The Function And Mechanism Of Mitochondrial-associated Membranes Tether Protein And Rab11a Protein In Resistance To PRRSV Infection

Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the most dangerous and challenging viral infectious diseases in pig industry worldwide.Due to the characteristics of viral susceptibility to mutation,antigenic diversity,suppression of host immune responses,the virus had strategies to escape the immune responses in vaccinated herds.Now effective measures to protect pigs from PRRSV are still lacking in pig industry.Therefore,we reveal the mechanism of host-PRRSV interaction,explore the key host genes that affect the replication of PRRSV,which can provide a reference for the development of new disease-resistant varieties and new vaccines against PRRS.Part 1: Mitochondrial fission and the MAMs were increased during PRRSV infection and modulation of MFN2 affected PRRSV replicationendoplasmic reticulum and mitochondria are organelles with important functions in cells,the physical contact site between the ER and mitochondria namely mitochondria-associated membranes(MAMs).It represents a hot spot for the intracellular signaling including mitochondrial fission,autophagosome formation,ER stress,and apoptosis.The synthesis of RNAs by PRRSV in host cells requires efficient and accurate assembly of replication and transcription complexes(RTC).The present study showed that the RTC may originate from the host ER or mitochondrial components.Besides,PRRSV has a complex interaction with the host by regulating processes such as mitochondrial fission,autophagy,endoplasmic reticulum(ER)stress,and apoptosis.However,the role of MAMs play in it are still unclear.Therefore,this study investigated the structural and functional changes of MAMs and mitochondria during PRRSV infection and their role in anti-PRRSV replication.(1)ROS levels increased and mitochondrial was dysfunction in PRRSV-infected cells: Flow cytometry revealed that the level of free radicals(ROS)in PRRSV-infected cells was increased(P <0.01);confocal microscopy observed that JC-1 in PRRSV-infected group changed from red fluorescence to green fluorescence,indicating that mitochondrial membrane potential(MMP)was decreased in infected group;microplate reader assay revealed that the content of intracellular ATP in infected group was significantly lower than control group from 12 h to 48 h post-infection(P <0.001 or P <0.01).(2)PRRSV infection induced mitochondrial fragmentation and activated the mitochondrial fission pathway: Confocal microscopy revealed that the proportion of fragmented mitochondria was significantly higher in Marc145 cells infected with PRRSV for 36 h than in controls(62.279% ± 3.66% vs 15.44% ± 2.66%)(P <0.001).The dynamic changes of mitochondria during PRRSV infection were further counted,and the results showed that the percentage of fragmented mitochondria in infected-cells was significantly higher than that in the control group,showing a time-dependent manner.Porcine alveolar macrophages(PAM)are the primary target cells for PRRSV under natural infection.Confocal microscopy showed that mitochondria were clustered around the nucleus and fragmented in PAM cells.Western Blot indicated that the mitochondrial fission pathway CDK1/p-Ser616 Drp1 was activated in PRRSV-infected Marc145 cells and PAM cells.These results indicated that PRRSV infection disturbed mitochondrial dynamics,increased mitochondrial fission and activated the mitochondrial fission pathway.(3)Increased mitochondria-associated membranes(MAMs)in PRRSV-infected cells: Transmission electron microscopy observed that the gap between the ER and mitochondria became smaller,some fragmented mitochondria were surrounded by the ER,and the area connecting the ER to mitochondria increased;Ds Red-Sec61β vector were constructed to trace the ER,and confocal microscopy observed that it colocalization with the mitochondrial marker protein(Tom20)was increased;In addition,PRRSV NSP2 protein was localized to the endoplasmic reticulum.Western Blot analysis revealed that both the ER and mitochondrial marker proteins were significantly increased at 12 h post-infection and significantly decreased at 36 h post-infection;MAMs marker protein significantly increased at 24 h and 36 h post-infection.These results indicated that PRRSV infection changed the content in ER and mitochondria,and increased the formation of mitochondria-associated membrane(MAMs).