In contrast to the IFN-β promoter, the IFN-stimulated response element (ISRE) promoter contains two ISREs which can be activated by IFN regulatory factor 3 (IRF3), IRF7, or both (22
). To elucidate the effect of DI RNA replication upon IRF3-mediated signaling, we used an ISRE promoter-driven luciferase reporter plasmid. DI particle infection of 293-pcDNA control cells led to a meager 1.7-fold increase in luciferase activity over that in the corresponding uninfected cells (Fig. ). However, DI particle infection of 293-NPeGFPL cells resulted in greater than 200-fold induction of luciferase activity over that in the uninfected 293-NPeGFP cells (Fig. ). Activation of IFN-β gene transcription requires coordinate actions of IRF3, NF-κB, and ATF-2/c-Jun transcription factors (27
). Nearly 40-fold induction of NF-κB promoter activity in 293-NPeGFPL cells infected with DI particles compared to that in similarly infected 293-pcDNA cells was observed (Fig. ). For direct verification of IFN-stimulated gene expression, we examined the expression of IFN-stimulated gene 56 (ISG56), one of the viral stress-inducible genes that are induced by IFNs, dsRNA, and virus infections (24
). Results (Fig. ) showed strong induction of ISG56 protein expression in 293-NPeGFPL cells infected with DI particles (lane 4), whereas in uninfected 293-NPeGFPL cells and in 293-pcDNA cells with or without DI particle infection, ISG56 protein was undetectable. In each of the above-described studies, DI RNA replication products were readily detected (data not shown). Taken together, the results from the above-described studies show that replication of DI RNA in 293-NPeGFPL cells potently activates IFN and IFN signaling.
FIG. 4. (A) Activation of the ISRE promoter by DI particle infection. The experiment was conducted as described in the legend to Fig. but using the ISRE promoter-driven luciferase gene. (B) Activation of the NF-κB promoter by DI particle (more ...)
In summary, we report here the establishment of stable cells expressing the VSV replication proteins. Although a stable cell line expressing Sendai virus replication proteins has been described previously (28
), this is the first description of a cell line constitutively expressing the VSV replication proteins. Using this cell line, we have shown that replication of DI RNA activates IFN and IFN signaling pathways. It has been reported previously that snap-back DI (±) particles of VSV activate IFN signaling. Furthermore, a preexisting molecule in the snap-back DI genome (presumably a dsRNA structure) has been proposed to be responsible for induction of IFN, as heat inactivation or UV treatment of DI particles did not inhibit IFN induction in chicken embryo fibroblasts and mouse L cells (15
). Contrary to these findings, our results revealed that replication of DI particle T RNA with a panhandle-type DI genome (16
) is required for IFN-β activation as well as IFN signaling. Mere entry and uncoating of DI particles or expression of only the viral replication proteins was not sufficient to induce IFN-β or IFN signaling. VSV DI genomes are synthesized in the form of nucleocapsid, and possible formation of dsRNA structures in infected cells has not been reported. Viral replication intermediates like dsRNA or single-stranded RNAs (ssRNAs) with triphosphorylated 5′ ends are sensed by both cytoplasmic sensors, e.g., RIG-I, MDA-5, and Nod2 (5
), and endosomal receptors (Toll-like receptor 3 [TLR3], TLR7/TLR8, and TLR9) (25
). However, HEK293 cells are deficient in TLRs and Nod2. Therefore, in these cells, IFN induction and signaling may be mediated by RIG-I and/or MDA-5 through recognition of DI RNA. RIG-I has been shown previously to be involved in detection of rhabdoviruses and paramyxoviruses, resulting in subsequent induction of IFN responses (11
). MDA-5 has also been shown to be involved in recognition of measles virus (12
) and Sendai virus (29
) DI particles. It would therefore be interesting to examine whether VSV DI RNA replication activates IFN through involvement of one or more of these cytoplasmic sensors.
Since infection of cells with VSV and many other negative-strand RNA viruses results in cytopathogenesis and cell death, it is difficult to study the long-term effects of virus replication on host cell functions in the context of virus infection. VSVs encoding mutant M proteins have been used to study IFN activation (2
), but these viruses are also cytopathic. Therefore, results from studies of the effects of VSV RNA replication on IFN activation and signaling in the context of VSV infection are difficult to assess. Our system will provide the opportunity to examine the effects of viral genome replication on host cell functions in the absence of the cytopathogenic effects of VSV M and G proteins.