key: cord-344740-st2yw090
authors: Lee, Changhee; Bachand, Aimee; Murtaugh, Michael P.; Yoo, Dongwan
title: Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins
date: 2004-12-08
journal: Vet Immunol Immunopathol
DOI: 10.1016/j.vetimm.2004.09.020
sha: 
doc_id: 344740
cord_uid: st2yw090

The porcine reproductive and respiratory syndrome virus (PRRSV) GP4 and GP5 proteins are two membrane-associated viral glycoproteins that have been shown to induce neutralizing antibodies. In the present study, the host cell gene expression profiles altered by the GP4 and GP5 proteins were investigated by the use of DNA microarrays. Sublines of Marc-145 and HeLa cells were established by stable transfection with open reading frame (ORF)4 and ORF5 of PRRSV, respectively, and differential gene expressions were studied using microarray chips embedded with 1718 human-expressed sequence tags. The genes for protein degradation, protein synthesis and transport, and various other biochemical pathways were identified. No genes involved in the apoptosis pathway appeared to be regulated in GP5-expressing cells. The microarray data may provide insights into the specific cellular responses to the GP4 and GP5 proteins during PRRSV infection.

For viruses to replicate, they must enter a host cell and utilize host cell biosynthetic machinery and energy supplies. Infected cells activate innate and adaptive immune responses, and host antiviral defense is switched on to eliminate invading viruses. Viruses may persist in infected cells when antiviral defenses are insufficient. The series of interactive processes cause the differential expression of cellular genes, and it has been of interest to understand how altered gene expression plays a role during virus infection. RT-PCR, RNase protection assays, and Northern and Western blot analyses are commonly used techniques to identify altered gene expression. RT-PCR in particular, has been used to study porcine reproductive and respiratory syndrome virus (PRRSV)-mediated altered gene expression for IFN-a, IFN-g, IL-10, IL-12, and TNF-a (Johnsen et al., 2002; Feng et al., 2003; Suradhat and Thanawongnuwech, 2003; Thanawong-www.elsevier.com/locate/vetimm Veterinary Immunology and Immunopathology 102 (2004) 189-198 nuwech and Thacker, 2003) . Such techniques, however, are often time-consuming and labor-intensive and lead to high degrees of experimental variation. For such reasons, little is known concerning the molecular changes in cells upon PRRSV infection.

The recent development of DNA microarray technology allows for the simultaneous assessment of mRNA transcription patterns for thousands of genes, and is commonly applied to determine patterns of differential gene expression (Schena et al., 1995) . Using this technique, it is now possible to define changes in gene expression that evaluate host cellvirus interaction and to obtain specific insights into the molecular nature of viral pathogenesis (Browne et al., 2001; Johnston et al., 2001) . Microarrays are particularly useful in studying whether cellular mRNAs, differentially regulated by each viral protein, play a crucial role for virus multiplication in the cell.

The PRRSV-2 (North American) genome contains nine open reading frames (ORFs). ORF1a and ORF1b code for two partly overlapping non-structural polyproteins that are predicted to be post-translationally processed to 13 cleavage products. These nonstructural proteins are believed to participate in viral genome replication and subgenomic mRNA transcription. ORFs 2-7 code for six structural proteins: GP2-GP5, membrane (M), and nucleocapsid (N) proteins (Meulenberg et al., 1995; Snijder and Meulenberg, 1998) . A small internal ORF is found within ORF2, which encodes the E protein (Wu et al., 2001) .