(4)PRRSV infection modulated the expression of key genes in the MAMs region,and interfering MFN2 inhibit the replication of PRRSV: We further detect the expression of key genes that enriched in the MAMs region.The results of q PCR assay showed that the m RNA expression levels of MFN2,IP3R2,VDAC1,IP3R1,and PACS2 were significantly increased at 36 h post infection(P <0.001).MFN2 is an endoplasmic reticulum-mitochondria tether,we further detect the effect of interfering MFN2 expression on PRRSV replication.The q PCR results showed that viral ORF7 m RNA in infected group was lower than those in the control group at 12 h,24 h,36 h and 48 h post infection(P <0.001).Western Blot results exhibited that interfering the expression of MFN2 leaded to reduced expression of intracellular viral NSP2 protein.It was further validated on PAM cells and the results showed that the intracellular PRRSV N protein and NSP2 protein were lower in the si MFN2-treated group than in the NC group.Part 2: The role and mechanism of Rab11 a gene in resistant to PRRSV replicationOur group previously conducted a transcriptomic analysis of the lung tissues of PRRSV-infected Tongcheng pigs(resistant pigs)and Landrace pigs(susceptible pigs)and found that the expression of Rab11 a was significantly higher in Landrace pigs than in Tongcheng pigs.The small GTPase Rab11 a is involved in regulating membrane trafficking events during autophagy as a transport endosome.Therefore,this study investigated whether Rab11 a affects the replication of PRRSV by participating in the autophagy pathway.(1)PRRSV infection induced complete autophagy and upregulated Rab11 a expression: Western Blot was used to detect the expression of autophagy marker proteins(LC3-II)in PRRSV-infected cells at different times during infection.The results showed that the expression of LC3-II was significantly increased from 24 h to 48 h post infection(P <0.001)and peaked at 36h;The expression of p62 was significantly decreased at 36 h and 48 h post infection(P <0.001).The intracellular LC3 in PRRSV-infected cells was punctate and colocalized with LAMP2(lysosomal membrane receptor protein)by confocal microscopy.These results suggest that PRRSV infection not only induces autophagosome formation,but also promotes the fusion of autophagosomes and lysosomes for degradation,Therefore,complete autophagy occurs in PRRSV-infected cells.This study further verified the expression of Rab11 a after PRRSV infection at the cellular level.The results of q PCR and Western Blot showed that PRRSV infection caused increased expression of Rab11 a m RNA and protein.Combined with previous sequencing results,PRRSV infection induced Rab11 a expression both in vitro and in vivo.(2)Rab11a was involved in PRRSV-induced autophagy process: Western Blot revealed a significant increase in LC3-II and p62 expression in the CQ(autophagosome-lysosome degradation inhibitor)-treated group(P <0.001).The expression of LC3-II reduced in the LY294002(early inhibitor of autophagy)treated group(P < 0.001).Western Blot analysis revealed significant increase in the expression of both LC3-II and p62 in si RNA-treated cells(P <0.01 or P <0.001).After interfering with Rab11 a,the expression patterns of LC3-II and p62 were consistent with the CQ-treated group.Taken together,Rab11 a may be involved in the process of autophagosome maturation when PRRSV infects cells.(3)Interfering with Rab11 a to inhibit the replication of PRRSV: Western Blot results showed that the intracellular viral NSP2 expression was significantly reduced in the LY294000 treated group(P <0.001).Intracellular viral NSP2 expression was significantly reduced in the CQ-treated group(P <0.01).These results indicated that blocking autophagy inhibits replication of PRRSV.The effect of interfering with Rab11 a on virus replication was next examined.The results showed that the m RNA expression of intracellular virus NSP2 and ORF7 were significantly reduced after Rab11 a interference(P <0.01 or P <0.001),and the expression of intracellular virus NSP2 protein was reduced(P <0.001).In summary,this study revealed that mitochondrial dysfunction and mitochondria fragmented in PRRSV-infected cells;PRRSV activated the mitochondrial fission pathway CDK1/p-Ser616Drp1;MAMs were increased in PRRSV-infected cells and interfering the MAMs tether protein MFN2 could inhibit PRRSV replication;Rab11a was involved in PRRSV-induced autophagy to favor PRRSV replication.The key host factors Rab11 a and MFN2 affecting PRRSV replication were screened to provide molecular targets for disease resistance breeding and development of prevention and treatment drugs.