The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol - PubMed (original) (raw)

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol

M Y Nakano et al. J Virol. 2000 Aug.

Abstract

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.

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Figures

FIG. 1

Fiber release from incoming Ad2 depends on intact F-actin and is inhibited by RGD peptides but not PKC inhibitors. HeLa cells were treated with the indicated inhibitors for 30 min at 37°C and then incubated with [35S]methionine-labeled Ad2 in the cold for 1 h and warmed for different times as indicated. Cell lysates were immunoprecipitated with antifiber antibodies under nondissociating conditions and fractionated by SDS-PAGE. [35S]methionine in hexon (Hex) and fiber (Fib) was quantitated and expressed as a ratio of hexon to fiber by using purified [35S]methionine-labeled Ad2 as a standard (100%). The data are representative of at least two independent experiments.

FIG. 2

Cell surface trypsinization assay of incoming [35S]methionine-labeled Ad2 in cells treated with actin and PKC inhibitors. HeLa cells were treated with various inhibitors for 30 min, and then [35S]methionine-labeled Ad2 was bound for 60 min in the cold. The cells were warmed for different times as indicated and treated with cold trypsin to probe for surface accessibility of hexon. The ratio of intact (Hex) to cleaved (Hex′) hexon was determined after SDS-PAGE (A to F) and plotted for each condition, taking the sum of the cleaved and uncleaved hexons from cells not treated with trypsin (−T) as 100% (G and H). Controls not treated with trypsin are shown in panels A, B, D, and F. Dashed lines in panels G and H indicate the total hexon radioactivity, and solid lines show the trypsin-resistant hexon determined by PhosphorImager analysis. Symbols for different drug treatments are used as indicated in panels G and H. Representative data from at least two independent experiments are shown.

FIG. 3

CLSM analysis of incoming Ad2-TR in cells treated with actin and PKC inhibitors reveals the actin and PKC requirements for nuclear transport of Ad2. TR-labeled Ad2 (red) was bound to HeLa cells pretreated with drugs or not pretreated and internalized for 0 min (A) or 60 min (B to E) as indicated in Materials and Methods. CLSM sections sampling the entire cell were generated for the TR channel and the Alexa 488 channel (plasma membrane Ca ATPase 1 [green]) and projected en face. The limits of the nucleus as determined by DAPI staining (results not shown) are indicated by a white trace. A single representative cell for each of the conditions is shown.

FIG. 4

Quantitative subcellular analysis of incoming fluorescently labeled wt Ad2 and _ts_1 Ad2 in cells treated with actin- or PKC-directed inhibitors. Ad2-TR was cold bound to inhibitor-treated or control HeLa cells and internalized for 0 min (open bars) or 60 or 70 min (solid bars) as described in Materials and Methods. The cells were fixed and analyzed for virus fluorescence in the cell periphery, the cytoplasm, and the nucleus by using an image deconvolution routine described previously (37). Results are shown as mean fluorescence values, with the corresponding SEM derived from the indicated number of analyzed cells (n). (A) Results for wt Ad2-TR in actin-inhibited cells. (B and C) Results for wt and _ts_1 Ad2-TR in cells not treated with drugs (solid bars) or treated with the PKC inhibitor PKC-myr (stippled bars) or a control myristoylated autocamptide (autocamp) directed against Cam kinase II (striped bars).

FIG. 5

Thin-section electron microscopy of wt Ad2-infected HeLa cells treated with actin- or PKC-directed inhibitors. Cells were treated with drugs as described in Materials and Methods and incubated with 50 μg of purified Ad2 per ml for 60 min in the cold. Unbound virus (approximately 98 to 99% of input virus) was washed off, and the cells were incubated with or without inhibitors for 30 min at 37°C, fixed, and processed for thin-section EM. (A) control cell infected without drugs. (B) CD-treated cell. (C) Jas-treated cell. (D) PKC-myr-treated cell. The inset in panel D shows an enlargement of a region proximal to the plasma membrane. Thin arrows indicate extracellular Ad2 particles, large arrows indicate cytoplasmic Ad2, small arrowheads show coated vesicles containing Ad2 (A) and also Ad2 particles within small vesicles (D), and the large arrowhead depicts Ad2 within a medium-sized vesicle (D), while ∗ indicates a large vesicle (D). Note the enrichment of Ad2 particles in plasma membrane invaginations of cells treated with the actin stabilizer Jas (C) and the localization of Ad2 within small vesicles of PKC-myr-treated cells (D). Bar = 500 nm.

FIG. 6

Quantification of Ad2 particles at the plasma membrane, in intracellular vesicles, and in the cytoplasm of HeLa cells treated with actin- and PKC-directed drugs or not treated. HeLa cells were infected and analyzed by thin-section EM as in Fig. 5. (A) Control cells. (B) CD-treated cells. (C) Jas-treated cells. (D) PKC-myr-treated cells. Viruses were counted in smooth (sh), invaginated (iv), and coated-pit (cp) regions of the plasma membrane, within small (s), medium (m), or large (l) vesicles, and also in the cytosol (solid bars). The number of Ad particles on the entire plasma membrane (open bars) and within the entire vesicle population (striped bars) is also indicated (tot). Mean values are expressed as the percentage of total virus particles, and the corresponding SEM values are indicated based on the analyzed number of cells (ranging from 11 to 14) and virus particles (ranging from 339 to 607). The corresponding number of electron micrographs (ranging from 90 to 179) is also indicated for each of the conditions.

FIG. 7

Dissociation of Ad2 from target cells requires functional dynamin. wt or _ts_1 Ad2-TR was bound in the cold to HeLa cells expressing either wt dynamin I (wt) or the GTP hydrolysis-defective K44A dynamin mutant. Following warming for 5 min (open bars) or 60 min (solid bars), cells were fixed and the cell-associated TR fluorescence was determined by FFT as described earlier (37). Results are expressed as mean values normalized to the corresponding cell areas including the SEM and the number of cells analyzed (n).

References

1. Bai M, Harfe B, Freimuth P. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J Virol. 1993;67:5198–5205. - PMC - PubMed
1. Baum S G, Horwitz M S, Maizel J V. Studies of the mechanism of enhancement of human adenovirus infection in monkey cells by simian virus 40. J Virol. 1972;10:211–219. - PMC - PubMed
1. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999;10:440–447. - PubMed
1. Bergelson J M, Cunningham J A, Droguett G, Kurt-Jones E A, Krithivas A, Hong J S, Horwitz M S, Crowell R L, Finberg R W. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science. 1997;275:1320–1323. - PubMed
1. Blumenthal R, Seth P, Willingham M C, Pastan I. pH-dependent lysis of liposomes by adenovirus. Biochemistry. 1986;25:2231–2237. - PubMed

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol - PubMed (original) (raw)