Research On The Diversity And Function Of Equine Infectious Anemia Virus Rev Gene

Equine Infectious Anemia Virus（EIAV）is a member of the lentivirus genus of retroviral family.It mainly infects Equine animals（horses,donkeys and mules）.The Infectious disease causing fever,Anemia,bleeding,jaundice,wasting,edema and heart failure in Equine animals is called Equine Infectious Anemia（EIA）.EIAV and human immunodeficiency virus type I（HIV-1）share similar genomic structure,molecular mechanism of viral replication and immune mechanism.EIAV is the only lentivirus that has successfully developed a attenuated vaccine.Therefore,EIAV is an important model virus to study the mechanism of lentivirus replication,pathogenicity and immune protection.EIAV is a lentivirus with the simplest genome structure.It can encode three structural proteins（Gag,Pol and Env）and three accessory proteins（Tat,S2 and Rev）.Rev is an adjuvant protein shared by all members of the lentivirus genus.It binds to a special second-order sequence（RRE）on the viral m RNA to transport the m RNA encoding viral structural proteins to the cytoplasm and promote the expression of viral structural proteins.The biological function of Rev mediated viral structural protein expression is abbreviated as nuclear export activity.The EIAVRev gene has high variability,and the variation of Rev is associated with disease outcome.To establish a method for detecting the nuclear export-activity of EIAVRev,in this study,the Gag-Pol coding region sequence and RRE sequence of EIAV were cloned into an expression vector,named p Gagpol-RRE,and was transfected into HEK293T cells with or without the Rev expression vector pc DNA-Rev _FDDV-HA.Western blot analysis showed that p Gagpol-RRE was not expressed alone and could only be expressed under the action of Rev,and the expression level of Gag⁵⁵ increased with the presence of the Rev in a dose-dependent manner.On the basis of pc DNA-Rev _FDDV-HA,the key site 36 of Rev nuclear export activity L was mutated into A by PCR method,and the Rev expression vector pc DNA-Rev _L36A-HA was constructed and co-transfected with p Gagpol-RRE in HEK293T cells.Western blot results showed that the expression of Gag⁵⁵could not be detected.These results indicate that the expression vector p Gagpol-RRE can be used to evaluate the nuclear export activity of EIAVRev.On this basis,we further identified that Rev derived from different EIAV strains could all mediate the expression of Gagpol.These results indicated that expression vector p Gagpol-RRE could be used to evaluate the nuclear export-activity of EIAVRev.In the study,we found that the EIAV attenuated vaccine strains had high frequency of deletion mutations in the non-essential region of Rev,and the number of deletion bases of these mutations were all three integer times,so they did not affect the translation of Rev.We constructed a Rev expression vectors with 27 aa deletions and EIAV infectious clones with 27 aa deletions,respectively.The transfected cells showed that the the deletion mutation did not affect the expression and the subcellular localization of Rev.The deletion mutation did not affect the expression of Gag-Pol,which the main structural gene of EIAV.that is,the Rev deletion mutation did not affect its nuclear export-activity.Rev deletion mutant did not affect the production of viruses,but aborted the replication of progeny virion in the target cells.These findings suggest that,in addition to the nuclear export function,the Rev of EIAV may also have other unknown functions,which are involved in the viral replication process.The nuclear-export activity of Rev depends on its own polymerization.In order to study the polymerization characteristics of EIAVRev,this study first verified the polymerization of Rev through Co-IP and bilimolecular fluorescence complementation experiments,and the laser confocal analysis showed that the polymerization occurred in the nucleus.Secondly,a split luciferase assay for quantitative detection of Rev polymerization was established in this study.The analysis of sites known to affect the nuclear-export activity of Rev shows that the system can detect the polymerization of Rev characteristically.On this basis,the conserved amino acid sequence sites of Rev of different EIAV strains were mutated.The split luciferase assay showed that 102V of Rev was the key site affecting its polymerization,and the nuclear-export activity assay of Rev was used to prove that the mutation of this site would cause the loss of nuclear-export activity of Rev.In conclusion,this study has established the evaluation Rev nuclear-export activity,the method was used to study the biological function of Rev_Δ122-148,which an,EIAVRev natural mutant,found that the deletion mutation did not affect the nuclear output activity of Rev,but caused the disruption of EIAV replication ability in target cells.The split luciferase technique was used to establish a method for detecting the multimerization of Rev protein,the 102V site in the conserved region of Rev was identified as the key site affecting its multimerization.