Expression and Regulation by Interferon of a Double-Stranded-RNA-Specific Adenosine Deaminase from Human Cells: Evidence for Two Forms of the Deaminase

Abstract

A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of ;6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific aden-osine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.