Proteolytic processing of the capsid precursor by trypsin is essential for porcine astrovirus infectivity and isolation in vitro

Porcine astroviruses (PAstV) have been prevalent worldwide, causing asymptomatic, intestinal or neurological clinical symptoms. However, the maturation mechanism and elements of the PAstV life cycle remains largely unknown, which poses an obstacle for PAstV isolation and pathogenic study. Previous studies have reported that PAstV’s isolation and replication in PK-15 cells requires the addition of trypsin, yet the detailed role of this protease has not been revealed. In this study, we found that trypsin could enhance the cytopathic effects and RNA replication of PAstV. The capsid precursor, of ∼90 kDa (VP90), could directly release into the extracellular culture media and subsequently processed by trypsin into four terminal products of about 25 (VP25), 27 (VP27), 30 (VP30) and 34 (VP34) kDa. This cleavage process was found to be essential for the infectivity of PAstV, as progeny viruses assembled from un-cleaved or incomplete processed capsid precursor protein lost its infectivity. Moreover, non-infectious progeny viruses regain infectivity after trypsin treatment. Unlike human astrovirus, which undergoes a "VP90-VP70" cleavage process, intracellular caspases were found to promote but are not required for PAstV-GX1 viral release. Virus purification confirmed that the VP34, VP30 and VP27 constitute mature, infectious viral particles. Importantly, sufficient processing under trypsin concentration, which produce VP27, VP30 and VP34, were proved to be components of PAstV mature virions. In general, PAstV's infectivity strictly depends on extracellular trypsin cleavage of its capsid precursor VP90 into VP25/27/30/34, bypassing the intracellular "VP90-VP70" pathway seen in Human Astrovirus—a novel maturation strategy in astroviruses.