宿主分子Tyro3对猪瘟病毒复制的影响及其与E2蛋白相互作用的研究

Tyro3 is a host factor that interacts with E2 glycoprotein and promotes classical swine fever virus replication

本研究室前期研究发现受体酪氨酸激酶家族(TAM)成员Mertk为猪瘟病毒(CSFV)的侵入因子。为研究TAM受体家族另外两个成员Tyro3及Axl对CSFV复制的影响,本研究设计并合成了针对Tyro3、Mertk、Axl的siRNA(siTyro3、siMertk、siAxl)和一条无关siRNA(siNC),将这3种siRNA分别转染PK-15细胞,24 h后以MOI 0.01感染CSFV-SM,48 h后经荧光定量PCR检测各分子的表达以及CSFV基因拷贝数。荧光定量PCR结果显示,3种siRNA分子均能够下调PK-15细胞中Tyro3、Axl和Mertk的转录水平,且下调Mertk、Tyro3后CSFV基因组拷贝数均极显著降低,下调Axl后CSFV基因组拷贝数却无显著变化。因此选择siTyro3转染PK-15细胞后以MOI 0.01CSFV-SM感染PK-15细胞,通过荧光定量PCR及间接免疫荧光试验(IFA)检测siTyro3的转录水平以及CSFV的复制情况,结果显示,Tyro3基因的转录水平、CSFV基因组拷贝数及病毒滴度均显著降低,表明下调Tyro3的表达能够抑制CSFV的复制。按常规方法包装制备Tyro3慢病毒Lenti-Tyro3-HA,将其转导PK-15细胞,传10代后每代均经荧光显微镜观察,并将第10代转导细胞分别经荧光定量PCR和western blot鉴定。结果可见转导的细胞每代均发出绿色荧光,荧光定量PCR和western blot结果显示,第10代转导细胞中Tyro3基因的转录水平极显著上升,且可检测到Tyro3的特异性条带。表明获得了稳定表达Tyro3蛋白的PK-15细胞系PK-Tyro3-HA。将CSFV-SM分别以MOI 1和0.1感染PK-Tyro3-HA细胞系后经荧光定量PCR检测,结果显示CSFV基因组拷贝数分别升高了1.5倍(MOI 1)和2.3倍(MOI 0.1),表明过表达Tyro3后能够促进细胞中CSFV的复制。将不同剂量的兔源Tyro3多克隆抗体(pAb)与PK-15细胞共孵育,封闭PK-15细胞表面Tyro3表位后感染rCSFV-Rluc,经海肾荧光素酶检测,结果显示,20μg/mL和40μg/mL的Tyro3 pAb孵育细胞后再感染rCSFV-Rluc组的海肾荧光素酶活性显著降低,表明Tyro3 pAb能够抑制CSFV的复制。将pCAGGS-Tyro3-HA分别与pCAGGS-E2-Flag、pCAGGS-Erns-Flag共转染HEK293T细胞48 h后,进行免疫共沉淀试验,结果显示Tyro3与CSFV E2囊膜糖蛋白存在相互作用。综上,本研究首次证实Tyro3通过与CSFV E2囊膜糖蛋白的相互作用促进CSFV在PK-15细胞中的复制,为进一步揭示CSFV与宿主细胞的相互作用提供参考依据。

Our previous study demonstrated that Mertk,a member of the TAM receptor,is a classical swine fever virus(CSFV)entry factor.To investigate the effect of Tyro3 or Axl,two other members of TAM receptor,on CSFV replication,siRNAs against Tyro3,Mertk,Axl(siTyro3,siMertk,siAxl)and control siRNA(siNC),were designed and synthesized,then the siRNAs were transfected into PK-15 cells to knockdown the protein expression level.At 24 hours post-transfection,the cells were infected with CSFV-SM at multiplicity of infection(MOI)of 0.01 for additional 24 hours(h).Then the cells were collected for examination of m RNA transcriptional level of Tyro3,Axl and Mertk,and cell supernatants were collected for determination of viral genomic copies by quantitative PCR.The results showed that the mRNA transcriptional level of Tyro3,Axl and Mertk was decreased in PK-15 cells,and downregulation of Mertk and Tyro3 level,but not Axl,was able to significantly decreased CSFV genomic copies.In addition,siTyro3 transfected PK-15 cells were infected by CSFV-SM(MOI 0.01),then the Tyro3 transcriptional level and viral replication were determined by q PCR and indirect immunofluorescence assay(IFA).The results showed that the mRNA transcriptional level of Tyro3 and CSFV genomic copies and viral titers were significantly decreased,which indicating that knockdown of Tyro3 inhibites CSFV replication.According to the conventional method,the lentivirus Lenti-Tyro3-HA was packaged and transduced into PK-15 cells.The cells were cultured for 10 passages and each passage was examined by fluorescence microscope.Furthermore,the mRNA transcriptional level and protein expression of Tyro3 in the 10th passage of lentivirus transduced cells was detected by qPCR and western blot analyses,and the results showed that the expression of Tyro3 was significantly increased compared with the control group(p<0.001),indicating that a PK-15 cell line stably overexpressing Tyro3protein was generated,termed PK-Tyro3-HA cells.Subsequently,PK-Tyro3-HA cells were infected with CSFV-SM at MOIs of 1 or0.1.The results showed that CSFV genomic copies were increased by 1.5-and 2.3-fold,respectively,which indicating that overexpression of Tyro3 promotes CSFV replication.PK-15 cells were pre-incubated with different doses of rabbit polyclonal Tyro3antibody to block the Tyro3 epitope on cell surface and then infected with rCSFV-Rluc.The Renilla luciferase assay confirmed that the Tyro3 polyclonal antibody with 20μg/mL and 40μg/mL can inhibit CSFV replication.For the Co-immuno-precipitation assay,HEK293T cells were co-transfected by pCAGGS-Tyro3-HA with pCAGGS-E2-Flag or pCAGGS-Erns-Flag for 48h.The results showed that Tyro3 interacted with CSFV E2 envelope glycoprotein.Taken together,we demonstrated for the first time that Tyro3interacts with CSFV E2 envelope glycoprotein and promotes the replication of CSFV in PK-15 cells,providing a reference for further revealing the interaction between CSFV and the host.