The Study On Haloarchaeal Temperate Virus SNJ2 And The Lysogenic System In Its Host Cells

Although limited in isolate number, archaeal viruses have already represented abundant diversity. The study of archaeal viruses is providing new insight into the origin and evolution of viruses. The documented haloarchaeoviruses are overwhelmingly isolated from environments, rather than from lysogenic host strains. However, pro viral regions of different viral lineages have been predicted to integrate in the genomes of archaea by informatics approaches. The temperate virus-host relationships in Archaea are less investigated compared with that in other two domains. Here in this study, we isolated a new pleolipovirus SNJ2 (Saline Natrinema sp. J7-1 virus 2) from the SNJ1-lysogenic host strain Natrinema sp. J7-1. Our study focused on the temperate virus SNJ2 and the virus-host relationships of SNJ2 and SNJ1 with their host strains.Extreme halophilic archaeal strain Natrinema sp. J7 was isolated from Yingcheng Salt Mine in Hubei Province. The laboratory derivatives Natrinema sp. J7-1 and J7-2 are identical strains except for their plasmid contents. Strain J7-1 harbors the plasmid pHH205, the provirus of SNJ1, which infects and forms plaques on the lawns of strain J7-2 that harbors plasmid pJ7-?. Based on the comparative sequence analysis of the two major capsid proteins (MCPs) and ATPase-specific motif as well as the presence of lipids in the virions, SNJ1 has been predicted to be the member of PRD1-adenovirus viral lineage in previous studies. However, due to the difficulties in virus purification, the electron micrographs of SNJ1 were failed to obtain. In this study, we observed the morphology of SNJ1 virions after improvement of virus purification and confirmed that it is an icosahedral virus with an internal membrane.During the purification process, we discovered a new pleomorphic, membrane-containing, temperate virus SNJ2 that co-induced with SNJ1 from strain J7-1. SNJ2 virus is dependent on the regulatory region of SNJ1 for efficient production. The SNJ2 virion contains a discontinuous, circular, double-stranded DNA genome of 16992 bp, in which both nicks and single-stranded regions are present preceded by a "GCCCA" motif. Based on different degrees of breakage, major interruptions, minor interruptions and intact sites are classified. SNJ2 encodes 25 putative ORFs, among which five form a cluster of conserved ORFs homologous to archaeal pleolipoviruses isolated from hypersaline environments. Two structural protein encoding genes in the conserved cluster were verified in SNJ2. Based on the organization and replication of genome, as well as the homology of the conserved cluster, SNJ2 was grouped to family Pleolipoviridae, genus Betapleolipovirus.The SNJ2 provirus site-specifically integrates into tRNAMet gene 3'distal of J7-1 chromosome with 14 bp direct repeat of the attachment sites. The viral genomes replicated at a low rate during cell growth. With mitomycin C treatment, the integrated SNJ2 provirus in polyploid J7-1 genomes was induced to excise and undergo a lytic cycle but at a low level, while, the circularized viral genome replicated largely. Through continuous induction, a "SNJ2-free" strain designated Natrinema sp. J7-3 was obtained. SNJ2 infects and integrates into the genome of strain J7-3 instead of replicating and releasing progeny to form plaques on its lawns. Setting SNJ2 nucleic acid sequence as the query, a total of 17 SNJ2-like proviruses were found integrate in the genomes of haloarchaea from 10 genera, suggesting that pleolipopro viruses are ubiquitous in haloarchaea. The majority of these pro viral regions contain the pleolipoviral conserved gene cluster, while the minority lost one or two conserved gene(s), which were supposed to be defective proviruses. Different from pleolipoviruses that isolated from environments, SNJ2-like proviruses contain a series of virus-host regulation genes, such as genes encoding for integrase, transcriptional regulator, repressor, Orcl/Cdc6 Rep. Furthermore, the ORFs acquiring through HGT from haloarchaeal genomes make the genomes of proviruses more redundant. Similar to SNJ2, SNJ2-like proviruses all site-specifically integrate into tRNA gene in host chromosome with direct repeats ranging from 12 to 59 bp.In the study of SNJ1 virus-host relationship, strain J7-1 that contains pHH205 showed superiority in growth when co-cultured with strain J7-2. Among the infected J7-2 cells, a quarter could form SNJ1-lysogenic cells to escape from being lysed, and we named this strain that contain two plasmids Natrinema sp. LJ7. Strain LJ7 could produce SNJ1 virus and partially immune to SNJ1 infection. The growth of LJ7 showed instability due to the self-lysis, but it can stably carry both plasmids in period of time. The wild type of SNJ1 formed turbid plaques on the lawns of strain J7-2, however, two SNJ1 mutants that produced clear plaques were obtained during virus propagation by infecting strain J7-2. The frequency of J7-2 cells to form lysogenic LJ7 was dramatically decreased when infecting by SNJ1-Mutant 1 and Mutant 2. This phenotype was caused by single-base mutations in the genomes of two mutants. Both of the mutation sites locate in the non-coding region in the upstream of ORF4 and ORF5 of putative MazE encoding genes. We speculate that this non-coding region may influence the transcription of antitoxin MazE encoding gene and further lead cell lysis by unbalancing the toxin-antitoxin system, or influence the transcription of downstream unknown lytic-lysogenic regulation genes.The study on temperate pleolipovirus SNJ2 and its virus-host relationship deepens our understanding on haloarchaeal temperate viruses. The putative SNJ2-like proviruses expend the pleolipoviral lineage and suggest that the number of temperate viruses of archaea is likely underestimated. The study on SNJ1 virus-host relationship shows that there may exist exquisite and complicated lytic-lysogenic regulation between virus and host or the virus itself. More efforts are needed to better understand archaeal temperate viruses and the relationship with their host.