THE DUCK HEPATITIS-B VIRUS POLYMERASE IS ACTIVATED BY ITS RNA PACKAGING SIGNAL, EPSILON

The epsilon stem-loop at the 5' end of the pregenomic RNA of the hepat itis B viruses is both the primary element of the RNA packaging signal and the origin of reverse transcription. We have previously presented evidence for a third essential role for epsilon, that of an essential cofactor in the maturation of the viral polymerase (J. E. Tavis and D . Ganem, J. Virol. 70:5741-5750, 1996). In this case, binding of epsil on to the polymerase is proposed to induce a physical alteration to th e polymerase that is needed for it to develop enzymatic activity. Thre e lines of evidence employing duck hepatitis B virus supporting this h ypothesis are presented here. First, an unusual DNA polymerase activit y employing exogenous RNAs (the trans reaction) that was originally di scovered with recombinant duck hepatitis B virus polymerase expressed in Saccharomyces cerevisiae yeasts was shown to be an authentic proper ty of the viral polymerase. The trans reaction was found to be templat e dependent reverse transcription of the exogenous RNA, The trans reac tion occurred independently of the hepadnavirus protein-priming mechan ism, yet it was still strongly stimulated by epsilon. This directly de monstrates a role for epsilon in activation of the polymerase. Second, the reverse transcriptase domain of the polymerase was shown to be ph ysically altered following binding to epsilon, as would be expected if the alteration was required for maturation of the polymerase to an en zymatically active form. Finally, analysis of 15 mutations throughout the duck hepatitis B virus polymerase demonstrated that the epsilon-de pendent alteration to the polymerase was a prerequisite for DNA primin g, reverse transcription, and the traits reaction.