The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed , and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its cochaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive Cterminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-tobe-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors. Reverse transcription in hepadnaviruses (hepatitis B viruses[HBVs]) is carried out by a novel virally encoded reverse transcriptase (RT) (27,30). The RT has the unique ability to initiate DNA synthesis de novo, using itself as a protein primer (18,19,35,39,43; for a review, see reference 9). This protein priming reaction requires the specific interaction between the RT and a short RNA signal, termed ε, located at the 5Ј end of the viral pregenomic RNA (pgRNA; the template for reverse transcription) (22,36). The product of protein priming is a three-to four-nucleotide DNA oligomer, representing the 5Ј end of the viral minus-strand DNA, covalently attached to the RT via an invariant tyrosine residue located at its N-terminal domain (19,21,32,34; for a review, see reference 11).The ability of the hepadnavirus RT to carry out specific RNA recognition and protein priming is reflected in its structural organization, which displays both similarities to, as well as differences from, conventional RTs encoded by retroviruses and other retroelements (4, 9, 23). As mentioned above, the N-terminal domain (the so-called terminal protein [TP]) bears the invariant primer tyrosine residue; the TP domain is conserved among all hepadnaviruses but absent from any other known RTs. The centr...

RNAs synthesized in vitro by purified Escherichia coli RNA polymerase from a bacteriophage 82 promoter are heterogeneous at the 5' end. We show that this heterogeneity results from variable addition of extra adenine residues, allowed by slippage of the initial oligonucleotide pppAAA-OH against its DNA template sequence TTT. Slippage backward by one base allows another A to be added, giving pppAAAA-OH, and this cycle can continue more than 20 times before it is ended by incorporation of UMP encoded by the fourth template base A. Slippage is abolished by mutation of the TTT template sequence to TGT and is sensitive to the concentrations of UTP and ATP in the reaction mixture. Analysis of deletions, substitutions, and point mutants implies that the slippage reaction requires only the existence of TTT at the initiation site of the template strand, although changes in neighboring nucleotides slightly affect its efficiency.