THE PROTEIN P-30, ENCODED AT THE GAG-PRO JUNCTION OF MOUSE MAMMARY-TUMOR VIRUS, IS A DUTPASE FUSED WITH NUCLEOCAPSID PROTEIN

A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30. The protein consists of tw o domains, the zinc-finger-containing nucleocapsid (NC) protein portio n with 95 residues and a C-terminal extension comprising 154 residues. The C-terminal domain shows similarity in sequence with the enzyme dU TPase from other sources. In this paper, we demonstrate that p30 is a functional dUTPase. Overproduction of the NC protein in Escherichia co il, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of rea dthrough. By a 1-base insertion, eliminating the gag stop codon and fu sing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed. The overproduced protein, pur ified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity. Analytical gel filtration and sequence homology to o ther dUTPases suggest a trimeric assembly of p30 subunits. MMTV thus p ossesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain o f dUTPase. (C) 1994 Academic Press, Inc.