Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations.
Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations.
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DOI：
10.1099/vir.0.82865-0
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发表时间：
2007-07
期刊：
影响因子：
--
通讯作者：
Sarand I
中科院分区：
文献类型：
--
作者：
Žusinaite E;Tints K;Kiiver K;Spuul P;Karo-Astover L;Merits A;Sarand I
塞姆利基森林病毒（SFV）的复制酶由四种非结构蛋白组成，分别命名为nsP1 - 4，并通过nsP1的一个两亲性肽段和棕榈酰化的半胱氨酸残基与细胞膜结合。研究发现，阻止nsP1棕榈酰化的突变也会减弱病毒的复制。将这些半胱氨酸替换为丙氨酸，或者将其缺失，会消除病毒的活性，这可能是由于nsP1和nsP4（复制酶的催化亚基）之间的相互作用被破坏。然而，在单个感染周期中，由于nsP1中第二位点突变的积累，病毒的复制能力得以恢复。这些突变导致nsP1 - nsP4相互作用恢复，但没有恢复nsP1的棕榈酰化。具有棕榈酰化位点突变的蛋白，以及除了棕榈酰化位点突变外还含有补偿性突变的蛋白，具有酶活性，并且至少部分定位于转染细胞的质膜上。有趣的是，删除包括nsP1棕榈酰化位点在内的7个氨基酸对病毒活性影响相对较小，并且对nsP1 - nsP4相互作用没有显著影响。同样，辛德毕斯病毒nsP1棕榈酰化位点的半胱氨酸变为丙氨酸对病毒复制只有轻微影响。综上所述，这些发现表明nsP1的棕榈酰化本身并不是决定其与细胞膜结合并形成功能性复制酶复合物能力的因素。相反，这些能力可能与nsP1的三维结构以及nsP1与病毒复制酶复合物其他成分相互作用的能力有关。
The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.
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