Antiviral RISC mainly targets viral mRNA but not genomic RNA of tospovirus

Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated, in vitro and in planta. RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic (viral and viral-complementary strands) RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group. Author summary Plants have evolved RNA silencing as the first line of defense against virus infection. Antiviral RNA silencing as well as viral counter-defense strategies have been extensively studied for ss(+)RNA plant viruses, but less for plant negative-strand (-)/ambisense RNA plant viruses (NSVs). Upon virus infection, RNA-induced silencing complexes (RISCs) are activated to bind and cleave invasive viral RNAs. Unlike ss(+)RNA plant viruses, genomic RNA molecules of plant NSVs are not naked and tightly associate with viral nucleocapsid protein into ribonucleoproteins (RNPs), the core infectious unit for NSVs. However, their viral mRNAs are not assembled into RNPs. Whether the assembly of genomic RNA into RNPs provides another counter-defense strategy to protect against antiviral RISC activity has not been determined for any NSV. In this study, using tospovirus as a representative plant NSV model system, it is demonstrated that antiviral RISC mainly targets viral mRNAs but not genomic RNA embedded in RNPs. Our findings suggest that the RNA targets of RISC differ between plant NSVs and ss(+)RNA plant viruses and that RNPs provide another counter-defense way for NSVs to (partially) evade/escape from cytoplasmic RISC-mediated degradation.

Automated summary

This study found that antiviral RNAi mainly targets tospovirus mRNAs, while their genomic RNA is protected in RNPs against RISC-mediated cleavage. These findings suggest that the RNA targets of RISC differ between plant NSVs and ss(+)RNA plant viruses, indicating a potential counter-defense mechanism for NSVs to evade RISC-mediated degradation.