The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Structure of a replication intermediate in the synthesis of Rous sarcoma virus DNA in vivo

Intermediates in the synthesis of Rous sarcoma virus DNA in vivo contain a short second strand of DNA (plus strong-stop DNA) synthesized by using the region near the 5' end of the first (minus) strand of DNA as the template. In this report, we show that the 3' end of plus strong-stop DNA is extended about 15 to 20 nucleotides beyond the 5' end of the minus-strand DNA template, probably copying a portion of the tRNATrp molecule that serves as primer for synthesis of the minus strand of DNA. The extra sequences present in plus strong-stop DNA may play a central role in the generation of the long terminal repeat present in mature forms of viral DNA.