Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

New England Biolabs, Inc., Ipswich, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 08/2011; 6(8):e23668. DOI: 10.1371/journal.pone.0023668
Source: PubMed


Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.

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Available from: William E Jack, Oct 10, 2015
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    • "gp16, belonging to the replication initiator (Rep) family and nicking one strand of the viral genomic termini, was proposed to be involved in the initiation of the DNA replication [19]. The Holiday junction resolving enzyme (Hjr) gp35 was suggested to resolve the concatemers of the replicative intermediates, producing monomeric copies with linear hairpin ends [20]. Taken together, the functions of many SIRV2 genes remain unknown and the knowledge of its biology and basic molecular processes such as DNA replication, recombination and maturation is still limited. "
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    ABSTRACT: The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterisation of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a ssDNA binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed a ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, SIRV2 gp19 was characterized and shown to possess a 5´ to 3´ ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction between the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination which may function in viral DNA replication/repair. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 03/2015; 427(12). DOI:10.1016/j.jmb.2015.03.013 · 4.33 Impact Factor
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    ABSTRACT: Linear viruses with double-stranded DNA genomes are classified into two families, Lipothrixviridae and Rudiviridae. The members of these two families, all of which infect hyperhermophilic members of the domain Archaea, differ significantly in the complexity of their virions as well as in their mechanisms of genome replication. However, recent structural and genomic studies have revealed a robust evolutionary link between members of the two families. To acknowledge this relationship we propose to unify the two families into the new taxonomic order "Ligamenvirales".
    Archives of Virology 01/2012; 157(4):791-5. DOI:10.1007/s00705-012-1229-7 · 2.39 Impact Factor
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    ABSTRACT: Archaeal viruses display unusually high genetic and morphologic diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus Rod Shaped Virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on formation of remarkable pyramidal structures on the host cell envelope. Using whole transcriptome sequencing we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one third of S. islandicus genes was differentially regulated. While expression decreased of genes involved in cell division, those playing a role in anti-viral defense were activated on large scale. Expression of genes belonging to Toxin-Antitoxin and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of anti-viral defense systems is expected to aid future studies aiming at detailed understanding of functions and interplay of these systems in vivo.
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