Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

Ildar Gabaev,James C Williamson,Thomas W.M Crozier,Thomas F Schulz,Paul J Lehner

doi:10.1016/j.celrep.2020.108249

Ildar Gabaev, James C Williamson + Show 3 more

Open Access

https://doi.org/10.1016/j.celrep.2020.108249

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Abstract

SummaryKaposi’s sarcoma herpesvirus (KSHV) is an oncogenic human virus and the leading cause of mortality in HIV infection. KSHV reactivation from latent- to lytic-stage infection initiates a cascade of viral gene expression. Here we show how these changes remodel the host cell proteome to enable viral replication. By undertaking a systematic and unbiased analysis of changes to the endothelial cell proteome following KSHV reactivation, we quantify >7,000 cellular proteins and 71 viral proteins and provide a temporal profile of protein changes during the course of lytic KSHV infection. Lytic KSHV induces >2-fold downregulation of 291 cellular proteins, including PKR, the key cellular sensor of double-stranded RNA. Despite the multiple episomes per cell, CRISPR-Cas9 efficiently targets KSHV genomes. A complementary KSHV genome-wide CRISPR genetic screen identifies K5 as the viral gene responsible for the downregulation of two KSHV targets, Nectin-2 and CD155, ligands of the NK cell DNAM-1 receptor.

Highlights

Kaposi’s sarcoma herpesvirus (KSHV) or HHV-8 causes Kaposi’s sarcoma (KS), a highly vascular tumor of lymphatic and blood vessels (Damania and Cesarman, 2013)
We used HuAR2T-tert, a cell line derived from conditionally immortalized human umbilical vein endothelial cells (May et al, 2010) that harbors latent recombinant KSHV encoding GFP and RFP (HuAR2T.rKSHV.219) and is engineered to report the presence (GFP) and reactivation (RFP) of virus (Vieira and O’Hearn, 2004)
KSHV is reactivated from these endothelial cells by delivery of exogenous KSHV-RTA to the latent population

Summary

Introduction

Kaposi’s sarcoma herpesvirus (KSHV) or HHV-8 (human herpesvirus 8) causes Kaposi’s sarcoma (KS), a highly vascular tumor of lymphatic and blood vessels (Damania and Cesarman, 2013). KS is one of the most common tumors in AIDS patients (Ganem, 2010) and is linked to two B cell malignancies: primary effusion lymphoma (PEL) and multicentric Castleman disease (Cesarman et al, 1995; Soulier et al, 1995). Primary KSHV infection results in latent viral infection, the default state with only few viral genes and microRNAs (miRNAs) expressed (Ganem, 2010; Schulz, 2006). The latent virus undergoes lytic reactivation that in vivo is triggered by viral co-infections or immunosuppression (reviewed in Aneja and Yuan, 2017). During lytic-stage KSHV infection, the repertoire of viral gene products is expressed in a temporal cascade, resulting in viral replication and the release of new virions

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