Mapping of sequences in the 5' region and 3' UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

Exploring the Diversity of Mechanisms Associated With Plant Tolerance to Virus Infection.

Tolerance is defined as an interaction in which viruses accumulate to some degree without causing significant loss of vigor or fitness to their hosts. Tolerance can be described as a stable equilibrium between the virus and its host, an interaction in which each partner not only accommodate trade-offs for survival but also receive some benefits (e.g., protection of the plant against super-infection by virulent viruses; virus invasion of meristem tissues allowing vertical transmission). This equilibrium, which would be associated with little selective pressure for the emergence of severe viral strains, is common in wild ecosystems and has important implications for the management of viral diseases in the field. Plant viruses are obligatory intracellular parasites that divert the host cellular machinery to complete their infection cycle. Highjacking/modification of plant factors can affect plant vigor and fitness. In addition, the toxic effects of viral proteins and the deployment of plant defense responses contribute to the induction of symptoms ranging in severity from tissue discoloration to malformation or tissue necrosis. The impact of viral infection is also influenced by the virulence of the specific virus strain (or strains for mixed infections), the host genotype and environmental conditions. Although plant resistance mechanisms that restrict virus accumulation or movement have received much attention, molecular mechanisms associated with tolerance are less well-understood. We review the experimental evidence that supports the concept that tolerance can be achieved by reaching the proper balance between plant defense responses and virus counter-defenses. We also discuss plant translation repression mechanisms, plant protein degradation or modification pathways and viral self-attenuation strategies that regulate the accumulation or activity of viral proteins to mitigate their impact on the host. Finally, we discuss current progress and future opportunities toward the application of various tolerance mechanisms in the field.

Expression and antiviral function of ARGONAUTE 2 in Nicotiana benthamiana plants infected with two isolates of tomato ringspot virus with varying degrees of virulence.

ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.