IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence

Abstract

Here, we report that specific manipulations of the cellular response to virus infection can cause prevention of apoptosis and consequent establishment of persistent infection. Infection of several human cell lines with Sendai virus (SeV) or human parainfluenza virus 3, two prototypic paramyxoviruses, caused slow apoptosis, which was markedly accelerated upon blocking the action of phosphatidylinositol 3-kinases (PI3 kinases) in the infected cells. The observed apoptosis required viral gene expression and the action of the caspase 8 pathway. Although virus infection activated PI3 kinase, as indicated by AKT activation, its blockage did not inhibit JNK activation or IRF-3 activation. The action of neither the Jak-STAT pathway nor the NF-kappaB pathway was required for apoptosis. In contrast, IRF-3 activation was essential, although induction of the proapototic protein TRAIL by IRF-3 was not required. When IRF-3 was absent or its activation by the RIG-I pathway was blocked, SeV established persistent infection, as documented by viral protein production and infectious virus production. Introduction of IRF-3 in the persistently infected cells restored the cells' ability to undergo apoptosis. These results demonstrated that in our model system, IRF-3 controlled the fate of the SeV-infected cells by promoting apoptosis and preventing persistence.

FIG. 1.

PI3K inhibition enhances apoptosis of cells infected with Sendai virus or hPIV3. (A) HT1080 cells were pretreated with the PI3 kinase inhibitor LY294002 (LY; 20 μM) or vehicle control for 30 min. Cells were then mock infected or infected with Sendai virus (80 hemagglutinating units/ml) as indicated. Six hours after virus addition, cells were fixed, permeabilized, and stained for DNA fragmentation using a TUNEL protocol. The same field of cells was imaged for phase contrast and TUNEL staining. (B) HT1080 cells were treated as described for panel A. After 6 hours, whole-cell extracts were prepared, and proteins (30 μg) were separated by SDS-PAGE and transferred to PVDF. A Western blot assay was performed with an antibody specific for the cleaved PARP protein (C-PARP). A Western blot assay against actin controlled for protein loading. (C) Cells were pretreated with the LY inhibitor and infected with the Sendai virus as described for panel A. Extracts were prepared at the indicated times, and Western blot assays for cleaved PARP and actin were performed as described for panel B. (D and E) A549 (D) and BEAS-2B (E) cells were pretreated with the PI3K inhibitor LY, infected with SeV, and stained as described for panel A. (F) A549 cells were pretreated with LY and stained as described for panel A. Cells were mock infected or infected with hPIV3 (multiplicity of infection, 2) as indicated.

FIG. 2.

Viral gene expression is needed for apoptosis. A. HT1080 cells were pretreated with LY294002 for 30 min. Cells were then mock infected (Con) or infected with Sendai virus that had been inactivated by UV treatment for 0, 30, or 60 seconds. Cells were lysed 6 h p.i., and Western blot assays to determine PARP cleavage were performed as described in the legend for Fig. 1B. B. HT1080 cells were treated as described for panel A. RNA was harvested 4 h p.i., and 561 and actin mRNA levels were determined by RNase protection assays.

FIG. 3.

Apoptosis is induced by the caspase 8 pathway. A. HT1080 cells were treated with LY294002 and Sendai virus as previously described. Whole-cell lysates were prepared, and Western blot assays were performed with antibodies against caspase 8 and caspase 9. The positions of the full-length (57- and 47-kDa) and cleaved (43/41- and 37/35-kDa) caspase 8 and caspase 9 are indicated. B. LY294002 pretreatment and Sendai virus infection of HT1080 cells were performed as described in the legend for Fig. 1. Cells were lysed at the indicated time points, and a caspase 8 Western blot assay was performed as described for panel A. C. HT1080 cells were pretreated with 100 μM of the general caspase inhibitor z-VAD, the caspase 8 inhibitor z-IETD, the caspase 9 inhibitor z-LEHD, or the DMSO solvent control 1 hour before virus addition. The PI3 kinase inhibitor LY294002 (50 μM) was added to all cells 30 min before virus addition. Cells were fixed and stained 6 h after infection.

FIG. 4.

