Abstract
The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.
Highlights
Kaposi’s sarcoma-associated herpes virus (KSHV) downregulates MHC class I molecules from the surface of infected cells through the action of virally encoded K3 and K5 ubiquitin E3 ligases, thereby allowing evasion of the host immune system [1]
Yeast endosomal sorting complex required for transport (ESCRT)-III consists of a core complex of four components (Vps2p, Vps20p, Vps24p and Vps32p/Snf7p) with four peripheral/regulatory proteins and it is the polymerization of Vps32p/Snf7p together with the co-assembly of Vps24p and Vps2p that drives intralumenal vesicle (ILV) budding from the endosome’s limiting membrane by a spring-like mechanism [12,13]
We found that the core ESCRT-III proteins VPS32B, VPS24 and VPS2A were required, the remaining core ESCRT-III protein VPS20/CHMP6 was not needed
Summary
Kaposi’s sarcoma-associated herpes virus (KSHV) downregulates MHC class I molecules from the surface of infected cells through the action of virally encoded K3 and K5 ubiquitin E3 ligases, thereby allowing evasion of the host immune system [1]. Following the stable expression of KK3 in HeLa cells (HeLa-KK3 cells), newly synthesized MHC class I molecules that have trafficked through the secretory pathway to the plasma membrane are polyubiquitinated, rapidly endocytosed by a clathrin and epsin/Eps R-dependent route and targeted for lysosomal degradation [1,4] This lysosomal targeting requires the endosomal sorting complex required for transport (ESCRT) proteins HRS and TSG101 [2,5]. For the VPS20 rescue experiment, myc-VPS20 containing three silent mutations in the region of sequence identity with oligo siRNA was cloned into pIRESneo and the resulting plasmid used to transfect HeLa cells with TransIT-HeLa Monster® followed by antibiotic selection of stably expressing cells Subsequent treatment of these cells with oligo or the ON-TARGET plus pool siRNA for VPS20 was as described above. Following uptake of FITC-w6/32 into HeLa-KK3 cells, 7839 gold particles were counted in total on six independently labelled EM grids from two separate experiments and after VPS20 depletion 1535 gold particles were counted on three independently labelled EM grids
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