experiments. Cell lines stably expressing viral envelope proteins would be useful for detailed molecular analysis of interactions between viral and host cell proteins and mechanisms underlying LDV susceptibility restriction. In this study, we established a stable mammalian cell system expressing both the LDV ORF 5-and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins) as viral envelope proteins and analyzed the expressed proteins by immunological methods.

Monkey kidney Cos7 cells [19] were obtained from Riken Cell Bank (RCB0539, Tsukuba, Japan) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine (conditioned medium). For transfection, approximately 5 £ 10 6 cells were suspended in 0.5 ml K-PBS (30 mM NaCl, 120 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 5 mM MgCl 2 ) and electroporated with 25 mg DNA with a Bio-Rad gene pulser (Bio-Rad Laboratories, California, USA). The electrical parameters were 220 V, 975 mF, and 100 V resistance.

Purified LDV type C (LDV-C) was prepared with 4 and 8-week-old SJL/J mice (Jackson Laboratory, Maine, USA) as described previously [20] . A polyclonal antibody (#36) against LDV M/VP-2 was obtained after immunizing rabbits with a synthetic polypeptide corresponding to the LDV-C ORF 6 C-terminal region coupled with the keyhole limpet hemocyanin (KLH) [21] . A monoclonal antibody against LDV VP-3 (MAb no. 36) has been described previously [22] .

To prepare expressed LDV-C ORF 5 and ORF 6 proteins, plasmid pcDNA3.1-VP3 and pcDNA6-VP2 were constructed as shown in Fig. 1 . Briefly, the entire LDV ORF 5 coding region plus a Kozak consensus sequence that is the ATG initiator codon [23] were isolated from LDV-C infected mouse sera by the reverse-transcription-polymerase chain reaction (RT-PCR). Viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The RNA was reverse transcribed by murine leukemia virus reverse immediate-early promoter/enhancer element ( P CMV). pcDNA-VP2 expresses the predicted ORF 6 protein as a fusion protein with the C-terminal myc epitope (derived from c-myc; EQKLISEEDL) and the 6 £ His-tag (HHHHHH-COOH). pcDNA3.1-VP3 expresses the predicted ORF 5 protein as a fusion protein with the Cterminal V5 epitope (derived from the paramyxovirus P and V proteins; SV5, GKPIPNPLLGLDST) and the 6 £ His-tag. P CMV, CMV promoter; MCS, multiple cloning site; SV40ori, SV40 promoter and origin; bsd R , Blasticidin resistance gene; neo R , Neomycin resistance gene; amp R , Ampicillin resistance gene. transcriptase (PERKIN ELMER, New Jersey, USA) according to the manufacturer's instructions. For ORF 5 cDNA amplification, the forward primer VP3F 5 0 -AAATTATGG GGGACGGTTATAACCTTGGTTTTGGCC-3 0 corresponding to nt 1 -31 of LDV ORF 5 with a Kozak consensus sequence (underlined) and reverse primer VP3R 5 0 -TTTCTTATCGTCATCGTCGGCCTCCCATTTTTCGGC-3 0 which was complementary to nt 625 -642 of ORF 5 with enterokinase recognition site (underlined) were utilized. The RT-PCR product was cloned into cDNA3.1/V5-His-TOPO (Invitrogen, NV Leek, The Netherlands) to yield pcDNA3.1-VP3. This expression vector has a single, overhanging 3 0 deoxythymidine (T) residue and a neomycin resistance cassette. ORF 5 cDNA may be expressed as a C-terminal fusion to the V5 epitope derived from the P and V proteins of the paramyxovirus, SV5, and the polyhistidine metal-binding tag (6 £ His-tag).

Plasmid pcDNA6-VP2 construction for LDV ORF 6 expression was carried out with the expression vector pcDNA6/Myc-His (Invitrogen), which contains the blasticidin resistance gene. The LDV-C ORF 6 coding region plus a Kozak consensus sequence was derived from infected sera by RT-PCR, as described above. For amplification of ORF 6 cDNA, the forward primer VP2F 5 0 -CGGGATCCATTATGGGAGGCCTAGAATTTTG-3 0 corresponding to nt 1-20 of LDV ORF 6 with a BamHI site (underlined) and a Kozak consensus sequence (double underlined) and reverse VP2R 5 0 -CGGGATCCTTTTGAGACATATTT CAAAA-3 0 which was complementary to nt 494-513 of ORF 6 with a BamHI recognition site (underlined) were utilized. The amplified ORF 6 cDNA was inserted into the pcDNA6/Myc-His BamHI sites. The inserted ORF 6 cDNA may be expressed with a C-terminal peptide encoding the Myc epitope derived from c-Myc and the 6 £ His-tag.

