Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death

Background: 3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus. Results: Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis. Conclusions: 3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.