The GP5 protein, consisting of 200 amino acids, is a major glycosylated structural component of the virion. It resembles a type I integral membrane protein with a putative endoplasmic reticulum (ER) translocational signal of 31 amino acids at its N-terminus. GP5 however lacks the typical C-terminal hydrophobic anchor sequence. Instead, a triple membrane spanning region is found in the middle of the protein between residues 65 and 130, leaving a large stretch of a 70 amino acid cytoplasmic tail at the C-terminus (Mardassi et al., 1995; Meulenberg et al., 1995) . GP5 exists as a heterodimer with the M protein in the virion (Mardassi et al., 1996) , and heterodimerization has been shown to be essential for virus infectivity in LDV (Faaberg et al., 1995) and EAV (Snijder et al., 2003) . The GP5 protein is reported to cause apoptosis (Suárez et al., 1996) and is able to induce neutralizing monoclonal antibodies in mice (Weiland et al., 1999; Ostrowski et al., 2002) .

The GP4 protein is a minor structural protein consisting of 178 amino acids (Murtaugh et al., 1995) , and as with GP5, is also able to induce neutralizing antibodies (Meulenberg et al., 1997; Weiland et al., 1999) . The electrophoretic migration of the mature protein incorporated into virions is 31-35 kDa, suggesting that the GP4 protein is heavily glycosylated during transport through the ER-Golgi complex (van Nieuwstadt et al., 1996) . Four potential N-glycosylation sites are found on the protein. The amino acid sequence shows that GP4 contains a putative Nterminal signal sequence at positions 1-22, and an additional hydrophobic sequence at positions 162-178 at the C-terminus. The hydrophobicity profile of GP4 suggests that it resembles a class I integral membrane protein. However, GP4 has a unique feature uncommonly seen in this class of proteins-the lack of a hydrophilic cytoplasmic tail on the carboxy terminus of the hydrophobic transmembrane region. This unique topology of GP4 is found to mimic a glycosylphosphatidylinositol (GPI) anchored protein (Ferguson and Williams, 1988) . Indeed, GP4 has been shown to be a GPI-anchored protein in our laboratory (Bachand, 2003) . The function of GPI anchors is poorly understood, but limited evidence suggests that they are involved in 'lipid rafts' (Varma and Mayor, 1998) or in cellular signal transduction (Jacobs et al., 2000) .

The present study was designed to examine regulation of specific host cell gene expression by two PRRSV structural proteins. We established two independent cell sublines to stably express the GP4 and GP5 proteins of the North American type PRRSV, and investigated their effect on host cell gene expression using DNA microarray technology.

Marc-145 cells (a subclone of MA 104 cells (Kim et al., 1993) ) were grown in DMEM containing 8% fetal bovine serum (Invitrogen), 50 units/ml of penicillin, and 50 mg/ml of streptomycin. HeLa-Tet-off cells were purchased from Clontech. These cells constitutively express the chimeric tetracycline transactivator (Gossen and Bujard, 1992) . HeLa-Tet-off cells and Marc-GP4 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum, 2 mM L-glutamine, 50 units/ml of penicillin, 50 mg/ml of streptomycin, and 100 mg/ml of G418 (Geneticin; Invitrogen). HeLa-GP5 cells were maintained in DMEM containing 10% serum, 2 mM L-glutamine, 50 units/ml of penicillin, 50 mg/ml of streptomycin, 100 mg/ml of G418, 1 mg/ml of doxycycline (Clontech), and 100 mg/ml of hygromycin B (Invitrogen). All cells were maintained at 37 8C with 5% CO 2 in a humidified incubator.

DNA was manipulated according to standard procedures (Sambrook and Russell, 2001) . The ORF4 gene of the North American type PRRSV strain ATCC VR2332 was PCR-amplified from the parental plasmid pGEM3zf-ORF4 (Wootton et al., 2000) and subcloned into the EcoRI and HindIII sites of the pCI-Neo mammalian expression vector (Promega) to generate pCI-Neo-ORF4. The GP5 expression plasmid pTRE-hyg-ORF5 was constructed by subcloning the VR2332 ORF5 gene from the parental plasmid pGEM3zf-ORF5 (Wootton et al., 2000) into pTRE-hyg (Clontech) using the BamHI site such that the ORF5 gene was placed under control of the tetracycline-responsive element along with the hygromycin-resistance gene.