Cos7 cells stably expressing LDV envelope proteins were generated by cotransfection of linearized plasmids pcDNA-VP2 and pcDNA-VP3, followed by subsequent selection with both 400 mg/ml Geneticin (G418; Gibco BRL, Rockville, MD, USA) and 5 mg/ml blasticidin S (Kaken Seiyaku Co., Ltd, Japan). Resistant colonies were typically visible within 2 -3 weeks under selection. Individual colonies were picked and amplified to confluence in 6-well plates for testing protein expression. At this stage, selection pressure was lifted, but conditioned medium described above was still utilized. Forty clones were screened for expression of the 6 £ His-tagged proteins by Western blotting, as described below. Clones expressing high levels of proteins were selected and expanded for further analysis.

For screening cell clones stably expressing LDV ORF 5 and ORF 6 proteins, the 40 individual clones expanded as described above were analyzed by Western blotting. Confluent cell monolayers collected from each clone were harvested at 72 h after passage, and the cells were lysed with lysis buffer composed of 8 M urea, 2% TritonX-100, 5% 2-mercaptoethanol, and EDTA-free protease inhibitor cocktail (Roche Diagnostics Co., Ltd, Germany). Lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Bedford, USA). Blots were reacted with a monoclonal antibody raised against 6 £ His-tagged proteins (diluted 1:1000, Qiagen) followed by incubation with HRP-conjugated sheep anti-mouse IgG (diluted 1:5000, Amersham Biosciences, Buckinghamshire, UK). Immunoreactions were visualized with the ECL Western blotting analysis system (Amersham Biosciences).

To analyze co-expression of LDV ORF 5 and ORF 6 proteins in the established clones, each expanded clone was harvested within five passages of the initial establishment and lysed as described above. Cell lysates were purified by histidine-tagging HiTrap chelating columns charged with Ni 2þ ions (Amersham Biosciences) according to the manufacturer's instructions with a few modifications. In brief, lysates were loaded onto columns equilibrated with start buffer composed of 20 mM phosphate, 0.5 M NaCl, and 8 M urea. Then, the loaded column was washed with start buffer. Although the manufacturer recommends using start buffer containing 10 mM imidazole to prevent nonspecific binding of cellular proteins, buffer without imidazole was used in this study because of the inhibition of specific binding and decreased recoveries of 6 £ His-tagged specific proteins in response to imidazole (data not shown). Bound protein was eluted with 20 mM phosphate buffer containing 0.5 M NaCl, 8 M urea, 0.5 M imidazole, and 50 mM EDTA.

Purified LDV virion protein prepared as previously described [24, 25] and expressed protein purified as described above were separated by SDS-PAGE on 12.5% polyacrylamide gels and electroblotted onto a PVDF membrane. Blots were reacted with rabbit antibody #36 (diluted 1:100) against LDV-C ORF 6 as reported previously [21] or monoclonal antibody no. 6 against LDV VP-3 [22] . The secondary antibody was HRPconjugated donkey anti-rabbit IgG (diluted 1:10000, Amersham Biosciences) or HRPconjugated sheep anti-mouse IgG (1:5000, Amersham Biosciences) as described above.

LDV ORF 5 protein extracted from established stable clones was analyzed for N-glycosylation by treatment with N-glycanase. In brief, affinity-purified protein extracted from each clone was desalted with PD-10 desalting columns (Amersham Biosciences) and concentrated. The protein was then diluted to a final volume of 50 ml with buffer containing 50 mM sodium phosphate (pH 6.7), 10 mM EDTA, 1% (v/v) Nonidet P-40, 1% (v/v) 2-mercaptoethanol, and protease inhibitor cocktail as described above. The protein was incubated with and without 50 U of PNGase F/ml (New England BioLabs, Beverly, USA) at 37 8C for 18 h and analyzed by Western blotting with monoclonal antibody no. 6 against LDV VP-3 as described above.

