Human herpesvirus 6A (HHV-6A) and HHV-6B alter E2F1/Rb pathways and E2F1 localization and cause cell cycle arrest in infected T cells

Abstract

E2F transcription factors play pivotal roles in controlling the expression of genes involved in cell viability as well as genes involved in cell death. E2F1 is an important constituent of this protein family, which thus far contains eight members. The interaction of E2F1 with its major regulator, retinoblastoma protein (Rb), has been studied extensively in the past two decades, concentrating on the role of E2F1 in transcriptional regulation and the role of Rb in cell replication and cancer formation. Additionally, the effect of viral infections on E2F1/Rb interactions has been analyzed for different viruses, concentrating on cell division, which is essential for viral replication. In the present study, we monitored E2F1-Rb interactions during human herpesvirus 6A (HHV-6A) and HHV-6B infections of SupT1 T cells. The results have shown the following dramatic alterations in E2F1-Rb pathways compared to the pathways of parallel mock-infected control cultures. (i) The E2F1 levels were elevated during viral infections. (ii) The cellular localization of E2F1 was dramatically altered, and it was found to accumulate both in the cytoplasmic and nuclear fractions, as opposed to the strict nuclear localization seen in the mock-infected cells. (iii) Although E2F1 expression was elevated, two exemplary target genes, cyclin E and MCM5, were not upregulated. (iv) The Rb protein was dephosphorylated early postinfection, a trait that also occurred with UV-inactivated virus. (v) Infection was associated with significant reduction of E2F1/Rb complexing. (vi) HHV-6 infections were accompanied by cell cycle arrest. The altered E2F1-Rb interactions and functions might contribute to the observed cell cycle arrest.

FIG. 1.

E2F1 expression during HHV-6A (U11O2) and HHV-6B (Z29) infections. (A) Western blot analyses of proteins from mock-infected and virus-infected cells, employing antibodies to E2F1, HHV-6, P41, and γ-tubulin. The antibody used for P41 is the MAb 9A5 of N. Balachandran (8, 13). (B) Quantitative representation of the results. Shown are E2F1 and P41 protein accumulation relative to the protein level at the highest point of accumulation of the HHV-6A samples, at 72 hpi. The P41 and E2F1 levels were corrected for loading variations by the intensity of the γ-tubulin staining.

FIG. 2.

E2F1 is increased in the infected cells. Cultures of 4 × 10⁶ cells were infected with HHV-6B (Z29) and reacted with primary antibodies for E2F1 (rabbit), P41 (mouse), and Cytox orange for nuclear localization. Cye2 (anti-rabbit) and Cye5 (anti-mouse) secondary antibodies were used to mark E2F1 in green and P41 in purple. Each time frame contains a Cye5 field (P41), Cye2 field (E2F1), Cytox orange field, bright field, and a merging of fields 1 to 3.

FIG. 3.

Alterations in E2F1 cytoplasmic (cyto) and nuclear (nuc) localization. (A) Recovery of E2F1 and P41 proteins in the cytoplasmic or nuclear fractions. (B) Quantitative representation of the results shown relative to the highest level of E2F1 expression in the cytoplasmic and nuclear fractions. The E2F1-nuc analyses represent averages from four experiments, and the E2F1 cytoplasmic data represented the averages from two experiments. All differences were statistically significant by t tests.

FIG. 4.

Rb hypophosphorylation in HHV-6A- and HHV-6B-infected SupT1 cells. (A) Western blots of mock-infected and virus-infected nuclear lysates were reacted with antibodies to Rb and pRb proteins. (B and C) Graphic representation of the results relative to the highest level of Rb or pRb protein found in the samples. The intensities of Rb and pRb were corrected for loading variations by the intensity of eIF2α. The measurements for the Rb were based on results from two experiments. The comparison of the pRb in the mock infection versus that of HHV-6B (Z29) infection was based on results from a single experiment. pRb in the cytoplasmic fractions of mock-infected and virus-infected cells was not detected (data not shown). The quantitations of pRb in the mock and HHV-6A (U1102) infections were based on results from three separate experiments. nuc, nuclear.

FIG. 5.

Alterations of pRb and E2F1 in mock-infected and virus-infected cells. Graphic representations of E2F1 and pRb in the nuclei of mock-infected and virus-infected cells are shown. pRb and E2F1 values are shown relative to the highest protein level detected: E2F1 in the 96-hpi sample of HHV-6B infections and pRb at 72 hpi in the mock-infected cells.

FIG. 6.

Dephosphorylation of Rb in cells infected with inactivated virus. (A) The viruses were irradiated by UV or were untreated, and Western blots from mock-infected and virus-infected cell lysates were reacted with antibodies to the pRb and P41 proteins (9A5 MAb). Two exposures of the blots (low-exp and high-exp) are shown for the pRb and the P41 proteins. (B) Graphic representation of the results relative to the highest pRb levels (mock-infected cells). The intensities of pRb were corrected for loading variations by the intensity of eIF2α. nuc, nuclear.

FIG. 7.

Levels of cyclin E and MCM5 proteins in HHV-6A (U11O2)-infected SupT1 cells. (A) Western blots of proteins prepared from the cytoplasmic (cyto) and nuclear (nuc) fractions of mock-infected and HHV-6A-infected cells tested with cyclin E, MCM5, and eIF2α antibodies. (B) Quantitative representation of the results for the nuclear fractions relative to the highest level of cyclin E and MCMS protein expression in the mock-infected cultures. The data are based on results from a single experiment for cyclin E and the averages from two experiments for MCM5.

FIG. 8.

E2F1 can initiate cyclin E expression in SupT1 cells. (A) Western blots tested with E2F1, cyclin E, and eIF2α antibodies after transfection of SupT1 cells with an E2F1-expressing plasmid. (B) Graphic representation of the results relative to the highest level of protein expression in panel A.

FIG. 9.

Reduced Rb and MCM5 complexes with E2F1 in the infected cells. (A) E2F1 was immunoprecipitated with anti-E2F1 antibody, starting with 500 μg lysate. Proteins, which were coprecipitated, were tested using antibodies to Rb, MCM5, and γ-tubulin. (B) Graphic representation of the results relative to the highest level of protein expression in the mock-infected cells. The quantitative analyses were based on results from two independent experiments. The differences of Rb and MCM5 between mock-infected and virus-infected cells were statistically significant, as determined by t tests.

FIG. 10.

Effect of the infection on the cell cycle. (A) FACS analyses of mock-infected and virus-infected cells prepared at 24 and 48 hpi and labeled with the P41 9A5 MAb (gray line). (B) Cell cycle analyses at 48 hpi. (C) Graphic representations of the FACS results based on results from three separate experiments. The differences in the ratios of G₂/M phases between mock-infected and virus-infected cells at 24 and 48 h were statistically significant, as determined by t tests. Ap, apoptotic cells.