Vaccinia virus gene F3L encodes an intracellular protein that affects the innate immune response

Abstract

The Vaccinia virus BTB/kelch protein F3 has been characterized and its effects on virus replication in vitro and virus virulence in vivo have been determined. The loss of the F3L gene had no effect on virus growth, plaque phenotype or cytopathic effect in cell culture under the conditions tested. However, the virulence of a virus lacking F3L in an intradermal model was reduced compared with controls, and this was demonstrated by a significantly smaller lesion and alterations to the innate immune response to infection. The predicted molecular mass of the F3 protein is 56 kDa; however, immunoblotting of infected cell lysates using an antibody directed against recombinant F3 revealed two proteins of estimated sizes 37 and 25 kDa.

Fig. 1.

Plaque phenotypes of VACVs lacking each BTB/kelch protein. (a) Plaques produced on confluent BS-C-1 cells by vF3, vΔF3, vF3-rev infection (top row) vA55, vΔA55, vA55-rev (middle row) and vC2, vΔC2 and vC2-rev (bottom row). Infected cells were overlaid with DMEM/2.5 % fetal bovine serum/1.5 % carboxymethylcellulose for 2 days at 37 °C before being stained with crystal violet. (b) Higher magnification detail of plaque edges under phase-contrast microscopy.

Fig. 2.

(a) VACV-induced lesions (mm) in C57BL/6 mice infected intradermally with the indicated viruses. The horizontal black line indicates the time points where vΔF3 lesions were significantly smaller than vF3 and vF3-rev. (Student's t-test, P<0.05). (b) Percentage of NK1.1⁺ CD3⁻ cells present in infected ears at 4 days p.i and TCRγδ⁺ cells 6 days p.i. Asterisks indicate significance (Student's t-test, P<0.05), bars represent mean percentage of cells present (n=4)±sem. These data are representative of two separate experiments.

Fig. 3.

Characterization of the F3 protein. Cells were infected at 5 p.f.u. per cell or mock-infected, with or without MG132 (10 μM), and lysates were analysed by immunoblotting with (a, b) anti-F3 IgG (1 : 1000). (b) Intentionally overdeveloped image of (a), to reveal the 25 kDa band more clearly. (c) Immunoblot of lysates from cells infected with vF3, vΔF3 or vF3-rev in the presence of MG132. (d) Anti-F13 mAb p37 (1 : 1000). (e) AraC (40 μg ml⁻¹) was added at time 0 as indicated and the blot was probed with anti-F3 antibody (1 : 1000). (f) Intentionally overexposed image of (e) to reveal the presence of the 25 kDa band.