To examine the cellular localization of expressed ORF 5 and ORF 6 proteins in stable clones, immunofluorescence analysis was performed as described previously [22, 25] , with some modifications. In brief, cell clones within five passages of initial establishment were harvested at 72 h after passage and fixed for 10 min with 2% paraformaldehyde in phosphate-buffered saline (PBS) at 4 8C with or without permeabilization by Triton X-100 for intracellular or cell surface staining [26] . Permeabilized and intact cells were incubated with LDV-C ORF 6 peptide-specific rabbit antibody #36 (diluted 1:100) and monoclonal antibody no. 6 against LDV VP-3 (not diluted) at 4 8C overnight. After washing with PBS, the cells were incubated with tetra-methylrhodamine isothiocyanate (TRITC)-conjugated swine anti-rabbit IgG (H þ L) (1:10, DAKO Co. Ltd, Denmark), fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG (H þ L) (1:10, DAKO Co. Ltd), and 4 0 ,6diamino-2-phenyl-indole (DAPI; 2 mg/ml, Roche Diagnostics Co. Ltd) which stains DNA 1 h at room temperature. The cells were washed again with PBS, and mounted preparations were observed with an LSM510 laser-scanning confocal microscope equipped with an Axiovert-100M (Carl Zeiss, Oberkochen, Germany).

To establish cell lines stably expressing LDV ORF 5 and ORF 6 proteins, Cos7 cells were cotransfected with pcDNA-VP2 and pcDNA-VP3. Forty clones resistant to both G418 and blasticidin were selected and screened for 6 £ His-tagged protein expression. Four of the 40 clones expressed the 6 £ His-tagged protein, of which two were high-level expressers (clones 4-D and 4-H). These two clones were shown to stably express ORF 5 and 6 proteins, however, expression was reduced significantly by the 20th cell passage, even in the continuous presence of selective antibiotics (data not shown).

Because they express high levels of 6 £ His-tagged protein, clones 4-D and 4-H were used for analysis of LDV ORF 5 and ORF 6 protein expression. After extraction and purification of the expressed proteins in clones 4-D and 4-H by histidine-tagging HiTrap chelating columns as described in Section 2, the expression rate levels and molecular weight of each protein in the clones were determined by Western blotting. The peak levels of 6 £ His-tagged ORF 5 and ORF 6 protein expression were observed 72 h after passage (data not shown). As shown in Fig. 2 , purified His-tagged ORF 5 protein was detected as a cluster of bands approximately 30 -45 kD (lanes 4 and 6) . These bands comprise fulllength ORF 5 plus the V5 epitope and 6 £ His-tag sequences. In addition, 23 and 24.5 kD bands, derived from full-length ORF 6 plus, the c-myc epitope and 6 £ His-tag sequences, were also detected (Fig. 3 , lanes 5 and 6).

To examine whether expressed ORF 5 protein was glycosylated, the effects of N-glycanase were investigated. After N-glycanase digestion, the protein band shifted to 28 kD, which represents full-length ORF 5 plus the V5 epitope and 6 £ His-tag sequences (Fig. 2, lanes 5 and 7) . These results suggest that the size of unglycosylated ORF 5 protein is 28 kD and that the other protein bands were due to different degrees of glycosylation.

Indirect immunofluorescence analysis with permeabilization of clone 4-D revealed that 100% of the cells of the clone were fluorescence-positive, albeit at different relative intensities, whereas the parent Cos7 cells were negative except for nuclei of all cells stained with DAPI (Fig. 4) . LDV ORF 5 protein was distributed throughout the cytoplasm, showing characteristic localization around the perinuclear region and Golgi complex (Fig. 4, VP3 ). ORF 6 protein was also distributed throughout the cytoplasm (Fig. 4, VP2) . The nuclei were detected by DAPI staining (Fig. 4, DNA) . In addition, ORF 5 and ORF 6 proteins appeared to be colocalized in most cells (Fig. 4, merge) . The clone 4-H staining data was identical to that of clone 4-D (data not shown). 

To examine ORF 5 protein cell surface expression, clone 4-D was analyzed by immunofluorescence analysis without permeabilization. As shown in Fig. 5 , fluorescence was observed on the edge of unpermeabilized cells, suggesting that expressed ORF 5 protein is present on the cell surface. The clone 4-H staining data was identical to that of clone 4-D (data not shown). In contrast, ORF 6 protein cell surface expression was unverified because there are no antibodies that react with cell surface M/VP-2. In our previous study [21] , a rabbit antibody that reacted consistently with virion M/VP-2 in infected macrophages was generated, however, the antibody reacts only with the M/VP-2 cytoplasmic domain.

