The Mechanism Of Vesicular Sorting Protein 35 (VPS35) Regulating PDCoV Proliferation

Porcine deltacoronavirus（PDCoV）is an emerging porcine enteric coronavirus that causes diarrhea,vomiting,dehydration and even death in piglets.PDCoV has cross-species transmission potential.In addition to pigs,PDCoV also infects cattle,mice,poultry and human,posing a serious threat to the development of livestock development and human health.PDCoV-encoded accessory protein NS6 plays an important role in the infection and pathogenesis of the virus.However,the specific mechanism by which NS6interacts with host proteins to participate in the regulation of viral replication is still unclear.Retromer plays an important role in the retrograde of intracellular proteins.After binding to endosomes,retromer recognizes and sorts various cargo proteins and transports them from endosomes to trans-Golgi network（TGN）or cytoplasmic membrane,avoiding their degradation in lysosomes.A variety of viruses employ retromer to enter cells,assemble virions,and promote their proliferation.This study identified the interaction between PDCoV NS6 and VPS35,the core component of retromer,and explored the mechanism by which NS6 hijacks VPS35 to promote viral proliferation.The specific research content is as follows:1.PDCoV NS6 interacts with retromerUnder the condition of PDCoV infection,immunoprecipitation and mass spectrometry analysis were performed using NS6 monoclonal antibody.It ws found that NS6 had a potential interaction with VPS35.To verify this result,co-immunoprecipitation assay was conducted under the conditions of NS6-overexpression and PDCoV infection,and the results confirmed the interaction between NS6 and VPS35.Because VPS35 forms a cargo selective complex together with both VPS26 and VPS29,we further explored whether NS6 interacted with VPS26 and VPS29.Co-IP assay were carried out under the condition of overexpression and PDCoV infection,and the results showed that PDCoV NS6 interacted with both VPS26 and VPS29.These results suggested that PDCoV NS6can interact with retromer（VPS35,VPS26 and VPS29）.2.Effect of VPS35 on the proliferation of wild-type PDCoV and recombinant PDCoV with NS6 gene deletionBecause VPS35 is the core component of retromer and plays most important roles,the biological significance of the interaction between NS6 and VPS35 was further studied.To analyze the effect of VPS35 on wild-type PDCoV proliferation,LLC-PK1 and IPI-2I cells were transfected with a VPS35-specific small interfering RNA（si RNA）interference（si VPS35）,and then infected with PDCoV for 12 h.The samples were collected and real-time fluorescent quantitative RT-PCR（RT-q PCR）,TCID₅₀and western blot analysis were performed.The results showed that knockdown of VPS35 inhibited PDCoV proliferation,but did not affect the adsorption and invasion of PDCoV,indicating that VPS35 played a role after the virus entring into cells.In order to determine the role of NS6 protein in VPS35-mediated regulation of PDCoV proliferation,a recombinant PDCoV with NS6 deletion was constructed and named r CHN-HN-2014-ΔNS6.LLC-PK1cells were transfected with si VPS35 and then infected with r CHN-HN-2014-ΔNS6.After12 h,cell samples were collected for RT-q PCR,TCID₅₀and western blot analysis.The results showed that knockdown of VPS35 did not significantly inhibit the proliferation of r CHN-HN-2014-ΔNS6,indicating that the regulation of PDCoV proliferation by VPS35was NS6-dependent.3.Golgi apparatus and late endosomal/lysosomal transport pathways play important roles in PDCoV infectionPrevious reports have shown that VPS35 can regulate the retrograde process of cargo proteins from endosomes to trans-Golgi network.We speculated that the role of VPS35 in promoting PDCoV proliferation may be related to its cargo transport function.LLC-PK1cells were treated with Golgicide A,Brefeldin A,and Retro-2,which target the Golgi and endosomal transport pathways,and then infected with PDCoV.Western blot,RT-q PCR and TCID₅₀results showed that all three inhibitors could inhibit the proliferation of PDCoV.LAMP2 is a marker protein of late endosomal/lysosome.When LAMP2 was knocked down in LLC-PK1 and IPI-2I cells,the proliferation of PDCoV was significantly inhibited,indicating that late endosomal/lysosome played an important role in the PDCoV infection.Indirect immunofluorescence assay showed that NS6 co-localized with Golgi apparatus and late endosomal/lysosome after PDCoV infection.These results suggested that the Golgi and late endosomal/lysosomal transport pathway were crucial for PDCoV infection.4.Knockdown of VPS35 causes a selective endosome-to-Golgi retrieval defect of NS6To further analysize whether knockdown of VPS35 resulted in retention of NS6protein in lysosomes and caused its degradation,LLC-PK1 cells with VPS35 knockdown were infected with PDCoV or transfected with expression construction encoding NS6protein,followed by treatment with protein synthesis inhibitor cycloheximide（CHX）.Western blot results showed that knockdown of VPS35 significantly accelerated the degradation of NS6 protein.Meanwhile,LLC-PK1 cells with VPS35 knockdown were infected by recombinant PDCoV with the replacement of NS6 gene by GFP（r CHN-HN-2014-ΔNS6-GFP）and then treated with CHX.After CHX treatment,cells were collected for detecting the intracellular GFP protein expression.The results showed that compared with the wild-type PDCoV group,knockdown of VPS35 had a minor effect on degradation of GFP.These results suggested that VPS35 interacted with NS6,resulting in the reduced degradation of NS6 protein.We further detected the localization of NS6protein in Golgi apparatus and late endosomal/lysosome with or without VPS35knockdown by indirect immunofluorescence assay.The results showed that in cells with VPS35 knockdown,the co-localization of NS6 protein with trans-Golgi network decreased,but its co-localization with late endosomes increased.Thus,VPS35knockdown selectively inhibited the retrieval of NS6 protein from late endosomes to trans-Golgi network,thereby accelerating the degradation of NS6 protein and inhibiting PDCoV proliferation.5.The positive regulatory effect of VPS35 on porcine enteric coronaviruses is conservedTo investigate whether VPS35 could affect the proliferation of other porcine enteric coronaviruses,LLC-PK1 cells were transfected with si VPS35 and then infected with swine acute diarrhea syndrome-coronavirus（SADS-CoV）,transmissible gastroenteritis virus（TGEV）and porcine epidemic diarrhea virus（PEDV）.RT-q PCR and western blot results showed that knockdown of VPS35 inhibited the proliferation of SADS-CoV,TGEV and PEDV,suggesting that the positive regulatory effect of VPS35 on porcine enteric coronaviruses was conserved.