| Summary: | 碩士 === 國立成功大學 === 醫學檢驗生物技術學系 === 102 === SUMMARY
Enterovirus 71 (EV71) usually caused hand-foot-mouth disease (HFMD) in children under 5 years old. In some cases, EV71 caused serious neurological disorders and lead to death. Although several receptors/attachment molecules for EV71 have been reported, the binding, entry, and infection mechanisms of EV71 remain ambiguous. There are two well-studied cellular receptors for EV71, scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1). In our previous studies, we have identified several EV71-interacting proteins from RD cells by glycoproteomic techniques. Selenoprotein S (SelS), a member of selenoprotein family which plays a role in inflammation and metabolic diseases, was one of the EV71-interacting proteins. This study aims to investigate the roles of SleS in the early stage of EV71 infection. We found that EV71 viral particles directly interacted with SelS. Knocked down SelS expression reduced the binding and infection of EV71 to RD cells and IL-1β production. These results suggested that SelS may participate in EV71 attachment during the early stage of infection, as well as the subsequent inflammation responses. We are also trying to map the EV71 binding epitope on SleS protein. Our findings not only indicate a new drug target for anti-EV71 drug development, but also offer novel information for dissecting EV71 infection mechanism.
Key words: Enterovirus 71(EV71), Selenoprotein S(SelS), human scavenger receptor class B 2 (SCARB2)
INTRODUCTION
Enterovirus 71 (EV71) belongs to human enterovirus species A of the genus Enterovirus within the family Picornavirus, which is a major causative agent of hand-foot-mouth disease (HFMD). Infection of EV71 to central nerve system (CNS) may cause serious neurological disorders in children less than 2 years old. Till now, there are no antiviral drugs or vaccines specific for EV71. EV71 genome includes P1, P2 and P3. The P1 region encodes VP1 toVP4 proteins formed a closed icosahedron capsid. The capsid protein VP1 contributes to virulence and neurotropism and interacts with host receptors. There were 2 receptors identified in 2009, human scavenger receptor class B 2 (SCARB2) and human P-selectin glycoprotein ligand-1 (PSGL-1). SCARB2 is the main cellular receptor that can be recognized by all of EV71 strains. Also, it has been reported as receptors or cofactors of Coxsackievirus A7, A14 and A16. Furthermore, SCARB2 is involved in internalization and uncoating of EV71 infection. PSGL-1 Is expressed on myeloid cells and stimulated T lymphocytes. Different from SCARB2, not all EV71 strains can bind to PSGL-1. Blocking of these proteins by specific antibody cannot entirely block the infection of EV71 to cells. In our previous studies, we have identified some EV71 interacting glycoproteins by glycoproteomic approaches, and selenoprotein S (SelS) was one of them. Sels is a member of selenoprotein family, involves in inflammation and metabolic diseases. SelS has a transmembrane domain and localized in the ER membrane and on the cell surface. It can promote cell survival regulating the ER stress as well as inflammation. However, whether SelS involves in virus infection is still unclear. Thus, this study aims to investigate the roles of SleS in the early stage of EV71 infection by biochemical and virological analyses. We will also try to map the epitope of EV71 on SleS.
MATERIALS AND METHODS
Cell culture and virus propagation: The RD (Rhabdomyosarcoma) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 1% penicillin streptomycin and 10% fetal bovine serum (FBS) and maintained in 37˚C incubator equilibrated with 5% CO2. EV71 viruses were propagated in RD cells grew in DMEM with 1% penicillin streptomycin and 2% fetal bovine serum, incubated at 35 ˚C. The virus titter was determined by plaque forming assay and CCID50. Viral supernatants were precipitated by PEG/NaCl solution, and purified by sucrose gradient.
Enzyme-linked immunosorbent assay (ELISA). Serial diluted BSA and EV71 viral particles were coated in 96-well microtiter plates with coating buffer (0.1 M Na2HPO4) at 4 °C for 16 h. Recombinant human SleS protein (10 ng/mL, Abnova, Taiwan) was added into each well and incubated at 37 °C for 2 h. After washing out unbound proteins with 0.5% Tween20 in PBS, primary antibodies (anti-SleS antibody, 1:100) were added into each well and incubated at 37 °C for 2 h. After washing, HRP conjugated secondary antibody was added into the well and incubated at 37 °C for 1 h followed by adding HRP substrates (WBKLS0500, Millipore). The binding signals were detected using an ELISA reader (EnVision, PerkinElmer).
Binding assay and infection assay: The expression of SelS expression on RD cells was knocked down by transfecting SleS specific si-RNA. The knockdown efficiency was evaluated by RT-PCR, flow cytometry and western blot. The RD-sh-SelS, vehicle and wild type cells were infected with EV71 in 4˚C for 3h, and the bound EV71 was detected by flow cytometry. These cells were also infected with EV71 in 37˚C for 1h, and the total viral RNA was analyzed by Q-PCR.
Peptide mapping: Biotin conjugated SleS peptides were synthesized by solid phase technology. The binding of EV71 with SleS peptides were evaluated by AlphaScreen, VOPBA and ELISA assays, and verified with plaque assay.
RESULTS AND DISCISSIONS
In the ELISA results, SelS bound to EV71 particles in a dose dependent manner accompany with the increased protein concentration, and vice versa. The expression of SleS on RD cells was significantly reduced measured by fluorescence microscopy, RT-PCR, flow cytometry and western blot. It should be noted that the viability of transfected RD cells was reduced as a consequence of si-RNA treatment within 48 h. The binding ability of EV71 to RD-sh-SleS cells was also dropped compared with vehicle and wild type cells. In addition, we found that the expression of IL-1β was decreased in RD-sh-SleS cells. The mapping of EV71 binding sites was still ongoing.
CONCLUSIONS
These results suggested that SelS may involve in the binding of EV71 to host cells during the early stage of infection. It should be further investigated whether SleS influence the inflammation responses after virus internalization. The mapping of the EV71 binding epitope on SleS protein will also be accomplished. Our findings not only indicate a new drug target for anti-EV71 drug development, but also offer novel information for the investigation of EV71 infection mechanism.
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