Overexpression of the nucleocapsid protein of Middle East respiratory syndrome coronavirus up-regulates CXCL10

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory diseases in humans and has a high mortality rate. During infection, MERS-CoV regulates several host cellular processes including antiviral response genes. In order to determine if the nucleocapsid protein of MERS-CoV (MERS-N) plays a role in viral-host interactions, a murine monoclonal antibody was generated so as to allow detection of the protein in infected cells as well as in overexpression system. Then, MERS-N was stably overexpressed in A549 cells, and a PCR array containing 84 genes was used to screen for genes transcriptionally regulated by it. Several up-regulated antiviral genes, namely TNF, IL6, IL8, and CXCL10, were selected for independent validation in transiently transfected 293FT cells. Out of these, the overexpression of MERS-N was found to up-regulate CXCL10 at both transcriptional and translational levels. Interestingly, CXCL10 has been reported to be up-regulated in MERS-CoV infected airway epithelial cells and lung fibroblast cells, as well as monocyte-derived macrophages and dendritic cells. High secretions and persistent increase of CXCL10 in MERS-CoV patients have been also associated with severity of disease. To our knowledge, this is the first report showing that the MERS-N protein is one of the contributing factors for CXCL10 up-regulation during infection. In addition, our results showed that a fragment consisting of residues 196-413 in MERS-N is sufficient to up-regulate CXCL10, while the N-terminal domain and serine-arginine (SR)-rich motif of MERS-N do not play a role in this up-regulation.

Keywords: CXCL10; Middle East Respiratory Syndrome coronavirus; nucleocapsid.

Figure 1. Characterization of mAb 7H6

(A) 293FT cells were transiently transfected with empty vector, FLAG-tagged full-length MERS-N, and its N- and C-terminal fragments. Western blot analysis was performed using mAb 7H6 and anti-FLAG antibody. (B) 293FT cells were transiently transfected with empty vector or FLAG-tagged N of different HCoVs. Western blot analysis was performed using mAb 7H6 or anti-FLAG antibody. (C) Vero E6 cells were mock infected or infected with MERS-CoV (multiplicity of infection of 1) and stained with mAb 7H6 at 2 days post-infection. (D) 15-mer peptides with ten amino acids overlapping sequences of the C-terminal fragment of MERS-N were generated and their binding to mAb 7H6 was analyzed by ELISA. Three independent experiments were performed and a representative data is shown. The results are mean values with error bars showing S.D. of triplicate wells. (E) Peptides between N376-410 were aligned and the minimal binding sequence was mapped (denoted in a box). (F) 293FT cells were transiently transfected with empty vector, FLAG-tagged MERS-N and mutant MERS-NΔ391-395. Western blot analysis was performed using mAb 7H6 and anti-FLAG antibody.

Figure 2. Selection of genes for downstream validation

(A) A549 cells were transduced with lentiviral virus to express MERS-N and LacZ. Cells were subsequently selected in blasticidin, and protein expression was analyzed at day 2 and 10 post-selection by IFA using mAb 7H6. (B) RNA transcripts were obtained from A549-transduced cells expressing MERS-N or LacZ at day 2 and 10 post-selection and assayed using an 84-gene antiviral PCR array. Gene regulation was analyzed with a cutoff of 4-fold change and genes up-regulated on both day 2 and 10 post-selection are represented in a Venn diagram. The intersection shows common genes up-regulated on both days.

Figure 3. Expression of the four selected antiviral genes in 293FT cells

(A) 293FT cells were transiently transfected with empty vector and FLAG-tagged MERS-N. Western blot analysis was performed using anti-FLAG antibody. (B) RNA transcripts from transiently transfected 293FT cells were assayed by RT-qPCR with TaqMan probes to analyze the mRNA fold changes of TNF, IL6, IL8, and CXCL10. mRNA expression of genes was first normalized against GAPDH and subsequently, MERS-N up-regulation was normalized against levels in vector-transfected cells. (C) Cell supernatants were harvested and CXCL10 secretion was evaluated using ELISA. (D) The secretion of CXCL10 by MERS-N was normalized against the vector. The results of these experiments were expressed as mean ± S.D. (error bars) of five independent experiments. Asterisk (*) indicates statistical significance of P<0.05 when compared with vector-transfected cells at the respective time-points.

Figure 4. Expression of CXCL10 in A549 cells

(A) A549 cells were transiently transfected with empty vector and FLAG-tagged MERS-N. Western blot analysis was performed using anti-FLAG antibody. (B) RNA transcripts from transiently transfected A549 cells were assayed by RT-qPCR with TaqMan probes to analyze the mRNA fold changes for the expression of CXCL10. mRNA expression of CXCL10 was first normalized against GAPDH and then normalized against levels in vector-transfected cells. (C) Cell supernatants were harvested from the transiently transfected cells, and CXCL10 secretion was evaluated using ELISA. (D) The secretion of CXCL10 of cells overexpressing MERS-N was normalized against the vector. The results of these experiments were expressed as mean ± S.D. (error bars) of six independent experiments in triplicates. The asterisk (*) indicates statistical significance of P<0.05 when MERS-N is compared with vector control.

Figure 5. MERS-N regulates CXCL10 through its C-terminal fragment

(A) 293FT cells were transiently transfected with empty vector, FLAG-tagged N, N1-195, and N196-413. Western blot analysis was performed using anti-FLAG and anti-GAPDH antibodies. (B) RNA transcripts were obtained from transiently transfected 293FT cells and assayed by RT-qPCR to analyze the mRNA fold changes of CXCL10. (C) Cell supernatants were harvested from transiently transfected 293FT cells, and CXCL10 secretion was evaluated using ELISA. The results of these experiments were expressed as mean ± S.D. (error bars) of three independent experiments in triplicates. Asterisk (*) indicates statistical significance of P<0.05 when compared with vector-transfected cells at the respective time-points.