Effects of genomic length on translocation of hepatitis B virus polymerase-linked oligomer

Accurate translocation of the polymerase-linked oligomer to the acceptor si te (DR1*) in reverse transcription is crucial for maintaining the correct s ize of the hepatitis B virus (HBV) genome, Various sizes of foreign sequenc es were inserted at different sites of the HBV genome, and their effects on accurate translocation of polymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in these insertion muta nts: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicated that the minus strand of RC and type I DL form was elong ated from DR1*, while the minus strand of the type II DL form was elongated from multiple internal acceptor sites (WS), such as IAS2, These IASs were also found to be used by wild-type HBV but with a very low frequency. Mutat ion of IAS2 by base substitution abrogated polymerase-linked oligomer trans ferring to IAS2, demonstrating that base pairing also plays an important ro le in the function of IAS2 as a polymerase-linked oligomer acceptor site. D ata obtained from our insertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site and the acceptor is imp ortant. The polymerase-linked oligomer prefers to translocate to an accepto r, DR1* or IAS2, which are ca. 3.2 kb apart. However, it will translocate t o both DR1* and IAS2 if they are not located 3.2 kb apart. These results su ggest that the polymerase-linked oligomer may be able to scan bidirectional ly for appropriate acceptor sites at a distance of 3.2 kb. A model is propo sed to discuss the possible mechanism of polymerase-linked oligomer translo cation.