key: cord-1010659-xnvko0yc
authors: Kanzawa, Noriyuki; Nishigaki, Kazuo; Hayashi, Takaya; Ishii, Yuichi; Furukawa, Souichi; Niiro, Ayako; Yasui, Fumihiko; Kohara, Michinori; Morita, Kouichi; Matsushima, Kouji; Le, Mai Quynh; Masuda, Takao; Kannagi, Mari
title: Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-κB activation
date: 2006-12-22
journal: FEBS Lett
DOI: 10.1016/j.febslet.2006.11.046
sha: d65e23d9cd5d984bd31df5c4363d376dbcc527d9
doc_id: 1010659
cord_uid: xnvko0yc

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.

Severe acute respiratory syndrome (SARS) is characterized by dyspnea with rapidly progressing changes on radiography in the later stages of the illness [1, 2] . Pathological findings of the lungs in SARS resemble those in acute/adult respiratory distress syndrome (ARDS) associated with various clinical conditions [3] . It has been proposed that ARDS is the outcome of an uncontrolled inflammatory response and that nuclear factor kappa B (NF-jB) is a critical transcription factor involved in the pathogenesis of ARDS [4, 5] .

SARS coronavirus (SARS-CoV) genome contains open reading frames (ORFs) for several accessory proteins without sequence similarity to known coronavirus proteins. These include 3a (originally called X1 or ORF3), 3b (X2 or ORF4), 6 (X3 or ORF7), 7a (X4 or ORF8), and 8b (X5 or ORF11) [6] [7] [8] . Here, we investigated whether these accessory gene products of SARS-CoV were able to induce inflammatory responses.

DNA fragments corresponding to the SARS-CoV 3a/X1, 3b/X2, 6/ X3, 7a/X4, and 8b/X5 genes (identical to the Urbani strain, GenBank accession number AY278741) were amplified by polymerase chain reaction (PCR) from cDNA from the Hanoi 01-03 strain [9] of SARS-CoV using the specific primers 5 0 -CACCATGGATTTGTT-TATGAGA-3 0 (forward) and 5 0 -CAAAGGCACGCTAG-TAGTCGTCG (reverse) for 3a/X1; 5 0 -CACCATGATGCCAACTA-CTTTGTTTGC-3 0 (forward) and 5 0 -ACGTACCTGTTTCTTCC-GAAACG-3 0 (reverse) for 3b/X2; 5 0 -CACCATGTTTCATCTT-GTTGACTTCC-3 0 (forward) and 5 0 -TGGATAATCTAACTCCAT-AGGTTC-3 0 (reverse) for 6/X3; 5 0 -CACCATGAAAATTATTCT-CTTCCTG-3 0 (forward) and 5 0 -TTCTGTCTTTCTCTTAATGGT-GAAGC-3 0 (reverse) for 7a/X4; 5 0 -CACCATGTGCTTGAAGAT-CCTTGTAAG-3 0 (forward) and 5 0 -ATTTGTTCGTTTATTTAAA-ACAACAAG-3 0 (reverse) for 8b/X5. Each amplified fragment was inserted into the pENTRä/D-TOPO vector (Invitrogen, Carlsbad, CA), and was subsequently transferred into a modified lentivirus expression vector (pLenti6/V5-DEST, Invitrogen) containing an intron of human beta-globin (Fig. 1A ) by site-specific recombination using the Gateway Cloning System (Invitrogen). They were designated as pLenti/V5/X1, pLenti/V5/X2, pLenti/V5/X3, pLenti/V5/X4, and pLenti/V5/X5, respectively. An enhanced green fluorescent protein (EGFP)-expressing vector (pLenti/V5/GFP) was also constructed as a control.

