ORF4 Encoded By Temperate Haloarchaeal Virus SNJ1 Is The Master Regulator Of Virus Life Cycle

Viruses that can only live by parasitizing in cells play an important role in the nutritional cycle and the evolution of host cells.They are abundant and can infect all three domains of life,in many circumstances outnumbering cells by 10-100-fold.In various extreme environments where archaea are the dominant population,viruses are considered to be the most important "predator".Observation by electron microscope also confirmed the high concentrations of viruses,with virus-like particles reaching up1.3 × 1010/m L.Compared with the widely studied bacteriophage(about 8500 strains),only about 120 strains of archaeal virus have been sequenced and reported.At present,most of the isolated archaea viruses are lytic viruses,and the study of archaeal temperate viruses is still in its infancy.Here we reported the life cycle regulation of lysogenic virus SNJ1(Saline Natrinema sp.J7-1 virus 1)in extremely halophilic archaea Natrinema sp.J7-1.SNJ1’s genome is double-stranded circular DNA,belonging to Sphaerolipoviridae family.In the lysogenic state,the provirus existed as a plasmid(formerly known as p HH205)in cytoplasm.When induced with mitomycin C(MMC),a large number of progeny viruses were synthesized and released outside the cell.In the early experiments of J7-1,we accidentally obtained a SNJ1 cured strain named Natrinema sp.CJ7.When SNJ1 infected CJ7 and J7-1,plaques were only observed on the double-layer plate of CJ7,while J7-1 not.This indicated that SNJ1 has established immunity against the superinfection of the same virus in J7-1,that is,"superinfection immunity".The mechanism of superinfection immunity has been thoroughly studied in eukaryotic viruses and bacteriophages.However,only two archaeal viruses φH and SIRV2 have been reported,and the mechanism and regulators are still unknown.The preliminary work confirmed that the expression of SNJ1’s 1-656 bp by p FJ6 vector immunized CJ7-F strain(CJ7 uracil auxotrophic strain)against SNJ1.1-656 bp contains two cotranscribed genes,orf3 and orf4.In this study,we constructed frameshift mutation of these two genes,and proved that only ORF4 was necessary for superinfection immunity,while ORF3 showed no effect.ORF4 encoded by SNJ1 contains 68 amino acid residues.Bioinformatics analysis revealed that it has homology with transcriptional regulators of the Spo VT/Abr B family.ORF4 may form a dimer through a swapped hairpin domain to act as transcriptional regulator.The truncation of ORF4 demonstrated that the N-terminal 33 amino acids of ORF4 were necessary and sufficient for superinfection immunity.In order to elucidate the mechanism of superinfection immunity,SNJ1 was incubated with cells with or without SNJ1(CJ7 or J7-1,respectively)and cells with or without ORF4 expression(CJ7-F/p FJ6-MCS or CJ7-F/p FJ6-Hpro-orf4).Then we examined the effects of ORF4 on different stages of SNJ1’s life cycle: adsorption,genome entry,DNA replication and viruses release.It was found that the adsorption efficiencies of SNJ1 to these four strains were similar,indicating that ORF4 did not affect the adsorption process.However,after 1 h of infection,the intracellular copy number of SNJ1 genomic DNA(g DNA)was suppressed by ORF4.Viral g DNA in cells without ORF4 was about 10-fold higher than cells with ORF4.As the infection continues,the copy number of viral g DNA in cells containing ORF4 has been at a low level.After 9 h of infection,it was only 2% of the strain without ORF4.This showed that ORF4 significantly inhibited the DNA replication of SNJ1.Finally,compared with CJ7 and CJ7-F/p FJ6-MCS strains,the release of progeny viruses in the J7-1 and CJ7-F/p FJ6-Hpro-orf4 strains decreased by approximately 100 times.In addition,ORF4 was also responsible for SNJ1 lysis-lysogeny switch.When ORF4 was expressed,SNJ1 was in a lysogenic state and the genome was stably maintained at about two copies per chromosome.However,in the absence of ORF4,the copy number of genomes was increased about 10 times.ORF4 mutant virus can no longer lysogenize in CJ7 cell.It illustrated that ORF4 maintained the lysogenic state of SNJ1 by inhibiting DNA replication.Mitomycin C(MMC)induction turned SNJ1 to lytic state,indicating that ORF4 seems to be inactivated after MMC induction,thus activating the lytic cycle.Next,in order to explore the way ORF4 inhibited genome replication,we analyzed the regulatory relationship of ORF4 and the genes in the minimal replication region(orf5-rep A)of SNJ1.Through promoter activity detection,we found that ORF5 and ORF6 were transcriptional activators of orf7-rep A.And the results in Haloferax volcanii showed that ORF4 inhibited the transcription of orf5-orf6.Therefore,when ORF4 was expressed,the genome replication was suppressed,and SNJ1 maintained in the lysogenic state and immune to the superinfecting viruses.We identified two SNJ1-like plasmids through bioinformatics,namely the p NVE19 plasmid of Natrinema versiforme strain BOL5-4 and the unnamed5 plasmid of Haloterrigena jeotgali strain A29.Both the DNA sequence and the protein sequence of these two plasmids are homologous to SNJ1.So,we speculate that they may be two proviral genomes belonging to Sphaerolipoviridae family.ORF4 is highly conserved among SNJ1-like proviruses,with a protein identity of over 76%,which implies that ORF4-like protein is also a master regulator of life cycle in these proviruses.In conclusion,ORF4 is the first protein which simultaneously regulates the superinfection immunity and lysis-lysogeny switch in archaeal virus.This article reveals the regulatory mechanism of ORF4 on SNJ1’s life cycle,enriching the understanding of archaea virus and its interaction with host.