Deciphering the mechanism of defective interfering RNA (DI RNA) biogenesis reveals that a viral protein and the DI RNA act antagonistically in virus infection

Potato mop-top virus (PMTV) produces a defective RNA (D RNA) encompassing the 5=-terminal 479 nucleotides (nt) and 3=-terminal 372 nt of RNA-TGB (where TGB is triple gene block). The mechanism that controls D RNA biogenesis and the role of D RNA in virus accumulation was investigated by introducing deletions, insertions, and point mutations into the sequences of the open reading frames (ORFs) of TGB1 and the 8-kilodalton (8K) protein that were identified as required for efficient production of the D RNA. Transient expression of RNA-TGB in the absence of RNA-Rep (which encodes the replicase) did not result in accu-mulation of D RNA, indicating that its production is dependent on PMTV replication. The D RNA could be eliminated by dis-rupting a predicted minus-strand stem-loop structure comprising complementary sequences of the 5 = TGB1 ORF and the 3 = 8K ORF, suggesting intramolecular template switching during positive-strand synthesis as a mechanism for the D RNA biogenesis. Virus accumulation was reduced when the 8K ORF was disrupted but D RNAwas produced. Conversely, the virus accumulated at higher titers when the 8K ORF was intact and D RNA production was blocked. These data demonstrate that the D RNA inter-feres with virus infection and therefore should be referred to as a defective interfering RNA (DI RNA). The 8K protein was shown to be a weak silencing suppressor. This study provides an example of the interplay between a pathogen and its molecular parasite where virus accumulation was differentially regulated by the 8K protein and DI RNA, indicating that they play antagonistic roles and suggesting a mechanism by which the virus can attenuate replication, decreasing viral load and thereby enhancing its effi-ciency as a parasite