Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world [1, 2].

A preponderance of evidence has recently emerged to indicate that G6PD deficiency affects cells other than that of erythrocytes [3-13]. Mouse embryonic stem cells with disrupted G6PD gene are extremely sensitive to H2O2 and to the sulfhydryl group-oxidizing agent diamide [14]. In addition, there appears to be a strong somatic cell selection against G6PD-null cells, suggesting an important role of G6PD in the development and/or survival of oocytes [14]. We have shown that G6PD-deficient human foreskin fibroblasts (HFF) underwent growth retardation and accelerated cellular senescence during their serial cultivation [5]. Moreover, we have also shown that sodium nitroprusside (SNP), a nitric oxide donor, stimulated growth of normal HFF but induced apoptosis in G6PD-deficient HFF [4]. These findings indicate that G6PD-deficiency renders cellular redox status abnormal thus affecting cells besides red cells. However, how G6PD-deficiency may affect viral infectivity has not been thoroughly studied. Oxidative stress has been found to affect viral proliferation and virulence [15-22]. Consumption of high iron diet resulted in elevated oxidative stress and co-elevation of coxsackie B3 virus titers in mice [20]. Increase in oxidative stress and inadequate antioxidant response were also related to the severity of liver damage and replication

status of virus in hepatitis B virus infection [17, 19]. Moreover, selenium- and vitamin-E deficiency converted benign strain of coxsackie B3 virus to virulent strain and caused myocarditis due to oxidative-induced mutations in the viral genome [20, 22, 23]. Similar to what was found for coxsackie B3 virus, host deficiency in selenium led to an increase in influenza virus mutation and resulted in a more virulent phenotype [21]. Although accumulating evidence suggests that cellular redox status plays an important role in affecting the pathogenicity of influenza virus and coxsackie B3 virus, how the cellular redox status may affect the proliferation and pathogenecity of other virus besides these two types of virus remains largely undefined. How oxidative stress may affect viral infection to airway cells has not been clearly defined. In addition to a large intracellular sources of oxidants including mitochondrial electron transport system, cytochrome P450 reactions and the nitric oxide synthase system, pulmonary cells are exposed to approximately 8000 liters of oxygen rich air per day as well as toxic particles such as ozone and other oxidants [24]. Recent studies indicate that diesel exhaust enhanced influenza virus infections in respiratory epithelial cells [25, 26]. Human coronavirus 229E (HCoV 229E), a common pathogen for respiratory tract infection, belongs to large, enveloped RNA virus in the order Nidovirales [27] and with high affinity toward airway cells [28, 29]. The genome is typical of coronaviruses containing genes for replicase, spike, small

envelope, membrane, and nucleocapsid in 5 to 3 directions [30]. For a long time these viruses are known to cause only relatively mild clinical problem such as common cold [31, 32]. Recent identification of a novel coronavirus as causative agent of Severe Acute Respiratory Syndrome (SARS) catches much attention [33, 34]. However, how oxidative stress can affect coronavirus has not been investigated. Therefore, the objective of the current study is to delineate whether oxidative stress can affect HCoV 229E infection toward cells by using G6PD-deficient cells as a model for cells with increased oxidative stress. In this study, we used HCoV 229E to infect G6PD-deficient fibroblasts and G6PD-knockdown A549 cells. Both G6PD-deficient and G6PD-knockdown cells exhibited enhanced susceptibility to virus-induced cell death. The enhanced virus-induced cell death was not caused by the increase in HCoV 229E receptor, CD13, but by the increase in viral gene expression and in viral particle production. Moreover, ectopic expression of G6PD in G6PD-deficient fibroblasts or antioxidant treatment could attenuate the increase in susceptibility to HCoV 229E infection. This result demonstrates that G6PD deficiency enhanced the HCoV 229E infection in an oxidative stress-dependent manner, and the phenomena could be modulated by altering the host redox status.

Results G6PD-deficient fibroblasts exhibited enhanced susceptibility to HCoV 229E-induced cell death G6PD deficiency is a common disease worldwide, but there is no information linking G6PD deficiency with viral pathology. To investigate whether G6PD deficiency could affect viral infection, G6PD-deficient fibroblasts (HFF1) and normal fibroblasts (HFF3) were subjected to HCoV 229E infection and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [35]. We found that 0.025 to 0.1 M.O.I. of virus to infect cells was a suitable concentration range of virus inoculum and also found that the cell viability of G6PD-deficient and normal fibroblasts was different under this concentration range after 72 h post-infection (Fig. 1A). Then we further compared the susceptibility of normal and G6PD-deficient fibroblasts to HCoV 229E at 0.025 to 0.1 M.O.I. range and at different time points of post-infection. We found that the cell viability was significantly (p< 0.05) lower (8%) in HFF1 than that in HFF3 at 0.1 M.O.I after 48 h post-infection (Fig. 1B), and the difference in cell viability expanded to 18% after 72 h post-infection when the cells were infected with 0.1 M.O.I. HCoV 229E (Fig. 1C). These data show that G6PD-deficient fibroblasts had increased susceptibility to HCoV 229E-induced cell death, especially at 72 h post-infection.

