Adenovirus E3-6.7K inhibits apoptosis through an interaction with the cellular protein calcium-modulating cyclophilin ligand (CAML)

Adenovirus is an important etiological agent which causes acute respiratory and gastrointestinal infections worldwide. The virus encodes several proteins involved in suppressing the host antiviral response. One of these is the E3-6.7K protein which confers transfected cells with resistance from a variety of apoptotic stimuli and maintains endoplasmic reticulum (ER) Ca 2+ homeostasis, however, the mechanism of action is unknown. In this study, evidence is provided that E3-6.7K inhibits apoptosis and ER Ca²⁺ efflux through an interaction with Ca²⁺-modulating cyclophilin ligand (CAML), a cellular protein that regulates intracellular Ca²⁺. E3-6.7K was shown to interact with both mouse and human CAML in a yeast two-hybrid system. A direct interaction with human CAML was confirmed by an in vitro co-immunoprecipitation assay. In addition, immunofluorescence microscopy revealed that the two proteins co-localize in an ER-like pattern in transfected cells. Furthermore, yeast two-hybrid assays indicated that the interaction between the two proteins is localized to the N-terminal domain of CAML and to a 22 amino acid region near the C-terminus of E3-6.7K, termed the CAML-binding domain (CBD). Mutational analysis of the CBD showed that an interaction with CAML is required for E3-6.7K to inhibit thapsigargin-induced apoptosis and ER Ca²⁺ efflux. These findings suggest that E3-6.7K targets CAML as a mechanism to alter ER Ca²⁺ homeostasis which consequently protects cells from apoptosis. E3-6.7K has been shown to be an integral membrane protein. Most membrane proteins have only one orientation in a membrane bilayer. E3-6.7K can adopt three different topologies in microsomal membranes in a cell-free translation system: a type II orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K), the opposite type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K) and the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). In this study, the topology of E3-6.7K in cells was explored. Distinct topological forms were found, as both the N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. Based on the location of the CBD, the most probable binding partner of CAML is NtmE3-6.7K.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat