Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases

Abstract

The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32-to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.Rotavirus is an icosahedral virus belonging to the family Reoviridae and has a genome composed of 11 double-stranded RNA segments (21). One characteristic feature of rotavirus infection is the formation of punctate perinuclear structures called viroplasms 2 to 3 h into the infectious cycle (36). Viroplasms are sites of viral RNA replication and packaging of genome segments into progeny virions. Several rotavirus proteins (VP1, VP2, VP3, VP6, NSP2, NSP5, and NSP6) have been found in viroplasms during infection (25,47). Expression of NSP2 and NSP5 is reportedly required and sufficient for viroplasm formation (19,22). However, it has also been shown that expression of N-terminally tagged NSP5 alone results in the formation of viroplasm-like structures (32).NSP5 contains 198 amino acids with a predicted molecular mass of approximately 21 kDa. NSP5 is highly phosphorylated in infected cells resulting in a series of posttranslationally modified isoforms that range from 26 to 35 kDa (2). The initial modification that results in the shift from 21 to 26 kDa is unknown, but the appearance of 28-and 32-to 35-kDa isoforms from a 26-kDa precursor has been ascribed to O...

Phylogenetic analysis of nonstructural protein 5 (NSP5) gene sequences in porcine rotavirus B strains

“…2). Although the positions and mechanisms of phosphorylational modification of the NSP5 protein are still unclear, NSP5 is known to be activated by complex hyperphospholyration of serine residues among RVAs (Poncet et al, 1997;Afrikanova et al, 1998;Eichwald et al, 2002Eichwald et al, , 2004bSen et al, 2006;Bar-magen et al, 2007;Sotelo et al, 2010). While this Table 2 NSP5 sequence identities (%) in nucleotide (upper right) and amino acids (lower left) among genotypes grouped by phylogenetic analysis.…”

“…NSP5 has a high number of serine and threonine residues and various posttranslational modifications, like O-glycosylation and polyphosphorylation (Welch et al, 1989;Gonzalez and Burrone, 1991). Although there are some reports on the phosphorylational modification mechanisms of NSP5 protein involving autokinase and cellular kinases, the role of NSP5 phosphorylation is still unclear and elusive (Afrikanova et al, 1998;Eichwald et al, 2002Eichwald et al, , 2004bSen et al, 2006;Bar-Magen et al, 2007). NSP5 also binds to ssRNA and dsRNA without sequence specificity (Vende et al, 2002).…”

Rotavirus Induces Formation of Remodeled Stress Granules and P Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production

“…Interestingly, the phosphatase PPM1A, which negatively regulates host stress response pathways and antiviral defense mechanisms (84,85), is upregulated and is also localized to the VM in rotavirus-infected cells. The enhanced viral protein expression and progeny virus yield in cells in which PPM1A protein expression is downregulated by siRNA suggest that its phosphatase activity is detrimental to virus growth (86). The PP2C family phosphatases play important roles in several cellular processes, including nuclear transport, and their activities are regulated by posttranslational modifications, such as phosphorylation and N-myristoylation (87)(88)(89)(90).…”

Structural Organisation of the Rotavirus Nonstructural Protein NSP5

“…12 Cellular kinases, such as casein kinase I and II, have also been reported to phosphorylate NSP5. [13][14][15] Moreover, the interaction of NSP2 with NSP5 has been shown to stimulate NSP5 phosphorylation. 16 The role of NSP5 phosphorylation is still elusive, even though it was suggested to be involved in regulating the fate of mRNA between translation and replication 1 7 and in RNA selective encapsidation.…”