Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo.

Abstract

In rotavirus-infected cells, the non-structural proteins NSP5 and NSP2 localize in complexes called viroplasms, where replication and assembly occur. Recently, we have demonstrated direct interaction of NSP5 with NSP2, and as a consequence of that, up-regulation of NSP5 hyperphosphorylation. To investigate a possible structural role for the NSP2-NSP5 interaction, we analysed the cytoplasmic distribution of the two proteins in transfected cells by immunofluorescence using specific antibodies. Here we report that NSP2 and NSP5 can drive the formation of viroplasm-like structures (VLS) in the absence of other rotaviral proteins and rotavirus replication. Several NSP5 deletion mutants were constructed and expressed in combination with NSP2. Both the N-and C-terminal domains of NSP5 were found to be essential for VLS formation. Only one mutant, with an internal deletion of residues 81-130, was able to interact with NSP2 to form VLS. Analysis of the phosphorylation capacity of the different mutants in vivo indicated that hyperphosphorylation of NSP5 is necessary, but not sufficient, for VLS formation. Our results suggest a role for the non-structural protein NSP5 in the structure of viroplasms mediated by its interaction with NSP2.

Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo

“…The formation of viral inclusion bodies, or viroplasms, is a common feature of double-stranded (ds)RNA viruses from the family Reoviridae with genomes composed of 10-12 segments (Brookes et al, 1993;Fabbretti et al, 1999;Fukushi et al, 1962;Petrie et al, 1984;Rhim et al, 1962;Shikata & Kitagawa, 1977;Touris-Otero et al, 2004). These inclusion bodies are mainly composed of viral dsRNA, viral proteins and partially and fully assembled viral particles (Dales et al, 1965;Fabbretti et al, 1999;Isogai et al, 1998;Rhim et al, 1962;Silverstein & Schur, 1970;Touris-Otero et al, 2004;Wei et al, 2006).…”

“…These inclusion bodies are mainly composed of viral dsRNA, viral proteins and partially and fully assembled viral particles (Dales et al, 1965;Fabbretti et al, 1999;Isogai et al, 1998;Rhim et al, 1962;Silverstein & Schur, 1970;Touris-Otero et al, 2004;Wei et al, 2006). In addition, the inclusion bodies have been shown to contain microtubules and thinner 'kinky' filaments suggested to be intermediate filaments (Dales, 1963;Dales et al, 1965;Spendlove et al, 1964).…”

“…In addition, the inclusion bodies have been shown to contain microtubules and thinner 'kinky' filaments suggested to be intermediate filaments (Dales, 1963;Dales et al, 1965;Spendlove et al, 1964). Although the formation mechanism of these inclusions is largely unknown, they are thought to play an important role in viral infections because they are probable sites of viral genome replication, protein synthesis and virus assembly (Fabbretti et al, 1999;Isogai et al, 1998;Petrie et al, 1984;Rhim et al, 1962;Wei et al, 2006).…”

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

“…In addition, in viroplasms, NSP5 interacts with other viral proteins such as NSP2 (Poncet et al, 1997;Afrikanova et al, 1998), VP1 (Afrikanova et al, 1998) and VP2 (Berois et al, 2003). The in vivo interaction with NSP2 leads to NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs) (Afrikanova et al, 1998;Fabbretti et al, 1999). We have recently shown that the hyperphosphorylation is the consequence of a cellular kinase that is activated by NSP5 itself in a process resembling autophosphorylation (Eichwald et al, 2002).…”

“…An NSP2-encoding cDNA fragment, obtained by PCR amplification using specific primers to incorporate NcoI and BamHI restriction sites at the N and C termini, respectively, was cloned into pVOTE.1 (Ward et al, 1995) to obtain the construct pVOTE.1/NSP2. Constructs pT 7 v-NSP5, pT 7 v-D1, pT 7 v-D2, pT 7 v-D3, pT 7 v-D4, pT 7 v-DT, pT 7 v-D1/D2 and pT 7 v-D4T have been described previously (Afrikanova et al, 1998;Fabbretti et al, 1999;Eichwald et al, 2002). The internal deletion plasmids pT 7 v-D2/D4T, pT 7 v-D2/D3 and pT 7 v-D3/DT were obtained by PCR using specific internal primers and cloned into KpnI and BamHI restriction sites in pcDNA3 (Invitrogen).…”