Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Abstract

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Figure 1

IMV and EEV sensitivity to neutralization by complement. Purified IMV and fresh EEV (final dilution of 10⁴) were derived from RK₁₃ cells infected with VV strain IHD-J and were assayed for their sensitivity to neutralization by rabbit complement (final serum concentrations of 1/10, 1/20, and 1/30) as described in Materials and Methods. For IMV, 1/30* represents purified IMV assayed in the presence of inactivated supernatant from VV-infected cells (final dilution of 10³). The number of plaques obtained with active complement (hatched bars) are expressed as the percentage of the number of plaques obtained with heat-inactivated complement (control, solid bars). Data represent the average ± SD for triplicate measures. The average number of plaques obtained for each of the controls was about 200.

Figure 2

Effect of host cell species on the resistance of EEV to complement toxicity. Purified IMV (IMV, A and B) and fresh EEV (final dilution of 10⁴ for RK₁₃-derived EEV and 2 × 10³ for HeLa-derived EEV) (C and D) derived from RK₁₃ (A and C) or from HeLa (B and D) cells infected with VV IHD-J strain, were assayed for their resistance to neutralization by human complement (final serum concentrations of 1/10, 1/20, and 1/30) as described in Materials and Methods. The number of plaques obtained with active complement (hatched bars) is expressed as percentage of the number of plaques obtained with heat-inactivated complement (control, solid bars). Data represent the average ± SD for triplicate measures. The average number of plaques for the controls was about 200.

Figure 3

Detection of cellular proteins in VV virions by immunoblotting. Purified IMV (3 μg/lane), purified EEV (3 μg/lane), and mock-infected HeLa cell (HeLa) (1.2 × 10⁴ cells per lane) extracts were immunoblotted as described in Materials and Methods.

Figure 4

Detection of CD46 (A), CD55 (B), and CD59 (C and D) on VV virion surface by immunogold labeling. HeLa cells infected with WR (5 pfu/cell) for 14 h (A–C) or HeLa cells inoculated on ice with purified WR IMV (20 pfu/cell) (D) were fixed and treated as described in Materials and Methods for preembedding immunogold labeling. (Bar = 100 nm.)

Figure 5

(A) Surface expression of CD55 and CD59 by SVAREC. Hygro (negative control), CD55+, CD59+, CD55+/CD59+ SVAREC, and HeLa cells were analyzed for expression of CD55 (α-CD55) or CD59 (α-CD59) by fluorescence-activated cell sorter analysis as described in Materials and Methods. (B) Resistance of EEV derived from SVAREC to human complement. Fresh EEV (final dilution of 5 × 10³) derived from Hygro, CD55+, CD59+, CD55+/CD59+ SVAREC, or HeLa cells infected with IHD-J were assayed for their resistance to human complement (final serum concentration of 1/30) neutralization as described in Materials and Methods. The number of plaques obtained with active complement (hatched bars) is expressed as the percentage of the number of plaques obtained with heat-inactivated complement (control, solid bars). Data represent the average ± SD for triplicate measures. The average number of plaques for the controls was about 200.