Abstract
Enterovirus 71 (EV71), a member of the Picornaviridae family, may cause serious clinical manifestations associated with the central nervous system. Enterovirus 3C protease is required for virus replication and can trigger host cell apoptosis via cleaving viral polyprotein precursor and cellular proteins, respectively. Although the role of the 3C protease in processing viral and cellular proteins has been established, very little is known about the modulation of EV71 3C function by host cellular factors. Here, we show that sumoylation promotes EV71 3C protein ubiquitination for degradation, correlating with a decrease of EV71 in virus replication and cell apoptosis. SUMO E2-conjugating enzyme Ubc9 was identified as an EV71 3C-interacting protein. Further studies revealed that EV71 3C can be SUMO (small ubiquitin-like modifier)-modified at residue Lys-52. Sumoylation down-regulated 3C protease activity in vitro and also 3C protein stability in cells, in agreement with data suggesting 3C K52R protein induced greater substrate cleavage and apoptosis in cells. More importantly, the recombinant EV71 3C K52R virus infection conferred more apoptotic phenotype and increased virus levels in culture cells, which also correlated with a mouse model showing increased levels of viral VP1 protein in intestine and neuron loss in the spinal cord with EV71 3C K52R recombinant viral infection. Finally, we show that EV71 3C amino acid residues 45-52 involved in Ubc9 interaction determined the extent of 3C sumoylation and protein stability. Our results uncover a previously undescribed cellular regulatory event against EV71 virus replication and host cell apoptosis by sumoylation at 3C protease.
Highlights
Usually causes mild symptoms in childhood, such as herpangina or exanthema known as hand, foot, and mouth disease [1]
To further differentiate bands derived from multiple mono-sumoylation or from mono-sumoylation utilizing poly-SUMO chain formation, a SUMO-1 mutant (11KR) incapable of forming a polychain was subjected to in vitro sumoylation
3C was modified by SUMO-1 more effectively than SUMO-2 in vitro compared with PML conjugated by SUMO-1 or SUMO-2 to a similar extent
Summary
Usually causes mild symptoms in childhood, such as herpangina or exanthema known as hand, foot, and mouth disease [1]. Sumoylation at Lys-52 promoted 3C protein ubiquitination and degradation, correlating with a reduction of 3C substrate cleavage, apoptosis induction, and EV71 virus production in cells as well as EV71-elicited neurovirulence in a mouse model.
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