Simian Virus 40 T/t Antigens and Lamin A/C Small Interfering RNA Rescue the Phenotype of an Epstein-Barr Virus Protein Kinase (BGLF4) Mutant

Abstract

The Epstein-Barr virus (EBV)-encoded viral protein kinase, EBV-PK (the BGLF4 gene product), is required for efficient nuclear viral egress in 293 cells. However, since EBV-PK phosphorylates a number of different viral and cellular proteins (including lamin A/C), the relative importance of each target during lytic viral replication remains unclear. We show here that an EBV PK mutant (PKmut; containing stop codons at residues 1 and 5 in EBV-PK) is highly defective for release of infectious virus from 293 cells but not 293T cells. Furthermore, the phenotype of the PKmut in 293 cells is substantially reversed by expression of the simian virus 40 (SV40) large (T) and small (t) T antigens. Efficient rescue requires the presence of both SV40 T/t proteins. We show that 293T cells have a much higher level of constitutive lamin A/C phosphorylation than do 293 cells over residues (S22 and S392) that promote phosphorylation-dependent nuclear disassembly and that both large T and small t contribute to enhanced lamin A/C phosphorylation. Finally, we demonstrate that knockdown of lamin A/C expression using small interfering RNA also rescues the PKmut phenotype in 293 cells. These results suggest that essential roles of EBV-PK during lytic viral replication include the phosphorylation and dispersion of lamin A/C.The Epstein-Barr virus (EBV) BGLF4 gene product, EBV-PK, is homologous to the cytomegalovirus (CMV) UL97 kinase and belongs to a conserved family of herpesviral protein kinases. EBV-PK is the only protein kinase encoded by the EBV genome (6) and is expressed as an early lytic viral protein during viral reactivation (11). EBV-PK is also packaged into the virion tegument and thus is delivered in an active form to cells immediately after virus penetration (3). In the context of the intact virus, several recent studies indicate that a major and essential role of EBV-PK is to promote viral egress from the nucleus during lytic infection (10,13,41). Although EBV-PK phosphorylates a number of different viral and cellular proteins in vitro and when overexpressed in cells, the relative importance of the potential EBV-PK cellular and viral targets in the context of the intact viral genome has not been well studied.The nuclear EBV-PK protein is a serine/threonine kinase that shares many targets with cyclin-dependent cellular kinases. Indeed, half of the EBV-PK targets identified by a protein array in a recent study were also found to be in vitro substrates for the cellular kinase CDK1/cyclin B (63). Overexpression of EBV-PK in cells has been shown to result in phosphorylation of a number of different cellular proteins, including the cell cycle regulatory proteins p27 (19) and pRB (C. V. Kuny and R. F. Kalejta, unpublished data); nuclear lamin A/C (32); interferon regulatory factor 3 (IRF3) (54); cellular translational elongation factor 1␦ (24, 26); MCM4 (part of the MCM4-MCM6-MCM7 cellular replication origin binding complex) (29); and histone protein H2AX (52). The ability of EBV-PK to phosphorylate and inactivate two key cell cycl...

A Conserved Gammaherpesvirus Protein Kinase Targets Histone Deacetylases 1 and 2 To Facilitate Viral Replication in Primary Macrophages

“…Gammaherpesvirus kinases as regulators of lytic infection. All gammaherpesviruses encode a protein kinase that supports lytic gene expression, DNA replication, nuclear egress, and reactivation from latency (28,32,(38)(39)(40)(41). However, the role of gammaherpesvirus kinase in the context of de novo lytic infection has not been fully appreciated, in spite of the fact that gammaherpesvirus kinases are virion components (42).…”

Viral Serine/Threonine Protein Kinases

“…As an example to support this idea, mutation of the CHPK kinase of VZV does not substantially affect viral growth in cell culture, whereas it is essential for replication in human T cells and human thymus/liver xenografts in SCID-hu mice (150). Also indicative of the caution needed when interpreting results from immortalized cell lines is that SV40 T antigens have been reported to induce lamin A/C phosphorylation in 293T cells, thereby compensating for the defect in viral egress observed for a CHPK-null EBV recombinant in 293 cells (137).…”

The Cellular Ataxia Telangiectasia-Mutated Kinase Promotes Epstein-Barr Virus Lytic Reactivation in Response to Multiple Different Types of Lytic Reactivation-Inducing Stimuli

“…Therefore, we determined if EBV-PK is required for hydrogen peroxidemediated EBV lytic reactivation. AGS cell lines stably infected with wild-type (WT) B95.8 virus, or a previously described EBV-PK mutant virus (66), were generated and shown to have the expected phenotype in regard to expressing the hyperphosphorylated form of the BMRF1 protein (an easily observed EBV-PK-dependent form of BMRF1) (15,31) (Fig. 5).…”