Functional and Structural Dynamics of Hepadnavirus Reverse Transcriptase during Protein-Primed Initiation of Reverse Transcription: Effects of Metal Ions

Hepadnaviruses (hepatitis B viruses [HBVs]) are retroid viruses that replicate a small (ca. 3-kb) DNA genome via an RNA intermediate, the pregenomic RNA (pgRNA). The hepadnavirus reverse transcription pathway shows both similarities to and differences from that of classical retroviruses, including the mechanisms of nucleocapsid assembly and the initiation of DNA synthesis (40, 42, 45). To carry out their unique life cycle, the hepadnaviruses encode a specialized reverse transcriptase (RT) protein that displays a number of unique properties distinct from those of its retrovirus counterparts (23). Chief among these is its ability to initiate DNA synthesis de novo without the help of any DNA or RNA primer. Instead, a specific tyrosine residue within RT itself is used as the primer to initiate viral minus strand DNA synthesis (the so-called protein priming reaction) (31, 32, 52, 53, 56, 58). The unique ability of the hepadnavirus RT to carry out protein priming by using itself as a protein primer is reflected in its novel structural organization (11, 22, 23, 37). RT consists of four domains: from the N to the C terminus, they are the terminal protein (TP), the spacer, the RT, and the RNase H domains. TP is conserved among all hepadnaviruses but absent from any other known proteins. It is within TP where the aforementioned primer tyrosine residue is located. The functionally dispensable spacer connects TP to the central RT domain. Both the RT and RNase H domains share sequence homology with conventional RTs, including the YMDD active site motif in the RT domain and the catalytic D residues in the RNase H domain. Based on sequence alignment, structure modeling, and some genetic and biochemical data, the RT domain can be further divided into the finger, palm, and thumb subdomains, as in virtually all DNA and RNA polymerases (14, 41). Although no viral protein other than RT itself is required for protein priming in hepadnaviruses, there is an absolute requirement for a specific RNA template, the short RNA stem-loop structure, called ɛ, located on pgRNA (36, 53). The ɛ RNA bears two inverted repeat sequences and can fold into a conserved stem-loop structure, with a lower and an upper stem, an apical loop, and an internal bulge (16, 17, 22, 25, 28). Using the internal bulge of ɛ as the obligatory template and the specific tyrosine residue within its TP domain as the protein primer, RT synthesizes a 3- to 4-nucleotide (nt)-long DNA oligomer that is covalently attached to RT through the tyrosine residue (52, 53). Following the synthesis of this short DNA oligomer, the nascent DNA-RT complex is translocated from ɛ to an acceptor site near the 3′ end of pgRNA, from which reverse transcription continues (1, 2, 49, 51). Rather than serving simply as a passive template for protein priming, ɛ plays an active role in stimulating the enzymatic function of RT by inducing conformational changes in the latter (47, 48) while also undergoing significant conformational changes itself upon binding to RT (8, 10). Thus, a functional ribonucleoprotein (RNP) complex that is active in protein priming is formed only after both RT and ɛ undergo structural changes via an induced-fit mechanism. Adding to the importance of RT-ɛ interaction in hepadnavirus replication is its absolute requirement in packaging both RT and pgRNA into replication-competent nucleocapsids (4, 6, 18), where most, if not all, viral DNA synthesis occurs. As only a single hepadnavirus RT molecule is believed to be packaged into the viral nucleocapsid (5, 57) and thus able to carry out the complete synthesis of the viral DNA following nucleocapsid assembly, it seems plausible that a single RT polypeptide can serve both as the primer and as the catalyst for DNA synthesis. This is in contrast to other known protein priming reactions, where the protein primer and the DNA or RNA polymerase activity reside on separate polypeptides (e.g., in adenoviruses and picornaviruses) (39). As a result of this unique protein priming mechanism, RT remains covalently attached to the 5′ end of the viral minus strand DNA during the entire process of reverse transcription. The direct physical linkage of the primer and the catalyst likely imposes considerable constraint on the RT structure as it carries out protein priming and the subsequent series of reactions to produce the characteristic, partially double-stranded, relaxed circular DNA from the single-stranded pgRNA template. The intricate conformational gymnastics that RT likely undergoes during these complex reactions, and the additional factors likely involved, are poorly understood at present due to the dynamic nature of these processes and a lack of any high-resolution RT structures. Recent success in reconstituting hepadnavirus protein priming under cell-free and chemically defined conditions has provided experimentally tractable systems for elucidating the requirements for and the mechanisms of this complex reaction. In particular, a cellular chaperone complex consisting of heat shock protein 90 (Hsp90) and its cofactors (Hsp70, Hsp40, Hop, and p23) has been identified as a critical host factor required for protein priming by helping to establish and maintain an RT conformation competent for ɛ binding and protein priming (7, 20, 21, 24, 26, 27, 43). Interestingly, by removing the N-terminal third of TP, most of the spacer, part of the RT domain (the putative thumb subdomain), and the entire RNase H domain, we have recently isolated a truncated RT protein from the duck HBV (DHBV), MiniRT2, which no longer strictly depends on the host chaperones for protein priming (55). Strikingly, MiniRT2 is able to initiate protein priming by covalently attaching the first nucleotide (dGMP) to the primer tyrosine residue in TP but is unable to carry out any additional DNA synthesis to complete protein priming (54). This result suggests that protein priming can be further divided into two distinct steps: the covalent attachment of the first nucleotide to RT (initiation) and the subsequent addition of 2 or 3 nt to the initiating nucleotide (polymerization). The failure of MiniRT2 to carry out polymerization suggests either that it lacks the necessary sequences required for this step of protein priming or that an RT conformational change, which MiniRT2 fails to undergo, has to occur for the transition from initiation to polymerization during protein priming. Virtually all DNA and RNA polymerases require a divalent metal ion for catalysis, the choice of which is known to affect polymerase activities significantly (3, 12, 29, 35, 38, 50). We have tested potential effects of metal ions on hepadnavirus RT functions and report here that the choice of metal ions, Mn2+ versus Mg2+, had dramatic effects on protein priming by MiniRT2 and the full-length DHBV RT as well. Most dramatically, the presence of Mn2+, in contrast to that of Mg2+, allowed MiniRT2 to transition from the initiation to the polymerization stage of protein priming. The metal ions also affected the protein priming efficiency, the selection of nucleotide substrates, the template requirement, and the sensitivity of the protein priming reaction to a pyrophosphate analog RT inhibitor. The multiple effects of the metal ions on protein priming were most likely mediated by RT structural differences induced by the different ions, as revealed by the differential protease digestion patterns.