A reporter plasmid expressing luciferase (jB-Luc) driven by five tandem NF-jB binding sites derived from IL-2 receptor a [10] was provided by Dr. Junichi Fujisawa (Kansai Medical School, Osaka, Japan). Reporter plasmids for the wild-type (-133-Luc) and various mutant (AP-1-Luc, NF-jB-Luc, and NF-IL6-Luc) interleukin 8 (IL-8) promoters [11] were provided by Dr. Naofumi Mukaida (Kanazawa University, Kanazawa, Japan). The HA-tagged expression vectors Abbreviations: ARDS, acute/adult respiratory distress syndrome; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; IL-8, interleukin 8; JNK, Jun N-terminal kinase; NF-jB, nuclear factor kappa B; ORF, open reading frame; RANTES, regulated on activation normal T cell expressed and secreted; SARS-CoV, severe acute respiratory syndrome coronavirus HA-JNK1 and HA-JNK3 were provided by Dr. Hidenori Ichijo (The University of Tokyo, Tokyo, Japan). A control plasmid expressing renilla luciferase driven by the cytomegalovirus promoter (phRL-CMV) was purchased from Promega (Madison, WI).

Cell lysates were prepared in lysis buffer (20 mM Tris-Hcl [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100 and protease inhibitor cocktail [CALBIOCHEM, La Jolla, CA]), and proteins (20 lg) were separated by electrophoresis on a Tris-Glycine minigel (Invitrogen), transferred to nitrocellulose filters, and reacted with antibodies followed by visualization with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometoric analysis was performed on scanned filters using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/).

Anti-V5 antibody (Invitrogen) and anti-phospho Jun N-terminal kinase (JNK) antibody (Cell Signaling Technology, Beverly, MA) were used for Western blotting. Immunoprecipitation was performed with anti-HA (Roche) or anti-V5 antibodies as previously described [12] .

Luciferase-expressing various reporter plasmids (200 or 300 ng) together with renilla luciferase-expressing phRL-CMV (20 ng) were co- transfected with vectors expressing SARS-CoV genes into HEK293T or A549 cells (2 · 10 5 cells) using Lipofectamine2000 (Invitrogen) or Fugene6 (Roche), respectively. Luciferase and renilla luciferase activities were measured from cell lysates 30 or 40 h after transfection, using the Luciferase assay system (Promega) and the Renilla luciferase assay system (Promega), respectively.

The amounts of IL-8 and regulated on activation normal T cell expressed and secreted (RANTES) in the culture supernatants of A549 cells were measured by Quantikine human IL-8 and RANTES ELISA kits (R&D Systems), respectively, 48 h after transfection.

Lentivirus expression vectors for SARS-CoV 3a/X1, 3b/X2, 6/X3, 7a/X4, and 8b/X5 genes were constructed as shown in Fig. 1A . These expression vectors were transfected into HEK293T cells, and expression of coding proteins with predicted sizes were confirmed by Western blotting (Fig. 1B) . The expression level of 3b/X2 protein was always lower than the other SARS-CoV proteins tested in this system. Anti-V5 antibody detected two bands of 3a/X1 protein presumably due to a posttranslational modification as recently reported [13] .

3.2. Activation of NF-jB and JNK by SARS-CoV accessory proteins We first examined effects of SARS-CoV genes on NF-jB, the major transcription factors activated in ARDS [5] , using a reporter plasmid expressing luciferase (jB-Luc) [10] . As shown in Fig. 2 , expression of 3a/X1 and 7a/X4 significantly enhanced NF-jB mediated transcription (9.1 and 3.5-folds, respectively) in HEK293T cells (P < 0.05). The effects of 3b/ X2, 6/X3 and 8b/X5 were not significant compared to the GFP control. We also determined the effect of SARS-CoV genes on mitogen-activated protein kinases that are also associated with chemokine production [14, 15] . HA-tagged JNK1 expressed in HEK293T cells was markedly phosphorylated by 3a/X1and 7a/X4 but not by 3b/X2, 6/X3, or 8b/X5 ( Fig. 3A and B) . 3a/X1 and 7a/X4 also activated JNK3 (Fig. 3C) . There was no obvious activation of ERK and p38a by any SARS-CoV genes tested (data not shown).

