Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response
Figure 5
Identification of the region required for IRE1 binding and degradation.
(A) Schematic representation of the mutant M50 proteins used in the following experiments. Proline-rich (P) sequence, transmembrane (TM) domain, and the peptide recognized by the M50-specific antibody (ab) are indicated. Numbers on the right indicate amino acid positions. The HSV-1 UL56 protein was used as an unrelated control protein. 56TM is an M50 mutant containing the TM domain of HSV-1 UL56. (B) NIH-3T3 cells were cotransfected with plasmids coding for IRE1-HA (1 µg) and the proteins shown in panel A (2 µg). After 24 h, IRE1 levels were analyzed by immunoblot using an anti-HA antibody. M50 mutants and UL56 were detected with M50- and Flag-specific antibodies. (C) 293A cells were cotransfected with expression plasmids for IRE1-HA and full-length (fl.) or mutant M50. IRE1 was immunoprecipitated (IP) with an anti-HA antibody, and coprecipitating M50 proteins were detected by immunoblot using an M50-specific antibody. The same proteins were detected in whole cell lysates (WCL). LC, antibody light chain.