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. 1998 Feb;72(2):1577-85.
doi: 10.1128/JVI.72.2.1577-1585.1998.

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

C S Chung  1 , J C Hsiao, Y S Chang, W Chang
Affiliations

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

C S Chung et al. J Virol. 1998 Feb.

Abstract

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

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Figures

FIG. 1
FIG. 1
(A) Soluble heparin blocks vaccinia virus infection of cells. BSC40 cells were infected with vaccinia virus in the presence of different GAGs (heparin [HP], chondroitin sulfate [CS], and dermatan sulfate [DS]) at various concentrations (0, 1, 2, 5, and 10 μg/ml) for 60 min at 37°C. The cells were then washed with PBS and overlaid with agar for plaque assays. The numbers of plaques obtained from infection without GAGs, within the range of 100 to 150 plaques, were used as 100%. The data were obtained from an average of four plates. (B). Heparin blocks vaccinia virus binding to cells. BSC40 cells were infected with vaccinia virus at a MOI of 10 PFU per cell in the presence of different GAGs (50 μg/ml), as shown above the lanes, at 4°C for 30 min, washed with PBS, and lysed in gel sample buffer containing 5% 2-mercaptoethanol. The samples were separated by SDS-PAGE (10% polyacrylamide) and transferred for Western blot analysis with an antiserum (1:100) previously raised against purified virions. The arrow indicates the position of a viral 35-kDa protein encoded by the D8L gene. (C). Heparin analogs fail to block vaccinia virus infection. BSC40 cells were infected with vaccinia virus in the presence of heparin or its analogs CDSNAc-heparin, CDSNS-heparin, or NDSNAc-heparin as described in Materials and Methods. The cells were washed with PBS and overlaid with agar, and plaque numbers were determined after 3 days. The number of plaques obtained from infection without GAGs was used as 100%.
FIG. 2
FIG. 2
Undersulfation of GAGs by chlorate treatment blocks vaccinia virus binding. BSC40 cells were seeded in sulfate-free medium (A to C) or sulfate-reconstituted medium (D to F). In addition, 10 mM sodium chlorate was added to BSC cells in panels C and F to block sulfation in vivo. After 2 days, BSC cells were infected with vMJ360 at a MOI of 5 PFU per cell and fixed at 3 h p.i. for β-gal activity with X-Gal. (G) BSC40 cells were prepared as described above, infected with vaccinia virus at 4°C for 30 min, washed, and lysed immediately for Western blot analysis with an antiserum against a viral D8L protein, as marked by an arrow.
FIG. 2
FIG. 2
Undersulfation of GAGs by chlorate treatment blocks vaccinia virus binding. BSC40 cells were seeded in sulfate-free medium (A to C) or sulfate-reconstituted medium (D to F). In addition, 10 mM sodium chlorate was added to BSC cells in panels C and F to block sulfation in vivo. After 2 days, BSC cells were infected with vMJ360 at a MOI of 5 PFU per cell and fixed at 3 h p.i. for β-gal activity with X-Gal. (G) BSC40 cells were prepared as described above, infected with vaccinia virus at 4°C for 30 min, washed, and lysed immediately for Western blot analysis with an antiserum against a viral D8L protein, as marked by an arrow.
FIG. 3
FIG. 3
(A) Heparin blocks infections by other poxviruses. BSC40 cells were infected with Shope fibroma virus (SFV), myxoma virus (MXV), rabbitpox virus (RPV), cowpox virus (CPX), and vaccinia virus (VV) in the presence of heparin at various concentrations as shown at the bottom of the figure, and plaque numbers were determined as described in the legend to Fig. 1A. (B) MAb B2 blocked various poxvirus infections differently from the blocking effect of heparin. BSC40 cells were infected with different poxviruses in the presence of heparin (10 μg/ml) or hybridoma culture supernatant containing B2, and the plaque numbers were determined as described in the legend to Fig. 1A.
FIG. 4
FIG. 4
Vaccinia virus virions bind to the heparin-Sepharose beads in vitro. Different amounts of purified vaccinia virus virions, as indicated at the bottom of the figure, were incubated with heparin-Sepharose beads at 4°C for 60 min as described in Materials and Methods. The unbound virions were collected in the supernatant, and the titers were determined. The data shown on the y axis were obtained by the following formula % Bound = [1-(PFUsupernatant/PFUinput)] × 100.
FIG. 5
FIG. 5
(A) Purification of the recombinant A27L and A4L proteins. Both A27L (lanes 1 and 3) and A4L (lanes 2 and 4) recombinant proteins were expressed in BL21(DE3) as described in Materials and Methods and purified through a nickel column. The left panel is an SDS-PAGE gel stained with Coomassie brilliant blue, and the right panel is a Western blot probed with a MAb against a T7 tag (1:5,000) at the N terminus of these recombinant proteins. (B) Recombinant A27L protein binds to a heparin beads in vitro. Purified A27L (lanes 1 to 5) or A4L (lanes 6 to 10) protein was incubated with heparin-Sepharose beads (lanes 2, 3, 7, and 8) or control Sepharose beads (lanes 4, 5, 9, and 10) at 4°C for 60 min, and the supernatant was collected. The beads were washed three times, and the volumes of the bound (lanes 2, 4, 7, and 9) or supernatant (lanes 3, 5, 8, and 10) samples were adjusted so that equal proportions of each sample were loaded on a 15% polyacrylamide gel for SDS-PAGE. Purified A27L and A4L proteins are shown as controls in lane 1 and 6, respectively. The gel was transferred for Western blot analysis with a MAb against a T7 tag (1:5,000) at the N terminus of these recombinant proteins.
FIG. 6
FIG. 6
Recombinant A27L protein binds to the cell surface. Live BSC40 cells were incubated at 4°C for 30 min with PBS (A and F) or biotinylated A27L protein alone (B and G) or A27L protein previously mixed with heparin (C and H), condrointin sulfate (D and I), or dermatan sulfate (E and J) as described in Materials and Methods. The cells were washed three times and phycoerythrin-conjugated streptavidin was added for another 30-min incubation. After being washed, the cells were observed with a fluorescence microscope (A to E) or reverse-phase light microscope (F to J). All the images were analyzed as described in Materials and Methods.
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