3.3. Augmentation of IL-8 promoter activity by SARS-CoV accessory proteins We next examined whether SARS-CoV proteins were capable of activating the promoter of IL-8 that is a representative chemokine involved in ARDS [16] and regulated by NF-jB and MAP kinases including JNK [17] . It has been shown that the IL-8 level is elevated in the plasma of SARS patients [18] . When the reporter plasmids expressing luciferase under the control of the human wild-type IL-8 promoter (-133-Luc) [11] were co-transfected in HEK293T cells, IL-8 promoter activity was enormously augmented by expression of 3a/X1 (28.7-fold) and 7a/X4 (13.2-fold). The effects of 3b/X2, 6/X3, and 8b/X5 were not significant (Fig. 4A) . Augmentation of the IL-8 promoter activity by 3a/X1 and 7a/X4 was abolished by a mutation at the NF-jB site in the IL-8 promoter, indicating that this effect was mainly mediated through NF-jB (Fig. 4B ).

We next examined the effect of SARS-CoV gene products in human lung cancer-derived A549 cells [19] . The expression levels of SARS-CoV proteins in transiently transfected A549 cells were lower than those in HEK293T cells, but were detectable following immunoprecipitation with anti-V5 antibody (Fig. 5A) . In a reporter assay using A549 cells, 7a/X4 showed the greatest effect on the IL-8 promoter activity (11.2-fold) among tested (Fig. 5B) . Although 3a/X1 showed a 2.2-fold increase in IL-8 promoter activity at an early time point, such as 24 h after transfection (data not shown), it was no longer significant 40 h after transfection in A549 cells. A mutation at the NF-jB site affected 7a/X4-mediated activation of the IL-8 promoter (Fig. 5C) , indicating that the effect of 7a/X4 in A549 cells was also mainly mediated through the NF-jB site. A reporter assay using the jB-Luc plasmid showed that 7a/X4 enhanced NF-jB activity 9.3-fold while 3a/X1 enhanced it 2.8-fold in A549 cells (Fig. 5D) .

Thus, 3a/X1 and 7a/X4 were capable of activating NF-jB and IL-8 promoter, but such effects were predominantly elicited by 3a/X1 in HEK293T cells and by 7a/X4 in A549 cells.

SARS-CoV 3a/X1, and 7a/X4 Finally, we examined whether 3a/X1, and 7a/X4 actually induced inflammatory chemokine production in A549 cells. As shown in Table 1 , although A549 cells spontaneously produced IL-8, expression of 7a/X4 further increased the levels of IL-8 production. 3a/X1 also enhanced IL-8 production, but its statistical significance was variable (Table 1) . Production of another chemokine RANTES, that is controlled at least by NF-jB [20] and up-regulated in SARS-CoV-infected cells [21] , was significantly induced by 3a/X1 and 7a/X4 (Table 1) .

Both 3a/X1 and 7a/X4 proteins are expressed in SARS-CoVinfected cells [22] . 3a/X1 protein is a cell membrane-associated protein, potentially secreted and incorporated into the virion [23] [24] [25] . 7a/X4 protein is located in the cytoplasm [26] . Recent studies reported that over-expression of 3a/X1 and 7a/X4 induced apoptosis of the cell [27, 28] . Our results indicated that these cell-associated SARS-CoV accessory gene products could activate NF-jB and JNK, and might be strong candidates to induce pathological inflammatory responses in SARS. 

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Acknowledgement: We wish to thank Drs. Junichi Fujisawa (Kansai Medical School, Osaka, Japan), Naofumi Mukaida (Kanazawa University, Kanazawa, Japan), and Hidenori Ichijo (The University of Tokyo, Tokyo, Japan) for providing plasmids. This study was supported by the Special Coordination Funds for Promoting Science and Technology of Japan Science and Technology.