The predominant eIF4G-specific cleavage activity in poliovirus-infected HeLa cells is distinct from 2A protease
The predominant eIF4G-specific cleavage activity in poliovirus-infected HeLa cells is distinct from 2A protease
复制标题
DOI：
10.1006/viro.1998.9171
复制
发表时间：
1998-06-05
期刊：
影响因子：
3.7
通讯作者：
Lloyd, RE
中科院分区：
文献类型：
--
作者：
Bovee, ML;Marissen, WE;Lloyd, RE
人肠道病毒和鼻病毒在感染易感细胞后迅速并选择性地消除细胞mRNA的翻译。脊髓灰质炎病毒2A蛋白酶（PV 2A（prc））的表达足以通过直接或间接地通过细胞因子的活化切割eIF4G（以前的p220，eIF4 γ）来引起宿主翻译关闭。直接和间接裂解机制的证据都存在，然而，假定参与间接机制的因素尚未被纯化或定义。在这里，我们表明，占主导地位的eIF4G裂解活性的裂解物从感染的HeLa细胞是可分离的PV 2A（亲）的大小排阻色谱。2A（pro）分离成两个峰级分，其含有裂解源自脊髓灰质炎病毒多蛋白的肽底物的活性。这些峰2A（pro）级分由于直接切割反应的效率差，如预期的那样不切割eIF4G或eIF4G衍生肽。相反，含有峰eIF4G切割活性和仅痕量2A（pro）的级分有效地切割源自先前定位的eIF4G切割位点的肽底物，并且还切割源自脊髓灰质炎病毒1D2A区域的肽。通过四个色谱步骤高度纯化了主要的eIF4G切割活性，发现没有所有痕量的2A（pro）或其前体。来自感染细胞裂解物的2A（pro）的定量显示，在HeLa细胞中感染期间，2A（pro）没有达到相对于eIF4G的摩尔过量，如先前显示的，这是体外直接切割eIF4G所需的。此外，在病毒RNA复制的有效抑制剂2mM盐酸胍存在下感染HeLa细胞，抑制2A（pro）及其前体2ABC的积累低于可检测水平，但不能延迟体内eIF4G蛋白水解的发生。eIF4G切割活性在使用来自胍处理的细胞的级分的体外测定中仍然容易检测。因此，数据表明脊髓灰质炎病毒利用两种催化活性来确保eIF4G在体内的快速裂解。虽然不能直接测量，但2A（pro）可能确实切割细胞中的一部分eIF4G。然而，数据表明，可以被少量2A（pro）激活的细胞因子构成了感染细胞中eIF4G特异性切割活性的主体，并且负责体内观察到的快速和有效的eIF4G切割活性。(C)北京：科学出版社.
Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2A(prc)) is sufficient to cause host translation shutoff through cleavage of eIF4G (formerly p220, eIF4 gamma) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant eIF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2A(pro) by size exclusion chromatography. 2A(pro) separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2A(pro) fractions did not cleave eIF4G or an eIF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions, Conversely, fractions which contained peak eIF4G cleavage activity and only trace amounts of 2A(pro) efficiently cleaved a peptide substrate derived from the previously mapped eIF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant eIF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2A(pro) or its precursors. Quantitation of 2A(pro) from lysates of infected cells showed that during infections in HeLa cells, 2A(pro) does not reach molar excess over eIF4G, as previously shown to be required for direct eIF4G cleavage in vitro. Further, infection of HeLa cells in the presence of 2 mM guanidine-HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2A(pro) and its precursor 2ABC below detectable levels but was unable to delay the onset of eIF4G proteolysis in vivo. The eIF4G cleavage activity was still easily detectable in in vitro assays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of eIF4G in vivo. Although it was not directly measurable here, 2A(pro) likely does cleave a portion of eIF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2A(pro) constitutes the bulk of the eIF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient eIF4G cleavage activity observed in vivo. (C) 1998 Academic Press.