Golgi network targeting and plasma membrane internalization signals in vaccinia virus B5R envelope protein.
The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316.
Ward
B M
BM
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
Moss
B
B
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014616:Vaccinia virus / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C074512:42kDa protein, Vaccinia virus; D049452:Green Fluorescent Proteins