G6PD-knockdown A549 epithelial cells also showed enhanced susceptibility to HCoV 229E-induced cell death In order to investigate whether the enhanced susceptibility of G6PD-deficient fibroblasts to HCoV 229E infection was cell specific, G6PD-knockdown stable cells from human lung epithelial carcinoma cell line A549 were used. Three G6PD-knockdown stable cell lines, A549-5.8, A549-5.18 and A549-5.20, as well as a control cell line transfected with pCI-neo vector only, A549-5S-5, were selected. The G6PD activity of A549-5.8, A549-5.18 and A549-5.20 expressed only 18.6, 22.4 and 5.9% G6PD activity of the control cell line A549-5S-5, respectively (Fig. 2A). The decrease in G6PD expression was confirmed by western blot analysis of G6PD protein (Fig. 2B). Without virus infection, there was no difference for the growth rate among A549-5S-S, A549-5.8, A549-5.18 and A549-5.20 cells (data not shown). Then we determined the cell viability of G6PD-knockdown cells upon HCoV 229E infection. These G6PD-knockdown epithelial cells (A549-5.8, A549-5.18 and A549-5.20) showed significant increase in cell death induced by HCoV 229E infection as compared with control cells (A549-5S-5) at 24 h, 48 h and 72 h post-infection, respectively (Fig. 2C-E). These data demonstrate that enhanced susceptibility to HCoV 229E-induced cell death in G6PD-deficient fibroblasts was not cell-type specific, and the phenomena also could be found in G6PD-knockdown

epithelial cells. Enhanced virus-induced cell death could not be attributed to the increase in HCoV 229E receptor CD13 in G6PD-deficient cells Since HCoV 229E belongs to coronaviridae group I and needs receptors (CD13) to infect cells [36], we investigated whether HCoV 229E-induced cell death of G6PD-deficient cells could be attributed to the elevation of CD13 in these cells. Flow cytometry and western blot analysis revealed no significant difference in CD13 expression between G6PD-deficient and normal fibroblasts (Fig. 3A and 3B). Interestingly, G6PD-knockdown epithelial cells (A549-5.8, A549-5.18 & A549-5.20) expressed less CD13 on their cell surface than their control cells (A549-5S-5) (Fig. 3C and 3D), indicating that the enhanced susceptibility to HCoV 229E infection in G6PD-deficient fibroblasts and G6PD-knockdown epithelial cells could not be attributed to the increased expression of the viral receptor CD13. G6PD deficiency promoted HCoV 229E viral particle production and viral gene expression We next determined whether G6PD deficiency results in an increase production of viral particles in the following experiment. In this notion, viral particles were quantified by plaque assay at 24 and 48 h post-infection in G6PD-deficient and control cells. By using A549 plaque assay to determine the viral titer, the amount of

viral particles from G6PD-deficient fibroblasts (Fig. 4A) was found to be 3 fold more than that found in normal fibroblasts at 0.1 M.O.I. after 24 h post-infection (Fig. 4B). Interestingly, after 48 h post-infection, the difference in viral production between G6PD-deficient fibroblasts and normal fibroblasts was not as dramatic as that at 24 h post-infection, but the amount of viral particles from G6PD-deficient cells was still higher than their control ones. Similar findings were also observed in G6PD-knockdown A549 cells (Fig. 4C), and the amount of viral particles from G6PD-knockdown A549 were much higher than that from control A549-5S-5 both at 24 h and 48 h post-infection. To determine whether enhanced viral particle production in G6PD-deficient cells was caused by an increase in viral gene expression, RT-PCR technique was applied using HCoV 229E specific primers representing partial sequence of nucleocapsid. Since the actual nucleocapsid gene expression was not significantly different between G6PD-deficient and control cells at 2 h post-infection, the gene expression at 2 h post-infection was normalized to 1. In G6PD-deficient fibroblasts, viral gene expression at 4, 6, 8, 10 h post-infection was 2, 29, 470, 1601 fold higher than 2 h post-infection, respectively. However, in normal fibroblasts, viral gene expression at 4, 6, 8, 10 h post-infection was 1, 6, 47, 155 fold higher than 2 h post-infection, respectively (Table 1). Likewise, the viral gene expression was similar in A549 stable

clones with G6PD-knockdown (A549-5.8, A549-5.18 and A549-5.20) and showed much higher fold of increase than that in normal control (A549-5S-5) (Table 1). These data indicate that host cellular G6PD activity modulates viral gene expression. Ectopic expression of G6PD in fibroblasts ameliorated the enhanced susceptibility to HCoV 229E infection To further confirm that enhanced susceptibility to HCoV 229E-induced cell death as well as enhanced cellular viral production was modulated by cellular G6PD activity, G6PD-deficient cells (HFF1) were infected with G6PD-expressing retroviral vector, LGIN and LKGIN [5], and these cells were used to test the effects of G6PD replenishment on HCoV 229E-induced cell death and viral gene expression. LGIN and LKGIN expressed 11.1 and 12.8 fold more G6PD activity than their control LEIN (Fig. 5A), and the increase in G6PD activity was also confirmed by western blot data (Fig. 5B). It should be pointed out that the cell doubling time was not significantly different for the passages of LEIN, LGIN, and LKGIN chosen for the subsequent experiments. The expression of CD13 in LGIN and LKGIN was not significantly different from the control LEIN (Fig. 5B). However, when the susceptibility of these cells to HCoV 229E-induced cell death was compared, LEIN showed significantly decreased viability comparing to LGIN and LKGIN at 72 h post-infection (Fig. 5C). The viral nucleocapsid gene expression of HCoV 229E in LEIN, LGIN and LKGIN

