Abstract
BackgroundDefective interfering (DI) viruses are natural antivirals made by nearly all viruses. They have a highly deleted genome (thus being non-infectious) and interfere with the replication of genetically related infectious viruses. We have produced the first potential therapeutic DI virus for the clinic by cloning an influenza A DI RNA (1/244) which was derived naturally from genome segment 1. This is highly effective in vivo, and has unexpectedly broad-spectrum activity with two different modes of action: inhibiting influenza A viruses through RNA interference, and all other (interferon-sensitive) respiratory viruses through stimulating interferon type I.ResultsWe have investigated the RNA inhibitory mechanism(s) of DI 1/244 RNA. Ablation of initiation codons does not diminish interference showing that no protein product is required for protection. Further analysis indicated that 1/244 DI RNA interferes by replacing the cognate full-length segment 1 RNA in progeny virions, while interfering with the expression of genome segment 1, its cognate RNA, and genome RNAs 2 and 3, but not genome RNA 6, a representative of the non-polymerase genes.ConclusionsOur data contradict the dogma that a DI RNA only interferes with expression from its cognate full-length segment. There is reciprocity as cloned segment 2 and 3 DI RNAs inhibited expression of RNAs from a segment 1 target. These data demonstrate an unexpected complexity in the mechanism of interference by this cloned therapeutic DI RNA.
Highlights
Defective interfering (DI) viruses are natural antivirals made by most viruses
1/244 DI RNA interferes with packaging of segment 1 It was reported previously that the interference mediated by a DI virus occured at the level of packaging [57], and we investigated whether this was the case for DI 1/ 244
Using Seg 1-green fluorescent protein (GFP) plasmid with the transfection protocol described above, and a primer extension assay that detects Negative sense virion genomic RNA (vRNA), mRNA and Positive sense antigenome RNA (cRNA), we investigated 1/244 DI RNA mediated interference of RNA synthesis (Fig. 5)
Summary
Defective interfering (DI) viruses are natural antivirals made by most viruses. We have produced the first potential therapeutic DI virus for the clinic by cloning an influenza A DI RNA (1/244) which was derived naturally from genome segment 1 This is highly effective in vivo, and has unexpectedly broad-spectrum activity with two different modes of action: inhibiting influenza A viruses through RNA interference, and all other (interferon-sensitive) respiratory viruses through stimulating interferon type I. Viral replication frequently generates progeny genomes that contain extensive deletions [1,2,3,4]. This is thought to be a consequence of the polymerase detaching from the initial template and reattaching elsewhere on the same or different template. But not all, of these defective genomes are capable of interfering with the growth of the parental virus from which they were derived and are known as defective-interfering (DI)
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