The vaccinia virus fusion inhibitor proteins SPI-3 (K2) and HA (A56) expressed by infected cells reduce the entry of superinfecting virus

Abstract

The orthopoxvirus SPI-3 (K2) and A56 (hemagglutinin, HA) proteins interact and together prevent cell-cell fusion. SPI-3/A56 has been proposed to prevent the superinfection of previously infected cells by reducing virus-cell fusion. Binding of mature virions of vaccinia virus (VV) to VV-infected cells was unaffected by SPI-3 or A56 on the surface of infected cells. Entry of VV into infected cells was assessed using VV-P(T7)-luc carrying the luciferase reporter under T7 control. Cells infected with VV or cowpox virus (CPV) expressing T7 RNA polymerase and lacking SPI-3 and/or A56 were superinfected with VV-P(T7)-luc, and luciferase activity was measured. Inactivation of SPI-3 or A56 from the pre-infecting virus resulted in greater luciferase expression from the superinfecting VV-P(T7)-luc. Antibody against SPI-3 present during infection with wild-type CPV-T7 increased luciferase expression from superinfecting VV-P(T7)-luc. The SPI-3/A56 complex on the infected cell surface therefore appears to reduce the entry of virions into infected cells.

Figure 1. Binding of VV A5-YFP MV particles to uninfected and VV-infected CV-1 cells

(A – C) CV-1 cells were either uninfected (A) or infected with VV-T7 (B) or VV-T7 ΔA56 (C). At 16h p.i., VV A5-YFP was adsorbed for 1 h at room temperature. Unadsorbed virus was removed by washing, and the cells stained with DAPI. The images shown are merges of the DAPI blue channel (nuclei) and the FITC green channel (bound virions). (D) CV-1 cells in 12-well plates were mock-infected or infected with VV-T7 or with mutant derivatives lacking either SPI-3, A56, or both. At 16 h p.i., purified MV of VV A5-YFP was adsorbed to the cells at room temperature for 1 h. After washing to remove unadsorbed virions, cells were DAPI-stained, harvested, and transferred to a black 96-well plate to measure by fluorescence the amount of YFP (representing bound virus) and DAPI (number of cell nuclei). The results are expressed as the ratio of YFP/DAPI fluorescence. Error bars represent the standard deviation.

Figure 1. Binding of VV A5-YFP MV particles to uninfected and VV-infected CV-1 cells

(A – C) CV-1 cells were either uninfected (A) or infected with VV-T7 (B) or VV-T7 ΔA56 (C). At 16h p.i., VV A5-YFP was adsorbed for 1 h at room temperature. Unadsorbed virus was removed by washing, and the cells stained with DAPI. The images shown are merges of the DAPI blue channel (nuclei) and the FITC green channel (bound virions). (D) CV-1 cells in 12-well plates were mock-infected or infected with VV-T7 or with mutant derivatives lacking either SPI-3, A56, or both. At 16 h p.i., purified MV of VV A5-YFP was adsorbed to the cells at room temperature for 1 h. After washing to remove unadsorbed virions, cells were DAPI-stained, harvested, and transferred to a black 96-well plate to measure by fluorescence the amount of YFP (representing bound virus) and DAPI (number of cell nuclei). The results are expressed as the ratio of YFP/DAPI fluorescence. Error bars represent the standard deviation.

Figure 2. Expression of luciferase in CV-1 and in BSR-T7 cells following infection with VV-P_T7-luc, and the effect of AraC on luciferase expression by infected BSR-T7 cells

(A) CV-1 and BSR-T7 cells were infected with VV-PT7-luc at moi = 10, and luciferase activity determined at intervals. (B, C). Expression of luciferase by VV-P_T7-luc in BSR-T7 cells in the presence and absence of 40 µg/ml AraC. The time course from 0 to 6 h p.i. is shown (B), with an enlargement of the time course from 0 to 3h (C).

Figure 2. Expression of luciferase in CV-1 and in BSR-T7 cells following infection with VV-P_T7-luc, and the effect of AraC on luciferase expression by infected BSR-T7 cells

(A) CV-1 and BSR-T7 cells were infected with VV-PT7-luc at moi = 10, and luciferase activity determined at intervals. (B, C). Expression of luciferase by VV-P_T7-luc in BSR-T7 cells in the presence and absence of 40 µg/ml AraC. The time course from 0 to 6 h p.i. is shown (B), with an enlargement of the time course from 0 to 3h (C).

