The VP7 outer capsid protein of rotavirus induces polyclonal B-cell activation

Abstract

The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection.

FIG. 1.

Both homologous and heterologous rotavirus strains induce PP B-cell activation in mice. CD-1 mice were orally inoculated with uninfected gut homogenate and cell lysate, gut homogenate containing 10⁵ ID₅₀ of EC_wt, 10⁴ ID₅₀ of EDIM, or cell lysate containing 10⁸ PFU of RRV. Single-cell suspensions of the PP from each mouse (n = 5) were collected 2 days after inoculation and analyzed by flow cytometry for activated B lymphocytes (CD19⁺ CD69⁺). (A) Average percentage of activated B cells (CD19⁺ CD69⁺) out of total B cells (CD19⁺) in the PP ± standard deviation for each group. *, P < 0.05 by Student's paired t test compared to control mice. #, P < 0.05 by Student’s paired t test compared to EC_wt-inoculated mice. (B) Amount of antigen present in a fecal sample taken at the time of analysis as determined by ELISA.

FIG. 2.

Rotavirus activates B cells in vitro. Single-cell suspensions (2 × 10⁶), isolated from either the PP or spleens were stimulated in vitro with medium (white bars), rotavirus (black bars), or LPS (gray bars). Activation was quantified by flow cytometric analysis of CD69 expression on the B-cell populations. The data represent the average percentage of CD19⁺ CD69⁺ cells out of total CD19⁺ cells ± standard deviation (n = 3 to 5 mice). All cells were from CD-1 outbred mice unless otherwise noted. (A, B, and C) Representative flow cytometry dot plots of splenic lymphocytes harvested from one mouse and stimulated with either medium alone or 100-ng/ml RRV or LPS, respectively. Numbers in the upper-right-hand quadrant represent the CD19⁺ CD69⁺ population, and those in the bottom-right-hand quadrant represent the CD19⁺ CD69⁻ population. (D) PP and splenic single-cell suspensions were stimulated overnight with medium or 100-ng/ml RRV or LPS. (E) Splenic cells were stimulated with either medium alone (circles) or decreasing amounts (nanograms per milliliter) of RRV (squares). (F) Splenic cells were stimulated in vitro with medium or 100-ng/ml each of the indicated strains of rotavirus. (G) Splenic cells from the indicated inbred and outbred strains of mice were stimulated with medium or 1,000-ng/ml RRV or LPS. (H) Splenic single-cell suspensions depleted of the CD90⁺ T-cell population by using magnetic beads or from TCRKO mice were stimulated with either medium or 100-ng/ml RRV or LPS. (I) Both RRV and LPS (100 ng/ml) were pretreated with 10 μg of polymyxin B (PB) for 1 h at room temperature or boiled for 2 h prior to addition to splenic single-cell suspensions overnight. *, P < 0.05 compared to medium alone by Student's paired t test.

FIG. 3.

VP7 is necessary for the induction of B-cell activation by rotavirus in vitro. Splenic single-cell suspensions (n = 3 to 5) containing 2 × 10⁶ lymphocytes were stimulated overnight at 37°C in vitro with the indicated virus or VLPs. Cells were incubated with antibodies to CD19 and CD69. Double-positive cells were analyzed by flow cytometry. The data show the average percentage of activated B cells out of total B cells ± standard deviation. (A) Cells were treated with 100 ng/ml of RRV, inactivated RRV, or double-layered rotavirus particles. *, P < 0.05 compared to BSA control by paired Student's t test. (B) Cells were stimulated overnight with 100-ng/ml 2/4/6/7-, 2/6/7-, or 2/6-VLPs or Norwalk virus VLPs. *, P < 0.05 compared to both 2/4/6/7- and 2/6/7-VLP-treated cells. (C) Cells were stimulated overnight with 1,000-ng/ml SA11 Cl3 with glycosylated VP7 or an SA11 Cl28 mutant without glycosylated VP7. *, P < 0.05 compared to medium alone by Student's paired t test.