THE HEPATITIS-B VIRUS HBX PROTEIN IS A DUAL-SPECIFICITY CYTOPLASMIC ACTIVATOR OF RAS AND NUCLEAR ACTIVATOR OF TRANSCRIPTION FACTORS

The HBx protein of hepatitis B virus (HBV) is a transcriptional activa tor that is required for infection and may play an important role in H BV-associated hepatocarcinogenesis. Recently, we and others have shown that HBx stimulates the Ras-Raf-MAP kinase cascade, which leads to en hanced cell proliferation and the activation of transcription factors AP-1 and NF-kappa B, Other studies have shown that HBx can activate tr anscription by interacting directly with nuclear components of the tra nscription machinery, Therefore we examined the basis for the differen t reported activities of HBx, Here, we show that HBx is a complex prot ein, displaying independent activities in different intracellular loca tions, The intracellular distribution of HBx protein was first investi gated using scanning confocal laser immunomicroscopy and by genetic st udies, Our work has established that HBx expressed in cultured cells i s found authentically in both the cytoplasm and the nucleus, HBx is no t strongly associated with any intracellular structures, but some pref erential accumulation was observed near the cell surface, Next, HBx va riants were constructed containing a functional or mutant nuclear loca lization sequence, We show that when HBx is engineered to relocate exc lusively to the nucleus, it no longer activates the Ras-Raf-MAP kinase cascade, nor does it activate transcription factors AP-1 and NF-kappa B. Surprisingly, nuclear HBx fully retains the ability to stimulate H BV enhancer I, which is activated independently of the Ras and protein kinase C pathways, Therefore HBx protein stimulates signal transducti on pathways in the cytoplasm and transactivates transcription elements in the nucleus, Furthermore, SV40 T antigen is shown to induce the nu clear sequestration of HBx protein and to block its activation of NF-k appa B, demonstrating that HBx is regulated by proteins that alter its intracellular distribution. The conflicting functions of HBx protein in viral infection and possibly carcinoma may involve the regulation o f its differential distribution in the cell.