Figure 3. Expression of luciferase from VV-P_T7-luc depends on the multiplicity of superinfection

CV-1 cells infected with VV-T7 at moi = 5 were superinfected at 16 h p.i. with VV-P_T7-luc at multiplicities of 25, 50, 100, and 300 in the presence of AraC, and luciferase expression followed at intervals. The data for luciferase expression at 3h and at 4h post adsorption of VV-P_T7-luc were plotted as a function of multiplicity, and linear trendlines fitted (dotted lines). The R² values indicate how closely the values fit a linear trendline.

Figure 4. Comparison of luciferase expression after infection with VV-T7 or with derivatives lacking SPI-3 or A56, and superinfection with VV-P_T7-luc

CV-1 cells in 96-well plates were infected with VV-T7, VV-T7ΔSPI-3, VV-T7ΔA56, or VV-T7ΔSPI-3 ΔA56 at moi =5, and at 16 h p.i. superinfected with VV-P_T7-luc at moi = 100. Luciferase activity was determined at the indicated time points after adsorption of the superinfecting virus. Each time point shows the mean of 4 wells, plus and minus the standard deviation.

Figure 5. Luciferase expression after infection with VV-T7ΔSPI-3, transfection with plasmid DNAs carrying SPI-3 mutants, and superinfection with VV-P_T7-luc

96-well plates of CV-1 cells were infected with VV-T7ΔSPI-3 at moi = 5, transfected with the indicated plasmid DNAs, and at 16h p.i. superinfected with VV-P_T7-luc at moi = 100. Luciferase expression was measured at 2 h after adsorption of the superinfecting virus. Each bar shows the mean of 3 wells, plus and minus the standard deviation.

Figure 6. Luciferase expression following superinfection of cells infected with CPV-T7 derivatives

(A) CV-1 cells were infected with either CPV-T7 expressing the T7 RNA polymerase or with derivatives lacking SPI-3, A56, or both. At 16 h p.i. the cells were superinfected with VV-P_T7-luc, and luciferase activity determined at the indicated time points, as for the VV-T7 series in Figure 4. (B) Cells were infected with CPV-T7 or CPV-T7ΔSPI-3 in the absence or presence of 10 µg/ml purified anti-SPI-3 mAb. Superinfecting VV-P_T7-luc was added at 16h p.i. and luciferase activity determined at intervals after the end of the adsorption period. SPI-3 mAb was present during adsorption of VV-P_T7-luc and during subsequent incubation at 37°C in the treated wells. Error bars indicate the standard deviation.

Figure 6. Luciferase expression following superinfection of cells infected with CPV-T7 derivatives

(A) CV-1 cells were infected with either CPV-T7 expressing the T7 RNA polymerase or with derivatives lacking SPI-3, A56, or both. At 16 h p.i. the cells were superinfected with VV-P_T7-luc, and luciferase activity determined at the indicated time points, as for the VV-T7 series in Figure 4. (B) Cells were infected with CPV-T7 or CPV-T7ΔSPI-3 in the absence or presence of 10 µg/ml purified anti-SPI-3 mAb. Superinfecting VV-P_T7-luc was added at 16h p.i. and luciferase activity determined at intervals after the end of the adsorption period. SPI-3 mAb was present during adsorption of VV-P_T7-luc and during subsequent incubation at 37°C in the treated wells. Error bars indicate the standard deviation.

Figure 7. The effect of low pH treatment on cells infected with VV-T7 or VV-T7ΔSPI-3 ΔA56 and superinfected with VV-P_T7-luc

CV-1 cells were infected with VV-T7 or VV-T7ΔSPI-3 ΔA56 and at 16 h p.i. superinfected with VV-P_T7-luc. Cells were treated with pH 7 or pH 5 buffer for 3 min at room temperature either immediately before or after adsorption of VV-P_T7-luc, as indicated below the axis. Luciferase activity was measured at 2.5 h after the end of the adsorption period.