Transcriptional Regulation Mechanism Of Equine Herpesvirus 1 Neural Pathogenic Factor UL24

Equine herpesvirus 1(EHV-1) is a significant etiologic agent of abortion, paralysis, ataxia, and encephalomyelitis with neural symptoms. There are six regulatory proteins of EHV-1, including an immediately early protein(IE), four early proteins(IR2, EICP0 EICP22 EICP27) and a late protein(ETIF), which play important roles in viral gene expresssion regulation. Among the 76 genes of EHV-1, only eight proteins(gK, IR5, IE, IR2, EICP0, EICP22, EICP27, ETIF) have been characterized for their promoter region. Limited information known about the interaction of EHV-1 genes and regulatory proteins. ul24 is highly conserved in all herpesvirus, its coding protein is closely related to neural pathogenic. ul24 expression and regulation are correlated with the neural system diseases caused by EHV-1. At present, there is no report about transcriptional regulation mechanism of EHV-1 ul24. In this study, we take equine herpesvirus 1(EHV-1) ul24(ORF37) as the target to study the accurate location of ul24 promoter in EHV-1 genome and transcriptional regulation mechanism of ul24.In this study, two transcriptional start site(Tss1, Tss2) were confirmed by 5’RACE, which located upstream of ul24 open reading frame and at 70118 bp and 70397 bp of the genome respectively. According to the sequence signature nearby transcriptional start site, we constructed recombinant plasmids(pUL24I, pUL24II-1, pUL24II-2, pUL24II-3, pUL24II-4, pUL24II-5, pUL24II-6, pUL24II-7, pUL24II-8). Among them, UL24 I and UL24II-5 are responsible for ul24 transcription initiation at Tss1 and Tss2, respectively, which were confirmed by dual-luciferase reporting system. At the same time, we constructed eukaryotic expression recombinant plasmids of EHV-1 regulatory proteins(p IE, pIR2, pEICP0, pEICP22, pEICP27, pEICP27-Flag, pETIF), which were tested by Western blotting. To detect the effect of regulation proteins on ul24 promoter activity, we co-transfect ul24 promoter recombinant plasmids and regulatory proteins, detecting by dual-luciferase reporting system. The results showed that regulatory proteins have similar effects on Ul24 promoters, EICP0 increased UL24 I and UL24II-5 promoter activity by 2.3-fold and 17.5-fold; ETIF also did it by 1.8-fold and 4-fold. In contrast, IR2 reduced UL24 I and UL24II-5 promoter activity by 43.4-fold and 26.3-fold; IE did it by 2.3-fold and 3.9-fold; EICP22 can strengthen IE to boost UL24 I promoter activity.Moreover, we use immune co-precipitation and laser-confoca to explore the mechanism of EICP22 protein enhancing ul24 promoter activity. The results showed that EICP22 protein enhanced ul24 promoter activity does not depend on its interaction with transcription factor TBP.This study not only revealed the new transcriptional regulation mechanism of ul24 but also provided theoretical clue and experimental evidence for subsequent research of neural conditioned pathogenicity.