Adenovirus internalization and infection require dynamin - PubMed (original) (raw)

Adenovirus internalization and infection require dynamin

K Wang et al. J Virol. 1998 Apr.

Abstract

The cell receptors that facilitate adenovirus internalization into cells have been identified; however, the infectious pathway of virus entry has not been established. Adenovirus entry and infection were examined in HeLa cells lacking or overexpressing mutant dynamin, a protein that specifically regulates clathrin-mediated endocytosis. Expression of mutant dynamin significantly reduced adenovirus internalization and gene delivery, indicating a functional requirement for this molecule. These findings are consistent with virus entry via the clathrin-coated pit pathway.

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Figures

FIG. 1

Detection of mutant dynamin expression by immunoblotting. tTA-HeLa cells stably transfected with the K44A dynamin mutant were cultured in the presence (+ tet) or absence (− tet) of tetracycline for 48 h and then solubilized in sodium dodecyl sulfate sample buffer. Lysates prepared from 105 induced or uninduced cells were separated on a sodium dodecyl sulfate–7%-polyacrylamide gel under reducing conditions. Following transfer of the proteins to a nitrocellulose filter (Immobilon P; Amersham), the filter was probed with an antihemagglutinin epitope tag monoclonal antibody (12CA5) and then incubated with a goat anti-mouse immunoglobulin antibody conjugated to alkaline phosphatase. The blot was then developed by addition of a chromogenic substrate (Nitro Blue Tetrazolium).

FIG. 2

Flow cytometric analysis of Ad-mediated gene delivery to control or mutant-dynamin-expressing HeLa cells. (A) tTA-HeLa cells were cultured in the presence (+ tet) or absence (− tet) of tetracycline for 48 h and then infected by incubation with Ad.RSV.GFP at a virus particle/cell ratio of 300 for 1 h at 37°C. The cells were then washed and recultured for 48 h in the presence or absence of tetracycline prior to flow cytometric analysis. Control cells (dotted lines) were incubated in medium without virus. (B) tTA-HeLa cells were cultured in the presence of tetracycline and then infected with Ad.RSV.GFP. The cells were then divided into two equal samples; one was cultured for 48 h in the presence of tetracycline (+ tet), and the other was cultured in the absence (− tet) of tetracycline. Both were then analyzed by flow cytometry.

FIG. 3

Dose-dependent delivery of GFP to HeLa cells by use of recombinant Ad. tTA-HeLa cells were incubated in the presence (circles) or absence (triangles) of tetracycline for 48 h and then infected with Ad.RSV.GFP at various particle/cell ratios. GFP expression was analyzed 48 h later by flow cytometry. The data are representative of at least three experiments.

FIG. 4

Ad binding to HeLa cells expressing or lacking mutant dynamin. Cells were incubated for 48 h in the presence (+ tet) or absence (− tet) of tetracycline and then assayed for binding of 125I-labeled Ad type 2 (Ad2) particles as previously described (11). Nonspecific virus binding, which was subtracted from the total, was determined by incubating cells with a 200-fold excess of unlabeled virus particles. The data are the mean ± the standard deviation of triplicate samples.

FIG. 5

Ad internalization and gene delivery into HeLa cells expressing or lacking mutant dynamin. (A) Internalization of 125I-labeled Ad was measured in HeLa cells grown in the presence (circles) or absence (triangles) of tetracycline as previously described (25). Following warming of the cells to 37°C for various lengths of time, uninternalized virus particles were removed by incubating the cells in trypsin-EDTA and then washing them with HEPES-buffered saline. The data are the mean ± the standard deviation of triplicate samples. (B) In parallel studies, Ad-mediated gene delivery was examined in cells grown in the presence (circles) or absence (triangles) of tetracycline. Cells were incubated at 4°C with Ad.RSV.GFP at a particle/cell ratio of 300 and then warmed to 37°C for various lengths of time. After removal of uninternalized virus particles with trypsin-EDTA, the cells were cultured in the presence of tetracycline for 48 h prior to flow cytometric analysis. The data are representative of two experiments. Ad2, Ad type 2.

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Adenovirus internalization and infection require dynamin - PubMed (original) (raw)