Generation of unique poliovirus RNA replication organelles.
Generation of unique poliovirus RNA replication organelles.
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DOI：
10.1128/mbio.00833-13
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发表时间：
2014-02-25
期刊：
影响因子：
6.4
通讯作者：
Jackson WT
中科院分区：
文献类型：
--
作者：
Richards AL;Soares-Martins JA;Riddell GT;Jackson WT
脊髓灰质炎病毒（PV）是小核糖核酸病毒与宿主细胞相互作用的模型，它与细胞膜结合复制其基因组RNA。PV复制膜的起源尚未确定。关于复制膜起源的假设主要基于病毒蛋白的定位，包括外壳蛋白复合物I （COPI）和/或COPII分泌途径囊泡的修饰和自噬膜的颠覆。在这里，我们使用一种针对双链RNA （dsRNA）的抗体，通过检测dsRNA复制中间体来识别复制复合体。dsRNA信号依赖于病毒基因组复制，并与病毒整体膜蛋白3A共定位，后者是RNA复制复合体的一部分。我们发现，在感染早期，dsRNA不与自噬囊泡标记物共定位，这使得自噬体不太可能促进PV RNA复制膜的产生。我们还发现dsRNA不与COPII外壳的标记物Sec31共定位，事实上，我们证明了PV感染期间蛋白酶体依赖性的全长Sec31缺失。这些数据表明，COPII囊泡不太可能是PV复制膜的来源。研究表明，高尔基体常驻g蛋白Arf1及其相关的鸟嘌呤核苷酸交换因子（GEF） GBF1在感染早期与dsRNA短暂共定位。在未感染的细胞中，Arf1形成COPI外壳，尽管在感染期间COPI外壳本身不与dsRNA共定位。磷脂酰肌醇-4-磷酸与肠病毒诱导的囊泡有关，在整个感染过程中与Arf1/GBF1紧密共定位。我们的数据指出，在为PV RNA复制产生独特复制表面的过程中，一些copi生成机制的非规范作用。小核糖核酸病毒是一种多种多样的人类疾病的主要病因，它们的基因组与细胞膜复制相关。有多种假设可以解释这些膜的性质和起源，并且完全了解宿主对膜重排的需求将为病毒基因组复制提供必要的新药物靶点。在这里，我们研究了模型小核糖核酸病毒，脊髓灰质炎病毒，并表明从高尔基体到内质网逆行运输所需的细胞机制的一些组分（但不是全部）暂时存在于病毒RNA复制的位点。我们还发现，存在于PV RNA复制膜上的全长Sec31蛋白在感染过程中以蛋白酶体依赖的方式丢失。这项研究有助于调和关于脊髓灰质炎病毒复制膜起源的多种假设，并指出已知的宿主细胞蛋白复合物可能成为抑制小核糖核酸病毒感染的药物靶点。
Poliovirus (PV), a model for interactions of picornaviruses with host cells, replicates its genomic RNA in association with cellular membranes. The origin of PV replication membranes has not been determined. Hypotheses about the origin of replication membranes, based largely on localization of viral proteins, include modification of coat protein complex I (COPI) and/or COPII secretory pathway vesicles and subversion of autophagic membranes. Here, we use an antibody against double-stranded RNA (dsRNA) to identify replication complexes by detection of dsRNA replication intermediates. dsRNA signal is dependent on virus genome replication and colocalizes with the viral integral membrane protein 3A, which is part of the RNA replication complex. We show that early in infection, dsRNA does not colocalize with a marker for autophagic vesicles, making it unlikely that autophagosomes contribute to the generation of PV RNA replication membranes. We also find that dsRNA does not colocalize with a marker of the COPII coat, Sec31, and, in fact, we demonstrate proteasome-dependent loss of full-length Sec31 during PV infection. These data indicate that COPII vesicles are an unlikely source of PV replication membranes. We show that the Golgi resident G-protein Arf1 and its associated guanine nucleotide exchange factor (GEF), GBF1, transiently colocalize with dsRNA early in infection. In uninfected cells, Arf1 nucleates COPI coat formation, although during infection the COPI coat itself does not colocalize with dsRNA. Phosphatidylinositol-4-phosphate, which is associated with enterovirus-induced vesicles, tightly colocalizes with Arf1/GBF1 throughout infection. Our data point to a noncanonical role for some of the COPI-generating machinery in producing unique replication surfaces for PV RNA replication. Picornaviruses are a diverse and major cause of human disease, and their genomes replicate in association with intracellular membranes. There are multiple hypotheses to explain the nature and origin of these membranes, and a complete understanding of the host requirements for membrane rearrangement would provide novel drug targets essential for viral genome replication. Here, we study the model picornavirus, poliovirus, and show that some, but not all, components of the cellular machinery required for retrograde traffic from the Golgi apparatus to the endoplasmic reticulum are transiently present at the sites of viral RNA replication. We also show that the full-length Sec31 protein, which has been suggested to be present on PV RNA replication membranes, is lost during infection in a proteasome-dependent manner. This study helps to reconcile multiple hypotheses about the origin of poliovirus replication membranes and points to known host cell protein complexes that would make likely drug targets to inhibit picornavirus infections.