Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Abstract

e Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasmlike structures. Rotavirus, a member of the Reoviridae family, is the major etiological cause of severe gastroenteritis of viral origin in infants and young children. The infective virion consists of a nonenveloped triple-layered particle (TLP). Inside the inner layer, composed by pentamers of the structural protein VP2, are contained the 11 double-stranded RNA (dsRNA) segments of the viral genome, the RNA-dependent RNA polymerase VP1, and the RNA capping enzyme VP3, altogether forming the core of the virus. Around the core is present a second intermediate layer, composed by the structural protein VP6, forming a double-layered particle (DLP) that is surrounded by the third outermost layer composed by the proteins VP7 and VP4 forming the fully assembled infectious TLP.Upon virus entry in the host cell, the outermost layer of the virus is lost and DLPs become active in transcribing the viral mRNA from the dsRNA genome, acting VP1 also as a transcriptase. Even though it has been shown in vitro that the minimal requirement for viral replication is represented by VP1 and VP2 (1, 2), in vivo replication and packaging occur in viral factories, called viroplasms (3). These structures are formed, apart from VP1 and VP2, also by the other structural proteins necessary for the formation of the DLPs, VP3 and VP6, and two nonstructural proteins, NSP2 and NSP5. Both nonstructural proteins are essential for viroplasm formation and virus replication (4-6), but while NSP2 has been proposed to be the molecular motor responsible of the packaging of rotavirus genome in newly synthesized cores (7,8), the role for NSP5 is less clear. The NSP5 protein, synthesized by the smallest segment...

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