were also determined by quantitative RT-PCR. G6PD-overexpressing fibroblasts (LGIN and LKGIN) showed significantly decreased viral gene expression comparing to their control, LEIN (Fig. 5D). Taken together, these data provide strong support to the notion that cellular G6PD activity modulated cellular susceptibility to viral infection. G6PD-knockdown epithelial cells suffered elevated oxidative stress after virus post-infection Since G6PD-deficient cells favored viral replication, the redox status of G6PD-deficient cell was determined by testing cellular ROS level using flow cytometric technique in combination with DCF staining and by quantifying cellular NADPH/NADP+, intracellular GSH level using HPLC method. In the basal condition, G6PD deficient fibroblasts (HFF1) and G6PD-knockdown epithelial cells (A549-5.8) had lower NADPH/NADP+ ratio and intracellular GSH level than that in control cells (HFF3 and A549-5S-5) (Table 2). All these data confirmed that G6PD-knockdown cells had less reducing power than their control ones. Concommitant with the decrease in cellular reducing power, G6PD-knockdown epithelial cells showed significant higher production of ROS by DCF-staining (Fig. 6) than that in their control upon viral infection. Taken together, the diminishment in reducing power and enhanced ROS production were indicative that G6PD-knockdown epithelial cells

exhibited higher oxidative stress than the control cells after viral infection. Antioxidant had protective effect against viral infection We then evaluated whether ectopic application of antioxidant provides a protective effect against virus infection in G6PD-knockdown cells. Toward this end, the antioxidant, -lipoic acid, was applied in culture medium for 5 h before virus infection. A549 cells pre-treated with antioxidant were significantly less susceptible to virus-induced cell death than control cells after 48 h post-infection at 0.1 M.O.I. (Fig. 7A). ROS production of these epithelial cells was determined and the cells that pretreated with antioxidant produced less ROS following virus infection than cells without antioxidant pretreatment (Fig. 7B). In addition, viral gene (nucleocapsid) expression in G6PD-knockdown cells that treated with or without antioxidant was determined by Q-PCR. After using 0.1 M.O.I. virus to infect cells, the viral gene expression in G6PD-knockdown cells pretreated with antioxidant was lower than that in cells without antioxidant pretreatment (Fig. 7C). Together, these data support the notion that the susceptibility to HCoV 229E infection was associated with the cellular redox status.

Discussion A preponderance of evidence has indicated that viral infection increases oxidative stress in host cells [37-40]. However, only limited information is available concerning how the redox status can affect viral behavior in the host cells [15, 41]. For example, in selenium- and vitamin E-deficient mice the virulent of coxsackie B3 virus has been changed causing myocarditis [18]. Our current study, using G6PD-deficient cells as a model system, clearly indicates that increased oxidative stress renders G6PD-deficient cells more susceptible to viral infection than their controls (Table 1 and Fig 6). Such abnormality can be ameliorated by the antioxidant agent such as lipoic acid. These data provide additional support to the notion that the redox status of the host plays an important role to affect viral infectivity. Increased viral infection in G6PD-deficient cells could be, in part, attributed to increase viral receptor in these cells or due to the enhanced production of viral particles inside these cells. HCoV 229E belongs to group I coronavirus and infects human cells through receptor aminopeptidase N (CD13) [36, 42]. Since G6PD-deficient cells do not express higher CD13 on their cell surface than their control as demonstrated by flow cytometry and western blot (Fig. 3), one can rule out the possibility that enhanced susceptibility to HCoV 229E infection of

G6PD-deficient cells is due to an increase in human receptor CD13 on these cells. On

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the other hand, enhanced production of viral particles in G6PD-deficient cells was clearly supported by increased plaque formation (Fig. 4) and by elevated viral gene (nucleocapsid) expression (Table 1) following virus infection. Thus, G6PD-deficiency provides a more suitable milieu for viral replication than that provided by non-G6PD-deficient cells. One condition which favors viral replication is high oxidative stress. G6PD-knockdown cells produce more ROS than normal counterparts (Fig. 6) and have lower cellular GSH content (Table 2) than their control cells during viral infection. The low GSH content in G6PD-knockdown cells has been related to low NADPH to NADP+ ratio in these cells (Table 2). Since these changes in redox status of G6PD-knockdown cells are accompanied by an increase in viral nucleocapsid gene expression in these cells (Table 1), these findings are consistent with the postulate that increased oxidative stress in G6PD-knockdown cells promotes viral gene expression. Increasing evidence shows that ROS play important roles in regulating signal transduction and controls cellular physiology [43-45]. It has also been shown that virus infection could up-regulate promoter, like NF-B, or transcription factor, like STAT3, to influence cellular gene expression as a consequence of increase in oxidative stress [46-48]. Altering redox status can also affect proinflammatory cytokines production [49, 50] and thus influences the antiviral mechanism of

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G6PD-deficient cells. Since increased susceptibility of G6PD-knockdown cells to HCoV 229E infection has been correlated with the ROS production, then antioxidants pretreatment should be useful to attenuate the phenomena. Indeed, when G6PD-knockdown cells are pretreated with lipoic acid, the enhanced susceptibility of these cells to virus infection can be attenuated (Fig. 7). Lipoic acid is synthesized by eukaryotic cells and is not considered as a vitamin. Lipoic acid and its reduced form dihydrolipoic acid are involved in defense against oxidative stress and apoptosis [51, 52]. Our finding that lipoic acid could diminish ROS production in G6PD-knockdown cells (Fig. 7) supports the postulate that oxidative stress contributes to the enhanced susceptibility of these cells to HCoV 229E infection. Moreover, this finding also suggests that antioxidant treatment may have particular health benefit to G6PD-deficient subjects against viral infection. All in all, our findings provide strong support to the notion that redox status of host cells modulates the infectivity of viral pathogen. Our findings also have major medical implication. Unpublished observation in our laboratory indicates that G6PD-deficient individuals are more prone to hepatitis viral infection. This unpublished observation together with the findings reported in the current article reveal that enhanced oxidative stress in G6PD-deficient individuals may increase their

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susceptibility to viral infection. Moreover, our findings also suggest that antioxidants intakes may be helpful to G6PD-deficient subjects against viral infection.

