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.

FIG. 1.

Creation of stable 293 and 293T cell lines latently infected with WT or PKmut viruses. (A) 293 cells latently infected with WT or PK-mutant EBV (PKmut) were transfected with a BZLF1 (Z) expression vector to induce lytic infection, in the presence or absence of a cotransfected EBV-PK expression vector, and immunoblots performed to examine expression of EBV-PK, BZLF1, and β-actin. (B) 293 cells latently infected with WT or PKmut were transfected with a BZLF1 (Z) expression vector to induce lytic infection, in the presence or absence of a cotransfected EBV-PK expression vector, and immunoblotting was performed to examine the expression of BMRF1, BZLF1, and β-actin. The hypo- and hyperphosphorylated forms of BMRF1 are indicated.

FIG. 2.

The WT and PKmut viruses have a similar level of lytic viral protein expression following BZLF1 transfection in 293 and 293T cells. 293 cells latently infected with WT or PKmut EBV were transfected with a BZLF1 (Z) expression vector to induce lytic replication. The expression level of the indicated lytic viral proteins was determined by performing immunoblot assays on cellular lysates 2 days after transfection. The kinetic class (immediate-early [IE], early, or late) is indicated next to each viral protein examined. β-Actin was used as a loading control.

FIG. 3.

Viral DNA replication of the PKmut virus is not impaired in 293 cells. 293 WT or PKmut cells were transfected with a BZLF1 (Z) expression vector, in the presence or absence of a cotransfected WT or mutant (catalytically inactive) EBV-PK expression vector. Genomic DNA was isolated 3 days later and an EBV “terminus assay” was performed to detect the fused versus linear forms of the viral genome. (A) Southern blot hybridization was performed with a probe to the EBV termini. The positions of the fused viral termini (which are derived from both the fused termini in the latent viral episome plus the fused termini in the replicated viral concatemer DNA), and the cleaved termini (derived only from the replicated linear genome) are indicated. (B) Hybridization was performed with a probe against BZLF1 to document equal level of transfected BZLF1.

FIG. 4.

The PKmut is highly impaired for release of infectious viral particles in 293 cells and this defect is rescued by expression of EBV-PK in trans. 293 WT or PKmut cells were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic viral replication, in the presence or absence of a cotransfected WT PK expression vector or catalytically inactive (PK K102I) vector. The virus titer was quantitated by infecting Raji cells with various amounts of the supernatant at 72 h posttransfection and counting the number of GFP-positive cells by using a fluorescence microscope. The results from three independent experiments are shown. The average virus titer of WT virus-infected 293 cells transfected with BZLF1/BRLF1/gp110 is set as 100 (± the standard deviation).

FIG. 5.

The HCMV and KSHV kinases do not rescue the phenotype of the PKmut in 293 cells. (A) 293 PKmut cells were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic viral replication, in the presence or absence of a cotransfected HA-tagged expression vectors for EBV-PK, CMV-PK (UL97), or KSHV-PK (ORF36). The titer of virus was determined as described before. The average virus titer of 293 PKmut cells transfected with BZLF1/BRLF1/gp110 is set as 1. (B) Immunoblot analysis of the transfected HA-tagged EBV-PK, CMV UL97, and KSHV ORF36 proteins in the 293 cells used to derive the virus in the experiments in panel A is shown, along with transfected BZLF1 and cellular β-actin levels.

FIG. 6.

The PKmut is not defective for release of infectious virus in 293T cells. (A) 293T cells infected with WT or PKmut virus were transfected with BZLF1/BRLF1/gp110 expression vectors to induce lytic viral replication, and the titer of virus was determined 3 days later. The results from two independent experiments are shown (expressed as the number of infectious viral particles released per ml of supernatant as measured by the green Raji cell assay). (B) Immunoblots were performed on 293T EBV WT or 293T PKmut cells, transfected with or without BZLF1, to examine the level of BMRF1 hyperphosphorylation and endogenous EBV-PK expression. β-Actin was used as a loading control.

FIG. 7.

The PKmut phenotype in 293 cells is partially rescued by the SV40 large T/small t combination. (A) 293 PKmut cells were transfected with BZLF1/BRLF1/gp110 expression vectors in the presence or absence of a vector expressing both large and small SV40 T antigens (p129 LTag [T/t]), a vector that only expresses the SV40 small t antigen [pRSV-t(t)], or a vector that only expresses the large T antigen [pRSVBneodl1440(T)]. Virus titers were quantitated after 3 days. Values are the average green Raji units per ml ± the standard deviation relative to cells transfected with BZLF1/BRLF1/gp110 (set as 1). (B) The level of BALF4 (gp110), BZLF1, BRLF1, large T antigen, and β-actin in the 293 PKmut cells transfected with various vectors in Fig. A was examined by immunoblotting. (C) 293 PKmut cells were transfected with BZLF1/BRLF1/gp110 expression vectors in the presence or absence of a vector expressing WT SV40 large T and small t antigen (T/t) or a T/t vector in which the pRB-binding domain of large T antigen has been mutated (8). Virus titers were quantitated after 3 days. Values are the average green Raji units per ml ± the standard deviation relative to cells transfected with BZLF1/BRLF1/gp110 alone (set as 1). (D) The levels of large T antigen, BZLF1, and β-actin in the transfected cells in panel C are shown.

FIG. 8.

Knockdown of cell cycle regulatory protein p27 increases production of PKmut virus in 293 cells. (A) 293 cells infected with WT or PKmut were treated twice with control siRNA (Si-C), or siRNA directed against the p27 protein (Si-p27) prior to transfecting the cells with BZLF1/BRLF1/gp110 to induce lytic infection. The virus titer for each condition is shown (normalized to 1 for the amount of each virus induced in the presence of the control siRNA). (B) Western blot of the total p27 level in 293 WT and PKmut cells (note that 5 μg of total cell protein was loaded on the WT cell p27 blot, whereas 20 μg was loaded on the PKmut cell p27 blot). The level of BZLF1 and β-actin was also determined by immunoblotting analysis. (C) 293 cells infected with PKmut EBV were transfected with BZLF1 in the presence or absence of an EBV-PK expression vector. Western blotting was performed to examine the expression of p27 and β-actin.

FIG. 9.

Decreasing lamin A/C greatly enhances production of the PKmut virus in 293 cells. (A) 293 cells infected with WT or PKmut EBV were treated twice with control siRNA (Si-C), or siRNA directed against the lamin A/C proteins (Si-lamin) prior to transfecting the cells with BZLF1/BRLF1/gp110 to induce lytic infection. The virus titer for each condition is shown (normalized to 1 for the amount of virus induced in the presence of the control siRNA). (B) Western blot analysis of total lamin A/C, BZLF1, and β-actin in control siRNA- and lamin A/C siRNA-treated cells.

FIG. 10.

Lamin A/C is constitutively phosphorylated in 293T cells, but not 293 cells. (A) Equal amounts of whole-cell lysates from 293 or 293T cells were subjected to immunoblotting analysis with the phosphorylation-specific anti-lamin A/C antibodies (Ser-22 or Ser-392) or anti-total lamin A/C antibody. (B) 293 EBV PKmut cells were transfected with expression vectors for BZLF1, EBV-PK, SV40 small t antigen, or SV40 large T antigen as indicated. After transfection, cells were cultured in 0.1% serum medium for 18 h. Cell lysates were fractionated as described in Materials and Methods. The protein from the soluble fraction of cells was analyzed by immunoblotting assays with antibodies directed against phosphorylated lamin A/C (Ser-22) and tubulin. An antibody that recognizes total lamin A/C was used for immunoblot analysis of the nuclear matrix fractions.