Echovirus 1 infection depends on biogenesis of novel multivesicular bodies

Abstract

SummaryNon-enveloped picornavirus echovirus 1 (EV1) clusters its receptor a2b1 integrin and causes their internalization and accumulation in a2b1 integrin enriched multivesicular bodies (a2-MVBs). Our results here show that these a2-MVBs are distinct from acidic late endosomes/lysosomes by several criteria: (i) live intra-endosomal pH measurements show that a2-MVBs are not acidic, (ii) they are not positive for the late endosomal marker LBPA or Dil-LDL internalized to lysosomes, and (iii) simultaneous stimulation of epidermal growth factor receptor (EGFR) and a2b1 integrin clustering leads to their accumulation in separate endosomes. EGFR showed downregulation between 15 min and 2 h, whereas accumulation of a2b1 integrin/EV1 led to an increase of integrin fluorescence in cytoplasmic vesicles further suggesting that EV1 pathway is separate from the lysosomal downregulation pathway. In addition, the results demonstrate the involvement of ESCRTs in the biogenesis of a2-MVBs. Overexpression of dominant-negative form of VPS4 inhibited biogenesis of a2-MVBs and efficiently prevented EV1 infection. Furthermore, a2-MVBs were positive for some members of ESCRTs such as Hrs, VPS37A and VPS24 and the siRNA treatment of TSG101, VPS37A and VPS24 inhibited EV1 infection. Our results show that the non-enveloped EV1 depends on biogenesis of novel multivesicular structures for successful infection.

Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

“…In CV-1 monkey kidney cells, virus moves rapidly to caveolin-positive structures within the cell (35); infection appears to involve cholesterol, caveolin, and dynamin, but perturbation of actin dynamics has no effect (35). In SAOS-␣2 cells, a human osteosarcoma cell line engineered to express VLA-2, EV1 first enters tubular-vesicular structures within the cytoplasm and ultimately appears in unusual multivesicular bodies, which accumu- late both caveolin and fluid-phase markers (32,36). Agents that interfere with macropinocytosis inhibit infection of SAOS-␣2 cells, either by preventing the internalization of virions (as seen with dominant negative CtBP1) or by preventing traffic to multivesicular bodies (as seen with EIPA) (32,33).…”

Coxsackievirus A9 Infects Cells via Nonacidic Multivesicular Bodies

“…We previously showed that the last component of ESCRTs, the ATPase Vps4 (35), inhibits EV1 infection and EV1-MVB function (15). Therefore, we also wanted to examine the role of Vps4 in CVA9 infection.…”

“…In order to gain more accurate information of the pHs of the endosomes along the infectious CVA9 pathway, we performed an intraendosomal pH measurement assay (15,16). In the assay, a pH-stable AF-555 conjugate and a pH-sensitive fluorescein isothiocyanate (FITC) conjugate were introduced to forming CVA9-positive endosomes.…”

“…Intraendosomal pH measurements were conducted as previously described (15). Briefly, CVA9 was first bound to A549 cells on ice, followed by incubation with rabbit polyclonal anti-CVA9 antibody.…”

“…However, the cellular structures used by CVA9 for internalization and uncoating are still poorly known. Recently, we showed that another enterovirus, EV1, does not enter the host cell in acidic endosomes (15,16). Instead, EV1 enters multivesicular bodies (MVBs) that morphologically resemble proportion of infected cells that reacted with the anti-CVA9 antibody was then calculated.…”