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Experimental procedures Reagents and antibodies Dulbeccos modified Eagles medium (DMEM), trypsin, penicillin, streptomycin and amphotericin B were purchased from Invitrogen (Carlsbad, CA, USA). The G6PD antibody was from Genesis Biotech (Taiwan). The anti-actin and anti-CD13 antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Lipofectamine 2000 (LF2000) transfection reagents and DCFH-DA were from Invitrogen (Carlsbad, CA, USA). Antibiotic G418 sulfate and -lipoic acid were from Promega (Madison, WI, USA). Cell culture Primary human foreskin fibroblasts prepared from G6PD-deficient individual (HFF1) and non-G6PD deficient individual (HFF3) as well as G6PD overexpressing fibroblasts (LGIN & LKGIN) were described previously [10]. Lung epithelial carcinoma cell line A549 was from American Type Culture Collection (ATCC). All cells were cultured in DMEM supplemented with 10% FCS, 100 units/ml of penicillin, 100 units/ml of streptomycin, and 0.25 mg/ml of amphotericin B at 37 in a humidified atmosphere of 5% CO2 with or without 300 g/ml G418 dependent on transfection or not. The cells were sub-cultured at a ratio of 1:8 before the cultures were confluent. Human fetal lung fibroblast (MRC-5) was purchased from ATCC

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(CCL-171) and maintained in minimum essential medium (MEM) supplemented with 10% FCS and antibiotics. Generation of G6PD-knockdown A549 cells by RNAi technique All possible G6PD-RNAi sequences were confirmed by transient transfection. RNAi expression vector pTOPO-U6 had the EcoRV and BbsI sites for insertion of RNAi sequences [53]. In addition, BglII and DraIII cloning sites were designed for release of the complete RNAi expression cassette and for insertion into pCI-neo [54] expression vector. For the plasmids G6PD143, the complementary oligonucleotides G6PD-143S (5- ACACACATATTCATCATCGAA GCTTGGATGATGAATATGTGTGT-3) and G6PD143AS (5-GGATACACACA TATTCATCATCCAAGCTTCGATGATGAATATGTGTGT-3), were annealed. The annealing of G6PD-143S/G6PD143AS generated sites corresponding to the blunt end and the overhang that matched the EcoRV- and BbsI- digested pTOPO-U6. The ligation between the annealed oligo-nucleotides and pTOPO-U6 at the EcoRV and BbsI cloning sites generated pTOPO G6PD-143. pTOPO G6PD-143 was tested by transient transfect into K562 to determine the protein expression of G6PD. Complete RNAi expression cassette was removed by digestion of BglII and DraIII and was inserted into pCI-neo mammalian expression vector for stable transfection as described previously. All cells that stably transfected with vector only (A549-5S-5) or
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G6PD-RNAi (A549-5.8, A549-5.18 & A549-5.20) were grown in 300 g of G418/ml. Infection with HCoV 229E Strain 229E of human coronavirus was kindly provided from Dr. Lai MM (Academia Sinica, Taiwan) and propagated in monolayer of MRC-5 cells and purified by centrifugation. The virus titer was determined by plaque assay [55]. Virus pools were aliquoted, quick frozen on dry ice, and stored at -70 until used. G6PD activity G6PD activity was measured at 340 nm by the reduction of NADP+ in the presence of glucose-6-phosphate as described [56]. In brief, cells were collected by centrifugation at 500 g at 4 for 10 min. Cell pellets were re-suspended in 1 ml of extraction buffer (20 mM Tris-HCl (pH 8.0), containing 3 mM MgCl2, 1 mM EDTA, 0.02% (w/v) -mercaptoethanol, 0.1% triton X-100 and 1 M -amino-n-caproic acid) then chilled immediately in an ice bath and disrupted by sonication. Cell lysate was centrifuged at 13,000 g at 4 for 15 min, and the supernatant was used for the assay. A typical assay mixture consisted of 50 g of protein in 1 ml of G6PD assay buffer (50 mM Tris-HCl pH 7.8, with 50 mM MgCl2, 4 mM G6P, and 4 mM NADP+). Change in absorbance at 340 nm was monitored spectrophotometrically. Virus infection and MTT assay For each MTT assay, 2 104 cells were seeded in a 12-well dish. Twenty-four

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hours later, the culture was subjected to HCoV 229E infection. Cells were infected with HCoV 229E at different M.O.I.. For quantification of the degree of cell death in cell culture, we employed the viability MTT assay. At different times after infection (24 h, 48 h & 72 h p.i.), 10% tetrazolium was added to the medium and incubated at 37 for 4 h. The reaction was terminated by dimethyl sulfoxide solution and the absorbance was determined at 490 nm and 650 nm in an ELISA microplate reader (Spectramax 340PC384; Molecular Devices). Cell viability was calculated as percentage of control cells using the formula: (A490-A650) of treated cells 100/ (A490-A650) of control cells. Western blot analysis Cells were harvested and solubilized for 30 min at 4 in 50 mM Tris-HCl pH 7.5 containing 1% Nonidet P (NP)-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 g of aprotinin/ml and 1 g of leupetin/ml. Cell extracts were subjected to SDS-PAGE. Gels were electroblotted onto polyvinylidene difluoride membrane in Towbin transfer buffer (25 mM Tris-HCl, 192 mM glycine and 20% methanol). Membranes were treated for 1 h in blocking buffer, probed with first antibody overnight, and washed twice, followed by incubation with secondary antibody for 1 h. After an additional washing step, immunoblots were visualized using the ECL detection system.

