Preparation Of Keap1 Monoclonal Antibody And Its Application In Keap1-Nrf2 Pathway Regulation Of EIAV Replication

Equine infectious anemia virus(EIAV),an equine lentivirus,causes persistent infection characterized by recurring febrile episodes associated with EIA clinical signs.Unlike other lentivirus,most EIAVinfected horses turn to a life-long inapparent carriers by eliciting the immune control over virus replication.Therefore,as a well-established model for studies on lentiviruses and related host immune responses,understanding of the innate immnre response to equine EIAVinfection provides important information about the interaction between host immune defense and lentiviruses infection.In our previous research,we observed that multiple canonical pathways were coordinately regulated by EIAV infection.Except the antiviral pathways as well as the inflammatory pathways,Nrf2(transcription factor NFE2-related factor 2)antioxidant pathway were highly enriched upon EIAV infection.Based on the current research progress and previous data,we speculated Keap1-Nrf2 axis may play a key role in controlling EIAV infection.Elucidating the cellular innate defense during EIAV infection could facilitate the understanding of the interplay between immune control and EIAV infection.These results suggest that the Keap1-Nrf2 antioxidant pathway may be involved in the regulation of EIAV infection.In combination with the current research progress and previous results,we speculated that the interaction between EIAV and Nrf2 pathway contains important information about host inflammatory regulation and immune control of EIAV.We firstly generated a mouse monoclonal antibody against equine Keap1 by using the mature monoclonal antibody preparation technology developed in our laboratory.The Keap1 m Ab has high specificity and sensitivity and can be used in the following study.Then,transcriptomics,Real time PCR and other methods were used to determine the activation of Keap1-Nrf2 pathway upon EIAV infection in target cells,and the ARE-Luc activation system was used to screen the EIAV-encoded proteins responsible for activation.It was found that EIAVRev was the target protein that activated the Nrf2-ARE antioxidant pathway.The effect of Nrf2 activation on EIAV replication was further investigated by knockout of Nrf2 and induction of endogenous expression of Nrf2 by agonists.Activation of Keap1-Nrf2 pathway inhibited EIAV replication.Further studies showed that Keap1,a negative regulator of Nrf2,could bind to Rev,but Keap1 did not affect the expression of Rev.Furthermore,laser confocal analysis and nucleo-cytoplasmic separation of proteins revealed that Keap1 could bind to Rev and target it into the cytoplasm,limiting the nucleo-cytoplasmic shuttle ability of Rev.The nucleoplasmic separation of RNA isolated from EIAV pseudovirus-infected cells and Real-time PCR detection further confirmed that Keap1 affected the Rev-mediated RREdependent m RNA transport,thereby inhibiting the synthesis of virions.In summary,this study successfully prepared a horse-derived Keap1 monoclonal antibody,and found that the Keap1-Nrf2 pathway is activated by recognizing EIAV-Rev,and it is clear that the combination of Rev and Keap1 altered the subcellular location of Rev,It impacted Rev's nuclear and cytoplasmic shuttle ability and its ability to transport viral m RNA,thereby inhibiting EIAV replication.This study not only explained the molecular details of EIAV and Nrf2 antioxidant pathway regulation,and the molecular mechanism underlying Nrf2 pathway inhibits EIAV replication,but also laid an important foundation for further exploring the host's natural immune control of EIAV.