In this report, a mammalian expression system that expresses proteins that closely resemble the native LDV envelope proteins was described. Western blotting analysis combined with N-glycanase digestion revealed that the expressed ORF 5 protein was similar in size to native VP-3 and was N-glycosylated like native VP-3. This result is consistent with the native virion VP-3 size reported previously [12] . ORF 6 protein tagged 6 £ His residues was 23 and 24.5 kD, which is similar in size to native M/VP-2, but the reason for the two bands was unclear. It is possible that one band represents an ORF 6 plus 6 £ His-tag sequences degradation product. Moreover, Western blotting consistently yielded unidentifiable bands. These bands may comprise a 6 £ His-tagged specific protein cluster that was not relaxed sufficiently by 8 M urea treatment or a mammalian protein present due to incomplete purification.

Immunofluorescence analysis revealed that the localization pattern of expressed ORF 5 and ORF 6 proteins was similar to that of LDV VP-3 and M/VP-2 in infected macrophages [20, 21] . In addition, ORF 5 and ORF 6 proteins were colocalized in most cells. Because M/VP-2 appears to be associated with VP-3 in virions [14, 27] , colocalization of ORF 5 and ORF 6 proteins was expected. Therefore, an association of ORF 5 and ORF 6 proteins expressed in our established cell lines is necessary to be verified, however, we failed to detect the association by a co-immunoprecipitation assay using antibodies against LDV ORF 5 [22] and ORF 6 proteins [21] . Since the antibodies were unable to immunoprecipitate expressed ORF 5 and ORF 6 proteins solubilized in the lysis buffer described in Section 2, detailed experiments are required to select the suitable lysis buffer and condition for immunoprecipitation assay.

In general, the Western blotting and immunofluorescence analysis verified that the exogenously expressed forms of both ORF 5 and ORF 6 proteins were similar in size and form to the native virion proteins. We have established a mammalian expression system that expresses proteins closely resembling the native LDV envelope proteins. Application of this expression system will be useful for studies of the interaction between LDV envelope proteins and host cells.

Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses

Replication of lactate dehydrogenase-elevating virus in macrophages. I. Evidence for cytocidal replication

Morphological studies on equine arteritis virus

Morphogenesis of Lelystad virus in porcine lung alveolar macrophages

The coronavirus-like superfamily

Ultrastructural characteristics of porcine reproductive and respiratory syndrome virus propagated in the highly permissive MARC-145 cell clone

Intracellular synthesis, processing, and transport of proteins encoded by ORF 5 to 7 of porcine reproductive and respiratory syndrome virus

An infectious arterivirus cDNA clone: identification of a replicase point mutation which abolishes discontinuous mRNA transcription

Infectious transcripts from clones genome-length cDNA of porcine reproductive respiratory syndrome virus

Genetic manipulation of equine arteritis virus using full-length cDNA clones: separation of overlapping genes and expression of a foreign epitope

Structural proteins of equine arteritis virus

Structure and chemical -physical characteristics of lactate dehydrogenase-elevating virus and its RNA

The neutralization epitope of lactate dehydrogenase-elevating virus is located on the short ectodomain of the primary envelope glycoprotein

Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity

T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus

Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex

Characterization of regions in the GP5 protein of porcine reproductive and respiratory syndrome virus required to induce apoptotic cell death

The envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography

SV40-transformed simian cells support the replication of early SV40 mutants

Comparison of the ability of lactate dehydrogenase virus and its virion RNA to infect murine leukemia virus-infected or -uninfected cell lines

Production of virusspecific antiserum corresponding to sequences in the lactate dehydrogenase-elevating virus (LDV) ORF6 protein

Replication of lactate dehydrogenase-elevating virus in cells infected with murine leukaemia viruses in vitro

At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

Replication of lactate dehydrogenase-elevating virus in various species cell lines infected with dual-, ampho-and xenotropic murine leukaemia viruses in vitro

Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GP I anchor

Lactate dehydrogenase-elevating virus and related viruses

We thank Ms A. Mitsumoto and Ms M. Kaminishi for their excellent technical assistance and Mr R. Iritani and Ms A. Kinoshita for their help with the bleedings. Additionally, we are grateful to Dr Y. Shirakawa, Dr Y. Harada, and Mr A. Kawano for their helpful suggestions.