Human herpesvirus 6B induces cell cycle arrest concomitant with p53 phosphorylation and accumulation in T cells

Abstract

We studied the interactions between human herpesvirus 6B (HHV-6B) and its host cell. Productive infections of T-cell lines led to G1/S- and G2/M-phase arrest in the cell cycle concomitant with an increased level and enhanced DNA-binding activity of p53. More than 70% of HHV-6B-infected cells did not bind annexin V, indicating that the majority of cells were not undergoing apoptosis. HHV-6B infection induced Ser20 and Ser15 phosphorylation on p53, and the latter was inhibited by caffeine, an ataxia telangiectasia mutated kinase inhibitor. Thus, a productive HHV-6B infection suppresses T-cell proliferation concomitant with the phosphorylation and accumulation of p53.

FIG. 1.

HHV-6B induces cell cycle arrest in MOLT 3 cells. (A) Percentages of uninfected MOLT 3 cells and MOLT 3 cells infected with HHV-6B diluted 1:8 (400 TCID₅₀) or 1:40 (80 TCID₅₀), determined at 24, 48, and 72 h postinfection. Cell counts (performed in triplicate) at the indicated time points were normalized to the number of cells at 0 h for each of the three separate samples. (B) Immunofluorescence staining of HHV-6B-infected cells (400 TCID₅₀). Cells were stained for the presence of HHV-6B p41. Left, immunofluorescence microscopy; right, light microscopy (same fields of view). (C) MOLT 3 cells were uninfected or infected with 400 TCID₅₀ of HHV-6B for 0, 1, 2, 3, 4, and 7 days. At each time point, cells were incubated with fluorescein isothiocyanate-conjugated annexin V to monitor the fractions of early apoptotic cells. The percentages of annexin V-binding cells were determined by flow cytometry. (D) Relative proliferation of MOLT 3 cells infected with HHV-6B diluted 1:8 (400 TCID₅₀), 1:40 (80 TCID₅₀), 1:200 (16 TCID₅₀), and 1:1,000 (3 TCID₅₀) and of SupT1 cells infected with HHV-6B diluted 1:8 (400 TCID₅₀) (open circles), as determined by [³H]thymidine incorporation. The top graph shows a kinetic analysis of the inhibition between 8 and 18 h. The data are representative of three independent experiments. (E) Cell cycle analysis of uninfected and HHV-6B-infected T cells. Cells were stained with 7-aminoactinomycin D and analyzed by flow cytometry. (F) [³H]thymidine incorporation of MOLT 3 cells infected with 400 TCID₅₀ of infectious or UV-inactivated HHV-6B. The cells were cultured for 3 days, with [³H]thymidine incorporation during the last 18 h.

FIG. 2.

HHV-6B induces p53 accumulation in HHV-6B-infected MOLT 3 cells. (A) Immunoblotting with anti-p53 and anti-β-tubulin antibodies of whole-cell extracts from uninfected and HHV-6B-infected (400 TCID₅₀) MOLT 3 cells. (B) Same as panel A, but with extracts from SupT1 cells that were uninfected or infected with HHV-6B for 48 h. (C) Immunoblotting with anti-p53 of nuclear and cytoplasmic fractions from uninfected and HHV-6B-infected (400 TCID₅₀) MOLT 3 cells at 48 h postinfection. (D) Determination of DNA-binding activity of p53 in uninfected and HHV-6B-infected (400 TCID₅₀) MOLT 3 cells. The p53 DNA-binding activity of γ-irradiated MOLT 3 cells (3 h) is shown in the inset.

FIG. 3.

Immunoblotting of whole-cell extracts from uninfected and HHV-6B-infected (400 TCID₅₀) MOLT 3 cells that were pretreated for 30 min with or without 5 mM caffeine. Extracts were prepared at 48 h postinfection, and immunoblots were probed with antibodies against p53, Ser15-phosphorylated p53, and Ser20-phosphorylated p53.