Analysis of Rotavirus Nonstructural Protein NSP5 Phosphorylation

Abstract

The rotavirus nonstructural phosphoprotein NSP5 is encoded by a gene in RNA segment 11. Immunofluorescence analysis of fixed cells showed that NSP5 polypeptides remained confined to viroplasms even at a late stage when provirions migrated from these structures. When NSP5 was expressed in COS-7 cells in the absence of other viral proteins, it was uniformly distributed in the cytoplasm. Under these conditions, the 26-kDa polypeptide predominated. In the presence of the protein phosphatase inhibitor okadaic acid, the highly phosphorylated 28- and 32- to 35-kDa polypeptides were formed. Also, the fully phosphorylated protein had a homogeneous cytoplasmic distribution in transfected cells. In rotavirus SA11-infected cells, NSP5 synthesis was detectable at 2 h postinfection. However, the newly formed 26-kDa NSP5 was not converted to the 28- to 35-kDa forms until approximately 2 h later. Also, the protein kinase activity of isolated NSP5 was not detectable until the 28- and 30- to 35-kDa NSP5 forms had been formed. NSP5 immunoprecipitated from extracts of transfected COS-7 cells was active in autophosphorylation in vitro, demonstrating that other viral proteins were not required for this function. Treatment of NSP5-expressing cells with staurosporine, a broad-range protein kinase inhibitor, had only a limited negative effect on the phosphorylation of the viral polypeptide. Staurosporine did not inhibit autophosphorylation of NSP5 in vitro. Together, the data support the idea that NSP5 has an autophosphorylation activity that is positively regulated by addition of phosphate residues at some positions.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles

“…However, although a mechanism of concerted phosphorylation involving several proteins of viroplasms might be necessary for subsequent viroplasm maturation and functioning, our data suggest that NSP5 hyperphosphorylation is necessary, but not sufficient, for viroplasm formation. Thus, treatment of uninfected cells expressing recombinant NSP5 with inhibitors of cellular phosphatases leads to NSP5 hyperphosphorylation, but not to VLS formation (36).…”

Nonstructural proteins involved in genome packaging and replication of rotaviruses and other members of the Reoviridae

“…The molecular basis of the phosphorylation of NSP5 has been the subject of many studies (Afrikanova et al, 1996;Poncet et al, 1997;Blackhall et al, 1998;Afrikanova et al, 1998;Torres-Vega et al, 2000;Eichwald et al, 2002). Despite these efforts, the source of the kinase activity responsible for the hyperphosphorylation of NSP5 has not been fully resolved.…”

“…In addition to studies providing results that NSP2 plays an important role in NSP5 phosphorylation, other studies have provided results that cellular kinases and phosphatases contribute to the protein's phosphorylation (Blackhall et al, 1997;Eichwald et al, 2002). For example, Blackhall et al (1998) have shown that the presence of staurosporine, a strong inhibitor of protein kinase C and a wide range of protein kinases, decreases the extent of NSP5 phosphorylation in the infected cell. These investigators also noted that the addition of the phosphatase inhibitor, okadaic acid, to infected cells results in an increase in the phosphorylated isoforms of NSP5.…”

Rotavirus Enfeksiyonunun Moleküler Patogenezi

“…Yapısal olmayan protein NSP5, Serin ve Threonin kalıntılarından zengin dimerik bir fosfoproteindir ve enfeksiyondan 2 saat sonra tespit edilebilir (10). NSP5'in görevi replikasyon ve paketleme işlemi sırasında NSP2 ile bağlanmayı düzenlemektir.…”