Marc-145 cells were grown to approximately 75% confluence in a 35 mm diameter dish and then transfected for 24 h with 1.5 mg pCI-Neo-ORF4 DNA using Lipofectin (Invitrogen) according to the directions of the manufacturer. After 24 h, the transfection solution was replaced with DMEM and incubated for 12 h to allow the cells to divide at least once. Cells were then trypsinized and seeded into fresh 35 mm diameter dishes at approximately 6 Â 10 4 cells per dish. The pCI-Neo-ORF4 plasmid contains a gene conferring neomycin resistance and allows for the selection of cells that have integrated ORF4 into the cellular DNA. Freshly seeded cells were selected for neomycin resistance using 1 mg/ml of G418 (Invitrogen). Selection continued over the course of approximately 3 weeks, with G418 being replaced at least every 4 days. When the majority of cells died, resistant colonies of cells were picked using cell cloning cylinders and amplified for further characterization.

To generate cells expressing GP5, the Tet-off inducible gene expression system was chosen to prevent cell death that might occur due to the GP5 protein expression since GP5 was reported to induce apoptosis. The Tet-off inducible cell system was purchased from Clontech. HeLa-Tet-off cells were transfected with pTRE-hyg-ORF5 for 24 h using Lipofectin according to the manufacturer's instruction (Invitrogen). The transfection solution was removed and the cells were grown for additional 24 h in DMEM. For the transfected HeLa-Tet-off cells, the medium contained 1 mg/ml of doxycycline (Clontech). At 24 h of incubation, 300 mg/ml of hygromycin B was added, and cells were further incubated for 2 weeks until the majority of cells died. Hygromycin-resistant cell colonies were picked using cell-cloning cylinders and amplified in 24-well tissue culture plates.

Cells were grown on microscope slide coverslips placed in 35 mm-diameter culture dishes in the maintaining medium. After 12 h, doxycycline was removed for 24 or 48 h to induce GP5 expression in GP5 expressing cells. Cells were washed twice in phosphate-buffered saline (PBS) and fixed immediately with cold methanol for 10 min. For immunofluorescence, cells on microscope coverslips were blocked using 1% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cells were then incubated with a 1:50 dilution of porcine anti-PRRSV hyperimmune sera for 2 h. The cells were washed five times in PBS and incubated for 1 h at room temperature with a 1:100 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-swine secondary antibody (KPL). Cells were washed five times in PBS and the coverslips were mounted on microscope glass slides in the mounting buffer (60% glycerol and 0.1% sodium azide in PBS). Cell staining was visualized by a fluorescent microscope (model AX70, Olympus).

Marc-GP4 and HeLa-GP5 cells were seeded in 100 mm-diameter cell culture dishes. HeLa-GP5 cells were grown in the presence of 1 mg/ml doxycycline. To induce GP5 protein expression, doxycycline was removed and incubation was continued. At 48 h post-seeding or 48 h of induction, cells were starved for 30 min in methionine-deficient medium (Invitrogen) and labeled for 5 h with 100 mCi/ml of EasyTag EXPRESS protein labeling mix ([ 35 S]methionine and [ 35 S]cysteine, specific activity, 407 MBq/ml) (Perkin-Elmer). After labeling, cells were harvested, washed twice with cold PBS, and lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40) containing 1 mM phenylmethyl-sulfonyl fluoride (PMSF). After incubation on ice for 20 min, cell lysates were centrifuged at 14,000 rpm for 30 min in a microcentrifuge (model 5415, Eppendorf), and supernatants were recovered. For immunoprecipitation, cell lysates equivalent to 1:15 of a 100 mm diameter dish were adjusted with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 10 mM EDTA, 0.1% SDS) to a final volume of 100 ml and incubated for 2 h at RT with 5 ml of swine anti-PRRSV hyperimmune serum. The immune complexes were adsorbed to 7 mg of protein-A Sepharose CL-4B beads (Amersham Biosciences) for 16 h at 4 8C. The beads were collected by centrifugation at 6000 rpm for 5 min, washed twice with RIPA buffer and once with wash buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl). The beads were resuspended in 20 ml of SDS-PAGE sample buffer (10 mM Tris-HCl [pH 6.8], 25% glycerol, 10% SDS, 0.12% (w/v) bromophenol blue) with 10% b-mercaptoethanol, boiled for 5 min, and analyzed by sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE). Gels were dried on filter paper and radiographic images were obtained using Phosphor-Imager (Molecular Dynamics PhosphorImager SI, Amersham Biosciences).