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Measurements of cell specific receptors for HCoV 229E Cell surface membrane receptors for CD13 were measured by flow cytometry as previously described [57]. In brief, cells were harvested and washed by FABS (1PBS, 1% FBS and 0.1% sodium azide), then probed with first antibody CD13 (1:50) on ice for 30 min and washed twice with FABS, followed by incubating with FITC-conjugated goat anti-mouse secondary antibody (dilution 1/1000) in the dark on ice for 30 min. Cells were washed twice with FABS and fluorescence was determined by flow cytometry (Becton Dickinson FACScan) followed by analysis with CELLQuest software. RNA isolation, RT and Q-PCR Total RNA was isolated from HCoV-infected cells by using RNeasy Mini Kit (Qiagene). RT-PCR was also performed with Superscript III reverse transcriptase (Invitrogen) and AmpliTaq Gold DNA polymerase (Applied Biosystems). To quantify the DNA fragment, two HCoV 229E specific oligonucleotide primers of nucleocapsid were used: 5 AGGCGCAAGAATTCAGAACCAGAG 3 and 5

AGCAGGACTCTGATTACGAGAAAG 3. To 1 l of the RT mixture the following was added: 25 l of 2 SYBR Green Mix buffer (ABI PCR master mix), 5 l of primer mixture (10 pmole each) and the total volume was adjusted to 50 l with water. The real-time quantitative polymerase chain reaction was carried out in a sequence

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detection system GeneAmp5700 (Applied Biosystems). The PCR conditions were optimized as follows: 95 for 10 min, and 40 cycles each of 95 for 30 s, 66 for 45 s, and 72 for 30 s. The intensity of fluorescence, which is a direct measurement of the amount of amplified product, is measured with each cycle. The threshold at which significant amplification of first detected is determined, and all samples are evaluated by determining how quickly each sample reaches this threshold. The cycle at which this threshold is achieved is recorded as the Ct value. Data were normalized with primers for a housekeeping gene, actin. A sample with no template was included to ensure the absence of primer dimmer. Plaque assay Lung carcinoma cell line A549 was used for plaque assay because HCoV 229E was propagated and could form cytopathic effect (CPE) in A549 cells as compared to MRC-5 cells. Ten-fold serial dilution of virus was made in DMEM without serum. 500 l of each dilution was used to infect A549 cells, grown in 6-well plate, for 1 h at 37. The virus inoculum was aspirated off and the monolayer was washed once with 1 ml of phosphate buffer saline. The monolayer was overlaid with 3 ml of the 1:9 mixture of 3% agarose and DMEM followed by incubation at 37 until plaques were developed. After 3 days post-infection, cells were stained with 0.5% crystal violet. Plaques were counted by direct observation and the titer of virus was calculated.

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Quantification of cellular NADPH and NADP+ A total of 1.25 107 cells were harvested and washed by 1PBS twice. To extract NADP+, 300 l cell pellet suspension was added to 100 l of 0.1 M HCl. While on ice, the cells were incubated for 1 min, and then neutralized with 100 l 0.1 M NaOH followed by the addition of 300 l Tris-HCl buffer (pH 6.8). The mixture was centrifuged at 3000 g for 10 min to remove insoluble material. For determination of NADPH, 300 l cell pellet was treated with 100 l 0.1 M NaOH then incubated on ice for 1 min. The suspension was neutralized with 100 l 0.1 M HCl and 300 l of 1 M Tris-HCl buffer (pH 11) was added. The mixture was centrifuged at 3000 g for 10 min to remove insoluble material. All these steps are done in the same day and followed by HPLC determination of NADP+ and NADPH. HPLC (Waters; model 2695) separation was carried out with a C18 3-m reversed-phase column. The mobile phase consisted of a gradient of buffer A (0.1 M KH2PO4, 5 mM tetrabutylammonium hydrogen sulfate, 2.5% (v/v) acetonitril, pH 6.0) and buffer B (0.1 M KH2PO4, 5 mM tetrabutylammonium hydrogen sulfate, 25% (v/v) acetonitril, pH 5.5). After injection of 20-50 l of sample, the column was initially eluted at 0.8 ml/min for 3 min with buffer A, followed by 2 min elution with buffer A containing 11% buffer B and finally a 25 min elution by a buffer gradient with buffer B gradually increased to 100%. Before the application of next sample, the column

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was re-equilibrated for 10 min with 100% buffer A. HPLC separations were performed at room temperature. Detection was done spectroscopically at 260 nm. The identities of peaks were confirmed by co-elution with standards. Quantitative measurements were made on the basis of the injection of standard solutions with known concentration. Intracellular GSH measurement Cells (4105) were acidified with 300 l of 1% (wt/vol) meta-phosphoric acid and centrifuged to precipitate the proteins. The supernatant was filtered and chromatographed on an Intersil 4.6 250 mm ODS3 column eluted with mobile phase containing 49 % of buffer A (10 mM NaH2PO4 and 0.3 mM octane sulfonic acid adjusted to pH 2.7 with phosphoric acid) and 51% of buffer B (10 mM NaH2PO4, 0.3 mM octane sulfonic acid, and 10% acetonitrile adjusted to pH 2.7 with phosphoric acid) and a flow rate of 0.8 ml/min. An ESA Coulochem II detector (Waters Alliance Systems, Milford, MA) was used for analysis. The guard cell was set at 950 mV, electrode 1 and 2 at 400 mV, electrode 3 at 650 mV, electrode 4 at 700 mV, electrode 5 at 800 mV, electrode 6 at 850 mV, electrode 7 at 900 mV and electrode 8 at 950 mV. Quantification was obtained by integration relative to the internal standard. Direct ROS determination by DCF staining ROS production was determined using 2,7-dichlorodihydrogluorescein diacetate

