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

Abstract

Summary Kaposi’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.

Human endogenous retroviruses in cancer: Expression, regulation and function (Review)

“…Kaposi's sarcoma is a classic example that HERVs transactivated by exogenous virus infection can promote carcinogenesis ( 124 ). KSHV infection is an important cause of morbidity of Kaposi's sarcoma in patients with AIDS ( 125 ). The mechanism through which KSHV upregulates the expression of HERVs has been clarified as aforementioned.…”

Natural Killer Cell Responses during Human γ-Herpesvirus Infections

“…In addition, KSHV also employs the direct modulation of NK cell recognition by inhibition of ligand upregulation for the activation of NK cell receptors, of migration of NK cells and by exploitation of certain KIR haplotypes. Along these lines, KSHV downregulates ligands for activating NK cell receptors, such as NKG2D, NKp44, NKp80 and DNAM-1 [96][97][98][99]. The viral ubiquitin ligase K5 downregulates the NKG2D ligands MICA and MICB, the NKp80 ligand AICL, and the DNAM-1 ligands Nectin-2 and CD155 [96,98].…”

“…Along these lines, KSHV downregulates ligands for activating NK cell receptors, such as NKG2D, NKp44, NKp80 and DNAM-1 [96][97][98][99]. The viral ubiquitin ligase K5 downregulates the NKG2D ligands MICA and MICB, the NKp80 ligand AICL, and the DNAM-1 ligands Nectin-2 and CD155 [96,98]. Furthermore, viral ORF54 downregulates NKp44 ligands of an unknown identity independently of ORF54 s UTPase activity [97].…”

CRISPR Interference Efficiently Silences Latent and Lytic Viral Genes in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Cells

“…Post-transcriptional gene silencing and genome editing through RNA interference (RNAi) and CRISPR-Cas9, respectively, have expanded the functional genomics toolkit to study KSHV genes’ function and virus–host interactions [ 9 , 16 , 17 , 18 , 19 , 20 ]. RNAi using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has been useful for depleting many KSHV transcripts.…”

“…However, RNAi has inherent limitations, including off-target effects, the competition with endogenous substrates for the cell’s RNAi machinery, and their inability to target nuclear transcripts [ 21 , 22 , 23 ]. CRISPR editing overcomes these limitations and has been recently used to generate single viral gene knockouts or as part of genetic screens in KSHV-infected cells [ 16 , 17 , 18 , 24 ]. Nevertheless, CRISPR editing is encumbered by the viral genome multiplicity naturally observed in KSHV-infected cells, ranging from a handful to over 100 viral genomes per cell [ 25 ].…”