Preliminary Study On The Replication Mechanism Of Temperate Halovirus SNJ2

SNJ2 is a temperate enveloped virus of the halophilic archaea Natrinema sp.J7-1,It belongs to the Pelolipoviridae family,Betapleolipovirus genus.The SNJ2 virus genome encodes 25 predicted ORFs with a genome size of 16,992 bp.SNJ2 provirus site-specifically integrates into tRNA^Met gene 3’terminal on J7-1 chromosome,integration site is 14 bp of direct repeat sequence.Its own site specific recombination system can mediate the excision and cyclization of the provirus from the host chromosome when starting lysis lifecycle as well as the integration of circular virus genome into the host chromosome to turn on the switch of the lysogenic state.However,how SNJ2 replicates remains unclear.This paper attempts to explore the replication mechanism of SNJ2 genome in host cells.Firstly,the E.coli-Natrinema sp.J7-1 shuttle vector pFSNJ2 containing the full length of SNJ2 genome was constructed.This vector was transformed into J7-3-F with the method of halophilic transformation.Afterwards,plasmids were extracted from the third generation of the above transformants and were then transformed into Escherichia coli.However,only one transformant was detected despite of several round repeats.Surprisingly,the enzyme-digested fragment size was smaller than that of pFSNJ2,indicating that pFSNJ2 was unstable even though it could replicate in free form in J7-3-F strain.attBOP was detected in the culture of J7-3-F/pFSNJ2 after several inoculations,which reveals that pFSNJ2 has been integrated into the host chromosome.After induction with MMC,cyclization joint attP’OP forming in excision cannot be detected in supernatant and sediment.We infer this happened due to exogenous integrated in host chromosome affecting provirus specifically excised from host chromosome.Therefore it was speculated that pFSNJ2 could not replicate in the form of plasmid.Previous study has confirmed that the orf1 encoding integrase(Int^SNJ2)is the key factor responsible for the specific integration and excision of provirus SNJ2.Based on this,we checked whether SNJ2 could replicate in the free form.Defective virus SNJ2Δorf1::pyrF were obtained after the treatment of MMC induction from the strain J7-1-FΔorf1::pyrF carring a vector for orf1 expression.As expected,attBOP and attP’OP were not detected because the defective virus had no integrase coding gene and therefore could not be integrated into the chromosome.Also,no stable host carrying SNJ2Δorf1::pyrF were detected,which suggested free-formed provirus is not that stable in host cells.As a control,complemention strain J7-3-F/pYCJ-2300 was infected with SNJ2Δorf1::pyrF,and attBOP and attP’OP were detected in the infected strain,indicating that SNJ2Δorf1::pyrF can be both integrated into the host chromosome and excised from the host chromosome with the presence of integrase.The above results indicated that the defective virus SNJ2Δorf1::pyrF could be adsorbed into and infect the host J7-3-F,but could not be integrated into the host genome due to the lack of integrase.The phenomenon that the above detective virus were easily lost showing it can not replicate in the free form,also suggesting that SNJ2 genome can only be replicated by integration into the host chromosome.SNJ2 replicated at a low rate during normal cell growth,after MMC induction,SNJ2undergoes a lytic cycle to release a large amount of virus particles.To understand the time of large-scale replication of SNJ2 in cells after induction of J7-1 by MMC,we determined the copy number of SNJ2 in each cell of J7-1 before and after MMC induction by Q-PCR.The results showed that when J7-1 was not induced,the SNJ2 genome mainly existed in the integrated state.After MMC induction,the intracellular free SNJ2 genome began to increase significantly at 4 h.It is speculated that the time of large replication of SNJ2 genome 4 h post induction.8 hours after induction,it observed that the amount of attBOP was less than that of attP’OP.However,attBOP level remained constant,but attBOB’and attP’OP levels were increasing and positively correlated,indicating that the increase in attP’OP was derived from the host chromosome.It strengthens the evidence that the SNJ2 genome can only replicate in large quantities after integration into the host chromosome.Meanwhile,Western Blot was used to detect the expression levels of SNJ2capsid protein VP13 at different periods.It was speculated that the time of large release of SNJ2 was about 12 h after J7-1 was reduced.In addition,bioinformatics analysis predicted that orf8 encodes putative Orc1/Cdc6replication initiation protein.orf8 was ascertained to transcribe alone by RT-PCR.After J7-1-F?orf8 was induced by MMC,we did not detect attP’OP in the supernate,It was speculated that the orf8 deletion affected the replication of SNJ2?orf8,and the virus could not be properly packaged and released to the extracellular.Q-PCR was used to quantitatively analyze the content of attP’OP in supernatant and cell precipitation of J7-1-F、J7-1-F?orf8 and J7-1-F?orf8/pFJ6-1502 after the induction by MMC.These results suggest that orf8 gene may be related to virus replication.In conclusion,this study made a preliminary inquiry on SNJ2 replication mechanism in host cell.We speculate SNJ2 genome replicate in a integration form rather than in the free style.It is speculated that the large amount of replication time of SNJ2 in cells is 4 h after J7-1 induction by MMC,and the time of release to extracellular is 12 h.orf8 transcript alone and is associated with virus replication.This study laid a certain experimental foundation for further revealing the biological characteristics of archaea virus,origin and replication of virus and evolution of creatures.