Data for Fig 1A–1C.

Aichi virus (AiV), a small non-enveloped RNA virus, hijacks the endoplasmic reticulum (ER)–Golgi cholesterol transport machinery to form cholesterol-rich replication sites originating from Golgi membranes. Interferon-induced transmembrane proteins (IFITMs) are antiviral restriction factors, whose involvement in intracellular cholesterol transport is suggested. Here, we describe the roles of IFITM1 in cholesterol transport that affect AiV RNA replication. IFITM1 stimulated AiV RNA replication and its knockdown significantly reduced the replication. In replicon RNA-transfected or infected cells, endogenous IFITM1 localized to the viral RNA replication sites. Further, IFITM1 interacted with viral proteins and host Golgi proteins, ACBD3, PI4KB, OSBP, which constitute the replication sites. When overexpressed, IFITM1 localized to the Golgi as well as endosomes, and this phenotype was also observed for endogenous IFITM1 early in AiV RNA replication, leading to the distribution of cholesterol at the Golgi-derived replication sites. The pharmacological inhibition of ER–Golgi cholesterol transport or endosomal cholesterol export impaired AiV RNA replication and cholesterol accumulation at the replication sites. Such defects were corrected by expression of IFITM1. Overexpressed IFITM1 facilitated late endosome–Golgi cholesterol transport without any viral proteins. In summary, we propose a model in which IFITM1 enhances cholesterol transport to the Golgi to accumulate cholesterol at Golgi-derived replication sites, providing a novel mechanism by which IFITM1 enables efficient genome replication of non-enveloped RNA virus.爱知病毒（Aichi virus, AiV）是一种小型无包膜RNA病毒，可劫持内质网（endoplasmic reticulum, ER）-高尔基体胆固醇转运系统，以源自高尔基体膜的结构组装富含胆固醇的病毒复制位点。干扰素诱导跨膜蛋白（interferon-induced transmembrane proteins, IFITMs）为一类抗病毒限制因子，已有研究提示其参与细胞内胆固醇转运过程。本研究阐明了IFITM1在调控影响爱知病毒RNA复制的胆固醇转运通路中发挥的作用。 实验结果表明，IFITM1可促进爱知病毒RNA复制，而敲低IFITM1则会显著抑制病毒复制水平。在转染病毒复制子RNA或感染病毒的细胞中，内源性IFITM1定位于病毒RNA复制位点。进一步研究发现，IFITM1可与病毒蛋白以及宿主高尔基体蛋白ACBD3、PI4KB、OSBP发生相互作用，而上述蛋白正是构成病毒复制位点的核心组分。 当IFITM1过表达时，其可同时定位于高尔基体与内体；该定位表型在爱知病毒RNA复制早期的内源性IFITM1中同样存在，最终促使胆固醇在高尔基体衍生的复制位点处富集。 对ER-高尔基体胆固醇转运或内体胆固醇输出进行药理学抑制后，爱知病毒RNA复制以及复制位点处的胆固醇积累均受到显著损害；而通过外源表达IFITM1可逆转上述复制缺陷。在不含任何病毒蛋白的情况下，过表达的IFITM1即可促进晚期内体-高尔基体的胆固醇转运过程。 综上，本研究提出如下工作模型：IFITM1可增强向高尔基体的胆固醇转运，使胆固醇在高尔基体衍生的复制位点处富集，从而揭示了IFITM1助力无包膜RNA病毒高效完成基因组复制的全新分子机制。