摘要
酵母作为基因组结构简单(只编码大约6000个基因)、基因中内含子少、基因组信息最完备的模式真核生物,能够支持一些病毒在其细胞中完成复制的大多数步骤(Jandaand Ahlquist1993;Naito et al.2007a;Panavas et al.2005b;Panavas and Nagy2003;Pantaleoet al.2003;Raghavan et al.2004),使其成为研究病毒复制的模式寄主。研究表明,酵母E3泛素连接酶Rsp5p中的ww结构域通过与番茄丛矮病毒(TBSV)复制蛋白p92pol结合并促进p92pol降解而抑制TBSV在酵母体内的复制(Barajas et al.2009a)。本课题以酵母作为模式寄主,探索寄主蛋白在正链RNA病毒复制过程中的作用机理。运用相关质粒分别表达正链RNA病毒番茄丛矮病毒(TBSV)、兽棚病毒(FHV)和野田村病毒(NoV)的复制蛋白和复制RNA,研究五个含有ww结构域的酵母蛋白质在酵母体内及体外对TBSV以及两种昆虫病毒FHV和NoV复制的影响。并通过烟草体内试验探究四个含有ww结构域的拟南芥蛋白质对番茄丛矮病毒属黄瓜坏死病毒(CNV)复制的影响。另外,运用蛋白质体外pulldown试验和膜酵母双杂交(MYTH,membrane yeast two-hybrid)试验研究含有ww结构域的目的蛋白与病毒复制蛋白之间的相互作用关系和相互作用位点。
本研究的主要成果如下:
(1)除Rsp5p之外,另外三个含有ww结构域的酵母蛋白对酵母中TBSV的复制具有强烈抑制作用,它们是Prp40p、Wwm1p和Ess1p。Rsp5p、Wwm1p和Prp40p的过量表达也能抑制酵母中FHV和NoV的复制。
(2)Prp40p和Ess1p的过表达抑制TBSV复制蛋白p92pol的积累,Rsp5p、Prp40p和Wwm1p的过表达抑制FHV和NoV的复制蛋白PrtA的积累。它们很可能与Rsp5p对TBSV复制的抑制具有相同机理,通过促进病毒关键复制蛋白(即RNA-dependent RNApolymerase,RdRp)的降解而抑制正链RNA病毒复制。
(3)Wwm1的下调表达、两个基因(Wwm1和Rsp5;Wwm1和Prp40;Wwm1和Ess1)同时下调表达以及多个基因下调表达(在敲除Wwm1的酵母中同时抑制Rsp5和Prp40的表达;在敲除Wwm1的酵母中同时抑制Rsp5、Prp40和Ess1的表达)都促进TBSV复制,并且提高p33和p92pol的积累水平。在体外复制实验中,Wwm1和所有两个基因和多个基因组合(同上)的下调表达都促进TBSV复制。基因下调试验表明,Rsp5p和Wwm1p是调节TBSV复制的主要因子,而Prp40p和Ess1p的作用微弱或存在基因间功能的互补。
(4)酵母中五个含有ww结构域的蛋白质(Rsp5p、Prp40p、Wwm1p、Urn1p和Ess1p)都能够与TBSV的复制蛋白p33和p92pol发生相互作用,并且Rsp5p与p33C的结合位点在RNA结合域上(即RPR基序)。并且这五个蛋白质也都能够与FHV和NoV的复制蛋白PrtA发生相互作用。
(5)在Rsp5p蛋白中,ww结构域的数量越多,对TBSV复制的抑制作用越强,三个ww结构域的共同作用抑制效果最强。
(6)在烟草中,三个含有ww结构域的拟南芥蛋白质对番茄丛矮病毒属CNV的复制具有抑制作用,它们是AtDRH1、AtPrp40c(At3g19840)和AtFCA。并且这三个蛋白的ww结构域都可以和CNV的复制蛋白p33发生相互作用。
本文对模式寄主酵母和烟草中九个含有ww结构域的蛋白质在影响四种正链RNA病毒复制中的作用进行了研究,结果显示具有ww结构域的七个寄主蛋白对至少其中一种正链RNA病毒的复制起到抑制作用,并且在含有1-3个ww结构域的Rsp5重组蛋白片段中,抑制作用与ww结构域的数量正相关。本课题就含有相同结构域的蛋白质家族对正链RNA病毒复制的影响进行了研究,可以作为基因组范围和蛋白质组范围筛选的有效补充,鉴定出在这些高通量方法中遗漏的与病毒复制相关的寄主因子,丰富了我们对正链RNA病毒复制的理解。
As a model eukaryote, yeast has a small genome with few introns. Yeast also has thehighest percentage characterized genes. Because certain viruses can complete most of thesteps required for intracellular replication in yeast cells (Janda and Ahlquist1993; Naito et al.2007a; Panavas et al.2005b; Panavas and Nagy2003; Pantaleo et al.2003; Raghavan et al.2004), yeast has become a model host to study virus replication. The ww domains in yeastRsp5E3Ubiquitin ligase have been shown to interact with TBSV replication protein p92pol.Overexpression of Rsp5p inhibits TBSV replication through the degradation of p92polby itsww domains (Barajas et al.2009a). This dissertation focuses on the effect of host proteins on(+) RNA virus in yeast model host. Selective plasmids were transformed to yeast to expressthe replication proteins and replication RNAs of (+) RNA viruses Tomato bushy stunt virus(TBSV), Flock house virus (FHV) and Nodamura virus (NoV). The functions of five wwdomain-containing yeast proteins in the replication of TBSV, two insect viruses FHV andNoV were tested in yeast and in vitro. Four Arabidopsis thaliana ww domain-containingproteins were transformed to Nicotiana Benthamiana to test their function in Cucumbernecrosis virus (CNV) replication. Moreover, in vitro pulldown assay and membrane yeasttwo-hybrid (MYTH) assay were performed to test the interaction between wwdomain-containing host proteins and virus replication proteins. The binding domain was alsodetermined.
