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. 2003 Aug;77(16):9008-19.
doi: 10.1128/jvi.77.16.9008-9019.2003.

Intracellular trafficking of a palmitoylated membrane-associated protein component of enveloped vaccinia virus

Affiliations

Intracellular trafficking of a palmitoylated membrane-associated protein component of enveloped vaccinia virus

Matloob Husain et al. J Virol. 2003 Aug.

Abstract

The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1(H79G), a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with alpha-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

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Figures

FIG. 1.
FIG. 1.
Intracellular distribution of VSVG-GFP and F13L-GFP in cells expressing Sar1H79G-HA. HeLa cells were transfected with plasmids that expressed VSVG-GFP or F13L-GFP in the presence or absence of plasmids that expressed Sar1H79G-HA. After 24 h, the cells were fixed and permeabilized, stained with an anti-HA MAb followed by rhodamine red-conjugated anti-mouse IgG, and examined by confocal microscopy. Bars, 10 μm.
FIG. 2.
FIG. 2.
Localization of F13L-GFP and markers of endocytosis. HeLa cells were transfected with pF13L-GFP or infected with vF13L-GFP. After 24 h of transfection or 18 h of infection, the cells were incubated with FM4-64 or TR-Tfn for 20 min at 37°C. The cells were then fixed and either directly analyzed by confocal microscopy or first permeabilized and stained with an anti-clathrin antibody followed by tetramethyl rhodamine isothiocyanate-conjugated anti-goat IgG. Arrows points to the colocalization of F13L-GFP with FM4-64, clathrin-staining vesicles, or TR-Tfn. Green, GFP; red, rhodamine, Texas red, or FM4-64; yellow, overlap of green and red. Bars, 10 μm.
FIG. 3.
FIG. 3.
Effects of chlorpromazine on the endocytosis of TR-Tfn and intracellular localization of F13L-GFP. HeLa cells were transfected with pF13L-GFP (upper panels) or infected with vF13L-GFP (lower panels). At 24 h after transfection or 18 h after infection, the cells were pretreated with chlorpromazine for 10 min and then incubated with TR-Tfn for 20 min in the continued presence of chlorpromazine. The cells were then fixed and viewed by confocal microscopy. GFP and Texas red fluorescence is shown in the left and right panels, respectively. Bars, 10 μm.
FIG. 4.
FIG. 4.
Coexpression of F13L with a trans-dominant-negative dynamin mutant. HeLa cells were transfected or cotransfected with plasmids expressing F13L-HA, functional Dyn 2 (ab)-GFP, or dominant-negative Dyn 2 (ab) K44A-GFP. After 24 h, cells were incubated with TR-Tfn for 20 min and then either fixed and examined by confocal microscopy or first permeabilized and stained with an anti-HA MAb followed by rhodamine red-conjugated anti-mouse IgG. Bars, 10 μm.
FIG. 5.
FIG. 5.
Coexpression of F13L with a dominant-negative Eps15 mutant. HeLa cells were transfected or cotransfected with plasmids expressing F13L-HA, D3Δ2, or EH21. After 24 h, the cells were incubated with TR-Tfn for 20 min and then either fixed and examined by confocal microscopy or first permeabilized and stained with an anti-HA MAb followed by rhodamine red-conjugated anti-mouse IgG. Bars, 10 μm.
FIG. 6.
FIG. 6.
Coimmunoprecipitation of F13L with α-adaptin. BS-C-1 cells infected with vF13LHAC were harvested and lysed. The lysate was incubated with an antibody to α-adaptin, clathrin, or γ-adaptin, and immune complexes were captured on protein A beads, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was probed with an anti-HA polyclonal antibody to detect the F13L protein. A sample of the cell lysate was also analyzed directly. The masses of marker proteins (in kilodaltons) are given on the left.
FIG. 7.
FIG. 7.
Effects of wortmannin and nocadazole on the internalization of TR-Tfn and intracellular localization of F13L-GFP. HeLa cells were transfected with pF13L-GFP or infected with vF13L-GFP. At 24 h after transfection or 18 h after infection, the cells were pretreated with wortmannin (Wort) or nocadazole (Noc) for 10 min and then incubated in the continued presence of the drug with TR-Tfn for 20 min. Cells were then fixed and analyzed by confocal microscopy. Arrows point to the overlap of F13L-GFP with TR-Tfn. Green, GFP; red, Texas red; yellow, overlap of green and red. Bars, 10 μm.
FIG. 8.
FIG. 8.
Effect of nocodazole treatment on the colocalization of F13L-GFP with LAMP2. HeLa cells were transfected with pF13L-GFP or infected with vF13L-GFP. At 24 h after transfection or 18 h after infection, cells were either left untreated or treated with nocodazole (Noc) for 30 min. The cells were then fixed, permeabilized, and stained with an anti-LAMP2 MAb followed by rhodamine red-conjugated anti-mouse IgG. Arrows point to the overlap of F13L-GFP with LAMP2. Green, GFP; red, rhodamine red; yellow, overlap of green and red. Bars, 10 μm.

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