Activation of PI3K is not required for activation of the JNK and TBK1 pathways. A. HT1080 cells were infected with Sendai virus, and whole-cell extracts were prepared at the indicated times. Proteins were separated by SDS-PAGE and transferred to PVDF. Western blot assays were performed with antibodies against AKT and activated, phosphorylated AKT (P-AKT). B. HT1080 cells were pretreated with the PI3 kinase inhibitor LY or vehicle control and subsequently mock infected or infected with Sendai virus as indicated. Whole-cell extracts were prepared 6 h p.i., and Western blot assays were performed with antibodies against JNK and activated, phosphorylated JNK (P-JNK). C. HT1080 cells were pretreated with LY294002 or vehicle control for 30 min and subsequently mock infected or infected with Sendai virus as indicated. Two hours postinfection, cells were fixed, permeabilized, and stained for IRF-3 using a specific antibody. D. HT1080 cells were pretreated with LY294002 and infected with Sendai virus. RNA was harvested 4 h after virus addition. 561 and actin mRNA levels were determined by RNase protection assays. E. HT1080 cells were transfected with the expression vector of an inactive catalytic subunit of PI3K (p110KD), these cells were mock infected or infected with SeV as indicated, and PARP cleavage and P-AKT were analyzed at 8 h p.i.

FIG. 5.

Functional Jak/STAT and NF-κB pathways are not needed for apoptosis. A. U4C cells, which lack JAK1 and the interferon response, were pretreated with the PI3 kinase inhibitor LY294002 for 30 min. Cells were then mock infected (control) or infected with Sendai virus in the absence or presence of LY as indicated. Six hours after virus addition, cells were fixed, permeabilized, and stained. B. 2f-SR cells, which constitutively express the IκB superrepressor and subsequently fail to activate NF-κB, were treated as for panel A.

FIG. 6.

IRF-3 is required for the induction of cell death during Sendai virus infection. A. P2.1 cells were pretreated with the PI3 kinase inhibitor LY294002 and subsequently mock infected or infected with Sendai virus as indicated. B. P2.1.17 cells were treated with LY294004 and infected with Sendai virus as for panel A.

FIG. 7.

TRAIL is not required for apoptosis by Sendai virus. A. HT1080 cells were infected with Sendai virus as described in Materials and Methods. Cells were collected at the indicated times, and RNA was isolated. The amount of TRAIL message was determined by quantitative reverse transcription-PCR. B. HT1080 and P2.1 cells were pretreated with LY or vehicle control and infected with Sendai virus. Cells were harvested after 6 h, and RNA was isolated. TRAIL message was determined as for panel A. C. HT1080 cells were preincubated with LY294002 (50 μM) and subsequently infected or mock infected with Sendai virus. After infection, cells were washed and placed in complete medium containing LY294002. Anti-TRAIL blocking antibodies or DMSO vehicle control was added to the medium. Cells were fixed after 24 h.

FIG. 8.

Abrogation of the RIG-I/IRF-3 pathway leads to persistent infection. A. HT1080/RIG-IC/SeV cells (P15) or their matching mock-infected control cells were plated on coverslips and stained with an antibody against Sendai virus. Nuclei were visualized by DAPI staining. B. U4C cells, which lack JAK1 and the interferon response but express normal levels of IRF-3, were infected with SeV, total proteins extracts were separated by SDS-PAGE, and Western blot assays were performed with an antibody against the Sendai virus C protein at the indicated times after infection. C. Cell lysates were prepared from the three persistently infected cell lines or their matched mock-infected controls. Proteins (40 μg) were separated by SDS-PAGE, and Western blot assays were performed with an antibody against the Sendai virus C protein. D. HT1080/siIRF-3 clone 1 cells were transfected with an IRF-3 expression plasmid. As the RNAi targeting vector recognizes the 5′ and 3′ untranslated regions, IRF-3 cDNA expressed from a plasmid is not repressed. After 3 hours, the transfection reagents were removed, and the cells were washed and placed in complete medium. Cell lysates were prepared after 2 days, and a C-PARP Western blotting was performed as described for Fig. 1B.

FIG. 9.

SeV-induced apoptotic pathway. Infection of cells with SeV activates IRF-3, which in turn induces expression of virus stress-inducible genes and causes host cell apoptosis. In addition, virus infection also activates the PI3 kinase pathway, which is needed for replication of virus and generation of antiapoptotic signal in the host cells. Blocking the PI3 kinase pathway by chemical inhibitors induces early apoptosis of the host cells. A suitable balance between the apoptotic and antiapoptotic signals dictates the fate of the infected cell; however, the apoptotic signal is determined by the activation of IRF-3.