An acidic cluster in the cytosolic domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the plasma membrane

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigme nt epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epith elial cells constitutively expressing gB. The cytosolic domain of gB contai ns a cluster of acidic amino acids, a motif that plays a pivotal role in ve ctorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internal ization and recycling to the plasma membrane in CMV-infected human fibrobla sts (HF) and ARPE-19 cells by using antibody-internalization experiments. I mmunofluorescence and quantitative assays showed that gB was internalized f rom the cell surface into clathrin-coated transport vesicles and then recyc led to the plasma membrane. gB colocalized with clathrin-coated vesicles co ntaining the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acid ic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking t he acidic cluster were impaired in internalization and failed to recycle. T hese findings, together with our earlier observation that the acidic cluste r is a key determinant for targeting gB molecules to apical membranes in ep ithelial cells, establish that this signal is recognized by cellular protei ns that participate in polarized sorting and transport in the early endocyt ic/recycling pathway.