The Biological Effects Of Coronavirus Papain-like Proteases On The Autophagy

BackgroundAutophagy is an intracellular conserved prcess that is important for the removal oflong-lived proteins mediated by lysosomes for the maintenance of cellular homeostasis.Coronaviruses (CoVs) are enveloped positive sense RNA viruses.Besides hCoV-229E,hCoV-OC43, several new human CoVs, hCoV-NL63, hCoV-HKU-1, SARS-CoV, anda new human coronavirus hCoV-EMC which emerged in2012, were identified. ThehCoV-NL63was first reported by the Holland scholar in the2004, which wasdistributed globally. The virus is one of the new found pathogens infected in the humanrespiratory tract. To date, there are no clinically anti-coronavirus specific drugs andvaccines. Autophagy is closly associated with the viral infection, which’s process isserved as the important target in the research of anti-virus drugs. The coronaviruspapain-like proteases (PLPs) had an important functions during the coronavirusreplication. It is not clear that the PLP regulate the autophagy and its possiblemechanisms. Based on the question, this study is to further explore the function of themembrane-located PLPs encoded by hCoV-NL63.ObjectiveTaking hCoV-NL63as a research model, the study aims to determine therelationship between the coronavirus infection and autophagy, which is to expand thefunctions of the PLP to support the improtant investigation values for the furtherexploring the antivirus drug and describing the pathogenic mechanism of the humannew coronavirus.MethodsFirst, the plasmids of the PLPs and pcDNA3.1-eGFP-LC3B were co-transfected into HEK293T cells and then the indirect immunofluorescence assay was used to detect theeGFP-LC3B positive autophagosome accumulation. Second, The Western blotting, theindirect immunofluorescence assay and the transmission electron microscopy were usedto ascertain whether PLPs induce autophagy. To further explore the mechanism, thewestern blotting assay was used to detect the autophagic substrate p62expression. Thelocalization of GFP-LC3and the mRFP-LC3autophagosome accumulation wasvisualized by confocal microscopy. Third, the plasmid of the pcMV-Myc-Beclin1wasconstructed. And the co-immunoprecipitation assay was used to detect the interaction ofPLP2-TM and Beclin1. The interacion of PLP2-TM and endogenous LC3was alsodetected. Meanwhile, the western blotting assay was used to detect the endogenousBeclin1expression in the different cell lines. The deubiquitination of Belcin1mediatedby PLP2-TM was investigated by the co-immunoprecipitation assay. In the end, theresults that PLP2-TM promoted the interaction between Beclin1and STING weredetected by co-immunoprecipitation assay.Results1. Coronavirus PLP2-TM was a novel autophagy-inducing protein encoded bycoronavirus: In this study, the indirect immunofluorescence assay was used todetermine whether PLPs induced the autophagy and the results showed that onlyPLP2-TM induced the eGFP-LC3B positive autophagosome accumulation and otherpapain-like proteases including PLP1, PLP1-TM and PLP2didn’t induced theautophagosome accumulation, indicating PLP2-TM alone induces the autophagy, whichis a novel autophagy-inducing protein encoded by coronavirus.2. PLP2-TM induced autophagic process: PLP2-TM promoted the eGFP-LC3Bpositive autophagosome accumulation in the HEK293T, Hela and MCF-7cells, whichalso induced the LC3-II expression in the different cell lines. PLP2-TM induced theeGFP-LC3B positive autophagosome accumulation and LC3lipidation in atime-dependent manner in HEK293T cells. The autophagosome double-membrane structures were observed in the PLP2-TM-transfected HEK293T cells by transmissionelectron microscopy and the positive control Rapamycin treated cells were observed theautolysosome single-membrane structures. The negative cells were not observed anymembrane-related structures in the HEK293T cells. The autophagy inhibitor3-MAinhibited the LC3-II expression induced by PLP2-TM, indicating that PLP2-TMinduced the authentic autophagy.3. PLP2-TM induced incomplete autophagy: In this study, PLP2-TM induced theLC3-II expression but not promoted the degradation of p62expression in the HEK293Tcells, while the positive control Rapamycin treated cells were detected the LC3-IIexpression and the degradation of p62expression, indicating that PLP2-TM induced theincomplete autophagic process. PLP2-TM induced both of the GFP-LC3andmRFP-LC3autophagosome accumulation, which was consistent with the resultsobserved in the positive control CQ-treated cells. The negative controlRapamycin-treated cells were only observed the mRFP-LC3autophagosomeaccumulation. The data showed that PLP2-TM induced the incomplete autophagicprocess.4. To further investigate the mechanism of the PLP2-TM-inducing autophagy：(1)PLP2-TM interacted with the LC3: In this study, co-localization of PLP2-TM andeGFP-LC3B positive autophagosome were observed by using the confocal microscopeand PLP2-TM coimmunoprecipitated with autophagy marker protein LC3-I,LC3-II.This data indicated that the autophagososme-like structure induced by PLP2-TM wasused for coronavrius replication during coronavirus infection.(2) PLP2-TM interactedwith the Beclin1: First of all, the vector of pcMV-Myc-Beclin1was constructed andthen the interaction of PLP2-TM and Myc-Beclin1was detected byco-immunoprecipitation assay. The PLP2-TM also interacted with the endogenousBeclin1. The data indicated that PLP2-TM activates autophagosome formation butblock its fusion with lysosomes through interacting with Beclin1.(3) PLP2-TM deubiquitinated Beclin1to promote Beclin1expression: PLP2-TM promotes theendogenous Beclin1expression in the HEK293T, Hela and MCF-7cells. And tofurther detect ubiquitination of Beclin1, the data showed that PLP2-TM mediateddeubiquitination of Beclin1in the HEK293T cells.5. PLP2-TM promoted the STING-Beclin1interaction: HEK293T cells weretransfected with Myc-Beclin1, HA-STING, PLP2-TM-V5as indicated for48hr. Thecell lysates were detected the interaction using the co-immunoprecipitation assay andthe results showed that PLP2-TM promoted the STING-Beclin1interaction but notpromoted the STING-LC3interaction. The data was partially to explain that PLP2-TMregulated STING mediated the innate immune response through theautophagosome-related molecule Beclin1to sequester the STING in the innate immuneresponse.ConclusionPLP2-TM, which is a novel autophagy-inducing protein encoded by coronavirus,deubiquitinates and interactes with the Beclin1to induce the incomplete autophagy.Meanwhile, PLP2-TM promotes the interaction of STING and Beclin1, indicating thatPLP2-TM sequesters the STING through the Beclin1located on the autophagosomemembrane, which may explain that PLP2-TM blocks the STING-mediated the signalingto negatively regulate antiviral innate immune.