CHARACTERIZATION OF ESSENTIAL DOMAINS FOR THE FUNCTIONALITY OF THE MHBS(T) TRANSCRIPTIONAL ACTIVATOR AND IDENTIFICATION OF A MINIMAL MHBS(T) ACTIVATOR

Integrated hepatitis B virus DNA derived from hepatocellular carcinoma s can express, in one third of the cases investigated so far, a transc riptional activator encoded from 3' terminal truncated surface (preS/S ) genes resulting in a C-terminally truncated middle surface protein ( MHBs(t). Since MHBs(t), in contrast to the secreted MHBs, is retained in the secretory pathway at the ER, the question as to whether the ret ention generates the transcriptional activator function was investigat ed. Through fusion of MHBs to the ER-retention signal KDEL, it was sho wn that the intracellular retention does not generate the transcriptio nal activator function. Tryptic digestions of microsomal vesicles reve aled that the amino terminal domain of MHBs(t) directs into the cytopl asmic compartment, whereas in MHBs this domain directs into the lumen of the ER. This structural difference appears to be why transcriptiona l activator function arises. Through deletion analysis it was shown th at non-membrane-associated MHBs(t) proteins are also functional activa tors. Non-membrane associated MHBs(t) proteins represent a second clas s of MHBs(t) proteins. These MHBs(t)-proteins are homogenously distrib uted all over the cell and show no difference in functionality as comp ared to the membrane-associated MHBs(t) proteins. MHBs(t53) (truncated at aa53) was shown to be a minimal activator of this class. Both clas ses of MHBs(t) proteins were found to form dimers; an amphiphatic alph a helix was identified within aa 41-52, which is involved in mediating the dimerization. The integrity of this domain was also revealed to b e a prerequisite for the functionality of the activator, suggesting a linkage between dimerization and functionality.