3D Multivesicular Structures: Triggered Permeability by Integrin-mediated Picornavirus Entry

Abstract

We elucidated the structural features of vMVBs (virus-induced multivesicular body) compared to antibody-induced control as mock infection. High-pressure cryo fixation of cells is employed to conduct the study with immunoelectron tomography during early virus entry. ILV (intraluminal vesicle) grew concurrently with the dramatic alterations in vMVB size and membrane integrity brought on by EV1 infection. ILV breakages started to appear at 2 hours post-injury, followed by vMVB limiting membrane rupture. These breakages likely enable effective replication and are connected to EV1 genome egress from vMVBs. The increased internalization of EV1 appears to be caused by a2b1-integrin clustering, where the cellular uptake further develops membrane compartments forming MVBs. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and ILVs at 2 and 3.5 hours post-infection (p.i.), in contrast to the control MVBs without the virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h, suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of the viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Here, the rupture of endosomes upon the entry of a non-enveloped enterovirus unveils how the viral genome is transferred to the cytoplasm for subsequent replications.