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(DDFH-DA; Invitrogen). After the cells were treated for 24 h with control media or virus inoculum, the cells were washed with PBS and then loaded for 30 min with DCFH-DA (20 M) in serum free medium. The acetoxymethyl group on DCF-DA is cleaved by nonspecific esterase within the cells, resulting in a nonfluorescent charged molecule that does not cross the cell membrane. Intracellular ROS irreversibly oxidize the DCFH-DA to dichlororluorescein (DCF), which is a fluorescent product. After treatment, the media was removed, and the cells were harvested and determined by flow cytometry (Becton Dickinson FACScan) and the data were analyzed with CELLQuest software. Statistical analysis Data are expressed as a mean SEM of several experiments. Statistical differences between the means of two groups were analyzed by the Students t test. A value of p < 0.05 was considered significant. Acknowledgements This project is supported by grants from Chang Gung University (CMRPD140041), from the National Science Council of Taiwan (NSC94-2320-B182-041), and by a grant from Ministry of Education (EMRPD150241). The technical support in RNAi plasmid construction from the RNAi core laboratory of Chang Gung University is appreciated.

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airway epithelia from the apical surface. J Virol 2000;74:9234-9 29. Chilvers MA, McKean M, Rutman A, Myint BS, Silverman M and O' Callaghan C. The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur Respir J 2001;18:965-70 30. Brian DA, Baric RS. Coronavirus genome structure and replication. Curr Top Microbiol Immunol 2005;287:1-30 31. Pene F, Merlat A, Vabret A, et al. Coronavirus 229E-related pneumonia in immunocompromised patients. Clin Infect Dis 2003;37:929-32 32. Chibo D, Birch C. Analysis of human coronavirus 229E spike and nucleoprotein genes demonstrates genetic drift between chronologically distinct strains. J Gen Virol 2006;87:1203-8 33. Yu CH, Liu le T, Liu S, et al. [Enhanced-real time PCR: A highly sensitive method for SARS-coronavirus detection.]. Beijing Da Xue Xue Bao 2006;38:211-3 34. Tan YJ, Lim SG and Hong W. Understanding the accessory viral proteins unique to the severe acute respiratory syndrome (SARS) coronavirus. Antiviral Res 2006;72:78-88 35. Massa EM, Farias RN. Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction. Biochim Biophys Acta 1983;746:209-15 36. Nomura R, Kiyota A, Suzaki E, et al. Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae. J Virol 2004;78:8701-8 37. Riva DA, de Molina MC, Rocchetta I, Gerhardt E, Coulombie FC and Mersich SE. Oxidative stress in vero cells infected with vesicular stomatitis virus. Intervirology 2006;49:294-8 38. Piccoli C, Scrima R, D' Aprile A, et al. Mitochondrial dysfunction in hepatitis C virus infection. Biochim Biophys Acta 2006;1757:1429-37 39. Wang T, Weinman SA. Causes and consequences of mitochondrial reactive oxygen species generation in hepatitis C. J Gastroenterol Hepatol 2006;21 Suppl 3:S34-7 40. Sezer S, Tutal E, Aldemir D, et al. Hepatitis C infection in hemodialysis patients: Protective against oxidative stress? Transplant Proc 2006;38:406-10 41. Ryan-Harshman M, Aldoori W. The relevance of selenium to immunity, cancer, and infectious/inflammatory diseases. Can J Diet Pract Res 2005;66:98-102 42. Wentworth DE, Tresnan DB, Turner BC, et al. Cells of human aminopeptidase N (CD13) transgenic mice are infected by human coronavirus-229E in vitro, but not in vivo. Virology 2005;335:185-97 43. Hasnain SE, Begum R, Ramaiah KV, et al. Host-pathogen interactions during apoptosis. J Biosci 2003;28:349-58 44. Kaimul Ahsan M, Nakamura H, Tanito M, Yamada K, Utsumi H and Yodoi J.
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Thioredoxin-1 suppresses lung injury and apoptosis induced by diesel exhaust particles (DEP) by scavenging reactive oxygen species and by inhibiting DEP-induced downregulation of Akt. Free Radic Biol Med 2005;39:1549-59 45. Liu T, Castro S, Brasier AR, Jamaluddin M, Garofalo RP and Casola A. Reactive oxygen species mediate virus-induced STAT activation: role of tyrosine phosphatases. J Biol Chem 2004;279:2461-9 46. Waris G, Livolsi A, Imbert V, Peyron JF and Siddiqui A. Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease. J Biol Chem 2003;278:40778-87 47. Agostini M, Di Marco B, Nocentini G and Delfino DV. Oxidative stress and apoptosis in immune diseases. Int J Immunopathol Pharmacol 2002;15:157-164 48. Waris G, Turkson J, Hassanein T and Siddiqui A. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol 2005;79:1569-80 49. Tse HM, Milton MJ, Schreiner S, Profozich JL, Trucco M and Piganelli JD. Disruption of innate-mediated proinflammatory cytokine and reactive oxygen species third signal leads to antigen-specific hyporesponsiveness. J Immunol 2007;178:908-17 50. Wu Y, Cui J, Bao X, et al. Triptolide attenuates oxidative stress, NF-kappaB activation and multiple cytokine gene expression in murine peritoneal macrophage. Int J Mol Med 2006;17:141-50 51. Rydkina E, Sahni SK, Santucci LA, Turpin LC, Baggs RB and Silverman DJ. Selective modulation of antioxidant enzyme activities in host tissues during Rickettsia conorii infection. Microb Pathog 2004;36:293-301 52. Biewenga G, de Jong J and Bast A. Lipoic acid favors thiolsulfinate formation after hypochlorous acid scavenging: a study with lipoic acid derivatives. Arch Biochem Biophys 1994;312:114-20 53. Tseng CP, Huang CL, Huang CH, et al. Disabled-2 small interfering RNA modulates cellular adhesive function and MAPK activity during megakaryocytic differentiation of K562 cells. FEBS Lett 2003;541:21-7 54. Huang CL, Cheng JC, Liao CH, et al. Disabled-2 is a negative regulator of integrin alpha(IIb)beta(3)-mediated fibrinogen adhesion and cell signaling. J Biol Chem 2004;279:42279-89 55. Kuo L, Masters PS. The small envelope protein E is not essential for murine coronavirus replication. J Virol 2003;77:4597-608 56. Chiu DT, Liu TZ. Free Radical and Oxidative Damage in Human Blood Cells. J Biomed Sci 1997;4:256-259
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Table 1. Increased viral gene (nucleocapsid) expression in G6PD-deficient fibroblasts (HFF1) and G6PD-knockdown cells (A549-5.8, A549-5.18 and A549-5.20) as compared to normal fibroblasts (HFF3) and vector-only controls (A549-5S-5) Post-infection /Viral gene expression (Fold) Cell HFF3 HFF1 A549-5S-5 A549-5.8 A549-5.18 A549-5.20 2h 1 1 1 1 1 1 4h 1.060.85 6h 6.340.81 8h 47.016.85 10 h 155.4243.44

