Properly folded nonstructural polyprotein directs the Semliki Forest virus replication complex to the endosomal compartment
Properly folded nonstructural polyprotein directs the Semliki Forest virus replication complex to the endosomal compartment
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DOI：
10.1128/jvi.77.3.1691-1702.2003
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发表时间：
2003-02-01
影响因子：
5.4
通讯作者：
Kääriäinen, L
中科院分区：
文献类型：
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作者：
Salonen, A;Vasiljeva, L;Kääriäinen, L
在被甲病毒感染的细胞中，产生正链RNA的晚期RNA合成发生在细胞质空泡（CPV）上，这些空泡是经过修饰的内体和溶酶体。CPV的胞质表面由规则的膜内陷或球状体组成，它们是RNA合成的位点（P.库贾拉，A.伊卡海莫宁，N.埃萨尼，H.维西宁，P.奥维宁和L.卡里亚宁，《病毒学杂志》75:3873 - 3884,2001）。为了了解CPV是如何产生的，我们以不同组合表达了塞姆利基森林病毒（SFV）的单个非结构蛋白nsP1到nsP4，以及它们的前体多聚蛋白P1234及其裂解中间体。从P123或P1234获得了nsPs复合物，这表明前体阶段对于聚合酶复合物的组装是必不可少的。为了阻止多聚蛋白及其裂解中间体的加工，使用了在nsP2的蛋白酶结构域活性位点具有突变C478A（用上标CA表示）的构建体。含有nsP1的未裂解多聚蛋白是膜结合的且被棕榈酰化，含有nsP3的那些则被磷酸化，分别反映了天然nsP1和nsP3的特性。类似地，含有nsP1或nsP2的多聚蛋白具有针对单个蛋白的酶活性，表明它们在前体状态下正确折叠。未裂解的P12(CA)几乎完全定位在质膜和丝状伪足上，就像单独的nsP1一样，而P12(CA)3和P12(CA)34则在细胞质囊泡上被发现，其中一些含有晚期内体标记物。在免疫电子显微镜下，这些囊泡类似于在SFV感染细胞中的CPV。我们的结果表明，单独的nsP1结构域负责非结构多聚蛋白的膜结合，而nsP1结构域与nsP3结构域一起将其靶向细胞内囊泡。
The late RNA synthesis in alphavirus-infected cells, generating plus-strand RNAs, takes place on cytoplasmic vacuoles (CPVs), which are modified endosomes and lysosomes. The cytosolic surface of CPVs consists of regular membrane invaginations or spherules, which are the sites of RNA synthesis (P. Kujala, A. Ikaheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kaariainen J. Virol. 75:3873-3884,2001). To understand how CPVs arise, we have expressed the individual Semliki Forest virus (SFV) nonstructural proteins nsP1 to nsP4 in different combinations, as well as their precursor polyprotein P1234 and its cleavage intermediates. A complex of nsPs was obtained from P123 or P1234, indicating that the precursor stage is essential for the assembly of the polymerase complex. To prevent the processing of the polyprotein and its cleavage intermediates, constructs with the mutation C478A (designated with a superscript CA) in the active site of the protease domain of nsP2 were used. Uncleaved polyproteins containing nsP1 were membrane bound and palmitoylated, and those containing nsP3 were phosphorylated, reflecting properties of authentic nsP1 and nsP3, respectively. Similarly, polyproteins containing nsP1 or nsP2 had enzymatic activities specific for the individual proteins, indicating that they were correctly folded in the precursor state. Uncleaved P12(CA) was localized almost exclusively to the plasma membrane and filopodia, like nsP1 alone, whereas P12(CA)3 and P12(CA)34 were found on cytoplasmic vesicles, some of which contained late endosomal markers. In immunoelectron microscopy these vesicles resembled CPVs in SFV-infected cells. Our results indicate that the nsP1 domain alone is responsible for the membrane association of the nonstructural polyprotein, whereas the nsP1 domain together with the nsP3 domain targets it to the intracellular vesicles.