Epstein-Barr virus BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes

Abstract

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.

FIG. 1.

BZLF1 and EBNA2 expression after EBV infection of BL-derived, EBV-negative Akata and Daudi cells and peripheral B lymphocytes. (A) The cells were infected with EBV carrying the EGFP gene. At the designated time of incubation, expression of BZLF1 and EBNA2 mRNAs was examined by RT-PCR. To secure the quality of the RNA sample, GAPDH mRNA was also amplified by RT-PCR. (B) The cells were infected with EBV carrying the EGFP gene. At the designated time of incubation, expression of the BZLF1 protein was examined by immunoblot analysis using a mouse monoclonal antibody (Dako Denmark A/S). EBV-positive Akata cells treated with anti-Ig antibody were used as a positive control (pc). The β-actin protein was detected to show the quantity of each protein preparation. (C) Detection of the BZLF1 protein by immunofluorescence assay. The cells were infected with EBV carrying the EGFP gene. At 12 h postinfection, the cells were harvested and subjected to the immunofluorescence assay. (D) Analysis of early and late gene expression in primary B lymphocytes. At the designated time of incubation, expression of BMRF1 and gp350/220 mRNAs was examined by RT-PCR. (E) EBV-negative Akata and Daudi cells were infected with wild type EBV. At the designated time of incubation, expression of BZLF1 and GAPDH mRNA was examined by RT-PCR. (F) Detection of defective heterogenous EBV DNA. DNA extracted from het EBV-positive P3HR1 cells or recombinant Akata EBV was subjected to PCR. The molecular size marker (HaeIII digestion of ΦX174 DNA) is indicated in base pairs.

FIG. 2.

(A) Effect of UV inactivation of EBV preparation on expression of BZLF1 and EBNA2 mRNAs after EBV infection. EBV-negative Daudi and Akata cells and primary B lymphocytes were infected with UV-treated EBV. At the designated time of incubation, expression of BZLF1 and EBNA2 mRNAs was examined by RT-PCR. (B) Effect of anisomycin, an inhibitor of protein synthesis, on expression of BZLF1 and EBNA2 mRNAs after EBV infection. EBV-negative Daudi cells were preincubated for 3 h in a medium containing 25 μg/ml anisomycin. The culture was then centrifuged, and the cell pellet was resuspended in virus solution containing anisomycin and incubated for 90 min. Thereafter, the cells were resuspended in fresh medium containing anisomycin, incubated for the designated time, and examined for expression of BZLF1 and EBNA2 mRNAs by RT-PCR. The effect of anisomycin on expression of the β-actin protein was examined by immunoblot analysis. (C) Effect of actinomycin D, an inhibitor of RNA synthesis, on expression of BZLF1 mRNA after EBV infection. EBV-negative Daudi cells were infected with EBV in the presence of 5 μg/ml actinomycin D, incubated for the designated time, and examined for expression of BZLF1 mRNA by RT-PCR.

FIG. 3.

Involvement of c-fos in expression of BZLF1 mRNA after EBV infection. (A) Effect of c-fos on gene expression from BZLF1 promoter. The BZLF1-luciferase reporter plasmid (pGVB2/BZLF1) and a c-fos-expressing plasmid or control plasmid were cotransfected into EBV-negative Daudi cells by lipofection. After 48 h of incubation, cells were harvested and luciferase (luc) activity was determined. The luciferase activities obtained from c-fos-transfected cells were divided by those from control plasmid-transfected cells, and the results were expressed as the mean relative activity (fold induction) ± standard error. (B) Expression of c-fos in c-fos-silenced EBV-negative Daudi cell clones. EBV-negative Daudi cells were transfected with the TransSilent Fos shRNA vector (Panomics), and c-fos-silenced cell clones were isolated by cultivation in a selective medium. Expression of the c-Fos protein in these cell clones was examined by immunoblot analysis using a rabbit polyclonal antibody (Calbiochem). The β-actin protein was detected to show the quantity of each protein preparation. (C) Effect of c-fos silencing on expression of BZLF1 mRNA and protein after EBV infection. c-fos-silenced, EBV-negative Daudi cells were infected with EBV. At the designated time of incubation, expression of BZLF1 mRNA and protein was examined by RT-PCR and immunoblot analysis. EBV-negative Daudi cell clones stably transfected with the TransSilent control vector (Panomics) were also infected with EBV and examined for BZLF1 mRNA and protein expression as a positive control. Effect of c-fos silencing on EBNA2 mRNA expression was examined by RT-PCR.