1.560.06 3.060.54 13.714.56 11.825.45

28.843.27 9.663.09

467.8889.41

1601.27349.69* N. D. N. D. N. D. N. D.

410.9449.19

68.297.17* 3586.70642.819* 59.837.19* 3315.55210.65*

9.162.45 258.0174.29* 5300.41313.08*

Data are the means SEM, n=4. Number at 4, 6, 8 and 10 h post-infection were normalized to that at 2 h post-infection. * p <.05, G6PD-deficient or knockdown cells vs their control cells at the same post-infection hour. N. D. not determined.

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Table 2. NADPH/NADP+ ratio and intracellular GSH in G6PD-deficient fibroblasts, G6PD-knockdown epithelial cells, and their control cells with or without viral infection. Cells HFF3 HFF1 (normal fibroblast) (G6PD-deficient) [NADPH]/[NADP+] 1.32 0.32 0.59 0.13* 2.63 0.19 2.09 0.37* GSH at basal condition (mole/g of protein) 35.73 1.99 27.97 2.17* 85.68 2.84 70.71 3.84* GSH at 48h post-viral infection (mole/g of protein) N.D. N.D. 66.26 0.86 49.50 1.53*

A549-5S-5 (vector only control) A549-5.8 (G6PD-knockdown)

Pyridine nucleotide expressed as NADPH/NADP ratio and total intracellular GSH amount in cells measured by HPLC. Values are means SD, n = 5. * Significantly different from their control and p <.05. N. D. not determined.

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Figure Legends Fig. 1 Increased cell death in G6PD-deficient fibroblasts following HCoV 229E infection. Different M.O.I. of HCoV 229E was added to the same passage of fibroblast cells (PDL 12) and after 48 h (B) and 72 h (A and C) post-infection the cell viability was determined by MTT assay. All the cell viability was determined by the absorbance of MTT assay and expressed as percent of the respective HCoV 229E-infected nonexposed control. Data are the means SEM, n=4. *p <.05, HFF1 (G6PD-deficient fibroblasts) vs HFF3 (normal fibroblasts) at the same virus titer.

Fig. 2 Increased cell death in G6PD-knockdown A549 epithelial cells following HCoV 229E infection. The indicated A549 vector and G6PD-knockdown cells were harvested for G6PD activity assay (A) and western blot of G6PD protein (B). G6PD activity was given in IU/mg of protein in cell lysate. Cell viability of G6PD-knockdown A549 and their control with HCoV 229E virus infection at different M.O.I. for 24 (C), 48 (D) & 72h (E) was shown. Data are the means SEM, n=4. *p <.05; **p <.01, vector only A549-5S-5 vs G6PD-knockdown A549-5.8, A549-5.18 or A549-5.20 epithelial cells.

Fig. 3 Cell surface CD13 receptor expression in G6PD-deficient and normal

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fibroblasts, as well as in G6PD-knockdown epithelial A549 and their vector only control. The amount of surface receptor CD13 of G6PD-deficient fibroblasts (HFF1), normal fibroblasts (HFF3), G6PD-knockdown epithelial cell line (A549-5.8, A549-5.18 & A549-5.20) and their control cells (A549-5S-5) was determined by flow cytometry (A and C), and by western blot (B and D) as described in Experimental procedures.

Fig. 4 Elevated viral particle production in G6PD-deficient cells as indicated by plaque assay. 0.1 M.O.I. of viruses were used for infection and plaque assay was applied for measurement of virus titer. (A) Viral particle production as visualized by plaque formation was higher in G6PD-deficient fibroblasts (HFF1) at 24 h post-infection comparing to normal fibroblasts (HFF3). (B) Virus production was found to be higher in HFF1 than in HFF3, especially at 24h post-infection. (C) Similar data were found that viral particle production was higher in

G6PD-knockdown A549 (A549-5.8, A549-5.18 and A549-5.20) than in their control (A549-5S-5). Data are the means SEM, n=4. *p <.05; **p <.01, HFF1 vs HFF3, vector only A549-5S-5 vs G6PD-knockdown A549-5.8 or A549-5.18 or A549-5.20 epithelial cells at 0.1 M.O.I. of HCoV 229E.

