Interaction Analysis Of ORF2 Protein And Viruse Promoters From Sulfolobus Virus STSV2

The virus can modulates the transcription of the host and its own genes by virus-encoded specific transcriptional regulatory proteins directly binding to the DNA or competitively binding to the host RNAP during infection,and initiates a virus-related life process.Although studies of bacteriophages and archaeal viruses have primarily revealed some regulatory mechanisms of viral?or phage?gene transcription during infection,but little is known about the regulation mechanisms of archaeal viral gene transcription during infection.In addition,all of the reported archaeal viral transcriptional regulators are functioned as repressors for the gene transcriptional regulation.So far,no viral regulatory proteins as transcriptional activator to regulate the gene transcription during infection was reported.Our previous studies have shown that ave shown that overexpression of the transcriptional regulator-encoded gene ORF2 of virus STSV2?Sulfolobus tengchongensis spindle-shaped virus 2?in Sulfolobus islandicus E233S can significantly increase the mRNA transcription levels of host and viral genes during viral infection,indicating the characteristics of a gobal transcriptional activator.However,the specific regulation mechanisms of ORF2 remains unclear.Therefore,the virus STSV2-encoded ORF2 protein was used in this study to analyze the binding sites of viral gene promoters,and analysis of the structure characteristics and function of STSV2 capsid protein gene ORF37 were simultaneously carried out.ORF2 protein is a transcriptional regulator with typical winged HTH?wHTH?domain.To analyze the interaction of ORF2 protein with DNA and specific sequence information of binding sites during transcription,the ORF2 gene was firstly amplified by PCR,which was further used to construct a recombinant prokaryotic expression plasmid pET32aORF2 by fusing his tag to the N-terminal of ORF2 protein.The resultant plasmid was further transformed into E.coli BL21?DE3?for expression.After inducing expression,the his-tagged ORF2 protein was purified by Ni²⁺affinity chromatograph.At the same time,the upstream⁵00bp promoter fragments of ORF2gene,ORF8 gene and ORF37 gene were amplified and labeled with biotin,respectively.Electrophoretic mobility shift assay?EMSA?was performed to analyze in vitro the interaction of ORF2 protein with these three promoters.The results showed that the ORF2 protein interacted with these three promoter fragments,indicating that the ORF2 protein is a global transcriptional regulator that promotes gene transcription.Further,based on the promoter fragment P37-500,two shorter fragments P37-216 and P37-144,upstream of the viral capsid protein-coding gene ORF37,were obtained for same EMSA analysis.The results showed that ORF2 also could bind to both two short fragments.In order to further determine the sequence information of ORF2 protein-binding promoter DNA,the DNA structure of the two fragments of P2-500,P8-500 and P37-500 bound to ORF2 protein was analyzed by DNase I footprint assay.The results showed that ORF2 protein and P2 have The two binding sites 47-81 and 130-147 have two binding sites 44-94 and 124-147 with P8,and three binding sites 46-94,118-145and 154-175 with P37.Analysis of all binding sites revealed that the number of bases per binding site was different,and the protein-binding promoter did not have a highly conserved sequence structure,but would have a preference for A/T;in addition,the most downstream binding site The inclusion of a portion of the BRE element enhances the recruitment of RNAP complexes to facilitate gene transcription.This is in contrast to the reported transcriptional regulation of the RHH domain of the archaeal virus that acts as a transcriptional repressor.To further determine the specific structural information of the ORF2protein-bound promoter,the promoter activities of P37-500,P37-216 and P37-144were analyze to identify the core promoter region and other elements.First,a reporter gene lacS was inserted at downstream of the araS promoter,based on the Sulfolobus expression vector pSeSD,to construct an archaeal viral promoter screening system.The three promoter fragments P37-500,P37-216 and P37-144 were used to replace the native araS promoter to constructed the promoter activity screening vectors pSeSDp37-500lacS,pSeSDp37-216lacS and pSeSDp37-144lacS,respectively,and further transformed the E233S strain.The structural information of the promoters and their activity were determined by the?-galactosidase activity of the transgenic strains.The analysis results showed that the P37-144 sequence contained core promoter elements such as TATA-box,BRE?Transcription factor B Identification Element?,PPE?Proximal Promoter Element?and Inr?Initiator element?.Promoter activity analysis showed that the three P37 promoter fragments were not inducible promoters,and the P37-500 promoter exhibited similar activity to that of the araS promoter,but the promoter activities of P37-216 and P37-144 were higher than that of the araS promoter.The activity of P37-144 is 2.1 times higher than that of P37-500,and the activity of P37-216 is 1.7 times higher than that of P37-500.These results indicated that some repressor elements might localized at the upstream of the-144bp site of the P37 promoter,but the specific sequence information is still needed to be further revealed.In summary,the S.oxysporum STSV2 transcriptional regulatory protein ORF2interacts with multiple viral gene promoters and is a universal transcriptional regulator that promotes gene transcription.It binds DNA without a more conserved sequence structure.However,there will be a preference for A/T,and some downstream BRE sites will be included in the downstream site to enhance the recruitment of RNAP complexes.However,the specific mechanism of action and related regulatory processes need further study.The core promoter element and activity analysis of the P37 fragment showed that the T37-144 contained the TATA-box,BRE,PPE and Inr core promoter elements,and the P37-144 fragment had the highest promoter activity and the P37-216 fragment.The lowest P37-500 proves that there may be some repressive elements upstream of P37-144.This study will help to reveal the regulatory mechanism of ORF2 to promote gene transcription,to lay the foundation for revealing the transcription process and other life activities of STSV2virus infecting host,and to improve the research on the origin and evolution of archaeal virus and the relationship between virus and host..