Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, Tochigi, Japan.
Laboratory for Drug Target Research, Integrated Bioscience Division, Institute of Agriculture, Shinshu Universitygrid.263518.b, Nagano, Japan.
J Virol. 2022 Mar 23;96(6):e0190621. doi: 10.1128/jvi.01906-21. Epub 2022 Feb 2.
Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family . Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.
戊型肝炎病毒（HEV）是一种具有单链正链 RNA 基因组的准包膜病毒，属于. 对 HEV 的分子方面的研究和药物筛选得益于生物发光报告基因的发现。然而，将大的外源基因插入病毒基因组后，其稳定性很难维持。目前，利巴韦林用于治疗需要抗病毒治疗的 HEV 感染患者。这有几个主要的缺点。因此，开发新型抗 HEV 药物非常重要。我们开发了一种系统，该系统由在 ORF1 的高变区（HVR）中携带小萤光素酶基因（nanoKAZ）的重组传染性 HEV 组成（HEV-nanoKAZ）。它在培养细胞中高效复制，遗传稳定，并且具有与亲本病毒相似的形态特征。膜相关的（eHEV-nanoKAZ）和非膜相关的（neHEV-nanoKAZ）颗粒都是感染性的。在 HEV 感染患者中循环于血液并附着于肝细胞的 HEV 颗粒是膜相关的；因此，eHEV-nanoKAZ 用于药物筛选。eHEV-nanoKAZ 系统至少涵盖了 HEV 进入抑制剂和 HEV RNA 复制抑制剂。鉴定了四种具有抗 HEV 活性的药物。在未感染和产生 HEV 的 PLC/PRF/5 细胞中证实了它们在培养细胞中的有效性。两种命中药物（阿奇霉素和利托那韦）强烈抑制了培养上清液中的 HEV 产生，以及 ORF2 蛋白的细胞内表达，因此可能是候选的新型抗 HEV 药物。开发并应用了 HEV-nanoKAZ 系统进行药物筛选，有望有助于研究 HEV 生命周期。生物发光报告病毒是分子病毒学研究中的重要工具。它们已被广泛用于研究病毒生命周期和开发抗病毒药物。对于药物筛选，使用生物发光报告病毒有助于缩短进行测定所需的时间。本研究中开发了一种包含 ORF1 的 HVR 中的 nanoKAZ 基因的重组传染性 HEV 的系统（HEV-nanoKAZ），并成功地将其应用于药物筛选，鉴定了四种具有抗 HEV 活性的命中药物。本研究的结果提供了支持该系统在更具变异性的 HEV 研究中使用的证据。此外，两种形式的病毒颗粒（eHEV-nanoKAZ 和 neHEV-nanoKAZ）都是感染性的，这将使它们能够应用于需要两种形式的病毒颗粒的 HEV 研究中，例如在研究未知的 HEV 受体和阐明对 HEV 进入很重要的宿主因子。
