Sendai virus defective-interfering genomes and the activation of interferon-beta

Abstract

The ability of some Sendai virus stocks to strongly activate IFNbeta has long been known to be associated with defective-interfering (DI) genomes. We have compared SeV stocks containing various copyback and internal deletion DI genomes (and those containing only nondefective (ND) genomes) for their ability to activate reporter genes driven by the IFNbeta promoter. We found that this property was primarily due to the presence of copyback DI genomes and correlated with their ability to self-anneal and form dsRNA. The level of IFNbeta activation was found to be proportional to that of DI genome replication and to the ratio of DI to ND genomes during infection. Over-expression of the viral V and C proteins was as effective in blocking the copyback DI-induced activation of the IFNbeta promoter as it was in reducing poly-I/C-induced activation, providing evidence that these DI infections activate IFNbeta via dsRNA. Infection with an SeV stock that is highly contaminated with copyback DI genomes is thus a very particular way of potently activating IFNbeta, presumably by providing plentiful dsRNA under conditions of reduced expression of viral products which block the host antiviral response.

Preference of RIG-I for short viral RNA molecules in infected cells revealed by next-generation sequencing

“…2D). Identification of copy-back DIs as a RIG-I PAMP agrees with previous characterization of these molecules as exceptionally good inducers of the IFN response (26).…”

“…The fact that CIP and TAP treatment led to complete loss of DI's ability to induce an immunostimulatory response is interesting and illustrates that the 5′ triphosphate (although a diphosphate has not been experimentally ruled out in this study) is required even in the context of a relatively long dsRNA region, such as SeV DI RNA. Our finding that DI subgenomic particles preferentially associate with RIG-I provides an explanation for the historical observation that viruses containing these particles act as superior IFN inducers (26,29). The approach we used in our work provides a powerful tool for analysis of RIG-Iassociated RNA from various viral infections in multiple cell types as well as other protein/RNA complexes.…”

“…Specifically, RNA mapping to a region of the genome between positions 14,932 and 15,384 was much more abundant in infected cells than RNA mapping to the rest of the SeV genome. Because it is known from previous studies that the SeV-C copy-back DI particle genome maps to precisely those positions, we concluded that the majority (≈95%) of viral RNA species present in infected cells at 24 h postinfection (hpi) are of a copy-back DI nature (26). Comparison of RIG-I-associated RNA with that of the control IP revealed that the RIG-I pulldown was specifically enriched in DI RNA, with RIG-I samples containing approximately seven times more DI RNA than control samples (Fig.…”

A Novel Role for Viral-Defective Interfering Particles in Enhancing Dendritic Cell Maturation

“…2a). Recent studies using overexpression of the SeV V protein further support a role for dsRNA in the enhancement of type I IFN induction by DI particles (45). The protein RIG-I has been shown to directly interact with dsRNA leading to induction of type I IFNs (21) and is a likely candidate molecule for involvement in DC maturation in response to virus as other known dsRNA binding proteins, TLR3 and PKR, have been shown to be dispensable for DC maturation (5,6,14,46).…”

The Double-stranded RNA Binding Domain of the Vaccinia Virus E3L Protein Inhibits Both RNA- and DNA-induced Activation of Interferon β

“…DI-H4 stocks were described previously (24). Primary antibodies used included mouse anti-Rig-I (Alexis), mouse anti-actin (Chemicon), mouse anti-HA (Berkeley Antibody Co., Inc.), and rabbit anti-Sendai P,V,C (homemade).…”