Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thoug ht to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of as sociation of chronic DHBV infection with hepatocellular carcinoma developme nt supports this belief. Here, we demonstrate that DHBV genomes have a hidd en open reading frame from which a transcription-regulatory protein, design ated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhanc es neither viral protein expression, intracellular DNA synthesis, nor virio n production when assayed in the full-length genome context in LMH cells. H owever, similar to mammalian hepadnavirus X proteins, DHBx activates cellul ar and viral promoters via the Raf-mitogen-activated protein kinase signali ng pathway and localizes primarily in the cytoplasm. The functional similar ities as,well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellul ar localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinoge nesis, and imply unique opportunities for deciphering of their still-enigma tic in vivo functions.