Protein-primed RNA synthesis by purified poliovirus RNA polymerase

Paul, Aniko V.; Boom, Jacques H. van; Filippov, Dmitri V.; Wimmer, Eckard

doi:10.1038/30529

Cited by 344 publications

(380 citation statements)

References 27 publications

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“…The identification of cryptic priming sites on RT also suggests that RT may be able to use other proteins, viral or host, as primers to initiate DNA synthesis. As mentioned above, the RNA-dependent RNA polymerase of poliovirus also uses the protein primer VPg, which is not covalently linked to the polymerase, to initiate viral RNA synthesis (32). Similar to what we found for the DHBV RT protein, the poliovirus RNA polymerase could also use priming site(s) on the polymerase protein itself to initiate RNA synthesis, i.e., self uridylylation or the covalent attachment of UMP to the RNA polymerase (37).…”

Section: Discussionsupporting

confidence: 58%

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: Discussionsupporting

confidence: 58%

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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“…A Comparison of the in Vitro Uridylylation Reaction in the Presence and Absence of an RNA Template-The presence of an RNA template is an absolute requirement for PV VPg uridylylation, in contrast to rabbit hemorrhagic disease virus (8,28). Our result suggests that under the experimental conditions used, the PVA system did not require a template.…”

Section: Resultsmentioning

confidence: 73%

“…Reaction conditions were essentially as described previously (8). The reactions were performed at room temperature (RT ϭ 22°C) for 25 min in a final volume of 30 l containing 0.75 Ci of [␣-32 P]UTP (3000 Ci/mmol; Amersham Biosciences), 2 g of PVA VPg, a varying amount of NIb (0 -0.3 g), 10 mM HEPES, pH 7.5, 2.5 mM MnCl 2 , 2 units of RNasin (Promega), and 0.5 g of poly(A) n (n ϭ 35-200; Amersham Biosciences) when required.…”

Section: Recombinant Protein Expression-(his)mentioning

confidence: 99%

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Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Puustinen

Mäkinen

2004

Journal of Biological Chemistry

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“…RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12)(13)(14), although in vitro it has been shown to lack specificity for viral RNA because it readily utilizes heterologous nonviral templates (9). Like poliovirus (15), the HCV RdRp is capable of initiating viral RNA synthesis in vitro by a primer-dependent mechanism (9,10,16). However, Flaviviridae RdRps have also been shown to initiate RNA synthesis by a de novo mechanism (12,13,(17)(18)(19).…”

mentioning

confidence: 99%

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

Johnston

Gutshall

et al. 2003

Journal of Biological Chemistry

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In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of ⌬21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNAbound form of ⌬21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.

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Protein-primed RNA synthesis by purified poliovirus RNA polymerase

Cited by 344 publications

References 27 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

Uridylylation of the Potyvirus VPg by Viral Replicase NIb Correlates with the Nucleotide Binding Capacity of VPg

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

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