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

Abstract

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed . Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) 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 stemloop structure, called ε, located on pgRNA (36, 53). The ε 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 ε 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...

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

“…Independent TP and RT domains are known to trans-complement each other to carry out the protein priming reaction using the TP domain as a protein primer to initiate DNA synthesis (1,25,27). When the priming reactions were carried out in the presence of Mn 2ϩ , which is known to stimulate priming relative to Mg 2ϩ (27), we found, surprisingly, that the RT domain, as well as TP, acted as a protein primer for initiating DNA synthesis (Fig. 3).…”

“…However, a truncated DHBV RT protein, MiniRT2, with deletion of the entire RNase H domain, the N-terminal third of the TP domain, and most of the spacer, retains ε RNA binding and protein priming activity but no longer requires the host chaperones (55). Recently, we reported that the divalent metal ions, Mg 2ϩ versus Mn 2ϩ , have a dramatic effect on protein priming, including the overall priming efficiency, the template and nucleotide specificity, and the transition from initiation to polymerization, further attesting to the multiple RT conformational transitions that occur during protein priming (27).…”

“…Both the TP and the RT domains are essential for RT-ε interaction and protein priming but the RNase H domain is dispensable (10,23,33,51). Also, isolated TP and RT domains, when combined together, are able to reconstitute a functional RT protein that is able to carry out protein priming in a trans-complementation reaction (1,23,25,27). Protein priming can be further subdivided into two different stages, requiring distinct RT conformations, the initial covalent attachment of the first nucleotide of the minus-strand DNA (a dGMP in DHBV) to RT (to the primer Y) called initiation and the subsequent addition of the remaining 2 to 3 nucleotides to the initiating nucleotide called polymerization (27,54).…”

“…Also, isolated TP and RT domains, when combined together, are able to reconstitute a functional RT protein that is able to carry out protein priming in a trans-complementation reaction (1,23,25,27). Protein priming can be further subdivided into two different stages, requiring distinct RT conformations, the initial covalent attachment of the first nucleotide of the minus-strand DNA (a dGMP in DHBV) to RT (to the primer Y) called initiation and the subsequent addition of the remaining 2 to 3 nucleotides to the initiating nucleotide called polymerization (27,54).DHBV RT-ε interaction and protein priming have been reconstituted in vitro using purified components. The fulllength RT requires the assistance of the host cell chaperone proteins in order to establish and maintain a conformation that is competent to recognize the ε RNA and to initiate protein priming (9,10,13,15,17,18,41,42).…”

“…Protein priming reactions were carried out as previously described (27,50). Briefly, 1 pmol of purified RT or 5 l of the in vitro translation reaction was mixed with 6 pmol DHBV ε RNA (10) or 1 g of yeast tRNA, 1ϫ EDTA-free protease inhibitor cocktail (Roche), 0.…”