Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation
Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation
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DOI：
10.1128/jvi.00306-08
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发表时间：
2008-09-01
影响因子：
5.4
通讯作者：
Matsuura, Yoshiharu
中科院分区：
文献类型：
--
作者：
Okamoto, Kiyoko;Mori, Yoshio;Matsuura, Yoshiharu
丙型肝炎病毒（HCV）核心蛋白已被证明定位于抗洗涤剂膜（DRM）中，这与包括小泡蛋白在内的经典筏状部分不同，尽管核心蛋白在DRM定位的生物学意义尚未确定。HCV核心蛋白通过信号肽酶从Ala管腔侧的前体多蛋白（191）上切割下来，然后在跨膜区域内由信号肽肽酶（SPP）进一步加工。在这项研究中，我们研究了SPP在HCV核心蛋白在DRM中的定位和病毒传播中的作用。在293T细胞中，SPP切割的HCV核心蛋白C端经质谱鉴定为Phe（177）。在核心蛋白切割位点上游的两个残基（Ile（176）和Phe(177)）中引入的突变取消了SPP加工和DRM部分的定位。表达显性阴性SPP或用SPP抑制剂L685,458处理，导致DRM部分加工核心蛋白水平降低。在持续感染JFH-1的细胞中，SPP抑制剂抑制了HCV RNA的产生。此外，在核心蛋白中携带spp抗性突变的突变型JFH-1病毒无法在容许细胞系中繁殖。这些结果表明SPP对HCV核心蛋白的膜内加工是HCV核心蛋白在DRM中的定位和病毒传播所必需的。
Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.