An RS motif within the Epstein-Barr virus BLRF2 tegument protein is phosphorylated by SRPK2 and is important for viral replication

Abstract

Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis, B cell lymphomas, and nasopharyngeal carcinoma. Many of the genes required for EBV virion morphogenesis are found in all herpesviruses, but some are specific to gammaherpesviruses. One of these gamma-specific genes, BLRF2, encodes a tegument protein that has been shown to be essential for replication in other gammaherpesviruses. In this study, we identify BLRF2 interacting proteins using binary and co-complex protein assays. Serine/Arginine-rich Protein Kinase 2 (SRPK2) was identified by both assays and was further shown to phosphorylate an RS motif in the BLRF2 C-terminus. Mutation of this RS motif (S148A+S150A) abrogated the ability of BLRF2 to support replication of a murine gammaherpesvirus 68 genome lacking the BLRF2 homolog (ORF52). We conclude that the BLRF2 RS motif is phosphorylated by SRPK2 and is important for viral replication.

Figure 1. BLRF2 C-terminus mediates interaction with host and EBV proteins.

(A) GST pull-down assay to confirm binding of putative BLRF2 interacting proteins identified in a yeast two-hybrid assay. Lysates from 293T cells transfected with GST-BLRF2 and the indicated GFP tagged proteins were captured with GST-agarose, washed, resolved by SDS page and proteins detected by western blotting with anti-GFP (upper panels) and anti-GST antibodies (lower panels). Input lysates (2%) are shown in the left panels. (B) Mapping of BLRF2 binding partners confirmed in (A) to the BLRF2 N-terminus (GST-BLRF2 aa1–130 (i)) or C-terminus (GST-BLRF2 aa42–162 (ii)).

Figure 2. Characterization of BLRF2 during EBV replication.

(A) Time course of EBV protein expression using whole cell lysates from P3HR1-ZHT cells (parental) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced with 4HT for 0, 24, 48 or 72 hours. Detection of tubulin serves as loading control. Endogenous BLRF2 is indicated with a solid arrowhead and FLAG-HA-BLRF2 with an open arrowhead. (B) Immunofluorescence microscopy to determine BLRF2 localization during EBV replication in P3HR1-ZHT cells (parental, top panel) or P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 (FLAG-HA-BLRF2, bottom panels), either uninduced (left) or induced with 4-Hydroxytamoxifen (4HT) for 48 hours (middle) or 72 hours (right). Anti-BLRF2 and anti-FLAG staining are shown in green and DNA staining is shown in blue. (C) Subcellular fractionation of EBV proteins and control cell proteins from P3HR1-ZHT cells stably expressing FLAG-HA-BLRF2 induced for replication with 4HT for 0, 24, 48 or 72 hours. Equal relative amounts of the cytosol (C), membrane and organelles (M), nucleus (N), chromatin bound (Ch) and cytoskeletal (Cs) fractions were probed for the indicated proteins. Tubulin served as a control for the cytosol fraction and Lamin B for the cytoskeleton fraction. As for panel A, endogenous and FLAG-HA tagged BLRF2 are indicated with filled and open arrowheads, respectively.

Figure 3. BLRF2 forms protein complex with SRPK2 and host RNA splice factors.

