Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase

Abstract

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase-either expressed in Escherichia coli or highly purified from HeLa cells-phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.

A targeted approach to identification of vaccinia virus postreplicative transcription elongation factors: Genetic evidence for a role of the H5R gene in vaccinia transcription

“…The H5 protein has a predicted molecular weight of 22 kDa, but runs anomalously at 34 kDa on SDS gels. H5 is a substrate for the vaccinia virus coded B1 serine/threonine protein kinase and is multiply phosphorylated in vivo (Beaud et al, 1995;Brown et al, 2000;Beaud and Beaud, 2000;Boyle and Traktman, 2004). The H5 protein is synthesized in abundance throughout infection (Rosel et al, 1986); at early times is it distributed diffusely throughout the cytoplasm while at later times it is concentrated into virosomes and is ultimately packaged into virions (Beaud and Beaud, 1997;Murcia-Nicolas et al, 1999;Domi and Beaud, 2000).…”

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

“…The vaccinia protein H5R has been shown by isoelectric focusing to exist in at least four differently charged forms [ 11 , 13 ], suggesting that there are at least least five different phosphorylation sites on the protein. Consistent with this are the results of experiments (not shown) in which we employed recombinant H5R in which Thr-84 and Thr-85 had been replaced by Ala residues, and found that this still served as a substrate for the B1R protein kinase.…”

“…The preparation of authentic vaccinia H5R protein and recombinant B1R protein kinase were as previously described [ 11 ]. In some experiments a trpE-H5R fusion protein (pATH11-Ag35) was used [ 19 ].…”

“…Three proteins which become phosphorylated during infection of cells with vaccinia virus have been shown to be substrates of the B1R protein kinase in vitro . Two of these are the ribosomal proteins Sa and S2 [ 9 , 10 ] and the third is the product of the H5R open reading frame of the vaccinia virus genome [ 10 , 11 ].…”

Temperature-sensitive mutants with lesions in the vaccinia virus F10 kinase undergo arrest at the earliest stage of virion morphogenesis

“…In this regard, it is provocative that we have found that the H5 protein is a good substrate for F10-mediated phosphorylation in vitro (data not shown). The H5 protein (3,10,11) is an abundant phosphoprotein which is a major component of virosomes and of the membranes of intracellular mature virions; antisera to H5 can neutralize virion infectivity. Immunogold electron microscopy has revealed that H5 is associated with crescents and immature virions, and this association appears to persist even when the D13 protein is mutated and hence absent from nascent virion membranes.…”