Study On The Lysogeny-Lysis Regulation In Haloarchaeal Temperate Pleolipovirus SNJ2

Temperate viruses are also known as lysogenic viruses,when their nucleic acid invaded into the host cell,they can choose to complete the whole life cycle immediately like a virulent virus,which is called productive infection.Alternatively,the viral genome can integrate into the host chromosome or exsit as an extrachromosomal episome,thus achieving a long-term“friendly coexistence”with host cell,known as the lysogenic state.When growing conditions are unfavorable,these temperate viral genomes（Known as proviruses）,which already coexist peacefully with host cell,are then transferred from the lysogenic state into the replication cycle,producing progeny virions and released from host cell.Proviral regions are frequently found in genomes of both bacteria and archaea,and play important roles in microbial ecology and genome evolution.For instance,upon induction,temperate viruses can promote horizontal gene transfer and have significant impact on the composition and physiology of microbial communities in diverse ecological niches.Therefore,understanding the regulatory mechanisms behind lysis-lysogeny decision of temperate viruses is of great importance,Many prokaryotic viruses are temperate and their reactivation is tightly regulated.However,except for a few bacterial model systems,the regulatory circuits underlying the exit from lysogeny are poorly understood,especially in archaea.SNJ2 virus belong to Betapleolipovirus,so far,no known replication-related proteins have been found in the genomes of member of this genus.However,genes encoding replication-related proteins have been identified in the genomes of viruses of other two genera of this family,namely theαandγgenera.Therefore,it is necessary to reveal the replication mechanism of Betapleolipovirus through specific experimental evidence.A comparative analysis of viral genome in state of integration and cyclization by German scientist Mike Dyall-Smith,found that a new CDS（coding sequeence）appeared around the att P site after cyclization,which closely connects orf19-25 at att L with orf3-1 at att R like a bridge.We performed a co-transcription analysis and transcription start site identification on this CDS to confirm that it is indeed a new ORF（open reading frame）and named it orf26.Further results showed that orf26 was co-transcribed with orf19-25 to form an operon and the transcription start site is located inside the Int^SNJ2 encoding gene.Deletion mutation analysis showed that deletion of orf19-26 genes significantly reduced the replication ability of SNJ2,suggesting that orf19-26 might be a key region in controlling the replication of SNJ2virus genome.Notably,deletion of them did not affect the up-regulation of int^SNJ2operon in response to mitomycin C（MMC）induction,but orf19-26 was not transcribed after deletion of int^SNJ2.The results of site mutation of Int^SNJ2 and heterologous complement indicate that the integrity of integrase coding sequence is important for viral replication,otherwise orf19-26 would not be able to initiate transcription.It is important that E.coli-Natrinema shuttle vector with Orf19-26 as replication element can successfully obtain transformants,which fully indicates that Orf19-26 is a key gene cluster related to viral replication.The lysogen-lysis decision of temperate virus SNJ2 relies on integrase encoded by the virus itself to integrate the genome into or dissociate from the host chromosome.Therefore,the transcription regulation of Int^SNJ2 is of great significance for the lifecycle decision of virus.Bioinformatics analysis revealed that SNJ2-encoded Orf4is a homologous ofφH1 repressor,which is a DNA-binding protein in the Winged helix-turn-helix family,binding to two short interrupted palindromes upstream of its start codon,and it was suggested to be a part of an auto-regulation system.The analysis of deletion mutation of Orf4 on SNJ2 provirus in J7-1-F strain showed that knockout strains of orf4 could not be obtained directly unless the strain carried a plasmid that expressed Orf4 in addition.Intriguingly,we observed more active SNJ2 replication in the J7-1-FΔorf4/Ptna-Orf4 strain than in the J7-1-F wild-type strain.However,overexpression of Orf4 in the J7-3-F strain（SNJ2 cured）can block infection or replication of SNJ2 virus.These results suggest that SNJ2 life cycle is finely regulated by Orf4.The identification of the Orf4 binding site indicated that Orf4 may bind to the palindromic sequence 5’-CGTACTTCCTAGTACG-3’as well as its upstream and downstream sequences in the P_Int promoter to suppress int^SNJ2transcription,thereby maintaining SNJ2 in the lysogenic state.To switch to the lysis state,two other SNJ2-encoded proteins,Orf7 and Orf8,are required.Orf8 is a homolog of cellular AAA+ATPase Orc1/Cdc6,orf7 encodes a small protein of 93-a mino-acids,rich inβ-strands,but no putative function could be confidently assigned to it.Deletion mutation was introduced to Orf7 and Orf8 in the provirus,respectively.The results showed that deletion of both completely blocked the transmission of DNA damage signal of SNJ2 virus.Combined with RNA-seq data,Orf8-his conjugate protein western detection,Orf8 heterologous complement and mass spectrometry detection results,it is speculated that Orf8 is activated upon MMC-induced DNA damage,possibly through posttranslational modification.Unlike orf8,the transcription of orf7 was significantly upregulated upon MMC induction.However,the transcription of orf7 was nearly completely aborted in the J7-1-FΔorf8 strain indicating that Orf8 is required for its transcription.In line with this,the transcription of orf7 was recovered in the J7-1-FΔorf8 strain when Orf8 was complemented in trans from a plasmid and upon MMC treatment.A series of amylase activity reporting systems based on the Orf7 promoter P₇ showed that activated Orf8 regulates orf7transcription by controlling the P₇promoter.Overexpression of Orf7 can bypass the requirement of Orf8 and counteract the function of Orf4 to derepress the P_Int promoter.Comparative genomics analysis revealed that the SNJ2-like Orc1/Cdc6-centered three-gene module is common in haloarchaeal genomes.A total of 31 Orc1-encoding proviruses were identified in three orders of Halobacteria,The genetic arrangement of orf4-,orf7-and orc1-like genes is conserved in 27 proviruses,resembling that in SNJ2.Importantly,function verification of 5 Orf4-like proteins from four genera showed that all five putative repressors were able to repress the promoter of their cognate integrases,confir ming that temperate pleolipoproviruses use a common mechanism to maintain lysogeny across different genera of haloarchaea.Collectively,our results uncover the first a virus-borne DNA damage signaling pathway controls the lysogeny-induction switch in a group of temperate pleolipoviruses,and the three-gene module is conserved in proviruses belonging to the Betapleolipovirus,suggesting that a common life cycle regulation strategy was used by theses proviruses.