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)