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. 2004 Jul 27;101(30):11099-104.
doi: 10.1073/pnas.0404310101. Epub 2004 Jul 19.

Reovirus oncolysis: the Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection

Kara L Norman  1 , Kensuke Hirasawa, An-Dao Yang, Michael A Shields, Patrick W K Lee
Affiliations

Reovirus oncolysis: the Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection

Kara L Norman et al. Proc Natl Acad Sci U S A. .

Abstract

Reovirus is a benign human virus that was recently found to have oncolytic properties and is currently in clinical trials as a potential cancer therapy. We have previously demonstrated that activation of Ras signaling, a common event in cancer, renders cells susceptible to reovirus oncolysis. In this study, we investigate which elements downstream of Ras are important in reovirus infection. By using a panel of NIH 3T3 cells transformed with activated Ras mutated in the effector-binding domain, we found that only the RasV12G37 mutant, which was unable to signal to Raf or phosphatidylinositol 3-kinase but retained signaling capability to guanine nucleotide-exchange factors (GEFs) for the small G protein, Ral (known as RalGEFs), was permissive to reovirus. Expression of the activated mutant of the RalGEF, Rlf, also allowed reovirus replication. Specific inhibition of the Ral pathway by using dominant-negative RalA rendered normally permissive H-Ras cells (cells expressing activated Ras) resistant to reovirus. To further identify elements downstream of RalGEF that promote reovirus infection, we used chemical inhibitors of the downstream signaling elements p38 and JNK. We found that reovirus infection was blocked in the presence of the p38 inhibitor but not the JNK inhibitor. Together, these results implicate a Ras/RalGEF/p38 pathway in the regulation of reovirus replication and oncolysis.

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Figures

Fig. 1.
Fig. 1.
Involvement of Ras effector pathways in reovirus susceptibility. (A) Infection of Ras effector binding domain mutant-expressing NIH 3T3 cells. NIH 3T3 cells expressing empty vector (pDCR), activated Ras (V12), or activated Ras with a point mutation in the effector binding domain (V12C40, V12G37, and V12S35) were infected with reovirus at a multiplicity of infection of 40 plaque-forming units per cell and pulse-labeled with [35S]methionine-containing medium for 5 h at 60–65 h after infection. The cells were then lysed, and reovirus proteins were immunoprecipitated from part of the lysate by using rabbit polyclonal antireovirus Ab. Immunoprecipitated proteins were resolved by 10% SDS/PAGE, followed by autoradiography. Migration of the three size classes of reovirus proteins (λ, μ, and σ) is indicated on the left. (B) Effect of the PI3-kinase inhibitor, LY294002, on reovirus infection of Ras-transformed NIH 3T3 cells. NIH 3T3 cells expressing activated Ras were infected with reovirus at a multiplicity of infection of 40 plaque-forming units per cell and treated with 20 μM LY294002 (LY) or left untreated (cntr.) for the duration of the experiment. Cells were metabolically radiolabeled with [35S]methionine at 43–48 h after infection, and lysates were prepared and analyzed as described in A. Reovirus protein migration (λ, μ, and σ) is indicated on the left.
Fig. 2.
Fig. 2.
Reovirus infection of cells with activated RalGEF. (A) Ral activity in A14 cells expressing empty vector, active Rlf–CAAX, or Rlf–CAAX with a point mutation in the catalytic domain (inact.). Cells were lysed and Ral–GTP as well as total Ral levels were assessed, as described in Materials and Methods. (B) Cytopathic effects induced by reovirus. Cells were infected with reovirus at a multiplicity of infection of 40 plaque-forming units per cell or mock-infected, and photomicrographs were taken at 72 h after infection. (C) Reovirus S1 RNA synthesis in infected A14, Rlf–CAAX (inact.), and Rlf–CAAX cells. Cells were infected with reovirus, and at various times after infection RNA was extracted and subjected to RT-PCR for reovirus S1 RNA and cellular GAPDH RNA (control). Reactions were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) Reovirus protein synthesis in A14, Rlf–CAAX (inact.), and Rlf-CAXX-expressing cells. Infected cells were pulse-labeled with [35S]methionine-containing medium for 5 h at 67–72 h after infection. After labeling, cells were lysed and reovirus proteins were analyzed by immunoprecipitation and 10% SDS/PAGE/autoradiography, as described in Fig. 1. The three size classes of reovirus proteins (λ, μ, and σ) are indicated on the right.
Fig. 3.
Fig. 3.
Reovirus infection of H-Ras cells expressing dnRal. (A) Down-regulation of Ral activity in H-Ras cells expressing dnRal. H-Ras cells were infected with pBabepuro (vector) retrovirus or retrovirus expressing dnRalA A26 (dnRal). The levels of Ral–GTP and total Ral were assessed as described in Materials and Methods. (B) H-Ras cells harboring vector alone or expressing dnRal were infected with reovirus. Infected cells were pulse-labeled with [35S]methionine-containing medium for 5 h at 67–72 h after infection, and reovirus protein immunoprecipitates were subjected to 10% SDS/PAGE, followed by autoradiography. Reovirus protein migration is indicated on the left. (C) Ras, AKT, MEK, and ERK activity in control (vector) H-Ras and dnRal/H-Ras cells. For Ras activity, serum-starved cell lysates were subjected to pull-down reactions by using GST–Raf Ras binding domain conjugated to glutathione beads, followed by Western blot analysis for Ras. To assess downstream signaling, cells were serum-starved overnight and cell lysates were examined by 10% SDS/PAGE and Western blot analysis for the phosphorylated form of each kinase. (D) Reovirus transcription in control (vector) H-Ras and dnRal/H-Ras cells. Cells were infected with reovirus, and at various times after infection, RNA was extracted and examined by RT-PCR for reovirus S1 RNA and cellular GAPDH RNA control.
Fig. 4.
Fig. 4.
Effect of the JNK inhibitor SP600125 on reovirus infection of H-Ras cells. NIH 3T3 cells and NIH 3T3 cells expressing activated Ras (Ras V12) were infected with reovirus in the presence of increasing concentrations of the JNK inhibitor SP600125. Cells were radiolabeled as described in Fig. 1 for 5 h at 43–48 h after infection, cell lysates were prepared, and immunoprecipitates were subjected to 10% SDS/PAGE and autoradiography. Migration of reovirus proteins is indicated on the right.
Fig. 5.
Fig. 5.
Effect of the p38 inhibitor SB203580 on reovirus infecton of H-Ras cells and Rlf–CAAX cells. (A) NIH 3T3 cells and NIH 3T3 cells expressing activated Ras (Ras V12) were infected with reovirus or mock-infected in the presence of increasing concentrations of the p38 inhibitor SB203580. Cells were radiolabeled as described in Fig. 1 for 5 h at 43–48 h after infection, cell lysates were prepared, and whole-cell lysate (Left) or immunoprecipitates (Right) were subjected to 10% SDS/PAGE and autoradiography. cntr., Control uninfected/untreated Ras cells. Migration of reovirus proteins (λ, μ, and σ) is indicated on the right. (B) Control A14 cells and A14 cells expressing activated Rlf–CAAX were infected with reovirus in the presence of increasing concentrations of the p38 inhibitor SB203580. Cells were radiolabeled as described above, cell lysates were prepared, and immunoprecipitates were subjected to 10% SDS/PAGE and autoradiography. Migration of reovirus proteins is indicated on the right.
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