Abstract
Kaposi’s sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV, known as human herpesvirus-8, or HHV8)is the infectious cause of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentricCastleman’s Disease (MCD) [1]
We demonstrated that tetracycline (Tet)-inducible gene expression was feasible in the zebrafish XT context, KSHV reactivation from latency was inefficient in this model
Both BCBL1 cells and TREx-BCBL1-regulator of transcriptional activation (RTA) cells successfully proliferated in the yolk sac, with BCBL1 cells increasing in number by 2.2-fold, and TREx-BCBL1-RTA cells increasing by 2.8 fold over three days (Figure 1C)
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV, known as human herpesvirus-8, or HHV8)is the infectious cause of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentricCastleman’s Disease (MCD) [1]. Kaposi’s sarcoma-associated herpesvirus (KSHV, known as human herpesvirus-8, or HHV8). Is the infectious cause of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric. KSHV is a gammaherpesvirus that achieves life-long infection of human hosts by establishing latency in immature B lymphocytes and promoting differentiation into a plasmablast-like cell type [2]. An essential feature of herpesvirus latency is reversibility, and periodic reactivation from latency enables lytic KSHV replication and spread to new hosts. KSHV latency is unstable in vivo and in vitro, with spontaneous expression of lytic antigens [3]. PEL is a rare disease that occurs most frequently in human immunodeficiency virus (HIV)-positive individuals, or otherwise immunosuppressed individuals. PEL develops as bloody effusions in body cavities, including pleural, peritoneal, and pericardial spaces, but can form solid extracavity lymphomas [5]. The current standard of care is EPOCH (Etoposide, Prednisone, Oncovin/vincristine, Cyclophosphamide, Hydroxydaunorubicin/doxorubicin) or CHOP with or without antiretroviral therapy [5]
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