Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

Abstract

The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.

Keywords: complement; decay accelerating factor (CD55); membrane cofactor protein (CD46); vesicular stomatitis virus; viral resistance; virus neutralization.

Figure 1

Levels of complement-regulatory protein CD55 remain unaltered, but CD46 levels decline in HeLa cells infected with vesicular stomatitis virus. (A–G) Whole cell lysates collected from mock- and VSV-infected HeLa cells at the indicated time points were subjected to immunoblotting to determine the expression of CD46 and CD55. Samples at the time point 0–3 h (A), 4–7 h (B), 8–11 h (C), 12–15 h (D), 16–18 h (E), 19–21 h (F) and 22–24 h (G), respectively. Anti-CD55 and CD46 antibodies were used to detect the levels of the corresponding proteins in the lysate at different time points. Virus infectivity was detected using a VSV anti-M antibody while actin served as the loading control. The levels of CD55 were maintained at all of the time points tested compared to the mock; however, the levels of CD46 declined significantly, starting from 15 h onwards (note the decreasing levels of CD46 in D,E, and the complete absence in F,G). The entire panel of blots is representative of three independent experiments. (H) The densitometry analysis of the CD55 and CD46 protein expression, normalized against the loading control. The data represents the mean + SEM of the three independent experiments.

Figure 2

VSV infection causes a decline in the surface expression of CD46 and not CD55. HeLa cells were infected with VSV (10 MOI) for the specified time points. The surface distribution of CD55 and CD46 was determined by staining the mock- and VSV-infected cells with anti-CD55 and CD46 primary antibodies, and by counter staining with AF488-labelled secondary antibody. The two panels, A and B, denote the histogram representing the fluorescence intensity on the x-axis and the cell count on the y-axis. The infection of the HeLa cells with VSV did not alter the surface level expression of CD55 (A) even until 24 h; however, a drastic reduction in the surface expression of CD46 (B) could be evidenced 6 h post infection.

Figure 3

Vesicular stomatitis virus infection leads to down-regulation of CD55 and CD46 transcripts. A comparative analysis of the relative levels of CD55 (A) and CD46 (B) mRNA in VSV and mock-infected HeLa cells at 6, 12, 18 and 24 h was carried out by RT-qPCR. The total RNA isolated from the mock- and VSV-infected cells was converted to cDNA. Equal concentrations of cDNA from all of the samples were used to analyze the gene expression at various time points post VSV-infection using Taqman gene expression assays. The fold change was calculated by the comparative Ct method (2^- ddCt). The statistical significance was calculated using Students t-test, with * p ≤ 0.01; ** p ≤ 0.001; **** p ≤ 0.0001, and ns = non-significance.

Figure 4

Cycloheximide chase assay to measure protein stability. (A) The HeLa cells were treated with Cycloheximide for the indicated times, and the whole cell lysate was subjected to immunoblotting in order to determine the stability of CD55 and CD46. β-actin was used as the loading control, and P53 served as the positive control. (B) Quantification of the immunoblot results by Image J software. The result represented is the average + SEM of four independent experiments obtained by normalizing the band intensity of the RCA against b-actin. The statistical significance was calculated using Student’s t-test.

Figure 5

Complement regulator CD55 is found in greater abundance than CD46 on VSV from HeLa cells. (A) Equal concentrations of protein in the purified virus (5 μg) were separated by SDS-PAGE and subjected to Western blotting. The proteins that have been probed are indicated in the right, and their corresponding molecular weights are in parentheses. (B) An ELISA specific to VSV was performed by coating wells with serially-diluted gradient-purified VSV. The adsorbed virus particles were detected using an anti-VSV-G antibody. Variability in the absorbance was observed even at similar concentration of viruses purified at varying time intervals. Across the samples, an absorbance of ~1.3 was found to be common; this is indicated by the lines drawn against the optical density. (y-axis) to the corresponding concentrations (x-axis).

Figure 6

CD55 confers greater resistance to VSV against complement compared to CD46. (A) Western blot depicting the level of expression of CD55 and CD46 in HeLa and A549 cells; β-actin served as the equal loading control. (B) The effect of NHS in neutralizing VSV grown in HeLa and A549 cells was assessed by a plaque reduction assay. The virus harvested at the indicated time intervals was incubated either with NHS or PBS (black bars). At all of the time points tested, the HeLa-grown viruses showed marked resistance to complement-mediated neutralization. The degree of neutralization of A549-grown VSV known to harbor less CD55 was significantly higher than that of HeLa-grown VSV at 12–18 h (p < 0.0001). The symbols in the graph represent * p < 0.05; ** p < 0.005; *** p < 0.0005). The additional symbols represent the comparison of significance between A549- and HeLa-grown VSV treated with NHS at the respective time ranges, where # p < 0.0005; ^$ p < 0.0005; ^ p < 0.0001; ^@ p < 0.005.