The DNA microarray used in this study was comprised of 1718 human expression sequence tag (EST) clones printed at the Microarray Centre (Toronto, Ont., Canada). The genes were arrayed in duplicate on one slide. Detailed information on the layout of the microarray is found at the Microarray Centre (http:// www.microarrays.ca/support/glists.html). At 48 h postinduction, total cellular RNAs were extracted from each line of established cells, Marc-GP4 and HeLa-GP5, and from their corresponding parental cells, Marc-145 and HeLa-Tet-off, respectively. Complementary DNAs were synthesized by reverse transcription using 500 units of SuperScript II (Invitrogen) in a total reaction volume of 50 ml. Briefly, 10 mg of total RNA was primed with the AncT primer (Sigma Genosys; 5 0 T(20)-V-N 3 0 ) and reverse transcription was carried out in the presence of 0.5 mM each of dATP, dCTP, and dGTP (Invitrogen), 0.15 mM dTTP, amino-allyl 0.15 mM dUTP, 10 mM DTT in 10 firststrand synthesis buffer (Invitrogen; 250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl 2 ). The mixture was heated at 65 8C for 5 min followed by 42 8C for 5 min. SuperScript II was added, and the reverse transcription reaction was further incubated at 42 8C for 2 h. The reaction was stopped by heating to 95 8C for 5 min, and the RNA template was degraded by the addition 10 ml of 1N NaOH followed by incubation at 65 8C for 5 min. The mixture was neutralized by the addition 10 ml of 1 M HCl and 5 ml of 1 M Tris-HCl (pH 7.5). The cDNA was purified using Microcon columns (Millipore) and labeled with Alexa dyes (Molecular Probes) at room temperature for 1 h in darkness. For each microarray, control cDNA from parental cell lines was labeled with Alexa 546 (Cy3 equivalent), whereas cDNA from Marc-GP4 or HeLa-GP5 cells was labeled with Alexa 647 (Cy5 equivalent). The fluorescent labeled cDNAs were purified again with the Qiaquick PCR purification kit (Qiagen) and precipitated by adding one volume of isopropanol and incubating on ice for 40 min. After rinsing with 70% ethanol, the labeled cDNA was resuspended in 2.5 ml of DNase-free, RNase-free water (Invitrogen). For hybridization, 40 ml of calf thymus DNA (10 mg/ml) was added to 80 ml of DIG Easy hybridization buffer (Roche) and the solution was heated at 65 8C for 2 min. Two 2.5 ml samples of labeled cDNA were combined with 30 ml of the hybridization solution as prepared above. This solution was then incubated at 65 8C for 3 min and pipetted onto the chip. The array chip was then covered with a 24 mm  30 mm coverslip and incubated at 37 8C for 16 h. The slides were washed three times in 1  SSC containing 0.1% SDS for 15 min at 50 8C, rinsed twice in 0.1  SSC for 5 min each at room temperature, and dried. Array chips were scanned on a GenPix 4000A scanner (Axon Instruments Inc., Union City, CA). The normalization of raw data and the analysis of the data sets were performed using GeneTraffic microarray data analysis software (Iobion Informatics, La Jolla, CA). The log 2 R/G (where R and G represent Cy5 and Cy3, respectively) normalized ratio was selected as the value, which was further calculated to a fold change in regulation.

