A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication

Abstract

The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5 end of the DNA, releasing a 3 DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed.Archaeal viruses display tremendous diversity of both morphology and genome content. Seven viral families from the Crenarchaeota have been described, all with linear or circular double-stranded DNA (dsDNA) genomes but with few genes in common (28). The Rudiviridae are a family of nonenveloped rod-shaped viruses infecting thermophilic crenarchaea (27). Members include SIRV1 and SIRV2 (24), ARV1 (36), and SRV1 (37), all of which have covalently closed linear dsDNA genomes of 30 to 40 kb encoding up to 50 open reading frames (ORFs). The genome arrangement, including the presence of inverted terminal repeats (ITRs), is reminiscent of those of the large cytoplasmic DNA viruses, such as the Poxviridae (3, 10). Replication of the poxvirus genome, which occurs in cytoplasmic DNA replication factories (29), results in the generation of head-to-head concatemers that are resolved by a Holliday junction-resolving enzyme (7, 12). The Rudiviridae also generate head-to-head intermediates, ruling out a rolling-circle mechanism of replication (24), and encode a resolving enzyme that is assumed to be required for the deconcatamerization of the replicated rudiviral genome (2). A replication model for rudiviruses has been postulated based on the observed similarities with the Poxviridae. This involves replication initiation by an unidentified nicking enzyme at a site that is 11 nucleotides (nt) from the covalently closed terminus, yielding a 3Ј-hydroxyl suitable for priming DNA synthesis (3).Small circular DNA viruses such as phiX174 and M13 in bacteria, geminiviruses in plants, and circoviruses in animals replicate by rolling-circle replication (RCR). RCR requires a replication initiator (Rep) protein, which nicks the circular DNA, forming a 5Ј DNA adduct and releasing a free 3Ј end to act as a primer for DNA synthesis. ...

Virus World as an Evolutionary Network of Viruses and Capsidless Selfish Elements

“…In particular, highly diverged RCREs are encoded in the genomes of the archaeal ds-DNA viruses of the family Rudiviridae (149). These viruses possess dsDNA genomes of approximately 35 kb with a covalently closed hairpin at each end, and the RCRE protein has been shown to initiate replication by introducing a nick near the hairpin apex and then to reseal the DNA molecule during concatemer resolution (149). The presence of an RCRE that mediates an RCR-like replication mechanism in these relatively large dsDNA viruses suggests that some of the large viral genomes, particularly those of archaeal viruses, might have evolved from small RCR elements via gene accretion.…”

Archaeal Extrachromosomal Genetic Elements

“…Based on these results and previous models for rolling-circle DNA replication in bacterial and eukaryal viruses, SIRV1 DNA replication is proposed to proceed as follows (Fig. 4) (52,88). The virus initiates DNA replication by introducing a single-stranded nick 11 nucleotides from either terminus, releasing a free 3= end of the DNA.…”

“…The genomes of rudiviruses are covalently closed at both ends (26), whereas those of other linear dsDNA viruses are linked to a protein (23,86,87), modified in an unknown fashion at the ends (14), or yet to be determined (79). Genome replication among archaeal viruses is best understood for SIRV1 (88). SIRV1 has a 32-kb linear dsDNA genome with 2,029-bp inverted terminal repeats (ITRs).…”

“…The viral DNA in ϳ5% of the mature virions had a singlestranded nick 11 nucleotides (nt) from the terminus of the genome, as expected for the replication mechanism for a genome of a similar type, in which DNA replication is initiated through the introduction of a nick near either end of the linear genome. Structural analysis has shown that ORF119 of SIRV1 encodes a member of the replication initiator protein (Rep) superfamily, which includes proteins known to initiate rolling-circle replication (RCR) of a range of viruses and plasmids (88). The SIRV1 Rep protein existed as a dimer in solution and was capable of sequence-specific cleavage of ssDNA in vitro.…”

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

“…By contrast, prokaryotic hairpin-containing replicons appear to replicate via different mechanisms. The archaeal rudiviruses encode an initiation endonuclease and are thought to use a rolling hairpin-like replication route (41). The bacteria in the genus Borrelia have been shown to contain the origin in the middle of their linear chromosomes (42).…”