A new family of rolling-circle replication endonucleases widespread in archaeal viruses and plasmids.

Archaea possess a remarkably diverse mobilome, but for many archaeal viruses and plasmids, even the basic processes, such as genome replication, remain poorly understood. Here, we characterize a previously uncharacterized family of putative rolling-circle replication endonucleases, termed Rep-Arvir, widespread among viruses and plasmids associated with phylogenetically diverse archaea, including halophiles, methanogens, and hyperthermophiles. We show that RepSNJ2, encoded by the temperate pleomorphic virus SNJ2, a model member of the Pleolipoviridae family, is essential for driving autonomous replication. Moreover, a conserved hairpin-forming DNA element downstream of repsnj2 likely functions as the recognition site for RepSNJ2 and origin of replication of SNJ2. Notably, the functional replication operon is restored only following the excision and circularization of the SNJ2 viral genome, representing an elegant regulatory mechanism controlling the lysogeny-replication switch. Leveraging this system, we constructed SNJ2-based shuttle vectors that enable stable gene expression and are compatible with other Natrinema plasmids. Structural modeling revealed that the Rep-Arvir family is distantly related to the bacterial Rep_trans family endonucleases, a relationship not recognizable at the sequence level. These findings provide evidence for a previously unrecognized replication mechanism in archaea, highlight deep evolutionary links between archaeal and bacterial replicons, and provide a versatile genetic platform for studying virus-host interactions in hypersaline environments.