Abstract:Phosphorylation of the polyomavirus major capsid protein VP1 was examined after in vivo 32p labeling of virus-infected cells. Two phosphorylated peptide fragments of VP1 were identified by protease digestion, high-performance liquid chromatography purification, mass spectrometry, and N-terminal sequencing. The peptides from residues 58 to 78 and residues 153 to 173 were phosphorylated on threonine. Site-directed mutations were introduced at these threonine sites, and mutant viruses were reconstructed. A threon… Show more
“…Of the 17 serine and 23 threonine residues in total present in the agno-1a polypeptide chain, 14 of these are in favourable context with regard to the consensus sequences (Pinna, 1990 ; Roack, 1991 ; Li & Garcea, 1994 ) of either protein kinase C (1), casein kinase II (4) and proline-directed protein kinases (PDPK) (9) (see Fig. 2 Fig.…”
The avian polyomavirus budgerigar fledgling disease virus (BFDV) encodes an unusual set of four agnoproteins in its late upstream region. Of the two pairs of these proteins, which overlap each other in two different reading frames, the p L1 -promoted agnoprotein-1a (agno-1a) is the dominant species and is able to support virus propagation in the absence of the other three polypeptides. Viral BFDV agno-1a, and also agno-1a expressed via an influenza virus vector, consists of a complex series of electrophoretically separable subspecies that can be reduced by phosphatase action down to a primary unphosphorylated protein with an apparent molecular mass of 31 kDa. Through peptide mass spectrometry and site-directed mutagenesis, the positions of four serine and three threonine residues have been determined as phosphate-accepting groups, which are partially modified by the combined action of three different cellular kinases. Since extensively phosphorylated agno-1a is required for its intracellular function, control over VP protein expression, and unphosphorylated agno-1a is observed as an additional component in the BFDV virion, both extreme subspecies appear to be drawn from that complex mixture, which also includes the intermediate stages of phosphorylation.
“…Of the 17 serine and 23 threonine residues in total present in the agno-1a polypeptide chain, 14 of these are in favourable context with regard to the consensus sequences (Pinna, 1990 ; Roack, 1991 ; Li & Garcea, 1994 ) of either protein kinase C (1), casein kinase II (4) and proline-directed protein kinases (PDPK) (9) (see Fig. 2 Fig.…”
The avian polyomavirus budgerigar fledgling disease virus (BFDV) encodes an unusual set of four agnoproteins in its late upstream region. Of the two pairs of these proteins, which overlap each other in two different reading frames, the p L1 -promoted agnoprotein-1a (agno-1a) is the dominant species and is able to support virus propagation in the absence of the other three polypeptides. Viral BFDV agno-1a, and also agno-1a expressed via an influenza virus vector, consists of a complex series of electrophoretically separable subspecies that can be reduced by phosphatase action down to a primary unphosphorylated protein with an apparent molecular mass of 31 kDa. Through peptide mass spectrometry and site-directed mutagenesis, the positions of four serine and three threonine residues have been determined as phosphate-accepting groups, which are partially modified by the combined action of three different cellular kinases. Since extensively phosphorylated agno-1a is required for its intracellular function, control over VP protein expression, and unphosphorylated agno-1a is observed as an additional component in the BFDV virion, both extreme subspecies appear to be drawn from that complex mixture, which also includes the intermediate stages of phosphorylation.
