SLFN12与原型泡沫病毒Tas蛋白相互作用抑制其激活病毒启动子

SLFN12 Interacts with the Tas Protein of the Prototype Foamy Virus to Inhibit the Trans-activation Activities of the Virus Promoter

Schlafen(SLFN)家族多个成员具有抗病毒活性,但Schlafen家族成员12(Schlafen12,SLFN12)的相关功能尚未见报道。本研究从B细胞cDNA文库中克隆获得SLFN12全长cDNA,亚克隆构建一系列真核表达质粒,分析SLFN12对原型泡沫病毒(prototype foamy virus,PFV)复制的作用。将SLFN12全长质粒与PFV的感染性克隆(pcPFV)共转染HEK293T细胞。结果显示,当两者的转染质量比为1∶3时,SLFN12过表达使以游离病毒颗粒(cell-free)或细胞共培养(cell-to-cell)方式感染的PFV滴度分别降低了95.21%(P<0.001)以及97.91%(P<0.01),同时病毒蛋白质的表达量也减少,说明SLFN12能够抑制PFV的复制。共转染pcPFV与SLFN12的AAA结构域截短质粒进行同样的实验,PFV滴度分别对应下降了61.88%(P<0.05)以及72.28%(P<0.01),表明AAA结构域参与了SLFN12抑制PFV复制的功能。随后,利用报告质粒系统探究SLFN12对PFV启动子转录活性的影响,观察到SLFN12不影响病毒内部启动子(internal promoter,IP)(P>0.05)及长末端重复序列(long terminal repeat,LTR)(P>0.05)的基础转录活性,但抑制泡沫病毒反式激活因子(trans-activator of spumaviruses,Tas)的表达。当SLFN12和Tas表达质粒的转染质量比为1∶1时,Tas对IP以及LTR的转录激活作用相较对照组,分别减弱了66.27%(P<0.01)以及73.91%(P<0.01)。同时,免疫共沉淀实验观察到,Tas蛋白与SLFN12蛋白之间存在相互作用,表明SLFN12通过与PFV Tas相互作用削弱其表达,从而干扰Tas对IP和LTR的反式激活来抑制PFV复制。本研究发现,SLFN12具有抗病毒活性,拓宽了对SLFN家族抗病毒活性的认识,对于研究泡沫病毒潜伏感染的机制也具有参考价值。

Several Schlafens(SLFNs)have antiviral activities,but the related functions of Schlafen12(SLFN12)have not been closely studied.In this study,a series of eukaryotic expression plasmids of SLFN12 were constructed after obtaining the full-length SLFN12 clone from the B cell cDNA library.Then HEK293T cells were co-transfected with SLFN12 plasmids and the full-length prototype foamy virus(PFV)cDNA construct(pcPFV)at the ratio of 1∶3 to assess the effect of SLFN12 on PFV replication.The results showed that the overexpression of SLFN12 decreased the titers of PFV transmitted in cell-free or cell-to-cell manners by 95.21%(P<0.001)and 97.91%(P<0.01),respectively,and diminished the expression of viral proteins,indicating that SLFN12 inhibited PFV replication.The SLFN12-AAA and pcPFV co-transfection experiments also showed that the titers of PFV were 61.88%(P<0.05)and 72.28%(P<0.01),lower than the control group,respectively,illustrating that the anti-PFV activities of SLFN12 were partly due to the AAA domain.Subsequently,the luciferase reporter system was used to explore the role of SLFN12 in trans-activator of spumaviruses(Tas)to trans-activate internal promoter(IP)and long terminal repeat(LTR)of PFV.It was observed that when the ratio of transfected SLFN12 and Tas plasmids was 1∶1,SLFN12 weakened the trans-activation activities of Tas to IP and LTR by 66.27%(P<0.01)and 73.91%(P<0.01),respectively,as a result of reduced Tas protein expression,rather than affecting the basic transcriptional activities of IP(P>0.05)and LTR(P>0.05).Furthermore,the co-precipitation of Tas and SLFN12 indicated that SLFN12 interacted with Tas.Overall,SLFN12 decreased the level of Tas by interacting with Tas and impairing the trans-activation activities of Tas to PFV IP and LTR,which facilitated SLFN12 resistance to PFV.In conclusion,this study found that SLFN12 had antiviral activities,which broadened the understanding of the antiviral activity of the SLFN family and are valuable for studying the mechanism of foamy virus latent infection.