To examine the effects of the PRRSV GP4 or GP5 protein on cell function, cells were first established to express the GP4 or GP5 proteins. Marc-145 cells were chosen to establish a cell line stably expressing the GP4 protein since they are cells susceptible to infection by PRRSV. For GP5 expression, we used the tetracycline-dependent inducible gene expression system (Tet-on/Tet-off) to avoid a possible cell death that may result from GP5 expression since the PRRSV GP5 protein was reported to induce cytotoxicity in African green monkey kidney cells (Suárez et al., 1996) . HeLa-Tet-off cells, that were previously transformed with the regulatory plasmid pTet-off and therefore expressing the tetracycline transactivator (tTA), were chosen for additional transformation with the ORF5 gene. The presence of tetracycline (Tet) or its derivative doxycycline (Dox) in the culture medium prevents binding of tTA to the promoter, while the removal of Tet or Dox induces gene transcription placed under control of the promoter. Marc-145 and HeLa-Tet-off cells were transfected with pCI-Neo-ORF4 and pTRE-ORF5-hyg, respectively, and to confer resistance of transformed cells, neomycin (G418) or hygromycin were added. Transfected HeLa-Tet-off cells were maintained in the presence of doxycycline to prevent the GP5 expression during selection. Antibiotic-resistant cell clones were selected and individually confirmed for presence and transcription of the ORF4 or ORF5 gene by PCR and RT-PCR, and designated Marc-GP4 and HeLa-GP5, respectively.

Marc-GP4 and HeLa-GP5 cells were further examined for their respected protein expression by immunofluorescence ( Fig. 1B and C) . The cytoplasmic fluorescence was evident in these cells when incubated with PRRSV-specific hyperimmune sera, whereas no fluorescence was detected in parental Marc-145 and HeLa-Tet-off cells (data not shown), indicating that Marc-GP4 and HeLa-GP5 cells expressed the GP4 and GP5 proteins, respectively. Every cell expressed GP4 or GP5 indicating a homogenous population of cells. The GP4 and GP5 protein expressions were further confirmed by radioimmunoprecipitation (Fig. 1D) . A specific band of 31 kDa protein was identified in Marc-GP4 cells (lane 4). This band was absent in the parental Marc-145 cells (lane 3) and was considered the GP4 protein. We were not, however, able to detect the same protein from PRRSV-infected cells, and this is probably due to the low abundance of GP4 in PRRSV-infected cells as it is a minor protein. The GP5 protein was readily produced in HeLa-GP5 cells (lane 6), and its migration was similar to that of the authentic GP5 protein seen in PRRSV-infected cells (lane 1).

To investigate the effects of the PRRSV GP4 and GP5 proteins on cellular gene expressions, a microarray DNA chip technology was employed. Total cellular RNAs were extracted at 2 days post-induction and were reverse transcribed. The cDNAs were labeled using fluorescent dyes and used for hybridization of the human 1.7k microarray chip. This chip contained probes designed to detect 1718 human transcripts. The slides were scanned and the obtained images were analyzed using GeneTraffic microarray data analysis software. The log 2 Cy5/Cy3 normalized ratio 1.00, which corresponds to a two-fold change in regulation, was initially chosen as the cut-off value as used for standard and further calculated to a fold change. When a fold-change calculated by log 2 Cy5/ Cy3 was consistently 2.0 or greater in two independent experiments (four hybridizations), the value was considered significant.

Transcription of a total of 16 cellular mRNAs was altered by the GP4 protein expression in Marc-GP4 cells. Of these mRNAs, six genes were up-regulated and 10 genes were down-regulated (Table 1 ). The classifications of altered genes included those involved in cell adhesion, cell growth, replication, transcription, and protein degradation, and other unknown functional pathways. Seven genes associated with synthesis, transport, and biochemical pathway were also identified.

