Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.
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DOI：
10.1371/journal.ppat.1001087
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发表时间：
2010-09-02
期刊：
影响因子：
6.7
通讯作者：
Weinman SA
中科院分区：
文献类型：
--
作者：
Wozniak AL;Griffin S;Rowlands D;Harris M;Yi M;Lemon SM;Weinman SA
丙型肝炎病毒（HCV） p7蛋白对病毒产生至关重要，也是一个有吸引力的抗病毒靶点。P7在人工脂质双分子层中重组时是一个离子通道，但通道功能尚未在体内得到证实，P7通道活性是否在病毒产生中起关键作用尚不清楚。为了评估p7对细胞器pH调节和病毒产生的贡献，我们在存在或不存在p7表达的天然细胞内囊泡中植入了荧光pH传感器。p7增加了囊泡中的质子（H+）电导，并能够快速平衡H+梯度。这种传导被病毒蛋白抑制剂金刚烷胺、金刚乙胺和六亚甲基酰胺阻断。使用活细胞pH指示剂的荧光显微镜显示，HCV感染和复制子rna p7的表达都减少了高酸性（pH<5）囊泡的数量，并将溶酶体pH从4.5提高到6.0。这些效应在未感染的细胞、不表达p7的亚基因组复制子细胞或含有通道失活p7点突变的病毒RNA电穿孔的细胞中不存在。酸化抑制剂巴菲霉素A1部分恢复了含有通道失活突变的病毒RNA电穿孔细胞的病毒生产，但在含有p7缺失RNA的细胞中没有。流感M2蛋白的表达也补充了p7突变体，证实了病毒生产需要H+通道活性。因此，暴露于酸性pH使细胞内HCV颗粒不具有传染性，而细胞外病毒粒子的感染性是酸性稳定的，不受低pH孵育的影响，进一步证明了p7诱导的酸化丧失的关键要求。我们得出结论，p7作为一个H+渗透途径，起到防止酸性细胞内室酸化的作用。这种酸化的丧失是生产性HCV感染所必需的，可能是通过在尚未表征的成熟过程中保护新生病毒颗粒而实现的。丙型肝炎病毒（HCV）是慢性肝病最常见的病因。目前的治疗只是部分有效，而且充满了副作用。因此，更好地了解病毒复制和新病毒颗粒的形成对于开发新的治疗靶点非常重要。HCV p7蛋白是一种病毒编码蛋白，是产生新病毒颗粒所绝对需要的。当重组为人工脂质膜时，其表现为离子通道，但其在感染细胞中的功能尚不清楚。我们已经研究了p7作为细胞内离子通道，阻止pH梯度在细胞内形成的可能性。我们已经证明p7具有这种功能，它会导致多个细胞内区室的酸度损失。我们证明这种碱化是成功生产病毒所必需的。直接抑制细胞内atp酶或用另一种离子通道替代p7都能够补偿p7的缺陷并允许活性病毒的产生。因此，HCV使用p7来阻止细胞酸化过程。这一认识将允许新的治疗药物靶向这一机制，并为感染期间肝脏发病机制提供新的见解。
The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process. The hepatitis C virus (HCV) is the most common cause of chronic liver disease. Current therapy is only partially effective and fraught with side effects. A greater understanding of viral replication and new virus particle formation is thus important for developing new therapeutic targets. The HCV p7 protein is a virally encoded protein that is absolutely required for the production of new virus particles. It behaves as an ion channel when reconstituted into artificial lipid membranes but its function in infected cells is unknown. We have examined the possibility that p7 functions as an intracellular ion channel, preventing pH gradients from developing inside the cells. We have shown that p7 serves this function and it causes a loss of acidity in multiple intracellular compartments. We demonstrate that this alkalinization is required for successful virus production. Either direct inhibition of intracellular ATPases or replacement of p7 with an alternative ion channel is able to compensate for a defect in p7 and allow active virus to be produced. Therefore, HCV uses p7 to prevent cellular acidification processes. This understanding will allow for the targeting of this mechanism with novel therapeutic agents, and offers new insights into the mechanisms of liver pathogenesis during infection.
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影响因子：
6.7
作者：
Steinmann E;Penin F;Kallis S;Patel AH;Bartenschlager R;Pietschmann T
通讯作者：
Pietschmann T
影响因子：
14.8
作者：
Kato, Takanobu;Date, Tomoko;Wakita, Takaji
通讯作者：
Wakita, Takaji
影响因子：
1.5
作者：
Ciampor, F.
通讯作者：
Ciampor, F.
DOI：
10.1073/pnas.1834545100
发表时间：
2003-09-30
影响因子：
11.1
作者：
Sakai, A;St Claire, MS;Bukh, J
通讯作者：
Bukh, J
影响因子：
7.6
作者：
StGelais, Corine;Tuthill, Tobias J.;Griffin, Stephen
通讯作者：
Griffin, Stephen