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

ABSTRACT: The oral cavity has previously been identified as 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 epithelial differentiation and morphogenesis. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host and viral gene expression compared to latency or lytic replication. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184257 | GEO | 2022/07/28

REPOSITORIES: GEO

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Project description:The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of KaposiM-bM-^@M-^Ys sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure that is characterized by a specific pattern of activating and repressive histone modifications that ultimately promote latent gene expression while suppressing lytic gene expression. To investigate the molecular events involved in the establishment of the latent chromatin structure during the pre-latency phase of KSHV infection, we performed a comprehensive epigenetic study to analyze the recruitment of chromatin regulatory factors onto the KSHV genome at various time-points following de novo infection of SLK and TIME cells. This showed that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and was accompanied by the temporary induction of a limited number of lytic genes. Interestingly, transient expression of the RTA protein facilitated the increases of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both the Polycomb Repressive Complex 2 and 1. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a continuously transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection vs. lytic replication of KSHV. Please see above. 16 hybridizations: ChIP and Input DNA

Project description:The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure that is characterized by a specific pattern of activating and repressive histone modifications that ultimately promote latent gene expression while suppressing lytic gene expression. To investigate the molecular events involved in the establishment of the latent chromatin structure during the pre-latency phase of KSHV infection, we performed a comprehensive epigenetic study to analyze the recruitment of chromatin regulatory factors onto the KSHV genome at various time-points following de novo infection of SLK and TIME cells. This showed that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and was accompanied by the temporary induction of a limited number of lytic genes. Interestingly, transient expression of the RTA protein facilitated the increases of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both the Polycomb Repressive Complex 2 and 1. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a continuously transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection vs. lytic replication of KSHV. Please see above.

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

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