Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends

Nucleic Acids Research, Oct 2016

Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional ‘brush-like’ structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.

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Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends

Nucleic Acids Research Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends Laura Mart´ınez-Alvarez 1 Stephen D. Bell 0 Xu Peng 1 0 Department of Molecular and Cellular Biochemistry, Department of Biology, Indiana University , Simon Hall MSB, IN 47405 , USA 1 Archaea Centre, Department of Biology, University of Copenhagen , 2200 Copenhagen N , Denmark Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional 'brush-like' structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms. - In contrast to cellular organisms, viruses carry DNA or RNA genomes, which can be double-stranded or singlestranded with linear or circular structures. Consequently, their replication strategies show great diversity (1). Whereas the replication of circular DNA replicons follows two general replication models: replication and replication, named after the characteristic architecture of their replication intermediates (RIs) under electron microscope (2), the picture is more complex for viruses with linear DNA genomes such as phages , T4, 20, PRD1 and some eukaryotic viruses including adenoviruses, herpesviruses and parvoviruses (3–6). These viruses have to solve the so-called ‘end-problem’ and the generation of concatemers is one of the strategies employed to solve it. While replication of some viruses (e.g. T7), generates concatemeric RIs with predominantly linear configuration, branched and complex concatemers have been observed for phage T4 and Herpes simplex virus-1 (HSV-1) (3,5). For T4, recombinationdependent replication is the mechanism of formation of networked concatemeric intermediates, while for HSV-1 neither the shape nor the mechanism of formation has been elucidated (3,5). For other viruses, such as poxviruses, the formation of multimeric RIs has been reported, but their structure is still unknown (7). Linear dsDNA with covalently-closed ends (hairpin ends) has been found in all three domains of life, mainly associated with viruses. These include the genomes of eukaryotic viruses such as poxviruses, African Swine Fever virus and Chlorella viruses; the prophage genomes of bacteriophages N15, KO2 and PY54; the plasmids and genomes of Borrelia, Agrobacterium tumefaciens and a group of mitochondria and plastids; and the genomes of Rudiviridae that infect hyperthermophilic archaea (8–10). Based on the telomere structure and results from biochemical studies of relevant proteins, different replication models have been proposed. However, in none of the cases is replication clearly understood. Archaeal viruses exhibit a strikingly wide variety of morphologies, and the rod-shaped Rudiviridae family represents one of the most common morphotypes in hot terrestrial environments (11,12). This family contains four isolated members: SIRV1, SIRV2, ARV and SRV (11); and two additional candidate members (SMRV1 and ARV2) identified in hot spring metagenomes (13,14). Rudiviruses have linear dsDNA genomes of 25–35 kb with hairpin ends and inverted terminal repeats (ITRs) of up to 2 kb. Among rudiviruses, Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is one of the most intensively studied, which has a genome of 35 498 bp with 1652 bp long ITRs (10). Previous biochemical and structural studies by Oke et al. identified a candidate replication initiation protein (Rep), conserved in Rudiviridae members. Rep can introduce a nick at the SIRV genomic terminus, forming a covalent adduct with the newly created 5 end and releasing a 3 -OH terminus presumably used for priming DNA replication. Based on the in vitro data, it was proposed that the Rep protein initiates a strand-displacement replication (15). In this work, we show that SIRV2 reinitiates stranddisplacement replication consecutively from a single parental template, leading to the formation of large replication intermediates with unusual ‘brush-like’ structures. Moreover, a minor portion of the circular dimeric intermediate undergoes strand-coupled replication. MATERIALS AND METHODS Cell culture and virus propa (...truncated)


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Laura Martínez-Alvarez, Stephen D. Bell, Xu Peng. Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends, Nucleic Acids Research, 2016, pp. 8799-8809, 44/18, DOI: 10.1093/nar/gkw636