Whole-genome sequencing of Kaposi sarcoma-associated herpesvirus (KSHV/HHV8) reveals evidence for two African lineages.

Kaposi sarcoma (KS)-associated herpesvirus (KSHV/HHV-8) was first sequenced from the body cavity (BC) lymphoma cell line, BC-1, in 1996. Few other KSHV genomes have been reported. Our knowledge of sequence variation for this virus remains spotty. This study reports additional genomes from historical US patient samples and from African KS biopsies. It describes an assay that spans regions of the virus that cannot be covered by short read sequencing. These include the terminal repeats, the LANA repeats, and the origins of replication. A phylogenetic analysis, based on 107 genomes, identified three distinct clades; one containing isolates from USA/Europe/Japan collected in the 1990s and two of Sub-Saharan Africa isolates collected since 2010. This analysis indicates that the KSHV strains circulating today differ from the isolates collected at the height of the AIDS epidemic. This analysis helps experimental designs and potential vaccine studies.

Evidence for Multiple Subpopulations of Herpesvirus-Latently Infected Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphomas (PEL) are traditionally viewed as homogenous regarding viral transcription and lineage of origin, but so far this contention has not been explored at the single-cell level. Single-cell RNA sequencing of latently infected PEL supports the existence of multiple subpopulations even within a single cell line. At most 1% of the cells showed evidence of near-complete lytic transcription. The majority of cells only expressed the canonical viral latent transcripts: those originating from the latency locus, the viral interferon regulatory factor locus, and the viral lncRNA nut-1/Pan/T1.1; however, a significant fraction of cells showed various degrees of more permissive transcription, and some showed no evidence of KSHV transcripts whatsoever. Levels of viral interleukin-6 (IL-6)/K2 mRNA emerged as the most distinguishing feature to subset KSHV-infected PEL. One newly uncovered phenotype is the existence of BCBL-1 cells that readily adhered to fibronectin and that displayed mesenchymal lineage-like characteristics. IMPORTANCE Latency is the defining characteristic of the Herpesviridae and central to the tumorigenesis phenotype of Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV-driven primary effusion lymphomas (PEL) rapidly develop resistance to therapy, suggesting tumor instability and plasticity. At any given time, a fraction of PEL cells spontaneously reactivate KSHV, suggesting transcriptional heterogeneity even within a clonal cell line under optimal growth conditions. This study employed single-cell mRNA sequencing to explore the within-population variability of KSHV transcription and how it relates to host cell transcription. Individual clonal PEL cells exhibited differing patterns of viral transcription. Most cells showed the canonical pattern of KSHV latency (LANA, vCyc, vFLIP, Kaposin, and vIRFs), but a significant fraction evidenced extended viral gene transcription, including of the viral IL-6 homolog, open reading frame K2. This study suggests new targets of intervention for PEL. It establishes a conceptual framework to design KSHV cure studies analogous to those for HIV.

Interleukin-1 Receptor-Associated Kinase (IRAK) Signaling in Kaposi Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1ß) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1ß stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-κB activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1ß. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.

Extracellular vesicles from Kaposi Sarcoma-associated herpesvirus lymphoma induce long-term endothelial cell reprogramming.

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi's Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection.

Epstein-Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus.

Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi's sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein-Barr virus (EBV); however, the requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV+ single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV+/EBV+ PEL cell lines with an EBV-specific CRISPR/Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein-Barr nuclear antigen 1 (EBNA-1) in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.