Serine protein kinase activity associated with rotavirus phosphoprotein NSP5

Abstract

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32 P i , only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28-to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [␥-32 P]ATP resulted in 32 P labeling of 28-to 35-kDa NSP5. The distribution of 32 P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

“…4.2(b)} shows that NSP5 is predicted to have long IDPRs in its N-terminal region, and within the C-terminal domain that contains the C X C motif (C171 and C174) responsible for the iron-sulfur cluster organization. There are various MoRF regions present in NSP5 protein of rotavirus SA11 and other strains that are involved in the functionality of potential protein kinase C phosphorylation sites (S-X-R/K-R/K-X-X-S and K/R-X-S) at serine residues 22, 30, 75, 100, and 136 [131] [91]. Rotavirus was also demonstrated to induce atypical stress granules and P-bodies during Table 3 The infection rate (in percentage) of different serotypes of rotavirus based on the geography and temporal regions.…”

Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories

“…that involves several distinct Ser residues [19,20]. The NSP5 117 hyperphosphorylation is a complex process, which gives rise to multiple 118 phosphorylation states ranging from the most abundant 28 kDa phospho-isoform, 119 up to the hyperphosphorylated 32-34 kDa states [19,20]. All these forms have 120 been found to be more stable in viroplasms, while chemical disruption of 121 viroplasms results in NSP5 de-phosphorylation [21].…”

“…109Biochemical evidence suggests that viroplasms are essential for RV replication 110 since the virus production is highly impaired upon silencing of either NSP2 or 111 NSP5 [12][13][14][15]. 112 Rotavirus NSP5, encoded by genome segment 11, is a small serine (Ser)-and 113 threonine (Thr)-rich non-structural protein that undergoes multiple post-114 translational modifications in virus-infected cells, including O-linked glycosylation 115 [16], N-acetylation [17], SUMOylation [18] and crucially hyperphosphorylation 116that involves several distinct Ser residues [19,20]. The NSP5 117 hyperphosphorylation is a complex process, which gives rise to multiple 118 phosphorylation states ranging from the most abundant 28 kDa phospho-isoform, 119 up to the hyperphosphorylated 32-34 kDa states [19,20].…”

“…Furthermore, we have investigated the mechanism of 54 INTRODUCTION threonine (Thr)-rich non-structural protein that undergoes multiple post-114 translational modifications in virus-infected cells, including O-linked glycosylation 115 [16], N-acetylation [17], SUMOylation [18] and crucially hyperphosphorylation 116 that involves several distinct Ser residues [19,20]. The NSP5 117 hyperphosphorylation is a complex process, which gives rise to multiple 118 phosphorylation states ranging from the most abundant 28 kDa phospho-isoform, 119 up to the hyperphosphorylated 32-34 kDa states [19,20]. All these forms have 120 been found to be more stable in viroplasms, while chemical disruption of 121 viroplasms results in NSP5 de-phosphorylation [21].…”

Phosphorylation events during viral infections provide potential therapeutic targets

“…Phosphorylation of a cellular kinase can also influence its interaction with other proteins — for example, tyrosine phosphorylation of specific residues within Src‐family kinases is required for Src's interaction with proteins' SH2 domains . In Table , rotavirus NSP5 , HCMV UL97 , EBV BGLF4 , and HSV‐1 UL13 have all displayed an ability to autophosphorylate, although the full effects of these autophosphorylations on protein activity remain under investigation. As reviewed in Michel and Mertens , HCMV UL97 is autophosphorylated, but there are conflicting data regarding the role of this autophosphorylation in UL97's ability to phosphorylate and interact with other proteins.…”