title: Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway.
abstract: Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.
mesh_terms: D000818:Animals; D001483:Base Sequence; D002459:Cell Fusion; D002462:Cell Membrane / Q000378:metabolism; D002678:Chimera; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D005796:Genes; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics; D006386:Hemagglutination Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics*; D007668:Kidney; D008854:Microscopy, Electron; D013539:Simian virus 40 / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral