The spleen necrosis virus int gene product expressed in Escherichia coli has DNA binding activity and mediates att and U5-specific DNA multimer formation in Vitro

Abstract

To facilitate the in vitro study of the spleen necrosis virus (SNV) int gene product, we expressed the viral int locus in an Escherichia coli expression vector. Antiserum made against the protein produced in bacteria precipitated a 44-kDa polypeptide from virus-infected chicken embryo fibroblasts. This result is consistent with the expected size of the SNV int polypeptide. In a protein blotting assay, the expressed protein binds strongly to DNA and was able to complex nonspecifically with both single- and double-stranded DNAs containing or lacking viral sequences. However, under solution conditions favoring transient DNA unwinding, DNA binding was confined to supercoiled molecules containing either the SNV att sequence (the viral cis-acting region required for integration) or the U5 region of the long terminal repeat alone. Under these conditions of specific binding, multimeric DNA species were formed by apparent intermolecular interaction between protein-DNA complexes. These data indicate that retroviral integration may require local DNA unwinding at the att site for complex formation between the int gene product and DNA. This complex may be an intermediate in the viral DNA insertion process.