Transcriptome meta-analysis reveals a dysregulation in extra cellular matrix and cell junction associated gene signatures during Dengue virus infection

Abstract

Dengue Viruses (DENVs) cause one of the most prevalent arthropod-borne viral diseases affecting millions of people worldwide. Identification of genes involved in DENV pathogenesis would help in deciphering molecular mechanisms responsible for the disease progression. Here, we carried out a meta-analysis of publicly available gene expression data of dengue patients and further validated the meta-profile using in-vitro infection in THP-1 cells. Our findings reveal that DENV infection modulates expression of several genes and signalling pathways including interferons, detoxification of ROS and viral assembly. Interestingly, we have identified novel gene signatures comprising of INADL/PATJ and CRTAP (Cartilage Associated Protein), which were significantly down-regulated across all patient data sets as well as in DENV infected THP-1 cells. PATJ and CRTAP genes are involved in maintaining cell junction integrity and collagen assembly (extracellular matrix component) respectively, which together play a crucial role in cell-cell adhesion. Our results categorically reveal that overexpression of CRTAP and PATJ genes restrict DENV infection, thereby suggesting a critical role of these genes in DENV pathogenesis. Conclusively, these findings emphasize the utility of meta-analysis approach in identifying novel gene signatures that might provide mechanistic insights into disease pathogenesis and possibly lead towards the development of better therapeutic interventions.

Figure 1. Pictorial representation of the meta-analysis approach adopted for this study.

Figure 2. Transcriptome meta-analysis of Dengue virus infected patients.

(a) A graph representing data sets sharing the number of common DEGs. (b) Cloud representation of common DEGs expressed across 7 data sets. (c) Heat map showing consistently expressed genes (meta-genes) in different data sets used in this study.

Figure 3. Relative mRNA expression levels of five candidate genes (ATOX1, OAS2, USP18, CRTAP and PATJ) in THP-1 cells infected with DENV.

Quantitative Real-time polymerase chain reaction (RT-qPCR) was carried out to quantify the relative expression of the above-mentioned candidate genes in THP-1 cells infected with DENV-1 (Hawaii) and DENV-2 (TR1751) at a multiplicity of infection (moi) of 5 for the indicated time points. The relative expression of each gene (a) ATOX1, (b) OAS2, (c) USP18 (d) CRTAP, (e) PATJ was normalized to housekeeping gene β-Actin. Results are representative of one of three independent experiments and the error bars represent the standard error of the means (SEM). P values were determined based on comparison with uninfected cells. Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc test to identify significance using Graph Pad Prism5 software. ***P < 0.001, **P < 0.01, *P < 0.05 were considered statistically significant.

Figure 4. Pathway networks of candidate genes that were validated in Dengue infected patient samples.

Pathway networks of genes USP18, CRTAP, ATOX1, PATJ were generated based on Reactome Pathway database using Cytoscape software. No pathway interactions were found for OAS2. Target genes with red colour indicate upregulation; while with green colour indicate down-regulation.

Figure 5. PATJ overexpression interferes with DENV-1 infection.

(a) Laser scanning confocal microscopy to visualize the number of DENV-1 infected Vero cells transfected with either pCAGGS (vector control) or pCAGGS-Patj-myc. Vero cells were transfected with equal concentrations of both the plasmids for 36 hours followed by infection of DENV-1 (moi 5) for another 18 hrs. Following incubation, the cells were fixed, permeabilized, and stained with anti-dengue monoclonal Ab (green). (b) Quantification of the number of DENV-1 infected cells in vector transfected and pCAGGS-Patj-myc transfected Vero cells. Infected cells were counted in at least ten different fields for each experimental condition using Image J software and the average number of infected cells per field were plotted. (c) Foci forming unit reduction assay (FFURA) on vector transfected and pCAGGS-Patj-myc transfected Vero cells was performed to determine the antiviral activity of PATJ on DENV-1 using DENV foci immunostaining method as described in Materials and Methods on the fourth day after infection. Arrows indicate representative DENV-1 foci. (d) Data from duplicate assays of three independent experiments were plotted. The percentage of foci reduction is represented in the graph. Statistical analysis was performed using Student’s t-test using Graph Pad Prism version 5 (Graph Pad Software Inc., San Diego, CA.). Error bars represent the standard error of the mean (SEM). ***P < 0.001, **P < 0.01, *P < 0.05 were considered statistically significant.

Figure 6. Over-expression of CRTAP gene inversely regulates DENV-1 infection.

(a) Laser scanning confocal microscopy to visualize the number of DENV-1 infected Vero cells transfected with either pCMV6 (vector control) or pCMV6-CRTAP-Myc-DDK. Vero cells were transfected with equal concentrations of both the plasmids for 36 hours followed by infection of DENV-1 (moi 5) for another 18 hrs. Following incubation, the cells were fixed, permeabilized, and stained with anti-Dengue monoclonal Ab (green). (b) Quantification of the number of DENV-1 infected cells in vector transfected and pCMV6-CRTAP-Myc-DDK transfected Vero cells. Infected cells were counted as mentioned in the previous figure. (c) Foci forming unit reduction assay (FFURA) on vector transfected and pCMV6-CRTAP-Myc-DDK transfected Vero cells was performed to determine the antiviral activity of CRTAP on DENV-1 as already described in material and methods. Arrows indicate representative DENV-1 foci. (d) Data from duplicate assays of three independent experiments were plotted. The percentage of foci reduction is represented in the graph. (e) Collagen estimation was done in vector transfected or pCMV6-CRTAP-Myc-DDK transfected cell lysates as described in material and methods. DENV-1 infection was given as indicated. Statistical analysis was performed using Student’s t-test using Graph Pad Prism version 5 (Graph Pad Software Inc., San Diego, CA.). Error bars represent the standard error of the mean (SEM). ***P < 0.001, **P < 0.01, *P < 0.05 were considered statistically significant.