“…In addition, the phosphorylated residues Thr-63 and Thr-156 of polyomavirus VP1 are located in the B-C and D-E loops, respectively. These two threonine residues have been shown to be exposed on the exterior viral surface (Li & Garcea, 1994). The construction of a virus with a Thr-156 to alanine mutation resulted in virions with an assembly defect (Li & Garcea, 1994).…”
Section: Discussionmentioning
confidence: 99%
“…These two threonine residues have been shown to be exposed on the exterior viral surface (Li & Garcea, 1994). The construction of a virus with a Thr-156 to alanine mutation resulted in virions with an assembly defect (Li & Garcea, 1994). The phosphorylated residues Ser-80, Ser-133 and Ser-327 of BK VP1 were known to be located in the B-C loop, D-E loop and C-terminal region (Dugan et al, 2007;Liddington et al, 1991).…”
Section: Discussionmentioning
confidence: 99%
“…The phosphorylated residues of VP1 of polyomavirus have been identified at Ser-66, Thr-63 and Thr-156 (Li & Garcea, 1994;Li et al, 1995). Mutation of Thr-156 results in an assembly defect in vivo (Garcea & Benjamin, 1983;Li & Garcea, 1994). Furthermore, Ser-66 of VP1 is the substrate of casein kinase II during phosphorylation (Li et al, 1995).…”
ROCBK virus (BKV) infection may cause polyomavirus-associated nephropathy in patients with renal transplantation. Recently, the phosphorylated amino acids on the structural proteins VP1, VP2 and VP3 of BKV have been identified by liquid chromatography-tandem mass spectrometry in our laboratory. In this study, we further analysed the biological effects of these phosphorylation events. Phosphorylation of the BKV structural proteins was demonstrated by [32 P]orthophosphate labelling in vivo. Site-directed mutagenesis was performed to replace all of the phosphorylated amino acids. The mutated BKV genomes were transfected into Vero cells for propagation analysis.The results showed that expression of the early protein LT and of the late protein VP1 by the mutants VP1-S80A, VP1-S80-133A, VP1-S80-327A, VP1-S80-133-327A and VP2-S254A was abolished. However, propagation of other mutants was similar to that of wild-type BKV. The results suggest that phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is crucial for BKV propagation.
INTRODUCTIONPolyomaviruses are non-enveloped small DNA viruses and have a circular dsDNA genome of approximately 5.2 kbp. The polyomavirus family includes murine polyomavirus, simian virus (SV40), human John Cunningham virus (JCV), BK virus (BKV), KI virus, WU virus and Merkel cell polyomavirus (MCV) (Allander et al., 2007;Eash et al., 2006;Feng et al., 2008;Gardner et al., 1971;Gaynor et al., 2007;Kanitakis, 2008; Pérez-Losada et al., 2006). The genomes of these polyomaviruses contain regulatory, early and late regions. The regulatory region includes the replication origin as well as a promoter and an enhancer that regulate the transcription of the early and late genes. The early region encodes the regulatory proteins large tumour antigen (LT) and small tumour antigen, which are associated with replication and the regulation of the early and late genes. The late region encodes three structural capsid proteins, VP1, VP2 and VP3, and the regulatory agonoprotein (Safak et al., 2001). The viral capsid is composed of the major structural protein, VP1, the minor structural proteins, VP2 and VP3, and a viral minichromosome (Christiansen et al., 1977). The viral capsid consists of 72 pentameric capsomeres, including 12 pentavalent and 60 hexavalent capsomeres (Liddington et al., 1991).The structural proteins of polyomavirus play important roles in the infection life cycle, such as recognition of host receptors, haemagglutination activity (Bolen & Consigli, 1979;Bolen et al., 1981) and encapsidation of the viral genome during maturation (Chang et al., 1993;Gasparovic et al., 2006;Kawano et al., 2006). Furthermore, interactions among the structural proteins are also crucial for virus entry, nuclear translocation, DNA packaging and assembly of viral progeny (Chen et al., 1998;Delos et al., 1995;Gasparovic et al., 2006;Gharakhanian et al., 2003;Kawano et al., 2006;Shishido-Hara et al., 2004). However, it has been found that the structural proteins are modified into several subspecies, as demonstrated by t...
“…Besides its mitogenic function in primary cells transfected with large-T antigen, middle-T antigen has been shown to increase the efficiency of virus encapsidation, presumably by maintaining high activity levels of kinases of the mitogen-activated protein kinase family (27,33). An accumulation of activated mitogenactivated protein kinases in the nucleus has been suggested to phosphorylate VP1, resulting in increased encapsidation of the virus (12,16). Dimerization and binding of 14-3-3 proteins to middle-T antigen may be means for fine-tuning the interactions of this viral protein with the cellular signalling network.…”
The oncogenic protein of polyomavirus, middle-T antigen, associates with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like dl8 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
