Downloaded from www.microbiologyresearch.org by IP: 54.237.57.119 On: Sat, 23 Apr 2016 22:57:31 Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1a is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication Michela Campagna, 1 Mauricio Budini, 2 Francesca Arnoldi, 1 Ulrich Desselberger, 1 Jorge E. Allende 2 and Oscar R. Burrone 1 Correspondence Oscar R. Burrone burrone@icgeb.org 1 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34011 Trieste, Italy 2 Instituto de Ciencias Biomedicas, Programa de Biologia Celular y Molecular, Facultad de Medicina, Universidad de Chile, Independencia 1027, 8380453 Santiago, Chile Received 13 February 2007 Accepted 19 June 2007 The rotavirus (RV) non-structural protein 5, NSP5, is encoded by the smallest of the 11 genomic segments and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus because, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is produced early in infection and undergoes a complex hyperphosphorylation process, leading to the formation of proteins differing in electrophoretic mobility. The role of hyperphosphorylation of NSP5 in the replicative cycle of rotavirus is unknown. Previous in vitro studies have suggested that the cellular kinase CK1a is responsible for the NSP5 hyperphosphorylation process. Here it is shown, by means of specific RNA interference, that in vivo, CK1a is the enzyme that initiates phosphorylation of NSP5. Lack of NSP5 hyperphosphorylation affected neither its interaction with the virus VP1 and NSP2 proteins normally found in viroplasms, nor the production of viral proteins. In contrast, the morphology of viroplasms was altered markedly in cells in which CK1a was depleted and a moderate decrease in the production of double-stranded RNA and infectious virus was observed. These data show that CK1a is the kinase that phosphorylates NSP5 in virus-infected cells and contribute to further understanding of the role of NSP5 in RV infection. INTRODUCTION Rotaviruses are non-enveloped, triple-layered particles (TLPs) that contain a genome of 11 segments of double- stranded RNA (dsRNA) encoding six structural proteins (VP1–VP4, VP6 and VP7) and six non-structural proteins (NSP1–NSP6). Early viral morphogenesis is concentrated in discrete cytoplasmic inclusion bodies called ‘viroplasms’, in which VP1, VP2, VP3 and VP6 accumulate, together with two of the non-structural proteins, NSP2 and NSP5. Both NSP2 and NSP5 are essential for virus replication. In cells in which NSP5 is depleted by specific RNA interference against NSP5 mRNA (Campagna et al., 2005; Lopez et al., 2005) or neutralized by anti-NSP5-specific intrabodies (Vascotto et al., 2004), there is no formation of viroplasms and no virus replication. Similar results were obtained by blocking NSP2 production using specific small interfering RNA (siRNA; Silvestri et al., 2004). Rotavirus NSP5, which is encoded by RNA segment 11, is a protein of 196–198 aa (depending on virus strain) with a high proportion of serine (21 %) and threonine (4.5 %). NSP5 has been shown to interact with NSP2 and the viral polymerase VP1 (Afrikanova et al., 1998; Arnoldi et al., 2007). NSP5 undergoes post-translational modifications that include cytoplasmic O-glycosylation (Gonzalez & Burrone, 1991) and a complex pattern of hyperphos- phorylation (Afrikanova et al., 1996; Blackhall et al., 1997). As a consequence, NSP5 from virus-infected cells appears, after SDS-PAGE, as two major bands of 26 and 28 kDa and a series of slower-migrating bands with apparent molecular masses between 30 and 34 kDa. It has been shown that the 26 kDa form of NSP5 is the precursor of the higher- molecular-mass forms, corresponding to species character- ized by increasing levels of phosphorylation (Afrikanova et al., 1996; Blackhall et al., 1997). Several studies that have addressed the mechanism of NSP5 phosphorylation Journal of General Virology (2007), 88, 2800–2810 DOI 10.1099/vir.0.82922-0 2800 0008-2922 G 2007 SGM Printed in Great Britain