Regulation Of The Lysis-lysogeny Decision Of Temperate Halovirus SNJ1

For all temperate viruses,a control of the lysogenic state versus the lytic life cycle is essential.The studies on the genetic regulation of phages provided mechanic insights into lysis-lysogeny decisions.However,the virus-host interaction in extreme environments hasn't yet been understood in detail,especially for the halovirus.SNJ1 is a temperate virus of a halophilic euryarchaeon Natrinema sp.J7-1,and it has been classified to a new virus family named Sphaerolipoviridae as the only member of the genus Betasphaerolipoviridae.It has an icosahedral,nearly spherical capsid and internal lipid membrane,which is different from the other two temperate haloviruses cpH and?CH1 reported before.SNJ1 can be induced from a halophilic euryarchaeon Natrinema sp.J7-1 and infects Natrinema sp.CJ7,but it cannot infect J7-1.The provirus of SNJ1 is a circular dsDNA identical to the previously described Natrinema sp.J7-1 plasmid pHH205 without any combination with the host chromosomes.Similar to bacterialphage X,SNJ1 could be induced by the Mitomycin C(MMC)treatment,but the predicted encoded proteins of SNJ1 had no homology with CI-like or CII-like proteins,which indicated that a newly strategy may be adopted to make a lysis-lysogeny decision.Here we introduce a small predicted protein gp4(68 amino acids),encoded by orf4 in the genome of SNJ1,involved in the lysis-lysogeny decision in the early infection stage and the construction of the super-infection exclusion of the host cell.In our previous study,the E.coli-Natrinema sp.J7 shuttle vector pYC-S based on the whole length of the SNJ1 genome and the E.coli replicon was successfully constructed.It is interesting that the supernatant of CJ7/pYC-S(treated with MMC)formed two phenotype plaques(clear plaques and turbid plaques)simultaneously after infecting CJ7,while SNJ1 could only form turbid plaques under the same condition.Compared with SNJ1,the viruses forming clear plaques was more lytic to the host cell,and they caused a violently growth retardation after infection.But there are no significant differences between SNJ1 and the viruses forming turbid plaques.Sequencing results showed that all the viruses forming clear plaques were with a partial suspension of orf4 region,while the viruses forming turbid plaques were not the case.Meanwhile,the transcription start site analysis showed that orf4 and orf5-6 were independent.Based on this,the possibility that the destroyed region would directly influence the transcript of the downstream genes were excluded.So,it's naturally for us to speculate that orf4 may be related to the lysis-lysogeny decision of SNJ1 in the early infection stage.To verify the hypothesis,we constructed two types of orf4 mutants(start code mutation and orf4 deletion)based on pYC-S and found that all the progeny viral particles from these mutants only cause clear plaques.Besides,we could hardly get a lysogenic strain of these two types of orf4 mutant clones.Overall,these results showed that orf4 is a key factor involved in the lysis-lysogeny decision of SNJ1.On the other hand,constitutive expression of orf4 in Natrinema sp.CJ7-F(pyrF deletion)with a newly E.coli-Natrinema sp.J7 shuttle vector pFJ6 could significantly reduce the plating efficiency of both SNJ1 and these new types pYC-S related viruses,which suggested that the host could be immune to the virus in the presence of orf4.Then,series of trunctated proteins were produced,and we found that the 33 aa in the N-terminal(residues 1-33)of gp4 could be founctional.Interestingly,SNJ1 was able to infect the immuned strains resulting in a lysogenic strain.Then the assay of the plasmid maintenance and the virus production ability of the lysogenic strains was carried out.The results revealed that constitutive expression of orf4 in Natrinema sp.CJ7-F didn't have any influence on the replication and the production of SNJ1 after lysogeny establishment.In all,we conclude orf4 may assistant the host to establish the super-infection exclusion by repressing the virus entry.What's more,the results of the sequence analysis by PSI-BlastP and HHpre on orf4 showed that the predicted protein gp4 encoded by orf4 has homology with some bacterial transcriptional regulators with a low identity and similarity,and these regulators were all predicted to be members of SpoVT-AbrB superfamily which contains a AbrB-like Swapped Hairpin secondary structure comprised of two pairs of ? sheet hairpin and a a helix.In conclusion,gp4 is likely to be a multifunctional regulator eligible to control not only the lysis-lysogeny regulation of SNJ1 but also many other genes related to the life cycle of SNJ1.