The uncoating of EV71 in mature late endosomes requires CD-M6PR

Abstract

Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30 min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.

Keywords: EV71; Late endosomes; Mannose 6-phosphate receptor; SCARB2; Uncoating.

Fig. 1.

EV71 is uncoated at 30 mai and is dependent on acidification. (A) Experimental protocol for infection of RD-hSCARB2 cells with light-sensitive EV71. The red arrow indicates virus addition, the green arrow indicates the recovery of the infected cells followed by the titration of the live virus, black arrowheads indicate the start of light irradiation, gray areas indicate the periods without the light irradiation, and yellow area indicates the periods with the light irradiation. The time after the infection with light-sensitive virus is indicated. (B) Virus titer of the recovered virus. Horizontal axis indicates the onset of light irradiation after infection. The top line represents the virus titer without irradiation. Error bars represent s.d. (C) In situ hybridization for RNA genome of EV71 and immunofluorescence for the capsid proteins after infection of RD-hSCARB2 cells with EV71 in the absence or presence of NH₄Cl. The time of fixation after infection is indicated. DAPI is in blue, EV71 RNA genome is in green, whilst EV71 capsid antigens are in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate EV71 RNA+capsid double-labelled clustered vesicles. Insets are 400% enlarged panels of the arrowhead areas. Representative images are shown. Scale bars: 10 µm. (D) The numbers of visible green dots corresponding to EV71 RNA genomes per cell were shown. Number of z stack sets were three except for the 30 min NH₄Cl+ sample (two). **P<0.01. Statistical significance was determined by a two-tailed, unpaired t-test. Error bars represent s.d.

Fig. 2.

EV71 co-distributes with hSCARB2 and M6PR, but not with LAMP1 until 40 mai. Co-localization of EV71 and hSCARB2 (A), EEA1 (B), M6PR (C), or LAMP1 is shown by immunofluorescence (D). RD-hSCARB2 cells were infected with EV71 and fixed 30 min (B-D) or 40 min (A) after infection followed by immunofluorescence. DAPI is in blue, EV71 antigen is in green, whereas hSCARB2 or other markers are in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate co-localization of EV71 with markers. Representative images are shown. Scale bars: 10 µm. (E) Kinetics of co-localization of EV71 with hSCARB2 or other markers. Times indicate the time after infection.

Fig. 3.

EGF co-localizes with LAMP1 at 30 mai. (A) Co-localization of EGF with M6PR, LAMP1 or SCARB2 is shown by immunofluorescence of EGF and M6PR, LAMP1 and SCARB2. RD-hSCARB2 cells were infected with EV71 and then fixed 30 min after the addition of EGF followed by immunofluorescence. DAPI is in blue, EGF is in green, whereas organelle markers are in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate co-localization of EGF with markers. Representative images are shown. Scale bar: 10 µm. (B) Quantification of co-localization of EGF with specific markers. Number of z stack sets was two except SCARB2 was three. ns, not significant, **P<0.01. Statistical significance was determined by a two-tailed, unpaired t-test. Error bars represent s.d.

Fig. 4.

EV71 co-distributes with RAB9 during the uncoating period. Co-localization of EV71 with RAB5, RAB7, or RAB9 is shown by immunofluorescence of EV71 and RAB5 (A), RAB7 (B), or RAB9 (C). RD-hSCARB2 cells were infected with EV71 and fixed 30 min after infection, followed by immunofluorescence. DAPI is in blue, EV71 antigen is in green, whereas marker antigens are in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate co-localization of EV71 with markers. Representative images are shown. Scale bars: 10 µm. (D) Kinetics of co-localization of EV71 with the different markers. Times indicate the time after infection.

Fig. 5.

EV71 particles are incorporated into LEs. (A) Transmission electron microscopy. RD-hSCARB2 cells were infected with EV71 and fixed at 20 or 30 mai. Times indicate the time after infection. Arrowheads indicate representative EV71-like particles in LE. (B) Immuno-electron microscopy. Gold particles label EV71 antigen. Arrowheads indicate representative gold particles existed in the lumen of single membrane compartments without ILV at 20 mai, and representative gold particles accumulated in single membrane organelles containing ILVs at 30 mai. The experiments were repeated two times and the same trends were observed. Scale bars: 200 nm.

Fig. 6.

EV71 translocates into BMP-enriched LE at 30 mai. (A) RD-hSCARB2 cells were infected with EV71 and then fixed 20 min or 30 min after the infection followed by immunofluorescence. DAPI is in blue, EV71 antigen is in green, whereas BMP is labelled in red. The panels on the right are enlarged 3D cross-section views of the dashed rectangles in the merged panels. Arrowheads indicate co-localization of EV71 with BMP. Representative images are shown. Scale bars: 10 µm. (B) Co-localization rate of EV71 with BMP at 20 or 30 mai. **P<0.01. Statistical significance was determined by a two-tailed, unpaired t-test. Error bars represent s.d.

Fig. 7.

CD-M6PR is essential for EV71 replication. (A) Western blotting of RAB5, RAB7, RAB9, CD-M6PR, CI-M6PR and clathrin heavy chain in cells in which these genes have been targeted by siRNA knockdown. The expression of β-actin was examined as loading control. Triangles indicate molecular weight markers. Black dots indicate the predicted molecular weight of the proteins. The numbers are the densities and the ratio of anti-markers/anti β-actin. (B) RD cells were treated with siRNAs targeting RAB5, RAB7, RAB9, CD-M6PR, CI-M6PR or clathrin heavy chain and then infected with EV71-GFP. Cells were fixed at 31 h after infection, followed by fluorescent imaging. GFP-positive cells were counted and their percentage against the total number of DAPI-positive cells plotted. *P<0.05. Statistical significance was determined by a two-tailed, unpaired t-test. Error bars represent s.d.