Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces

Abstract

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M(r) 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to down-regulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.

Fig. 1.

Amino acid sequence of ICP0 showing putative binding sites of CIN85 SH3 domains.

Fig. 2.

CIN85 interacts with ICP0 through its SH3 domains. (A) CIN85 pulls down ICP0. GST-tagged full-length CIN85 or GST bound to beads were reacted overnight with equal amount of lysates of HEp-2 cells mock infected or exposed to 10 pfu per cell of HSV-1(F), ΔU_L13, or ΔU_s3 mutant for 16 h. The pulled-down proteins and the corresponding input lysates were resolved by electrophoresis in a denaturing gel and reacted with anti-ICP0 monoclonal antibody. (B) SH3 domains of CIN85 pull down ICP0. HEp-2 cells were harvested 7 or 25 h after infection with wild-type virus. Equal amounts of infected cell lysates were reacted for 4 h at 4°C with GST-tagged SH3 domains of CIN85 (designated as GST-SH3–1, GST-SH3–2, or GST-SH3–3, respectively) absorbed to glutathione-agarose beads. The proteins adhering to the beads were electrophoretically separated in denaturing gels and reacted with anti-ICP0 monoclonal antibody as described in Materials and Methods.

Fig. 3.

CIN85 (A) and Cbl (B) are pulled down by domains of ICP0 containing SH3 domain binding sites. GST-ICP0 chimeric proteins bound to agarose beads as described in Materials and Methods were reacted with equal amounts of uninfected HEK293 cell lysates. The proteins bound to the beads were solubilized, separated in denaturing gels, transferred to a nitrocellulose membrane, and reacted with antibodies against CIN85 (A Upper) or GST (A Lower), Cbl (B Upper) or stained with Ponceau S (B Lower). The dots identify the bands that contain full-size GST-ICP0 chimeric proteins.

Fig. 4.

EGF-induced reporter gene expression is decreased in a dose-dependent manner by cotransfection with Cbl or ICP0. (A) Experimental design: HEK293 cells grown in 12-well plates were transfected with SRE-luciferase reporter gene construct, CMV-LacZ, expression vector pRK-EGFR, and variable amounts of plasmids expressing Cbl or ICP0. The empty expression vector MTS1 was added to the transfection mixture as needed to maintain a constant amount of transfected DNA. After 12 h, the cells were replenished with serum-free medium for 24 h and then incubated in medium containing 100 ng/ml EGF for 4, 8, or 12 h. The cells were then harvested, lysed, and the luciferase and β-galactosidase activities were assayed as described in Materials and Methods. Luciferase activity was normalized against β-galactosidase levels for each transfection, and EGF-induced fold increase in luciferase activity was quantified for every pair in the triplicate and was expressed as the average induction (fold increase) ± SD. (B) Cbl and ICP0 down-regulate EGF-induced reporter gene. EGF-induced luciferase activity in cells transfected with Cbl alone or with ICP0 is shown. (C) ICP0 down-regulates EGF-induced reporter gene. EGF-induced luciferase activity in cells transfected with increasing amounts of ICP0 alone or in combination with Cbl is shown.

Fig. 5.

Cell surface and total EGFR levels are affected by ICP0 or by wild-type virus. (A) Effect of EGF on EGFR on cell surface. Replicate HEK293 cells grown in T-25 flasks were stimulated or mock-stimulated with 100 ng/ml EGF at time intervals shown. EGFR on cell surfaces was biotinylated and detected as described in Materials and Methods. (B) EGFR in cells transfected with ICP0. Replicate cultures of HEK293 cell flasks were transfected with EGFR and MTS1-ICP0 or the same amount of empty vector MTS1. At 12 h after transfection, the cultures were replenished with serum-free medium. At 36 h after transfection, the cells were mock-stimulated or exposed to EGF for 20 or 50 min. The cell surface levels of EGFR were assayed as described above (Upper). An aliquot of the total cell lysate removed before immunoprecipitation served to measure the level of EGFR in the total cell lysate (Lower). (C) EGFR in cells infected with HSV-1(F). Replicate cultures of HEp-2 cells were incubated in serum-free medium for 24 h, then mock-infected or infected with HSV-1(F). At 4 h after infection, the cells were mock-stimulated or exposed to EGF for 30 or 60 min. The cell surface levels of EGFR were assayed as detected above.

Fig. 6.

Schematic representation of the endocytosis and degradation or recycling of receptor tyrosine kinases in mock-infected (A) or infected (B) cells. In mock-infected cells, binding of the ligand results in dimerization and autophosphorylation of the receptor, recruitment and phosphorylation of Cbl, interaction with the adaptor protein CIN85 and endophilin, endocytosis, and either ultimate recycling or degradation of the receptor. In infected cells, ICP0, CIN85, and Cbl may form a complex that promotes the degradation of receptor tyrosine kinases in a ligand-independent fashion. In addition, ICP0 may preclude recycling of ligand-activated receptors.