The cytosolic tail of class I MHC heavy chain is required for its dislocation by the human cytomegalovirus US2 and US11 gene products

Abstract

The US2 and US11 glycoproteins of human cytomegalovirus facilitate destruction of MHC class I heavy chains by proteasomal proteolysis through acceleration of endoplasmic reticulum-to-cytosol dislocation. Modification of the class I heavy chain was used to probe the structural requirements for this sequence of reactions. The cytosolic domain of the class I heavy chain is required for dislocation to the cytosol and for its subsequent destruction. However, interactions between US2 or US11 and the heavy chain are maintained in the absence of the class I cytosolic domain, as shown by chemical crosslinking in vivo and coprecipitation when translated in vitro. Thus, substrate recognition and accelerated destruction of the heavy chain, as facilitated by US2 or US11, are separable events.

Figure 1

Schematic representation of mutant and wild-type class I heavy chain. Truncated class I heavy chains (tailless A2) are not dislocated, yet associate with US2 and US11. The truncated heavy chains retain four amino acids that protrude into the cytosol to facilitate proper insertion in the membrane (21). The sizes of the proteasome particle and the class I protein are rendered approximately to scale.

Figure 2

Tailless A2 is stable in the presence of US2 and US11 while the endogenous class I heavy chains are destroyed. Cells were pulse labeled for 5 min and chased for the indicated times in the absence of proteasome inhibitors. US2-expressing cells (a), US11-expressing cells (b), or control cells (CC; c) transfected with tailless A2 (A2-cyt) were subjected to pulse–chase analysis. Immunoprecipitations were done with antibodies reactive with free heavy chains (αHC) or folded class I products (W6/32). The US2 and US11 molecules that coprecipitate in the W6/32 complex are indicated.

Figure 3

Presence of tailless A2 inhibits dislocation of endogenous MHC class I heavy chains. Cells were pulse labeled for 5 min and chased for the times shown, all in the presence of ZL₃VS. The bracket with + and - denotes the forms of heavy chain with and without glycan, respectively. In US11-expressing cells (a), full length class I heavy chain is lost (a decrease in W6/32-reactive material) or dislocated (an increase in −CHO form). The tailless A2 continues to fold (increasing W6/32) while no deglycosylated tailless A2 accumulates. There is increased recovery of US11 in the cells expressing tailless A2. In control cells (b), endogenous and tailless heavy chains mature at similar rates. Fractionation of US11-expressing cells transfected with tailless A2 (c) shows the presence of deglycosylated full length heavy chains in the cytosol and no accumulation of tailless A2 in the cytosol.

Figure 4

DSP crosslinking of cells expressing tailless A2 reveals an association between US11 or US2 and tailless A2. Cells exposed to the crosslinker DSP (see Materials and Methods) were lysed in Nonidet P-40 buffer. Folded MHC class I products were recovered by immunoprecipitation with the W6/32 antibody. A portion of this immunoprecipitate was loaded directly (first IP). The remainder was denatured in SDS without reductant and reprecipitated with αHC and either α-US2 or α-US11 antibodies. Reimmunoprecipitation with α-US11 antibody recovers tailless A2, indicative of an association between them (a, right lane). Likewise, US2 antibodies recover crosslinked tailless A2 (b, right lane). Reduction of the thiol-cleavable crosslinker before SDS/PAGE analysis dissociates the crosslinked molecules and yields the constituent polypeptides that then migrate at their characteristic molecular weights.

Figure 5

Tailless HLA-A2 associates with US2 and US11 in vitro. Full length and tailless A2 mRNAs were translated in a microsome-supplemented in vitro translation system together with β₂m, HLA-DR1α, and US2 or US11 mRNA in the presence and absence of 1 mM DTT, as indicated. A portion of the total translation mixture was loaded directly (left lanes). The remainder of the microsomes was lysed and subjected to immunoprecipitation with W6/32 antibody (right lanes).

Figure 6

Model for dislocation of class I heavy chains by US2 and US11. (a) The dislocation reaction is initiated by binding of US2 or US11 to the heavy chain with most intermolecular contacts within the ER lumen. (b) Once in the translocon, the class I heavy chain unfolds. The light chain of class I and MHC-bound peptide may then dissociate within the ER. Cytosolic factors (black box) bind to the cytosolic tail of the class I heavy chain and aid in its extraction. (c) N-glycanase (gray box) attacks the glycan when it emerges into the cytosol. Proteasome cleaves the class I heavy chain. (d) In the presence of proteasomal inhibitors, a portion of the heavy chain is deflected into the cytosol directly and accumulates as a deglycosylated intermediate.