Topology of epitope-tagged F13L protein, a major membrane component of extracellular vaccinia virions.
The protein encoded by the vaccinia virus F13L open reading frame is required for the wrapping of intracellular mature virions by cisternae derived from trans-Golgi or endosomal membranes and is an abundant, palmitylated component of the outer membrane of extracellular virions. To study the topology of the F13L protein, we constructed recombinant vaccinia viruses and plasmids that express the F13L protein with an N- or C-terminal HA epitope tag. The recombinant viruses formed normal-size plaques and the tagged proteins were incorporated into the two outer membranes of intracellular enveloped virions (IEV), indicating that the epitope-tagged proteins were functional. By selective permeabilization of the plasma membrane of infected or transfected cells, we demonstrated that the N- and C-termini of the F13L proteins in the outer IEV membrane, as well as cellular membranes, were oriented toward the cytoplasm. After fusion of the outer viral membrane with the plasma membrane, externalized virions retain the inner of the two IEV membranes. The N- and C-termini of the F13L protein were exposed on the inner surface of this extracellular viral membrane, consistent with the accepted model of biogenesis of the IEV membrane by a wrapping process. Using a coupled in vitro transcription and translation system modified by the addition of microsomes, we determined that the F13L protein associated posttranslationally with membranes. The N- and C-termini were susceptible to protease digestion and the protein could be extracted with sodium carbonate, consistent with a peripheral mode of association.
Husain
Matloob
M
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Weisberg
Andrea
A
Moss
Bernard
B
D002254:Carbonates / Q000494:pharmacology; D002462:Cell Membrane / Q000821:virology; D002463:Cell Membrane Permeability; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion; D016253:Microscopy, Immunoelectron; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014616:Vaccinia virus / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000502:physiology*
D002254:Carbonates; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C005686:sodium carbonate