Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection

Abstract

Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.

Figure 1

Kinetics of human rhabdomyosarcoma cell proliferation at 8 and 20 h following infection with two EV71 strains. Untreated control and inactivated EV71‐treated cells were included. Viable cell count (grey bars) was assessed by the trypan blue exclusion technique. Non‐viable cell counts are represented as unfilled bars. The dashed line depicts the starting number of cells at the point of virus inoculation.

Figure 2

Immunofluorescence microscopy of uninfected and EV71‐infected RD cells at 8 and 20 h p.i. Infected cells were detected by anti‐EV71 monoclonal antibody, while the cell nuclei were displayed by DAPI staining. 
A–D. Uninfected RD cells. 
E–H. RD cells mock‐infected with inactivated EV71 strain MS/7423/87. 
I–L. RD cells infected with EV71 strain MS/7423/87. 
M–P. RD cells infected with EV71 strain 5865/Sin/000009.

Figure 3

Western blot analysis of ODC1, DCAMKL1, APOB and CLU expression at 8 and 20 h p.i. Total protein extracts (20 µg each) of uninfected control (C), mock‐infected (I), EV71 MS/7423/87 strain‐infected (M), and EV71 5865/Sin/000009 strain‐infected (S) RD cells were tested against the relevant specific antibodies. The GAPDH housekeeping protein was included to ensure equal loading of protein samples.

Figure 4

RT‐PCR amplification of the p53 gene at 8 and 20 h p.i. A 353 bp fragment representing the wild‐type p53 transcript was amplified in uninfected control (C), mock‐infected (I), EV71 MS/7423/87 strain‐infected (M), and EV71 5865/Sin/000009 strain‐infected (S) RD cells. An additional 486 bp fragment corresponding to the p53 splice variant appeared more distinctly in cells infected with the two EV71 strains after 20 h. GAPDH (153 bp fragment) served to ensure equal amounts of starting template in the samples.

Figure 5

Identification of six differentially expressed proteins by two‐dimensional proteomic analysis of uninfected (U) and EV71 MS/7423/87 strain‐infected (I) RD cell samples at 8 and 20 h p.i. The arrowheads indicate the protein spots identified as differentially regulated at greater than twofold changes (P < 0.05) using Delta2D analysis.