Microarray analysis of HeLa-GP5 cells showed an expression profile of 20 cellular genes altered by the GP5 protein expression in HeLa cells, with an increase in transcription of 12 genes and a decrease of eight genes ( Table 2 ). The genes that were affected by the GP5 protein represented those involved in the immune response, cell adhesion and structure, signal transduction pathway, cell cycle and cancer signals, and other functions. Five additional genes involved in synthesis, replication, transcription, and protein degradation were found to be altered in GP5 expressing HeLa cells. Cellular genes involved in apopotis were expected to be regulated in HeLa-GP5, but none of these genes appeared to be regulated by GP5 expression, suggesting that the GP5 protein may be irrelevant to PRRSV apoptosis (see Section 4).

In this study, we performed a global analysis of the transcriptional response of cells to expression of two PRRSV proteins. We established cell lines stably expressing the GP4 or GP5 protein, both protein genes under the control of human cytomegalovirus (hCMV) immediate early promoter, and experiments were designed to assess patterns of gene regulation in these cells. In GP5-expressing HeLa cells, actin-related protein 1 (ARP1) homologs A and B were identified to be up-regulated. These genes encode a subunit of dynactin that binds to both microtubules and cytoplasmic dynein, and is involved in ER-to-Golgi transport (Lees-Miller et al., 1992) . This may implicate an important function of GP5 in the transport of the viral components. The bisphosphoglycerate mutase (BPGM) gene was down-regulated by GP5 by more than six-folds. BPGM is an erythrocyte specific trifunctional enzyme regulating the level of 2,3-BPG in red blood cells. 2,3-BPG is the main allosteric effector of hemoglobin, shifting the equilibrium between the oxy and deoxy conformation of hemoglobins by stabilizing the unliganded form.

Sickle cell anemia in humans is characterized by polymerization of deoxygenated hemoglobin mutants giving rise to deformed erythrocytes and vasoocclusive complications. 2,3-BPG has been shown to facilitate this polymerization in sickle cell anemia. In humans, deficiency of BPGM has been shown to be associated with anemia (Jacobasch and Rapoport, 1996) . RASSF2 was also down-regulated by GP5. RASSF2 is a new member of the RASSF1 family and shares the properties of Ras effector/tumor suppressors (Vos et al., 2003) . Similarly, cyclin D3 gene expression was found to be suppressed. D-type cyclins are the key regulators along with cyclin E for cell cycle progression from G1 to S phase. Complexes formed between cyclin D or cyclin E and their kinase partners are involved in phosphorylation of retinoblastoma protein, which ultimately leads to activation of E2F transcription factor and progression to S phase of the cell cycle. A recent study demonstrates a clear reduction of cyclin D3 and cell cycle arrest in G0/G1 phase in cells infected with mouse hepatitis coronavirus, a member of nidoviruses (Chen and Makino, 2004) . In cell expressing GP4, the majority of differentially expressed genes were involved in synthesis, transport, and biochemical pathways. This observation implicates that the GP4 protein may utilize or change host cell machinery to transport viral or cellular components to the cell surface. Zinc finger protein 36 (zfp36) and microsomal triglyceride transfer protein were readily up-regulated, while metallothineins, proliferating cell nuclear protein, and N-acylsphingosine amidohydrolase were down-regulated. Zinc finger protein 36-like 1 is a member of the tristetraprolin family of tandem CCCH finger proteins. Tristetraprolin can bind to AU-rich elements within the 3 0 -untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation (Stumpo et al., 2004) . Tristetraprolin-knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion (Taylor et al., 1996) . Microsomal triglyceride transfer protein is a protein complex required for the assembly of lipoprotein particles (Gordon et al., 1995) . It is noteworthy that GP4 has recently been shown to be a lipid-anchored protein (Bachand, 2003) . Metallothionein is a metal binding protein and has been shown to be regulated by a common virus infection (Ilback et al., 2004) . Coxsackievirus Btype 3 virus infection altered the normal physiological trace element balance in the liver, kidney, spleen, and increased metallothionein in these organs. This may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range. The function of N-acylsphingosine amidohydrolase is unclear.

