Cytoplasmic membrane-bound vesicles in echovirus 12-infected cells

LLC-MK2 continuous line of rhesus monkey kidney cells infected with echovirus 12 were incubated with or without the inhibitor of viral RNA synthesis, 2-(α-hydroxybenzyl)-benzimidazole (HBB) and were examined by light and electron microscopy at intervals during a single cycle of viral replication. In the absence of HBB, cytoplasmic membrane-bound vesicles were first seen by electron microscopy in cells 4–5 hours after infection and increased in number with time after infection. Cytopathologic changes, observed by phase microscopy, developed 5–6 hours after infection. The addition of HBB to cultures at the time of infection of 1–2 hours later prevented the development of cytoplasmic membrane-bound vesicles and cytopathogenesis over a 7-hour period, but did not prevent the virus-induced disaggregation of cellular polyribosomes. However, if HBB was not added until 3 or 4 hours after infection, the development of membrane-bound vesicles was not prevented. No viral hemagglutinating particles were produced during a period of time equal to three cycles of viral replication when HBB was added to cells at 3 hours or less after infection and only 10% of maximal viral yields were produced when the compound was added 4 hours after infection. Since cytoplasmic membrane-bound vesicles (seen by electron microscopy) and viral cytopathogenesis (seen by light microscopy) (1) develop at closely parallel times after infection, (2) are both probably not direct consequences of virus-induced disaggregation of host-cell polyribosomes, and (3) both require for development the initiation of active viral biosynthesis, it is concluded that the formation of membrane-bound vesicles is a major ultrastructural reflection of viral cytopathogenesis.