HIV Integrase Degradation By Engineered Chimeric Ubiquitin Ligases

In eukaryotic cells,the ubiquitin-proteasome pathway(UPP)plays important roles in many cellular events by targeting certain regulatory proteins for 26 S proteasome degradation.As a highly conserved 76 amino acid polypeptide,ubiquitin is conjugated to substrates via an enzymatic cascade by activating enzyme(E1),ubiquitinconjugating enzyme(E2)and ubiquitin ligase(E3).E3 s bind E2 and determine specificity of the substrate to be ubiquitinated.When linked through K48,the polyubiquitinated substrates are recognized and degraded by the 26 S proteasome complex in the UPP.Owing to its critical role in regulating protein degradation,the UPP plays important role in host immunity against pathogens including viruses.Since many viruses are also competent in hijacking the intracellular ubiquitin-proteasome system to escape from host immune responses,it would be also applicable for us to engineer cellular E3 s to instruct the host UPP to target more vital viral proteins.In this study,we aimed to develop chimeric E3 s which target viral proteins for degradation,thus achieving improved cell protection from viral infection.Considering its virulence and lack of vaccine,human immunodeficiency virus 1(HIV-1)has been chose to be our target for candidate viral proteins.HIV viral proteins are mainly composed of structural proteins,viral-specific enzymes and accessory proteins.Their functions are highly dependent on well-studied virus-host protein interactions,which are conserved in all fully functional viruses.This study attempted to design and construct artificial chimeric ubiquitin ligases(E3s)based on known human E3 s in order to manually target HIV-1 integrase for ubiquitin proteasome pathway-mediated degradation.Herein,a series of prototypical chimeric E3 s have been designed and constructed,original substrate-binding domains of these E3 s were replaced with host protein domains which interact with viral proteins.After functional assessment screening,LIXR1 was identified as the first chimeric E3 targeting HIV-1 NL4-3 integrase.Expression of LIXR1 in cells could decrease the protein level of HIV integrase.But further optimization experiments and analysis indicated that LIXR1 did not decrease the protein level of HIV integrase via UPP.After screening anew,146 LI was identified as a functional chimeric E3 which degraded HIV-1 NL4-3 integrase via UPP.Because 146 LI was failed to be expressed in E.coli.,it was further optimized to generate 146LIS(146LI short)which could effectively catalyze ubiquitination of HIV-1 NL4-3 integrase in vitro.146 LIS has been shown to induce K48-specific polyubiquitination and reduce protein level of HIV-1 NL4-3 integrase more effectively in cells.Lymphocyte cells with 146 LIS knock-in generated by CRISPR HDR showed remarkably decreased integration of HIV-1 NL4-3 viral DNAs and reduced viral replication without obvious cell cytotoxicity.This study proved that it is feasible to construct chimeric ubiquitin ligases to target a viral protein.The design of chimeric E3 146 LIS could be borrowed to generate more functional E3 chimeras to target other HIV viral proteins as well,or applied to new strategies which can protect people from other severe viruses and human diseases.