DNA-binding Proteins Specified by Herpesvirus Saimiri

Abstract

SUMMARYHerpesvirus saimiri-specific proteins from the nuclear fractions of productively infected owl monkey kidney cells were dissociated from virus and host DNA by treatment with 2 M-NaCI or separation on Urografin density gradients. Empty virus capsids remained intact and could be separated from major non-structural proteins (110K, 51K and 48K) and from a subset of structural proteins (130K, 29K and 12K), either by Urografin gradient sedimentation or differential centrifugation. The DNA in such soluble extracts of nuclear proteins was efficiently removed by spermine precipitation, together with the host cell histones and large fractions of the 130K and 12K structural proteins. Proteins in the spermine-soluble fraction were analysed by affinity chromatography on columns of single-stranded calf thymus DNA coupled to cellulose. Two major structural proteins (130K and 12K), whose synthesis was sensitive to phosphonoacetic acid (PAA), and one minor PAA-resistant structural protein (29K) bound to DNA-cellulose. The major PAA-resistant 11 OK non-structural protein and the PAA-resistant non-structural 51K and 48K phosphoproteins were efficiently released into the spermine-soluble fraction and also bound to DNA-cellulose as did the 76K protein and minor species of 42K, 39K, 34K, 25K and 21K. Virus-specific proteins were eluted from such columns by buffers containing 0.4 M-NaC1 or by heparin in lowsalt buffers. Polypeptides from virus particles, infected cell extracts, or samples of eluates from DNA-cellulose chromatography, were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose filters and probed for their ability to bind labelled polynucleotides. The non-structural 51K phosphoprotein, the 12K and 29K structural proteins and a 100K virion polypeptide all bound labelled DNA. However, the binding activities of the 130K protein from virions or purified by affinity chromatography and of the 11 OK polypeptide could not be demonstrated reproducibly after transfer from SDS gels to nitrocellulose. Comparisons of the present results on the properties of the herpesvirus saimiri-specified DNA-binding proteins with published accounts of the DNA-binding proteins of other herpesviruses, suggest some striking similarities with the DNA-binding proteins of the Epstein-Barr virus.

Bovine Parvovirus DNA-binding Proteins: Identification by a Combined DNA Hybridization and Immunodetection Assay

“…The specificity of these methods has been validated by comparing DNA binding to proteins on membranes with the same interaction studied by other means such a filter binding assays and affinity chromatography. These confirmatory studies have been carried out for human plasma fibronectin (Hoch, 1982), the lac repressor (Bowen et al, 1980) and several structural and non-structural proteins of herpesvirus saimiri (Blair & Honess, 1983). However, binding of adenovirus type 5 DNA to the terminal protein on a membrane was not detected (Russell & Precious, 1982).…”

Asynchronous Expression of the Immediate-Early Protein of Herpesvirus Saimiri in Populations of Productively Infected Cells

“…51K and 110K non-structural DNA-binding proteins and virus-specific thymidine kinase and DNA polymerase activities) and finally by the synthesis of progeny virus DNA and proteins of the third phase, the late gene products (e.g. 160K, 130K and 150K structural proteins : Randall et al, 1983Randall et al, , 1984aHoness et al, 1982;O'Hare & Honess, 1983a, b;Blair & Honess, 1983). However, the growth cycle of the virus and the synthesis of independently regulated virus proteins is relatively protracted in populations of productively infected cells (e.g.…”

Post-translational modification of the tegument proteins (VP13 and VP14) of herpes simplex virus type 1 by glycosylation and phosphorylation

“…These two polypeptides exhibit Mr polymorphism in different strains of HSV-1, the fluctuation in apparent Mr of one polypeptide always being reflected by the other (Pereira et al, 1976). Evidence has appeared which suggests that VP 13, VP 14 and VP22 associate with the nuclear matrix during the virus replication cycle (Pinard et al, 1987) and that they are able to bind HSV DNA (Blair & Honess, 1983). VP 13/14 has been shown to be the product of the gene UL47 (Whittaker et al, 1991) and it shares amino acid homology with gp 10, a glycosylated tegument protein of equine herpesvirus type 1 (EHV-1).…”