Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

Wang, Xingtai; Hu, Jianming

doi:10.1128/jvi.76.12.5857-5865.2002

Cited by 25 publications

(46 citation statements)

References 36 publications

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“…Therefore, the results obtained with both omission of dNTPs and inclusion of ddNTPs were consistent with the notion that the DNA polymerization product synthesized, under these conditions, from priming sites in the TP (mostly from the cryptic site or sites, see Fig. 7 below) and RT (exclusively from the cryptic site or sites) domains had the sequence d(GTA(A)), the same as the polymerization product previously shown for the authentic Y96 site (50,54), and thus was likely templated by the same ε internal bulge sequence (UUAC).…”

Section: Resultssupporting

confidence: 74%

“…Since the cryptic sites in the TP and RT domains were able to initiate DNA synthesis, we were interested in determining whether these sites could also support subsequent DNA polymerization. We have recently shown that MiniRT2 was able to carry out DNA polymerization, i.e., the extension of the single dGMP attached to the protein to form a short (several nucleotides long) DNA oligomer in the presence of Mn 2ϩ but not in the presence of Mg 2ϩ (27,54). Therefore, we performed transcomplementation in Cryptic site priming shared the same nucleotide preference as the authentic site priming.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Boregowda

Zhu

et al. 2011

J Virol

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Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a transcomplementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.The Hepadnaviridae family includes the hepatitis B virus (HBV), a global human pathogen that chronically infects hundreds of millions and causes a million fatalities yearly, and related animal viruses such as the duck hepatitis B virus (DHBV) (40). Hepadnaviruses contain a small (ϳ3-kb), partially double-stranded DNA genome that is replicated through an RNA intermediate, called pregenomic RNA (pgRNA) by reverse transcription (39, 43). The virally encoded reverse transcriptase (RT) is unique among known RTs in its structure and function (14). RT is composed of four domains. The Nterminal TP (terminal protein) is conserved among all hepadnaviruses but absent from all other known RTs and is required for viral reverse transcription. The spacer domain, which does not have any known role in RT functions, connects TP to the central RT domain and the C-terminal RNase H domain. Both the RT and the RNase H domains share sequence homology with other RTs, including the RT active-site motif YMDD and the catalytic RNase H residues (4, 5, 35, 57).A prerequisite for the initiation of reverse transcription in hepadnaviruses is the specific interaction between RT and a short RNA signal called ε located at the 5Ј end of pgRNA (33, 51). RT-ε interaction is required to activate the polymerase activity of RT and ε also serves as the template for the initial stage of viral reverse transcription (13,16,44,46,47,49).Instead of relying on an RNA primer to prime DNA synthesis, as is the case with most other RTs and DNA polymerases in general, the hepadnavirus RT uses a specific Y residue in the TP ...

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Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Boregowda

Zhu

et al. 2011

J Virol

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“…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).…”

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confidence: 99%

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

Wan

2008

J Virol

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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...

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Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

et al. 2022

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Hepatitis B virus (HBV) chronically infects >250 million people. It replicates by a unique protein‐primed reverse transcription mechanism, and the primary anti‐HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains compared to only two in most reverse transcriptases: the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviruses, HBV P has resisted structural analysis for decades. Here, we exploited computational advances to model P. The TP wrapped around the RT domain rather than forming the anticipated globular domain, with the priming tyrosine poised over the RT active site. The orientation of the RT and RNase H domains resembled that of the retroviral enzymes despite the lack of sequences analogous to the retroviral linker region. The model was validated by mapping residues with known surface exposures, docking nucleic acids, mechanistically interpreting mutations with strong phenotypes, and docking inhibitors into the RT and RNase H active sites. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses infecting rodent to fish hosts and a nackednavirus, but not in other non‐retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will advance mechanistic analyses into the poorly understood enzymology of HBV reverse transcription and will enable drug development against non‐active site targets for the first time.

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Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses

Cited by 25 publications

References 36 publications

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

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

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

Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

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