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. 2012 Sep;93(Pt 9):1876-1886.
doi: 10.1099/vir.0.043943-0. Epub 2012 May 23.

Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

Virginie Doceul  1 , Michael Hollinshead  1 , Adrien Breiman  1 , Kathlyn Laval  1 , Geoffrey L Smith  1
Affiliations

Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

Virginie Doceul et al. J Gen Virol. 2012 Sep.

Abstract

Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

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Figures

Fig. 1.
Fig. 1.
Ectopic expression of A33 and/or A36 does not affect virus spread. (a) Expression of A33 and/or A36 in the RK13 and CV-1 cell lines. Cell extracts were analysed by immunoblotting with antibodies against A33, A36 and tubulin. (b) Expression of A33 and A36 does not affect VACV plaque size in RK13 and CV-1. Cell monolayers were infected with VACV WR for 4 days and the diameter of 12 plaques was measured (see Methods). The relative plaque size is expressed as a percentage of the size obtained for the parental cell line. Data shown are the mean±sd, n = 3.
Fig. 2.
Fig. 2.
Expression of A33 and A36 does not prevent VACV entry. (a) HEK293 cells and EACK cells (HEK293 cells expressing A56 and K2) were infected with vLuc-WR EEV or IMV. Cells were lysed 1 h after infection and the luciferase activity was determined. (b) The same experiment was performed with the CV-1 v5, A33, A33–A36-7 and A33–A36-8 cell lines. Data are expressed in relative luciferase units (RLU) per 103 cells. The results shown are the mean±sd, n = 4, and are representative of three experiments.
Fig. 3.
Fig. 3.
A34 and/or B5 are critical on EEV particles for actin-tail formation. EEV preparations from RK13 cells infected with VACV strain WR, vΔA33, vΔA34, vΔA56, vΔB5, or VACV strain IHD-J were added to the surface of cells at 37 °C for 30 min. Cells were then fixed and stained with phalloidin and anti-F13 mAb to visualize actin tails and EEV particles. Data are expressed as actin tails detected per 106 virus particles and represent the mean±sd, n = 3.
Fig. 4.
Fig. 4.
B5 SCR4 is critical for actin-tail formation by superinfecting EEV. EEV preparations from VACV mutants lacking B5 SCR1–4 (vSCR0), 2–4 (vSCR1) or 4 (vSCR1–3) or with a P189S mutation in B5 SCR4 (vB5P189S) were added to RK13 A33–A36-4 cells for 30 min. Cells were then fixed and stained with phalloidin and an anti-F13 mAb to visualize actin tails and EEV particles. The results are expressed in number of actin tails detected per 106 virus particles and represent the mean±sd, n = 3.
Fig. 5.
Fig. 5.
Characterization of rVACV vB5P189S. (a) Immunoblot. Lysates from BSC-1 cells infected with WR, vΔB5 or vB5P189S at 2 p.f.u. per cell were immunoblotted with anti-B5 mAb. (b) vB5P189S forms small plaques. BSC-1 cells were infected with WR, vΔB5 or vB5P189S and plaque size was measured after 3 days. Data are expressed relative to VACV WR and are the mean±sd, n = 3. Bars, 2.5 mm. (c) Electron microscopy of HeLa cells infected with WR, vΔB5 and vB5P189S at 2 p.f.u. per cell for 8 h. Black arrows indicate IMV association with wrapping membranes and complete IEV. Arrowhead indicates a virus-tipped actin tail at the surface of cells infected with vB5P189S. Bars, 500 nm (all top row and bottom row left), 2 µm (bottom row middle and right). (d) CEV formation. BSC-1 cells were infected as in (a) and CEV were quantified on cells in 9–10 different fields for each virus. Data shown are the mean±sd of three experiments. (e) Actin-tail production. RK13 cells were infected as in (a) and 16 h p.i. cells were fixed, permeabilized and stained with phalloidin and an anti-D8 mAb. The number of actin tails present at the surface of 50 cells was determined for each virus and classified into five categories: 0, 1–5, 6–20, 21–40 or >40 actin tails. Data shown are the mean±sd, n = 2. (f) Extracellular virus formation. RK13 were infected at 5 p.f.u. per cell for 24 h and the number of infectious virions present intracellularly and extracellularly was determined by plaque assay. Data are shown as the percentage of total infectivity that was present in the medium and are the mean±sd, n = 3.
Fig. 6.
Fig. 6.
Incorporation of A34 into EEV. (a) Expression of IEV/EEV proteins in cell lysates. HeLa cells were infected at 2 p.f.u. per cell with the indicated viruses for 24 h. Protein lysates were prepared and analysed by immunoblotting using antibodies raised against F13, A36, B5 and A34. An anti-tubulin mAb was included as loading control. Note that B5 was not detected in cells infected with vSCR0 and vSCR1 because the rat mAB 19C2 recognizes B5 SCR2 (Law & Smith, 2001). (b) Incorporation of A34 into EEV. RK13 cells were infected at 3 p.f.u. per cell with the indicated viruses for 16 h. EEV were collected, lysed and analysed by immunoblotting.
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