Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

Richard T Timms,Duncan L Smith,Michael P Weekes,Gordon Dougan,Robin Antrobus,Iva A Tchasovnikarova,Paul J Lehner,Lidia M Duncan,Klaus Früh

doi:10.1371/journal.ppat.1003772

Abstract

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

Highlights

Manipulation of the cellular machinery of the host by viruses is essential to ensure their successful replication
This revealed an essential role for the poorly characterised protein proteolipid protein 2 (PLP2); K3 and K5 hijack PLP2 to facilitate their export out of the endoplasmic reticulum, which is necessary for ubiquitination and subsequent degradation of their substrates
We identified many new cell surface receptors targeted by K5, all of which are likely to be dependent on PLP2

Summary

Introduction

Manipulation of the cellular machinery of the host by viruses is essential to ensure their successful replication. K3 and K5 are two genes encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV) which were originally identified through their ability to degrade major histocompatibility complex class I (MHC-I) molecules [2,3]. These genes encode membrane-bound E3 ubiquitin ligases, which use their N-terminal RING-CH domain to direct the polyubiquitination and subsequent endolysosomal degradation of target immunoreceptors [4]. K3 seems primarily focussed on MHC-I, K5, with which it shares 40% amino acid identity, is more promiscuous and targets a variety of additional cell surface immunoreceptors for degradation. How a single ligase is able to target such a structurally diverse range of molecules for degradation, whilst retaining specificity, is not well understood

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Discussion

Conclusion