Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Abstract

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.

FIG. 1.

Expression of HCMV US2 C-terminal truncated proteins. (A) Schematic representation of C-terminal mutants of HCMV US2. Wild-type US2 (residues 1 to 199) contains an N-terminal signal sequence (SS) of 20 aa, followed by an ER-lumenal domain that includes a glycosylation site (Asn⁶⁸) and a disulfide bridge between Cys⁵² and Cys¹³³. The lumenal domain is followed by a predicted transmembrane domain (from residues 162 to 185) and a 14-residue cytosolic tail (residues 186 to 199). (B) Wild-type (WT) US2 and US2 mutants were expressed in His16 cells by infecting cells with Ad vectors for 12 to 16 h; the cells were radiolabeled with ³⁵S-labeled methionine and cysteine for 5 min, and US2 proteins were immunoprecipitated with rabbit polyclonal anti-US2 sera. Molecular sizes of marker proteins of 28.1 and 20.9 kDa are indicated on the left. (C) His16 cells infected with Ad vectors expressing various US2 were radiolabeled for 30 min, and then US2 proteins were immunoprecipitated and either treated with endo H (+) or not treated with endo H (−).

FIG. 2.

Expression of HCMV US2 N-terminal truncated proteins. (A) Schematic representation of a panel of US2 mutants truncated from the N terminus. The native signal sequence of US2 was replaced with the murine MHC class I K^b signal sequence. (B) Expression of N-terminal truncated mutants of US2 in His16 cells infected with Ad vectors and radiolabeled for 5 min with ³⁵S-labeled methionine and cysteine. US2 proteins were immunoprecipitated with rabbit polyclonal anti-US2 sera. Note that K^b50-199 and K^b60-199 were not stable, and the expression was not shown. (C) Cells infected with Ad vectors expressing mutant US2 proteins were radiolabeled for 30 min, US2 immunoprecipitated, and then either treated (+) or not treated (−) with endo H.

FIG. 3.

Degradation of class I and II proteins by US2 C- and N-terminal truncation mutants. His16 cells were infected for 12 to 16 h with Adtet-trans alone or coinfected with Adtet-trans (Trans) and AdtetUS2 (wild-type US2 [US2wt]) or Ad vectors expressing US2 mutants: 1-186, 1-160, 1-150, 1-140, 1-130, 1-120, or 1-110 (A) or K^b21-199, K^b28-199, or K^b40-199 (B). The cells were radiolabeled with ³⁵S-labeled methionine and cysteine for 1 min, and then the label was chased for 15 or 30 min. The cell extracts were immunoprecipitated with MAb HC10 (anti-class I HC, upper panels) or DA6.147 (anti-class II DR-α, lower panels).

FIG. 4.

Sequential immunoprecipitation of US2 C-terminal truncation proteins with MHC class I and II proteins. His16 cells were infected with Ad vectors expressing US2 proteins for 12 to 16 h, incubated with 35 μM proteasome inhibitor ZL₃VS for 60 to 90 min, and then radiolabeled with ³⁵S-labeled methionine and cysteine for 20 min in the presence of ZL₃VS. Cell extracts were made with 1% digitonin buffer, and US2 was immunoprecipitated directly with polyclonal anti-US2 antibodies (A), class I HC was precipitated with MAb W6/32 (B), or class II complexes were precipitated with MAb DA6-147. In panels B and C, the class I or II complexes were denatured in 1% SDS and reprecipitated with anti-US2 antibodies.

FIG. 5.

Sequential immunoprecipitation of US2 N-terminal truncations with MHC class I and II proteins. His16 cells were infected with Ad vectors for 12 to 16 h and incubated with ZL₃VS for 60 to 90 min, and the cells were labeled for 20 min and then lysed in 1% digitonin buffer. (A) An aliquot of the samples was immunoprecipitated directly with polyclonal anti-US2 antibodies. (B) MHC class I was precipitated with MAb W6/32, and precipitated proteins were denatured and reprecipitated with polyclonal anti-US2 antibodies. (C) Class II complexes were immunoprecipitated with MAb DA6.147, and the precipitated proteins were denatured and reprecipitated with anti-US2 antibodies.

FIG. 6.

Expression of US2/3 chimeric proteins and degradation of class I and II proteins. (A) Schematic representation of US2 and US3 including the homologous sequences: hr1, hr2, and hr3. US2/3-hr1 contains hr1 of US3 introduced into US2 in place of US2 hr1. US2/3-hr2 has hr2 of US3 introduced into US2 in place of US2 hr2. US2Δhr3 has a deletion of 4 aa (RCVP) in the hr3 region. (B) Expression of US2/3 chimeric proteins. His16 cells were infected with Ad vectors, the cells were radiolabeled for 5 min with ³⁵S-labeled methionine and cysteine, and US2/3 proteins were immunoprecipitated with anti-US2 antibodies. (C) Degradation of class I and II proteins by US2/3 chimeric proteins. His16 cells were infected with Ad vectors and labeled with ³⁵S-labeled methionine and cysteine for 1 min, and then the label chased for 15 or 30 min. MHC class I HC (upper panel or class II complexes (lower panel) were immunoprecipitated with MAb HC10 or DA6.147, respectively.

FIG. 7.

Binding of chimeric US2/3 chimeras to MHC I and II proteins. His16 cells were infected for 12 to 16 h with Ad vectors expressing chimeric proteins, and cells were incubated with ZL₃VS for 60 to 90 min then radiolabeled for 20 min. (A) Digitonin cell extracts were immunoprecipitated directly with anti-US2 antibodies. (B) Cell extracts were immunoprecipitated with anti-class I HC (W6/32), denatured, and then immunoprecipitated with anti-US2 antibodies. (C) Cell extracts were immunoprecipitated with anti-class II MAb DA6.147 and then immunoprecipitated with anti-US2 antibodies.