Abstract
In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5’ phosphate groups. RNAs lacking 2’-O-methylated 5’ cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5’ capping or 2’-O-methylation of the 5’ cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.
Highlights
Rotavirus (RV) is the primary etiological agent of severe dehydrating diarrhea in young children worldwide, and is responsible for almost half a million deaths annually [1,2]
We hypothesized that RV singlestranded RNA (ssRNA) transcripts, and not genomic double-stranded RNA (dsRNA), would likely be the primary immunostimulatory molecules generated during infection
Using an in vitrotranscription approach, we demonstrated that nascent RV ssRNA transcripts have IFN stimulatory potential, suggesting that they possess at least one pathogen-associated molecular patterns (PAMPs)
Summary
Rotavirus (RV) is the primary etiological agent of severe dehydrating diarrhea in young children worldwide, and is responsible for almost half a million deaths annually [1,2]. The virusencoded RNA-dependent RNA polymerase, RdRP, uses the minus-strand of the segmented dsRNA genome as a template to generate mRNAs, which are extruded into the cytoplasm. Some of these mRNAs are translated into viral proteins, while others accumulate in cytoplasmic inclusions called viroplasms where they are packaged into newly forming subviral particles. During this encapsidation step, RV plus-strand RNAs undergo a single round of minus-strand replication to generate a complete set of eleven dsRNA genome segments. Progeny subviral particles bud through the endoplasmic reticulum, where they mature into TLPs that exit the cell via lysis or exocytosis [6]
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