Topology of epitope-tagged F13L protein, a major membrane component of extracellular vaccinia virions

Abstract

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.

Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

“…The F13L protein is expressed as a cytoplasmic protein and associates transiently with membranes, probably in the Golgi network, where it becomes palmitoylated by membrane-associated transferases (30). The F13L protein then induces vesicle formation by a mechanism that is dependent on its phospholipase motif (28,29).…”

“…HeLa and BS-C-1 cells were grown and subcultured in Dulbecco's modified Eagle medium (DMEM) and Earle's modified Eagle medium (EMEM), respectively, supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin, at 37°C under 5% CO 2 . Recombinant vaccinia virus vF13L-GFP and plasmid pF13L-GFP, which contain an enhanced green fluorescent protein (GFP) coding sequence appended to the C terminus of the F13L ORF, and recombinant vaccinia virus vF13LHA C and plasmid pF13L-HA, which contain the F13L ORF with a C-terminal hemagglutinin (HA) epitope tag, have been described previously (29,30). pVSVG-GFP and pSAR1 H79G with an N-terminal HA tag were provided by Jennifer LippincottSchwartz.…”

“…The F13L protein localizes in the Golgi network as well as in endosomes and influences the intracellular location of other viral envelope proteins (26,28,29,55). Topological studies have indicated that the N and C termini of the F13L protein face the cytoplasmic side of the outer IEV membrane and the inner side of the inner IEV membrane, which is destined to become the CEV or EEV envelope (30,47,56).Studies with fluid-phase tracers have indicated that recycling between the endocytic and exocytic compartments is greatly increased following vaccinia virus infection, making it difficult to determine whether the membranes that wrap IMV are derived from endosomal or trans-Golgi cisternae (55, 65). Enhanced recycling could benefit virus replication by allowing recovery of viral proteins that are deposited in the plasma membrane either through the secretory pathway or by fusion with the outer IEV membrane.…”

“…The F13L protein localizes in the Golgi network as well as in endosomes and influences the intracellular location of other viral envelope proteins (26,28,29,55). Topological studies have indicated that the N and C termini of the F13L protein face the cytoplasmic side of the outer IEV membrane and the inner side of the inner IEV membrane, which is destined to become the CEV or EEV envelope (30,47,56).…”

Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

“…Of these, A36 (35), A56 (28), and B5 (11,19) are type I integral membrane proteins, A33 (25) and A34 (9) are type II integral membrane proteins, and F13 (17) and probably F12 (34) are peripheral membrane proteins. The F13 and B5 proteins are required for wrapping of the IMV, and the other proteins are involved in subsequent steps, including intracellular movement, actin tail formation, and virus spread (29).…”