Heat Shock Protein 90-Independent Activation of Truncated Hepadnavirus Reverse Transcriptase

All hepatitis B viruses replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase, P protein, that carries a unique terminal protein (TP) domain. To initiate reverse transcription, P protein must bind to a stem-loop, , on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an -templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. binding requires the chaperone-dependent conversion of inactive P protein into an activated, metastable form designated P*. However, how P* differs structurally from P protein is not known. Here we used an in vitro reconstitution system for active duck hepatitis B virus P combined with limited proteolysis, site-specific antibodies, and defined P mutants to structurally compare nonactivated versus chaperone-activated versus primed P protein. The data show that Hsp70 action, under conditions identical to those required for functional activation, transiently exposes the C proximal TP region which is, probably directly, involved in RNA binding. Notably, after priming and RNA removal, a very similar new conformation appears stable without further chaperone activity; hence, the activation of P protein is triggered by energy-consuming chaperone action but may be completed by template RNA binding.Hepatitis B viruses (HBVs), or hepadnaviruses, are small enveloped DNA-containing viruses (12, 34) that replicate through protein-primed reverse transcription of a pregenomic RNA (pgRNA) intermediate (37). This unusual mechanism (4) is reflected in the structural organization of their reverse transcriptase (RT), P protein, which in addition to the conserved RT and RNase H (RH) domains (45) contains a unique terminal protein (TP) domain of about 200 amino acids not found in any other RT (Fig. 1A). TP is connected to the RT domain by a functionally dispensable spacer. Binding of P protein to a 5Ј-proximal stem-loop, ε (Dε for duck HBV [DHBV]), on the pgRNA triggers both encapsidation of the pgRNA and initiation of reverse transcription. Using the hydroxyl group of a specific Y residue in TP (Y63 in HBV [24], Y96 in DHBV [44,47]) rather than a tRNA 3Ј end as starting point, P protein generates a 3-to 4-nucleotide, ε-templated DNA oligonucleotide ("priming"; Fig. 1B). After this initiation phase, ε is replaced as a template by the 3Ј-proximal DR1* element on pgRNA and, primed by the TP-linked DNA oligonucleotide, minus-strand DNA is elongated from there. The end product of reverse transcription is a partially doublestranded, relaxed circular DNA in which the minus strand is still covalently linked to TP. This mechanism appears to hold for all hepadnaviruses, but most current knowledge is derived from DHBV (32), whose P protein, in contrast to that of HBV (17), is capable of performing the priming reaction in appropriate cell-free systems (2,6,16,19,41).Initial studies with P protein in vitro-translated in rabbit reticulocyte lysate (RL) revealed a strict...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Chaperones Activate Hepadnavirus Reverse Transcriptase by Transiently Exposing a C-Proximal Region in the Terminal Protein Domain That Contributes to ε RNA Binding

Ståhl

Beck

Nassal

2007

“…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). 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).…”

mentioning

confidence: 99%

“…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. (55). For the trans-complementation assay, equimolar amounts (1 pmol each) of the TP and RT domains were used.…”

mentioning

confidence: 99%

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

Self Cite

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 ...

“…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 ε 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).…”

mentioning

confidence: 99%

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

Wan

2008

Self Cite

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...