Phosphorylation of Ser-80 of VP1 and Ser-254 of VP2 is essential for human BK virus propagation in tissue culture

Abstract

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...

The Human Fetal Glial Cell Line SVG p12 Contains Infectious BK Polyomavirus

“…Unfortunately, NCCR and full-genome sequencing shed little light on the origin of the contaminating virus, as BKPyV UT appears to be a relatively common variant, having been found in patient samples both in North Norway in 1990 (50) and in the United States in 2005 (51). Of note, this strain has also been used in several research labs (10,46,(72)(73)(74). Although we cannot pinpoint the infection temporally, we can be certain that BKPyV has been present in the cells at least since 2006 based on ATCC records.…”

Biology of the BKPyV: An Update

“…Key VP1 residues for BKPyV assembly and entry have been identified by site directed mutagenesis [ 50 , 51 ]. Phosphorylation of Ser-80 of VP1 has been shown to be crucial for BKPyV propagation [ 52 ]. The receptor-binding site of BKPyV has been predicted to be located in a shallow groove formed by the BC and HI loops of VP1 [ 4 , 50 ].…”

“…In contrast, the mutation of the start codons of the minor capsid proteins alone or together reduced infectivity by over 99% compared to the WT demonstrating that these proteins are essential to create infectious virions [ 54 ]. Phosphorylation of Ser-254 of VP2 has been suggested to be critical for BKPyV propagation [ 52 ].…”

Histone deacetylase III interactions with BK polyomavirus large tumor antigen may affect protein stability

“…Previously, several PTMs were found in BKPyV structural proteins and minichromosomes [ 22 , 23 ]. The phosphorylation of BKPyV structural proteins is essential for viral propagation [ 24 ], but BKPyV LT protein modification has not been fully investigated. Phosphorylation of LT affects SV40 replication efficiency and interferes with host signaling pathways [ 25 , 26 ].…”