Study On The Mechanism Of E3 Ubiquitin Ligase CRL4B^PRPF19 Regulating SARS-CoV-2 Replication

The ORF6 protein is proposed to play crucial biological role in the life cycle of SARS-CoV-2.On the one hand,the ORF6 protein is deeply involved in antagonizing the host’s natural immunity and ensuring stable virus replication;on the other hand,the ORF6protein can also localize lipid droplets,mediating interactions between lipid droplet and endoplasmic reticulum,lipid droplet and mitochondria,enhancing lipid transport amount and providing energy for virus replication.Studying the interaction between the ORF6protein and the host will help discover the key factors that regulate SARS-CoV-2replication in host cells and further elucidate the potential molecular mechanisms of new coronavirus infection and replication.Ubiquitination modification is widely involved in the arms race between viruses and hosts.Taking this as an entry point,this article uses immunoprecipitation combined with mass spectrometry analysis to identify factors in host cells that may mediate ubiquitination modification of ORF6 protein.Further screening revealed that the intracellular CRL4B^PRPF19 E3 ubiquitin ligase complex mediates ORF6ubiquitination modification and proteasome degradation.Mechanistically,it was found that the CRL4B^PRPF19 complex interacts with ORF6 and promotes K48-linked ubiquitination modification at the four lysine sites of ORF6.Further research showed that CRL4B^PRPF19alleviated the inhibition of the interferon pathway produced by ORF6 and inhibited viral infection.Finally,it was determined that the CRL4B activator,etoposide,can promote the degradation of ORF6 protein,inhibiting the replication of SARS-CoV-2 at the cellular level and mouse models.The main contents are as follows:1.Determine the degradation of ORF6 protein through the proteasome pathwayBy detecting the protein abundance of overexpressed SARS-CoV-2 accessory proteins in cells after treatment with the proteasome inhibitor MG132,it was determined that ORF6 was degraded through the proteasome pathway.This fact was further confirmed by treating cells with other proteasome inhibitors bortezomib and carfilzomib.2.Identify host factors that mediate the ORF6 degradationBy analyzing the results of mass spectrometry experiments,we identified factors that interact with ORF6 and the interactions become stronger after MG132 treatment.We further conducted functional analysis of these factors and screened out factors related to the ubiquitin-dependent degradation.By knocking down the above factors one by one,it was found that after knocking down CUL4B,DDB1,RBX1,and PRPF19,the stability of the ORF6 protein was enhanced,proving that CUL4B,DDB1,RBX1,and PRPF19 jointly form the CRL4B^PRPF19E3 ubiquitin ligase complex,mediating ORF6 degradation through the proteasome pathway.3.Interaction between CRL4B^PRPF19and ORF6The results of Co-IP experiments showed that CUL4B,DDB1,RBX1 and PRPF19interacted with ORF6.Fluorescence resonance energy transfer assay demonstrated that PRPF19 acts as a substrate recognition receptor and directly recognizes and binds to ORF6protein.4.CRL4B^PRPF19 promotes ORF6 K48-linked ubiquitination modificationThrough ubiquitination detection experiments,it was found that CRL4B^PRPF19promoted the K48-linked ubiquitination modification of ORF6.Knockdown of CUL4B,DDB1,RBX1,and PRPF19 reduced the level of ORF6 K48-linked ubiquitination.5.All four lysine sites of ORF6 are involved in ubiquitination of K48-linkedTo identify the ubiquitination sites in ORF6 induced by CRL4B^PRPF19,we analyzed the full length of ORF6 and found four lysine sites,which were subsequently substituted by arginine separately or in combination.We examined the stability of ORF6 mutants with or without MG132 treatment and it was found that the four sites were involved in ubiquitination modification and proteasomal degradation,and the ubiquitination of ORF6would disappear only after all four lysine sites were mutated.6.CRL4B^PRPF19promotes ORF6 degradation and helps restore the interferon pathwayORF6 protein is a potent interferon antagonist,and we found that ORF6overexpression suppressed IFN-dependent ISRE induction.However,overexpression of PRPF19 induced the degradation of ORF6,resulting in ISRE releasing and loss of IFN-antagonizing.Furthermore,we determined the m RNA levels of IFN-α,IFN-β and IFN-stimulated genes（ISGs）IFIT1,IFIT3 and ISG15 when ORF6 or ORF6 and PRPF19were cotransfected into cells.Similarly,PRPF19 enhanced ORF6 degradation,further upregulating the m RNA levels of IFN-α,IFN-β and ISGs compared with ORF6 alone.7.CRL4B^PRPF19 promotes ORF6 degradation and inhibits SARS-CoV-2 replicationThe replication of the Omicron strain or the Wuhan strain was detected after overexpression or knockdown of PRPF19 in cells,and it was confirmed that CRL4B^PRPF19antagonizes SARS-CoV-2 replication by promoting ORF6 degradation.8.Enhanced CUL4B activity by Etoposide suppresses SARS-CoV-2 replicationRecent studies showed that Etoposide can induce CUL4B neddylation and promote the ubiquitination process.We thus examined the influence of Etoposide on ORF6degradation and virus replication at the cellular level and in mouse experiments.It was confirmed that etoposide inhibits SARS-CoV-2 virulence and alleviated lung lesions such as alveoli shrinkage and pulmonary edema typically observed in control mice by promoting the degradation of the ORF6 protein.