Main results from this research are:
(1) In addition to Rsp5p, three yeast ww domain-containing proteins inhibit TBSVreplication in yeast. They are Prp40p, Wwm1p and Ess1p. Overexpression of Rsp5p, Wwm1por Prp40p also inhibits the replication of FHV and NoV.
(2) Overexpression of Prp40p or Ess1p inhibits the accumulation of TBSV p92pol.Overexpression of Rsp5p, Prp40p or Wwm1p inhibits the accumulation of FHV and NoVreplication proteins (Protein A, PrtA). They likely function in the same way as Rsp5p, whichinhibits (+) RNA replication by facilitating the degradation of the key replication protein(RNA-dependent RNA polymerase, RdRp).
(3) Downregulation of Wwm1, two ww genes (Wwm1and Rsp5, Wwm1and Prp40,Wwm1and Ess1) or several genes (Wwm1, Rsp5and Prp40; Wwm1, Rsp5, Prp40and Ess1) increases TBSV replication and the accumulation of p33and p92pol. From the replicase assayin vitro, downregulation of Wwm1or all the gene combinations leads to higher TBSVreplication. These downregulation experiments suggest that Rsp5p and Wwm1p are importantregulators of TBSV replication, while the other ww domain proteins have lesser effects orthey have redundant function in TBSV replication in yeast.
(4) Five yeast ww domain-containing proteins (Rsp5p, Prp40p, Wwm1p, Urn1p andEss1p) interact with TBSV p33and p92pol. The binding domain of Rsp5p and p33C is in theRNA binding domain (termed RPR domain). Moreover, all the five yeast ww proteins interactwith FHV and NoV replication protein PrtA.
(5) More ww domains within Rsp5p, more inhibitory function on TBSV replication. Thehighest inhibition appears when there are three ww domains in Rsp5p.
(6) Three Arabidopsis ww domain-containing proteins inhibit CNV replication in N.Benthamiana. They are AtDRH1、AtPrp40c (At3g19840) and AtFCA. All of them interactwith CNV replication protein p33.
This dissertation studied the inhibitory functions of nine proteins from yeast model hostand Arabidopsis on four (+) RNA viruses. Results show that seven of them inhibit at least one(+) RNA virus replication. The number of ww domains in Rsp5p correlates with the inhibitoryfunction. Protein family members with same domains are studied here to test their effect on (+)RNA replication. This could be an efficient way to identify hose factors which were missed inthe genome-wide and proteomics-wide screens, as a supplement of the genome-wide andproteomics-wide studies, and broaden our understanding on (+) RNA virus replication.
引文
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