Epstein-Barr virus-encoded protein kinase (BGLF4) is involved in production of infectious virus

Abstract

The Epstein-Barr virus (EBV) BGLF4 gene product is a protein kinase (PK). Although this kinase has been characterized and several of its targets have been identified, its biological role remains enigmatic. We have generated and assessed a BGLF4 knockdown phenotype by means of RNA interference and report the following: (i) BGLF4-targeting small interfering RNA effectively inhibited the expression of its product, the viral PK, during lytic reactivation, (ii) BGLF4 knockdown partially inhibited viral DNA replication and expression of selected late viral genes, (iii) the absence of EBV PK resulted in retention of the viral nucleocapsids in the nuclei, and (iv) as a result of the nuclear retention, release of infectious virions is significantly retarded. Our results provide evidence that EBV PK plays an important role in nuclear egress of the virus and ultimately is crucial for lytic virus replication.

FIG. 1.

EBV BGLF4-targeting siRNA (si-PK) abolishes expression of EBV PK and hyperphosphorylation of EA-D during viral lytic reactivation and reduces EBV infectivity. (A) The viral lytic program was induced in 293/EBV+ cells by the expression of EBV BZLF1 alone or in combination with si-NC (negative control) or si-PK. Whole-cell lysates were prepared at 48 h postinduction, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by immunoblotting. Tubulin served as a loading control. (B) Raji cells were coincubated with the supernatants collected at 48 h posttransfection, and the number of infected (GFP-expressing) cells was determined by fluorescence-activated cell sorting analysis. The graph represents results from three independent experiments and shows percentages of infected cells, normalized to negative (0%) and positive (100%) controls, ± standard deviations.

FIG. 2.

Effects of EBV PK knockdown on the course of the viral lytic program. 293/EBV+ cells were treated as described for Fig. 1. (A) Expression of early (EA-D) and late (gp350 and VCA) viral proteins was visualized by indirect IF microscopy with the respective antibodies. GFP expression indicates the total number of EBV+ cells. (B) Whole-cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the expression of the EBV BFRF1 and BFLF2 proteins was determined by immunoblotting. PC, positive control.

FIG. 3.

EBV PK knockdown causes nuclear retention of viral capsids. The lytic program was reactivated in 293/EBV+ cells as described for Fig. 1, and the cells were fixed in 2% glutaraldehyde at 48 h postreactivation. Ultrathin sections were analyzed by electron microscopy. The cells were transfected with BZLF1 only (A) to reactivate the viral lytic program or cotransfected with si-NC (B) or si-PK (C and D). Arrows point to capsids in the cytoplasm (B). Nu, nucleus; NM, nuclear membrane; PM, plasma membrane.