The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rollingcircle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980 T , suggesting their broad applicability as genetic tools for Natrinema species. IMPORTANCEArchaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980 T . This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts. Haloarchaea are the dominant microbes in hypersaline environments, such as salt lakes and salterns. Viruses infecting haloarchaea outnumber their hosts by several orders of magnitude (1). It has been speculated that in such a harsh environment with no other predators, interactions between viruses and their hosts constitute the main evolutionary driving force (2). About 90 haloarchaeal viruses have been discovered so far, a relatively small number compared to the ϳ6,200 reported bacteriophages (3, 4). Given that the characterization of bacterial and eukaryotic viruses has yielded remarkable insights into the physiology and cell biology of their hosts, studies on haloarchaeal viruses are expected to be equally revealing. However, thus far, research has been limited by the scarcity of suitable and genetically tractable virus-host models. Consequently, most of the current knowledge on viral entry, transcription, genome replication, assembly, and ...

For most archaeal viruses, the mechanisms of genome replication are poorly understood, while the nature and provenance of their replication proteins are usually unknown. Here we show that replication of the circular double-stranded DNA genome of the halophilic Natrinema virus SNJ1, a member of the family Sphaerolipoviridae, is associated with the accumulation of single-stranded replicative intermediates, which is typical of rolling-circle replication. The homologues of RepA, the only enzyme that is indispensable for SNJ1 genome replication, are widespread in archaea and are most closely related to bacterial transposases of the IS91 and ISCR family insertion sequences, as opposed to other viral rolling-circle replication initiation proteins. Our results provide insights into the replication mechanism of archaeal viruses and emphasize the evolutionary connection between viruses and other types of mobile genetic elements.

Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As lysis-lysogeny switch and superinfection immunity of archaeal viruses are poorly studied, we suggest that SNJ1 could serve as a model system to study these processes. IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1.These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

Haloarchaeon Natrinema sp. J7, the first reported archaeon harboring both plasmid and chromosome-based temperate viruses, is a useful model for investigating archaeal virus-host and virus-virus interactions. However, the lack of genetic tools has limited such studies. On the basis of the automatically replicating sequences of the J7 chromosome and the pyrF marker, we constructed seven vectors, six of which were confirmed to possess replication ability in a pyrF-deletion derivative of J7 (J7-F). Among these vectors, pFJ1, pFJ4, and pFJ6 could be transformed into the host strain with relatively high efficiency (approximately 103 colony-forming units/μg DNA) and were present at about one copy per chromosome. These three vectors could be stably maintained in J7-F without selection and were used for heterologous protein expression. Only pFJ6 was found to be present in the transformed cells in an exclusively episomal, nonintegrated state (one copy per chromosome). In contrast, some pFJ1 and pFJ4 DNA was probably integrated into the J7-F chromosome. In addition, pFJ6 was found to be compatible with pYCJ in J7 cells, suggesting that these two vectors could be used for further studies of virus-virus and virus-host interactions.