31

Fig. 5 Amelioration of virus-induced cell death and viral gene expression by ectopic expression of G6PD. HFF1 cells were infected with control (LEIN), or G6PD-expressing retroviruses (LGIN & LKGIN). Expression of G6PD activity by activity assay (A), CD13 and G6PD protein (B) by western blot are shown. (C) Cell viability of LEIN, LGIN & LKGIN after infection with HCoV 229E at different M.O.I. for 72h was shown. (D) After 8h post-infection viral gene expression (nucleocapsid expression) as determined by Q-PCR is significantly decreasing in G6PD over-expressed cells (LGIN & LKGIN) comparing to their control (LEIN). Data are the means SEM, n=4. *p <.05; **p<.01, LGIN or LKGIN vs LEIN fibroblasts.

Fig. 6 ROS production of G6PD-knockdown epithelial cells (A549-5.8) and control cells (A549-5S-5) in basal condition and after virus-infection. A549 cells were loaded with 20 M DCF-DA for 30 min after exposure to 0 (basal condition) or 0.1 M.O.I. of HCoV 229E after 48 h post-infection. Fluorescence was measured using Flow cytometry.

Fig. 7 Protective effect of antioxidants against virus infection at 48 h post-infection in G6PD-knockdown cells pretreated with antioxidant 5 h before

32

viral infection. (A) A consistent protective effect by antioxidant lipoic acid (LA) against virus-induced cell death was observed. (B) ROS production following virus infection was attenuated by antioxidant (0.1 mM LA) pre-treatment. (C) Viral gene (nuclecapsid) expression of antioxidant-pretreatment cells and control cells was determined by Q-PCR after 2, 4, 6, 8 and 10 h post-infection with HCoV 229E infection.

33

A
100 Cell viability (% of uninfected control) H FF 1 P D L 1 2 (7 2 h p .i.) H FF 3 P D L 1 2 (7 2 h p .i.)

80

60

40

20 0 .0 0 .5 1.0 M .O .I. 1 .5 2 .0

B
100 Cell viability (% of uninfected control) 80 60 40 20

C
Cell viability (% of uninfected control)

100 80 60 40 20
HFF1 PDL12 (72 h p.i.) HFF3 PDL12 (72 h p.i.)

HFF1 PDL12 (48 h p.i.) HFF3 PDL12 (48 h p.i.)

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

Fig. 1

34

A
2.5 G6PD activity (IU/mg) 2.0 1.5 1.0 0.5 0.0

B
A549 -5S-5 A549 -5.8 A549 A549 -5.18 -5.20

G6PD ** ** **
Cell

Actin

A549-5S-5 A549-5.8 A59-5.18 A549-5.20

C
100 Cell viability (% of uninfected control) 80 60 40 20
A549-5S-5 A549-5.8 A549-5.18 A549-5.20

D *
24 h post-infection

Cell viability (% of uninfected control)

100 80 60 40 20

48 h post-infection
A549-5S-5 A549-5.8 A549-5.18 A549-5.20

**

**

**

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

E
100 Cell viability (% of uninfected control) 80 60 40 20

72 h post-infection
A549-5S-5 A549-5.8 A549-5.18 A549-5.20

**

**

**

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

Fig. 2

35

A HFF1 (G6PD-deficient) (PDL12)

B PDL 12 HFF1 HFF3 CD13 Actin

HFF3 (control) (PDL12)

C A549-5S-5 (control)

A549-5.8 (G6PD knockdown)

D
A549 -5S-5 A549 -5.8 A549 A549 -5.18 -5.20

CD13 Actin

A549-5.18 (G6PD knockdown)

A549-5.20 (G6PD knockdown)

Fig. 3

36

Plaque formation unit (PFU*105/ml)

HFF1

35 30 25 20 15 10 5 0

24 h postinfection 48 h postinfection

HFF3

HFF1 Cell

HFF3

60 Plaque formation unit (PFU*107/ml) 50 40 30 20 10 0

24 h postinfection 48 h postinfection
** **

**

A549-5S-5 A549-5.8 A549-5.18 A549-5.20

Cell
Fig. 4

37

A
1.0 G6PD activity (IU/mg) 0.8 0.6 0.4 0.2 0.0 LEIN LGIN Cell LKGIN

B ** ** CD13 G6PD Actin

LEIN LGIN LKGIN

C
100 Cell viability (% of uninfected control) 80 60 40 20

D
Relative viral gene expression

7000 6000 5000 4000 3000 2000 1000 0 0 2 4 6 8 10 Time (Post-infection, h)

** ** ** **

LEIN PDL40 (72 h p.i.) LGIN PDL40 (72 h p.i.) LKGIN PDL40 (72 h p.i.)

LEIN LGIN LKGIN

**

**

0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 M.O.I.

12

Fig. 5

38

A549-5S-5 Normal condition Virus infection Normal condition

A549-5.8

Virus infection

Fig 6

39

A
100 Cell viability (% of uninfected control) 90 80 70 60 50 40

A549-5S-5 w/o antioxidant with antioxidant

Control 0.01 mM LA 0.1 mM LA

A549-5.8 w/o antioxidant

A549-5S-5 A549-5.8 A549-5.18 A549-5.20

with antioxidant

Cells

C
8000

Relative viral gene expression

7000 6000 5000 4000 3000 2000 1000 0 0

A549-5S-5 A549-5.8 A549-5S-5 with LA pretreatment A549-5.8 with LA pretreatment

10

12

Tim e (Post-infection, hour)

Fig. 7

40