Single-cell analysis of Kaposi's sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model.

The oral cavity is the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression. While KSHV is the etiologic agent of Kaposi’s sarcoma that frequently occurs in AIDS patients, there is not biologically relevant, reproducible in vitro culture system to study KSHV infection and spontaneous lytic replication. Here, we describe that a 3D human oral epithelial ALI culture that can support a robust KSHV infection and spontaneous lytic replication associated with oral epithelial differentiation. scRNAseq analysis identified considerable alteration of host gene expression in both KSHV-infected cells and -uninfected bystander cells. Our study provides insights into the early stages of KSHV infection in oral epithelial cells and serves as a useful blueprint for the investigation of KSHV infection and transmission.

口腔是卡波西肉瘤相关疱疹病毒（KSHV）传播的主要部位，但KSHV如何在口腔上皮中建立感染和复制仍不清楚。在此，我们报道了一种在气液界面（ALI）利用完全分化的三维（3D）口腔上皮类器官的KSHV自发裂解复制模型。该模型显示，当基底上皮细胞因损伤而暴露时，KSHV会感染口腔上皮。与二维（2D）细胞培养不同，3D口腔上皮类器官ALI培养可使KSHV自发裂解复制水平较高，其中裂解复制细胞在上皮类器官的表层富集。单细胞RNA测序（scRNAseq）表明，KSHV感染在不同上皮层的感染细胞和未感染细胞中诱导宿主基因表达发生剧烈变化，导致角质形成细胞分化和细胞死亡改变。此外，我们鉴定出一个独特的感染细胞群体，其在KSHV的K2 - K5基因位点含有裂解基因表达，并且与潜伏感染或裂解感染细胞相比，宿主基因表达不同。这项研究展示了一种体外3D上皮类器官ALI培养模型，该模型重现了口腔中KSHV的感染情况，其中KSHV经历依赖上皮分化的自发裂解复制，且存在一个携带独特病毒基因表达的独特细胞群体。 虽然KSHV是艾滋病患者中常见的卡波西肉瘤的病原体，但目前没有具有生物学相关性且可重复的体外培养系统来研究KSHV感染和自发裂解复制。在此，我们描述了一种3D人口腔上皮ALI培养，它能够支持强烈的KSHV感染以及与口腔上皮分化相关的自发裂解复制。scRNAseq分析确定了KSHV感染细胞和未感染的旁观者细胞中宿主基因表达的显著改变。我们的研究为口腔上皮细胞中KSHV感染的早期阶段提供了见解，并为KSHV感染和传播的研究提供了有用的蓝图。