Evidence that the 5 '-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA

Hepatitis B virus (HBV) replicates by reverse transcription of an RNA inter mediate, the pregenomic RNA. The first step of HBV genome replication is th e encapsidation of the pregenomic RNA encoding the encapsidation signal, te rmed epsilon, into the core particles, which is preceded by recognition and binding of HBV DNA polymerase to epsilon. The pregenomic RNA contains two identical epsilon elements due to its terminal redundancy: one near the 5' end and another near the 3' end. Despite the fact that both epsilon element s have an identical sequence, only the 5' epsilon, but not the 3' epsilon, is functional for encapsidation. To understand the molecular nature of this position effect, we made a series of lacZ RNA expression plasmids which co ntain the epsilon element at various positions from the 5' end of the trans cripts. Following transfection, the lacZ RNAs in cytoplasmic core particles were measured by RNase protection assay for encapsidation, The results ind icated that the lacZ RNAs with epsilon positioned up to 65 nucleotides from the 5' end were encapsidated, whereas the lacZ RNAs with epsilon positione d further downstream were not. Interestingly, the cap-free lacZ RNA transcr ibed by T7 RNA polymerase was not encapsidated, implying that the 5' cap st ructure is required for encapsidation of the pregenomic RNA. We hypothesize d that HBV DNA polymerase must somehow recognize the cap structure and/or i ts associated factors, as well as the 5' epsilon, for encapsidation to occu r.