A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro

Wang, Chen; Villion, Manuela; Semper, Cameron; Coros, Colin J.; Moineau, Sylvain; Zimmerly, Steven

doi:10.1093/nar/gkr397

Cited by 52 publications

(63 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Structurally, how HP accommodates ssDNA products during the template-independent transferase reaction, as opposed to the doublestranded template-primer complex during template-directed DNA synthesis, remains to be understood but presumably involves different HP conformations induced by Mn 2ϩ versus Mg 2ϩ . We note that a protein-primed transferase activity has been identified, tentatively, for one other polymerase, an RT-like protein carried by some bacteria (64). Interestingly, this bacterial protein, like HP, is able to synthesize DNAs of up to several hundred nt long.…”

Section: Figmentioning

confidence: 79%

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Jones

2013

J Virol

View full text Add to dashboard Cite

Hepatitis B virus (HBV) replication requires reverse transcription of an RNA pregenome (pgRNA) by a multifunctional polymerase (HP). HP initiates viral DNA synthesis by using itself as a protein primer and an RNA signal on pgRNA, termed epsilon (H), as the obligatory template. We discovered a Mn 2؉ -dependent transferase activity of HP in vitro that was independent of H but also used HP as a protein primer. This protein-primed transferase activity was completely dependent on the HP polymerase active site. The DNA products of the transferase reaction were linked to HP via a phosphotyrosyl bond, and replacement of the Y63 residue of HP, the priming site for templated DNA synthesis, almost completely eliminated DNA synthesis by the transferase activity, suggesting that Y63 also serves as the predominant priming site for the transferase reaction. For this transferase activity, HP could use all four deoxynucleotide substrates, but TTP was clearly favored for extensive polymerization. The transferase activity was highly distributive, leading to the synthesis of DNA homo-and hetero-oligomeric and -polymeric ladders ranging from 1 nucleotide (nt) to >100 nt in length, with single-nt increments. As with H-templated DNA synthesis, the protein-primed transferase reaction was characterized by an initial stage that was resistant to the pyrophosphate analog phosphonoformic acid (PFA) followed by PFA-sensitive DNA synthesis, suggestive of an HP conformational change upon the synthesis of a nascent DNA oligomer. These findings have important implications for HBV replication, pathogenesis, and therapy. H epatitis B virus (HBV)is an important human pathogen and a member of the family Hepadnaviridae, which comprises a group of pararetroviruses replicating a double-stranded DNA genome through an RNA intermediate called pregenomic RNA (pgRNA) by a multifunctional viral polymerase protein (HP), a specialized reverse transcriptase (RT) (1-4). Structurally, HP is divided into four domains, including (from the N to the C terminus) the terminal protein (TP), spacer, RT, and RNase H domains (5-7). Although the RT and RNase H domains are conserved with those found in other RT enzymes, TP is unique to hepadnaviruses, and the spacer has no known function other than serving as a tether between the TP and RT domains (5, 7-11). As with other RTs, HP has RNA-and DNA-dependent DNA polymerase activity. Furthermore, HP also serves as a protein primer; specifically, a highly conserved Y residue in its TP domain (Y63) is used as a primer to initiate viral minus-strand DNA [(Ϫ)-DNA] synthesis (12, 13). This protein-primed reverse transcription requires a specific HBV RNA template called epsilon (Hε), located at the 5= end of the pgRNA, which is specifically recognized by HP and is thought to be an integral component of the HP holoenzyme (14-21). HP-Hε interactions trigger conformational changes in both the protein and RNA of the resulting ribonucleoprotein (RNP) complex, which are thought to be required functionally for HP to gain catalytic activity a...

show abstract

Section: Figmentioning

confidence: 79%

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Jones

2013

J Virol

View full text Add to dashboard Cite

show abstract

“…Point mutations in the RT motifs, including the predicted catalytic aspartate residues, disrupt or abolish abortive infection, consistent with AbiK being a polymerase (95,96).…”

Section: Abikmentioning

confidence: 70%

“…Even without an RNA or DNA template, long DNAs were polymerized by AbiK having a "random" sequence, making AbiK's activity analogous to a terminal transferase (96). The newly synthesized DNA was covalently attached to the AbiK protein, because an amino acid of AbiK was the primer for polymerization.…”

Section: Abikmentioning

confidence: 99%

“…When ORF570 of ECOR30 was expressed on a plasmid in E. coli strain BL21(DE3), the strain was rendered resistant at levels of 10 -7 relative to infection by T5 phage, but there was no effect on infection by lambda, T2, T4, or T6 phages (106). The expressed ORF570 was reported to have RT activity in a biochemical assay, but the assays were with crude extracts under conditions where artifactual incorporation can occur (96).…”

Section: Abia and Abi-p2mentioning

confidence: 99%

See 1 more Smart Citation

An Unexplored Diversity of Reverse Transcriptases in Bacteria

Zimmerly

2015

Microbiol Spectr

Self Cite

View full text Add to dashboard Cite

Reverse transcriptases (RTs) are usually thought of as eukaryotic enzymes, but they are also present in bacteria and likely originated in bacteria and migrated to eukaryotes. Only three types of bacterial retroelements have been substantially characterized: group II introns, diversity-generating retroelements, and retrons. Recent work, however, has identified a myriad of uncharacterized RTs and RT-related sequences in bacterial genomes, which exhibit great sequence diversity and a range of domain structures. Apart from group II introns, none of these putative RTs show evidence of active retromobility. Instead, available information suggests that they are involved in useful processes, sometimes related to phages or phage resistance. This article reviews our knowledge of both characterized and uncharacterized RTs in bacteria. The range of their sequences and genomic contexts promises the discovery of new biochemical reactions and biological phenomena.

show abstract

“…Similarly, a tailed bacteriophage, podovirus J, was reported to adsorb to the sides of plasmid-encoded pili (26). Plasmids, on the other hand, might encode factors that abolish viral replication in a number of different ways (45,69,114,282). Viruses return the favor by encoding antirestriction proteins or evolving toward resistance to the detrimental effects of these factors (25,245,281).…”

Section: Genomic Relationship To Other Mobile Genetic Elementsmentioning

confidence: 99%

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

Krupovìč

Prangishvili

Hendrix

et al. 2011

Microbiol Mol Biol Rev

216

176

View full text Add to dashboard Cite

SUMMARY Prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet Earth with human beings (among others). However, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. An understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. The vast majority of characterized prokaryotic viruses belong to the order Caudovirales, double-stranded DNA (dsDNA) bacteriophages with tails. Consequently, these viruses have been studied (and reviewed) extensively from both genomic and functional perspectives. However, albeit numerous, tailed phages represent only a minor fraction of the prokaryotic virus diversity. Therefore, the knowledge which has been generated for this viral system does not offer a comprehensive view of the prokaryotic virosphere. In this review, we discuss all families of bacterial and archaeal viruses that contain more than one characterized member and for which evolutionary conclusions can be attempted by use of comparative genomic analysis. We focus on the molecular mechanisms of their genome evolution as well as on the relationships between different viral groups and plasmids. It becomes clear that evolutionary mechanisms shaping the genomes of prokaryotic viruses vary between different families and depend on the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the life cycle. We also point out that horizontal gene transfer is not equally prevalent in different virus families and is not uniformly unrestricted for diverse viral functions.

show abstract

A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro

Cited by 52 publications

References 40 publications

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

An Unexplored Diversity of Reverse Transcriptases in Bacteria

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

Contact Info

Product

Resources

About