We have observed consistent increases in the expression of cathepsin genes in both Marc-GP4 and HeLa-GP5 cells. Cathepsin is involved in protein degradation. Despite a specific role of cathepsin during virus replication remains to be determined, the up-regulated expression of the gene encoding protease may represent a cellular defense against expression of foreign proteins.

Interestingly, no pro-apoptotic genes were identified in HeLa-GP5 cells. This observation is contradictory to the previous report (Suárez et al., 1996) but is consistent with our recent finding that the GP5 expressing cells did not show any detectable level of cytotoxicity or cell death (Lee et al., 2004) . Zhang et al. (1999) have shown that PRRSV infection induced the expression of IFN-inducible gene Mx1 and an ubiquitin-specific protease in porcine alveolar macrophages. These genes were not identifiable in the present study. This difference may be due to the use of different cell types since Marc-145 or HeLa cells were used in the present study to express the single GP4 or GP5 protein rather than using the whole virus to infect porcine macrophages in the previous report.

The DNA microarray has allowed us to identify the differential effects of PRRSV proteins on cellular genes. Confirmatory studies are further required as to the significance of the genes that have been identified in the present study. The method of choice to measure and confirm the differential mRNA expression mediated by the GP4 and GP5 proteins is real-time quantitative RT-PCR. It is also possible that altered mRNA profiles may not necessarily reflect altered production of corresponding proteins (Gygi et al., 1999) . In this regard, additional techniques such as Western blot analysis or protein arrays may be needed to support our findings. Nevertheless, our data obtained from the microarray study will provide future insights into the understanding of host cell virus interactions and eventually of the pathogenic mechanisms of PRRSV and the host responses to PRRSV infection.

Characterization of PRRSV GP4

Altered cellular mRNA levels in human cytomegalovirus-infected fibroblasts: viral block to the accumulation of antiviral mRNAs

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

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

Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus

Cell surface anchoring of proteins via glycosyl-phosphatidylinositol structures

Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters

Correlation between protein and mRNA abundance in yeast

Metallothionein is induced and trace element balance changed in target organs of a common viral infection

Hemolytic anemias due to erythrocyte enzyme deficiencies

Dengue virus non-structural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction

Cytokine mRNA profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of IFN-g and IL-10 after viral clearance

Identification of genes involved in the host response to neurovirulent alphavirus infection

Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line

Characterization of the porcine reproductive and respiratory syndrome virus GP5 in stably expressing cells

A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility

Molecular analysis of the ORFs 3-7 of porcine reproductive and respiratory syndrome virus, Quebec reference strain

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

Characterization of proteins encoded by ORFs 2-7 of Lelystad virus

Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus

Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus

Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain

Molecular Cloning: A Laboratory Manual

Quantitative monitoring of gene expression patterns with complementary DNA microarray

The molecular biology of arteriviruses

Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity

Chorioallantoic fusion defects and embryonic lethality resulting from disruption of Zxp36L1, a gene encoding CCCH tandom zinc finger protein of the tristetraprol family

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virusinduced apoptosis

Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus

A pathogenic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin

Interleukin-10, interleukin-12, and interferon-g levels in the respiratory tract following mycoplasma hyopneumoniae and PRRSV infection in pigs

Proteins encoded by ORFs 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

GPI-anchored proteins are organized in submicron domains at the cell surface

RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4

Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate

A 10 kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b

Molecular responses of macrophages to porcine reproductive and respiratory syndrome virus infection

The authors are grateful to Ryan Dowling for his technical assistance for microarray analysis. This study was supported by NSERC, Ontario Pork, the Ontario Ministry of Agriculture and Food (Animal Program).