The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6

Abstract

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted α-helical structure. A deletion mutant lacking the 10 C-terminal aa (∆C10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the ∆C10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant ∆C10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains

“…The formation of these particular cytoplasmic structures reflects the abilities of the two nonstructural proteins to interact with each other. On the other hand, in the context of viral infection, the situation is more complex and localization can also involve the interaction of mutants with wild-type NSP5 (31). Contrary to what was observed with VLS formation, all NSP5 mutants containing regions 4 and T localized to viroplasms independently of the phosphorylation state.…”

“…6). This is most likely due to interaction with wild-type NSP5, which was shown to depend on the last 10 carboxy-terminal amino acids (31). We have now determined that localization to VLS was only obtained when ⌬2-EGFP was cotransfected with NSP2 into a cell line stably expressing NSP5 (MA104-C7) (2) and not when it was cotransfected into a cell not expressing NSP5 (data not shown).…”

“…We have previously reported that in vivo hyperphosphorylation of NSP5 is up-regulated by NSP2 (1). Based on indirect evidence, we and others have suggested that NSP5 has autophosphorylation activity (1,6,9,25,31).In this report, we address the phosphorylation, kinase activity, and viroplasm localization of NSP5. Five important aspects were characterized: (i) activation of an NSP5-specific cellular kinase(s) responsible for NSP5 hyperphosphorylation, (ii) NSP5 domains involved in kinase activation, (iii) NSP5 phosphorylation sites, (iv) NSP5 phosphorylation by casein kinase II (CKII), and (v) carboxy-terminal domains responsible for NSP5 localization to viroplasms.…”

“…We have previously reported that in vivo hyperphosphorylation of NSP5 is up-regulated by NSP2 (1). Based on indirect evidence, we and others have suggested that NSP5 has autophosphorylation activity (1,6,9,25,31).…”

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

“…NSP5 is a fosfo-glycoprotein that presents different isoforms that differ in the phosphorylation level with molecular masses from 28 to 32-34 KDa (Afrikanova et al 1996, Blackhall et al 1997, Poncet et al 1997). NSP2 and NSP5 are located in the viroplasms of rotavirus infected cells, interact with each other and also with other viral proteins, NSP2 with the viral RNA polymerase VP1, and NSP5 with VP2 and NSP6 (Aponte et al 1996, Berois et al 2003, Torres-Vega et al 2000. The co-expression of NSP2 and NSP5 in cells, in the abscence of other viral proteins, results in viroplasm like-structures, VLS (Fabretti et al 1999), however when expressed individually they produce a diffuse distribution, hence these proteins are considered the minimum components of the viroplasms.…”