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. 2004 Mar;78(5):2357-66.
doi: 10.1128/jvi.78.5.2357-2366.2004.

Vaccinia virus entry into cells is dependent on a virion surface protein encoded by the A28L gene

Tatiana G Senkevich  1 , Brian M Ward, Bernard Moss
Affiliations

Vaccinia virus entry into cells is dependent on a virion surface protein encoded by the A28L gene

Tatiana G Senkevich et al. J Virol. 2004 Mar.

Abstract

The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.

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Figures

FIG. 1.
FIG. 1.
Morphology of purified +A28-HA (+A28) and −A28-HA (−A28) virions. Sucrose gradient-purified IMV were adsorbed to grids, washed with water, and stained with 7% uranyl acetate in 50% ethanol for 30 s. Bar, 200 nm.
FIG. 2.
FIG. 2.
Western blot analysis of proteins from purified +A28-HA (+A28), −A28-HA (−A28), and wild-type (WR) virions. Equal OD260s of sucrose gradient-purified IMV were analyzed by Western blotting with antibodies indicated on the left.
FIG. 3.
FIG. 3.
Transcriptional activity of purified +A28-HA (+A28) and −A28-HA (−A28) virions. Purified virions were incubated with nonionic detergent and ribonucleoside triphosphates, and the incorporation of [α-32P]UMP into RNA was determined. The amount of virion protein was determined from the OD260.
FIG.4.
FIG.4.
Binding and cytopathic effects produced by purified virions. Replicate HeLa cell monolayers on coverslips were infected with 10 PFU per cell of purified +A28-HA (+A28) or the equivalent OD260 of purified −A28-HA (−A28) virions. After 1 h of adsorption at room temperature, the cells were washed three times and incubated for 10 min, 30 min, or 1.5 h at 37°C. Cells were stained with anti-L1 mouse MAb, followed by fluorescein isothiocyanate-conjugated goat anti-mouse antibody (green). DNA was stained with DAPI (blue) and F actin with Alexa Fluor 568 phalloidin (red). For L1 staining, cells were imaged by confocal microscopy as a series of optical sections and are displayed here as a maximum-intensity projection. The left and middle panels of each row depict the same cell. For the low magnifications in the right panels, only Alexa Fluor 568 phalloidin staining is shown.
FIG. 5.
FIG. 5.
Binding and penetration of cells by purified virions. Replicate HeLa cell monolayers on coverslips were infected with 10 PFU per cell of purified +A28-HA (+A28) or the equivalent OD260 of purified −A28-HA (−A28) virions in the presence of 300 μg of cycloheximide per ml. After 1 h of adsorption at 4°C, the cells were washed and either fixed or incubated further at 37°C for 2 h. Cells were double stained with anti-A4 rabbit polyclonal and anti-L1 mouse MAbs, followed by fluorescein isothiocyanate-conjugated goat anti-mouse (green) and Rhodamine Red-X-conjugated goat anti-rabbit (red) antibody, respectively. DNA was stained with DAPI (blue). Cells were imaged by confocal microscopy as a series of optical sections and are displayed here as a maximum-intensity projection.
FIG. 6.
FIG. 6.
Determination of early gene expression. (A) Early RNA. BS-C-1 cell monolayers were treated with AraC (40 μg/ml) for 2 h prior to infection with 5 PFU of purified +A28-HA (+A28) virions per cell, the equivalent OD260 of −A28-HA (−A28) virions, or mock infected (Un). Total RNA was extracted, resolved by agarose gel electrophoresis, transferred to a nylon filter, probed with 32P-labeled plasmid containing the A20R early gene of vaccinia virus, and analyzed by autoradiography. (B) Cells were infected as in panel A, except that AraC was not added, and were metabolically labeled with [35S]methionine from 2 to 5 h. Lysates were incubated with a MAb to the E3 protein and to protein A-beads. After the beads were washed, the bound proteins were analyzed by SDS-PAGE and autoradiography.
FIG. 7.
FIG. 7.
Fusion from within. BS-C-1 cell monolayers were infected with 5 PFU of vA28-HAi per cell in the presence (+) or absence (−) of IPTG for 18 h at 37°C. At that time, the medium was replaced with pH 7.4 or 5.5 buffer for 2 min at 37°C. The incubation was then continued in regular medium for 3 h at 37°C. The cells were fixed, stained with Hoechst reagent, and examined by phase-contrast and fluorescence microscopy.
FIG. 8.
FIG. 8.
Fusion from without. BS-C-1 cell monolayers were incubated with 200 PFU of purified +A28-HA (+A28) virions per cell or the equivalent OD260 of purified −A28-HA- (−A28) virions. After 1 h of adsorption at 4°C, the cells were washed and then treated for 2 min with pH 7.4 or 5.5 buffer and then incubated in regular medium with 300 μg of cycloheximide per ml for 3 h at 37°. Cells were fixed, stained with Hoechst reagent, and examined by phase-contrast and fluorescence microscopy.
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