Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection
Wai Fook Leong11Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 117597. and Vincent T. K. Chow1*1Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 117597.*E-mail
micctk@nus.edu.sg; Tel. (+65) 6874 6200; Fax (+65) 6776 6872.1Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 117597.
*E-mail
micctk@nus.edu.sg; Tel. (+65) 6874 6200; Fax (+65) 6776 6872.
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Summary
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.
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Introduction
Enterovirus 71 (EV71) was first identified in 1969 in California, when it was isolated from the feces of an infant suffering from encephalitis (Schmidt et al., 1974). Subsequently, EV71 was reported as the agent involved in severe neurological diseases such as meningitis, encephalitis, monoplegia and acute flaccid paralysis. The virus is also associated with non-neurological diseases like hand, foot and mouth disease (HFMD), herpangina and pulmonary oedema. Among young children, EV71 is a notable cause of central nervous system (CNS) disease that usually results in rapid clinical deterioration and death, the molecular pathogenesis of which is still elusive (Kehle et al., 2003). In Singapore, the major EV71 outbreak in 2000 involved 6402 cases with four deaths (Singh et al., 2002a), and prompted the closure of pre-school centres for about 2 weeks. In 2001, there were 5187 cases of HFMD with EV71 as the predominant agent, with 75% of afflicted children below the age of four.
Belonging to the Picornaviridae, which comprises a large complex family of small non-enveloped, positive-strand RNA viruses with a genome size of about 7–9 kb, EV71 is known to induce an apoptotic response via its viral proteins such as 2A (Kuo et al., 2002) and 3C (Li et al., 2002). This is also observed in the related and widely studied poliovirus (Barco et al., 2000; Goldstaub et al., 2000; Calandria et al., 2004), which belongs to the same genus as EV71. Apoptosis is a complex mechanism that involves a network of cross-talk and multiple specifically controlled pathways. The process may be triggered by the interactions of the pro-apoptotic stimuli with various sensors such as the receptor-mediated pathway through caspase 8, mitochondrial-related pathway through caspase 9 (Desagher and Martinou, 2000) and endoplasmic reticulum (ER) stress-triggered pathway through caspase 12 (Nakagawa et al., 2000). Apoptosis in viral infection is the host response to lyse prematurely in order to curtail the reproductive cycle of the virus (Clem and Miller, 1993). In addition, the significance of apoptosis is to enable macrophages to phagocytose dead cells in order to prevent dysregulated inflammatory reactions, and to initiate specific immune responses in the infected host (Sun and Shi, 2001). However, the immune response can also result in apoptosis of uninfected cells, which causes enhanced immunosuppression or specific organ toxicity (Ahr et al., 2004).
Besides apoptosis, the other host response is the development of canonical cytopathic effect (CPE) following productive poliovirus infection, which often leads to inflammation. Thus, the interplay of the two cell death processes highlights both apoptotic and antiapoptotic effects of poliovirus (Agol et al., 2000; Belov et al., 2003; Romanova et al., 2005). It is postulated that in early infection during which the poliovirus RNA genome is translated, sufficient quantities of pro-apoptotic proteins are synthesized to trigger an early apoptotic response. However, with the onset of viral replication, the apoptotic response is interrupted by the apoptosis-suppressing effect of the virus, which dominates the entire period of productive infection. Only at the later stages of infection such as after the development of CPE, can some signs of apoptosis be obvious (Carthy et al., 1998). Although many RNA viruses do not encode antiapoptotic genes and the mechanism of this apoptosis-suppressing effect is still unclear, enterovirus 2B protein which exhibits membrane permeabilizing activity (Agirre et al., 2002), may have a significant role. Campanella et al. (2004) also demonstrated that coxsackievirus 2B protein is able to suppress apoptotic host responses by manipulating intracellular calcium ion homeostasis.
In addition to the variability in apoptotic response, enteroviral infection instigates multiple cascades of host responses, especially mechanisms pertaining to its neuropathogenesis. These responses are also evident in infections with other viruses, e.g. dengue virus (Warke et al., 2003; Liew and Chow, 2004), herpes simplex virus (Kramer et al., 2003), JC virus (Radhakrishnan et al., 2003) and severe acute respiratory syndrome (SARS) coronavirus (Leong et al., 2005). Understanding the molecular basis of the host response to microbial infection particularly antiapoptotic responses, is essential for identifying targets to prevent disease and tissue damage resulting from the inflammatory response. In our study of EV71 infection of rhabdomyosarcoma (RD) cells, DNA microarray and two-dimensional (2-D) proteomic analyses were employed to probe into these molecular changes. The expression of the p53 tumour suppressor gene and its alternative splice variant in EV71-infected cells was also investigated.
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Results
Growth kinetics of EV71-infected RD cells
In order to expand the scope of study, two batches of RD cells, each comprising four experimental models, were analysed at 8 and 20 h post infection (p.i.). The four models included uninfected control, mock-infection control with UV-inactivated EV71 reference strain MS/7423/87, EV71 strain MS/7423/87 infection, and EV71 strain 5865/Sin/000009 infection. The cell growth kinetics did not differ much at 8 h p.i. However, at 20 h p.i., a decrease in viable cell population was apparent in both EV71-infected models (Fig. 1). Visible CPE was also observed in the infected cell culture, indicative of a full-blown infection. It was speculated that in poliovirus-infected cells, such CPE formation may be attributed to expression of the putative apoptosis-preventing effect (Agol et al., 2000). Viable cell counts for the uninfected and mock-infected controls were comparable without any potential ‘spillover’ effect from the inoculum itself, which may contain interleukins or interferons derived from the virus-infected cell culture. There was also no visible effect in the cells subjected to the UV-inactivated EV71. Thus, any evident changes that occurred in the infected cells were caused by the live virus itself.
Immunofluorescent analysis of EV71-infected RD cells
Immunostaining of the cells with EV71-specific monoclonal antibody at 8 h p.i. revealed only about 3–5% of the cells infected with either strain of EV71 (Fig. 2). Mature virions were reported to assemble in the cytoplasm at 12 h after infection (Rangel et al., 1998). At 20 h p.i., 50–60% of the cells were infected with either strain, especially those infected with strain 5865/Sin/000009 (Fig. 2). This observation reflected the virulence of the latter strain isolated from a fatal case of EV71 encephalitis during the outbreak in Singapore in 2000.
