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

Ståhl, Michael; Beck, Jürgen; Nassal, Michael

doi:10.1128/jvi.01196-07

Cited by 56 publications

(93 citation statements)

References 46 publications

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“…(2) This finding supports that the hypothesis that the pol-core interaction requires e binding by pol, and is in agreement with a pol-dependent cytoplasmic core retention. It further suggests that e binding changes the structure of pol aside from the already documented changes caused by pol-hsp70 and 90 interaction (Stahl et al, 2007). (3) The observation that pol becomes activated by binding to its RNA (Tavis & Ganem, 1996) supports the hypothesis that the supposed structural change of pol is essential for the viral life cycle.…”

Section: Discussionmentioning

confidence: 49%

“…Pol requires interactions with different heat-shock proteins (at least hsp40, hsp70 and hsp90; Beck & Nassal, 2003;Hu et al, 2004;Stahl et al, 2007) for binding to e. Subsequently the pol-e complex becomes encapsidated into the assembling capsids but only if core is phosphorylated at Ser 162 (Gazina et al, 2000). Conversion to DNA is facilitated by pol and occurs inside the capsid requiring phosphorylation of core at further sites (Gazina et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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Biopsies from patients show that hepadnaviral core proteins and capsids -collectively called core -are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that e, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

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Section: Discussionmentioning

confidence: 49%

Section: Introductionmentioning

confidence: 99%

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Deroubaix

Osseman

Cassany

et al. 2015

Journal of General Virology

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“…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).…”

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

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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“…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).…”

mentioning

confidence: 99%

“…Currently, very little is known about the structural details of hepadnavirus RT proteins, due to the lack of any high-resolution structural data. On the other hand, limited proteolysis, which leads to more-frequent cleavages at surface-accessible sites than at less accessible sites, has been used with some success to reveal the RT conformational maturation as induced by cellular chaperones (43) and ε binding (47,48). We therefore decided to adopt this approach in an attempt to directly demonstrate any RT structural differences induced by Mn 2ϩ versus Mg 2ϩ as well as the presumed RT structural transition from initiation to polymerization.…”

mentioning

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

Cited by 56 publications

References 46 publications

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus

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

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