Beclin1 Binds to Enterovirus 71 3D Protein to Promote the Virus Replication

Abstract

Enterovirus 71 (EV71) is the main pathogen causing hand-foot-mouth disease (HFMD) in infants and children, which can also lead to severe neurological diseases and even death. Therefore, understanding the replication mechanism of EV71 is of great significance for the prevention and control of EV71-induced diseases. Beclin1 (BECN1, a mammalian homologue of ATG6 in yeast) is an important core protein for the initiation and the normal process of autophagy in cells. In addition to its involvement in autophagy, Beclin1 has also been reported to play an important role in cancer and innate immune signaling pathways. However, the role of Beclin1 in EV71 replication remains elusive. Here, we primarily found that Beclin1 facilitates EV71 replication in human rhabdomyosarcoma (RD) cells and the autophagy was actually induced, but Beclin1 was not significantly affected at either mRNA level or protein level during early EV71 infection. Further studies discovered that Beclin1 could interacts with EV71 non-structural protein 3D mainly through its evolutionary conserved domain (ECD) and coiled-coiled domain (CCD), thus promoting the replication of EV71 in human rhabdomyosarcoma (RD) cells and human astroglioma (U251) cells. Collectively, we reveal a novel regulatory mechanism associated with Beclin1 to promote EV71 replication, thus providing a potential therapeutic target for the prevention and control of EV71-associated diseases.

Keywords: Beclin1; EV71; EV71 non-structural protein 3D; autophagy; enterovirus 71.

Figure 1

The expression level of Beclin1 was stably maintained during Enterovirus 71 (EV71) infection in rhabdomyosarcoma (RD) cells. (A,B) RD cells were transfected with GFP-mCherry-LC3 for 24 h, and then infected with EV71 for different times, or treated with Rapamycin (RAP) and Chloroquine diphosphate (CQ) as the complete and incomplete autophagy flux control, respectively. The images were visualized under Laser scanning confocal microscope. Bar = 10 μm (A), The graph shows the quantification of autophagosomes by taking the average number of dots in 50 cells (B). (C–F) RD cells were infected with EV71 (MOI = 1) for indicated time, the relative RNA levels of BECN1 (C) and EV71-VP1 (D) were determined by Real-time PCR, and the relative protein levels of Beclin1 and EV71 3D were detected by Western blot (E), and the LC3-II/LC3-I ratio was measured (F). (G–J) RD cells were infected with EV71 for 12 h at an indicated multiplicity of infection (MOI) (0, 1, 2, and 4), the relative RNA levels of BECN1 (G) and EV71 VP1 (H) were determined by Real-time PCR, the relative protein levels of Beclin1 and EV71-3D were detected by Western blot (I), and the LC3-II/LC3-I ratio was measured (J). Graph expressed as mean ± SD, ns, not-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

Beclin1 promotes EV71 replication in RD cells. (A and B) RD cells were transfected with Flag-vector or Flag-Beclin1 at the indicated concentrations for 24 h and infected with EV71 (MOI = 1) for 12 h. The RNA and protein levels of EV71 VP1 were detected by Real-time PCR with GAPDH as an internal control (A) and Western blot (B), respectively. (C and D) RD cells were transfected with Flag-vector or Flag-Beclin1 for 24 h and then infected with EV71 (MOI = 1) at the indicated times. The RNA and protein levels of EV71-VP1 were detected by Real-time PCR with GAPDH as an internal control (C) and Western blot (D), respectively. Graph expressed as mean ± SD, ns, not-significant; * p < 0.05; ** p < 0.01.

Figure 3

Knockdown of BECN1 attenuates EV71 replication in RD and U251 cells. (A,B) RD cells were transfected with pLKO.1-shNC, pLKO.1-shBeclin1-1, pLKO.1-shBeclin1-2, and the efficiency of knockdown was testified by Real-time PCR (A). The transfected RD cells were infected with EV71 (MOI = 1), and the protein levels of EV71-VP1 and Beclin1 were detected by Western blot with β-actin as an internal control (B). (C) RD cells were transfected with Flag-vector or Flag-Beclin1, pLKO.1-shNC or pLKO.1-shBeclin1 and infected with EV71 for 12h, and supernatants were collected for TCID50 assay. (D–F) The stable RD cells were infected with EV71 (MOI = 1) for different periods. The RNA and protein levels of EV71-VP1 were detected by Real-time PCR with GAPDH (D) and Western blot with β-actin as controls (E), and the LC3-II/LC3-I ratio was measured(F), respectively. (G–I) The stable U251 cells were infected with EV71 (MOI = 2) for different periods. The RNA and protein levels of EV71 VP1 were detected by Real-time PCR (G) and Western blot (H), and the LC3-II/LC3-I ratio was measured(I), respectively. Graph expressed as mean ± SD, ns, not-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

EV71 3D interacts and co-localizes with Beclin1 in the cytoplasm. (A) HEK293T cells were co-transfected with plasmid encoding Flag-Beclin1, and plasmids encoding EV71 non-structure proteins, HA-2A, HA-2B, HA-2C, HA-3AB, HA-3C and HA-3D, for 36 h, respectively. The cell lysates were immunoprecipitated with control IgG or an anti-Flag antibody, and then immunoblotted with the indicated antibodies. (B) HEK293T cells were co-transfected with plasmids encoding HA-3D and Flag-Beclin1. The cell lysates were immunoprecipitated with control IgG or an anti-Flag antibody or anti-HA antibody, and then immunoblotted with the indicated antibodies. (C) RD cells were transfected with plasmids encoding Flag-Beclin1 alone or HA-3D alone or co-transfected with Flag-Beclin1 and HA-3D for 24 h. The localization and distribution of Flag-Beclin1 protein (green), EV71 3D protein (red), and the nuclei (blue) were visualized under Laser scanning confocal microscope. Bar = 10 μm. (D) RD cells were infected with EV71 (MOI = 2) for 12 h, and the cells were lysed and immunoprecipitated with control IgG or an anti-Beclin1 antibody and then immunoblotted with indicated antibodies.

Figure 5

The coiled-coiled domain (CCD) and evolutionary conserved domain (ECD) of Beclin1 were essential for the facilitation of EV71 replication. (A) Schematic diagram of Beclin1 and its mutants. (B) HEK293T cells were co-transfected with plasmids encoding GFP-3D and Flag-vector as a negative control: Flag-Beclin1, Flag-Beclin1 BD, Flag-Beclin1 BD+CCD, Flag-Beclin1 BD+CCD+ECD, Flag-Beclin1 CCD+ECD, Flag-Beclin1 ECD. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (C and D) RD cells were transfected with plasmids expressing Flag-vector, Flag-Beclin1 and various mutants for 24 h and infected with EV71 (MOI = 2) for another 12 h. The expression RNA level of EV71-VP1 was detected by Real-time PCR with GAPDH as a control (C). The protein level of EV71-3D was detected by Western blot (D). Graph expressed as mean ± SD, ns, not-significant, ** p < 0.01.