Model of potential roles of autophagosome-amphisome in PV production and cell exit.
We have demonstrated that initiation of autophagy is required for viral RNA replication, while vesicle acidification is required for maturation of virus particles. In our model, by 2–3 h post-infection processed 3A protein integrates into single-membraned structures resembling secretory vesicles, which become the initial sites of viral RNA replication. 3-MA either inhibits formation of these single-membraned vesicles which act as the site of RNA replication, or inhibits the progress of these vesicles into double-membraned vesicles, which act as replication sites. As the peak of viral genome replication (3 h) passes, the autophagic machinery is engaged to change the single-membraned vesicles into double-membraned vesicles. As a result, some of RNA replication complexes and capsid proteins are trapped within the double-membraned vesicles, while some are cytosolic. As the amphisomes acidify, particle maturation is more efficient inside the vesicles. Maturation of the amphisomes can be inhibited using NH4Cl or bafilomycin A1. The mature virions, surrounded by two membranes, have a topological problem in exiting the cell. We propose three possible mechanisms for exit. [1] The outer membrane fuses with the plasma membrane, releasing a short-lived labile single-membraned vesicle containing virus, which eventually is released into the extracellular milieu. [2] The double-membraned vesicles lose one membrane, as occurs during maturation of cellular autophagosomes, and the single-membraned vesicles fuse with the plasma membrane. [3] Virus exits the vesicle into the cytoplasm due to breakup of the vesicle or another mechanism. Virus then exits the cell when lysis occurs at approximately 8 h post-infection.