(A) Schematic of two fractionation procedures tested to extract BLRF2 complexes. The cytosol (C) was removed by Digitonin extraction and split between two procedures. Procedure 1 was a three step process in which the membrane and organelles (M1) were collected after Triton X-100 (TX-100) lysis, the soluble fraction after NP40 lysis (S1) and the remaining insoluble pellet (P1). Procedure 2 was only two steps in which the soluble fraction (S2) was collected after NP40 lysis and the remaining insoluble pellet (P2). (B) Western blot analysis of the fractions obtained using procedure described in (A). BLRF2 extraction was monitored using rabbit polyclonal anti-BLRF2 antibody. Endogenous BLRF2 is indicted with a filled arrowhead and FLAG-HA-BLRF2 with an open arrowhead. Fraction composition was also assessed by western blotting for control cell proteins BRG1 (nuclear and chromatin bound), lamin B (cytoskeleton) and tubulin (cytoplasmic). (C) Silver stain gel of 10% of the final elutions from a tandem affinity purification of FLAG-HA-GFP and FLAG-HA-BLRF2 stable P3HR1-ZHT cell lines. Molecular weights of size markers are shown (left). (D) Network representation of interacting proteins identified in TAP-MS and Y2H generated by Pathway Palette and the BioGrid database. EBV proteins are shown as stars and host proteins as circles. The bait (BLRF2) is shown in pink. Interacting proteins are colored based on the technology that identified them (TAP – green; Y2H – yellow; Both – blue). Edges are colored based on the type of evidence used to infer the interaction (Co-complex - blue edges; Binary – red). EBV protein interactions are all binary as described in the text and host-host interaction data is derived from the Biogrid database. Only the connected components are shown. The table shows enrichment of KEGG Pathways for proteins identified by TAP-MS.

Figure 4. SRPK2 phosphorylates BLRF2.

(A) Schematic of BLRF2 indicating the central dimerization domain (aa 66–122) and the C-terminal RS motif. ClustalW alignment of the C-termini of BLRF2 (aa 123 to 162) and its homologs from the rhesus and marmoset lymphocryptoviruses and murine gammaherpesvirus 68. Degree of conservation is shown at the bottom (* - identical, : - high,. - moderate). A conserved basic domain containing the putative RS motif is highlighted by the box. (B) GST pull-down of 293T cells transfected with GST-BLRF2 wild-type (WT) or GST-BLRF2 SRS-ARA mutant (ARA) in the presence or absence of GFP-SRPK2. Western blot analysis using anti-GFP (top panels) and anti-GST (lower panels) antibodies. Input lysates (1%) are shown in the left panels. (C) Western blots of transfected 293T whole cell lysates probed with anti-Phospho-SR antibody (left) and anti-GST antibody (right). (D) Western blots of GST pull-downs from 293T cells transfected with GST-BLRF2 wild-type or ARA mutant. Phosphorylated GST-BLRF2 is shown in the left panel with anti-Phospho-SR antibody and total GST-BLRF2 level is shown by anti-GST antibody (right panel). (E) Western blot of GST pull-downs of 293T cells transfected with GST-BLRF2 wild-type and ARA mutant along with increasing amounts of SRPK2-GFP. Phosphorylated protein is detected by anti-Phosho-SR antibody (top panels), anti-GFP antibody showed SRPK2-GFP (middle panels) and anti-GST antibody showed total BLRF2 (bottom panels). Input lysates (1%) are shown in the left panels.

Figure 5. BLRF2 rescues ORF52 null MHV68 replication, but the BLRF2-ARA mutant cannot.

(A) Complementation assay measuring MHV68 virion release by quantitative PCR into supernatants of 293T cells co-transfected with replication defective MVH68 ORF52 null BAC and empty vector, wild-type FLAG-BLRF2, or FLAG-BLRF2-ARA mutant. Viral DNA was quantified four days post-transfection and the results shown are representative of two independent experiments performed in triplicate. Western blot with anti-flag antibody shows BLRF2-WT and –ARA expression levels.

Figure 6. The BLRF2 ARA mutant exhibits increased cytoplasmic localization compared to wild-type BLRF2.

(A) Immunofluorescence microcopy showing BLRF2 localization in HeLa cells transfected with BLRF2 wild-type or ARA mutant. Cells were stained with anti-BLRF2 antibody (green) and Draq5 DNA staining (blue). Examples of predominantly nuclear (left panels), mixed nuclear and cytoplamsic (middle panels), and predominantly cytoplasmic staining (right panels) are shown. (B) Summary of immunofluorescence analysis described in (A). The percentage of cells observed with predominantly nuclear (N) (blue), cytoplasmic (C) (red) or mixed (N and C) (gray) BLRF2 staining are shown.