Expression of the Arabidopsis Xrn4p 5′–3′ exoribonuclease facilitates degradation of tombusvirus RNA and promotes rapid emergence of viral variants in plants

Abstract

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.

Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast

“…The recRNA nature of the multimeric RNAs was confirmed by reverse transcription-PCR, which selectively amplifies only the recRNA (7,42). Based on these data, we conclude that the cell-free replicase preparation is capable of authentic RNA recombination, generating recRNAs comparable to those seen in vivo in yeast and plant cells (4,7,42).…”

“…Similar-size RNAs have also been detected in vivo in yeast and plant cells during replication of DI-72(ϩ) repRNA (7,42). Sequencing the in vivo-generated longer-than-full-length RNA species has conclusively demonstrated the recombinant nature of these RNAs, which consisted of either a full-length head-to-tail dimer or the shorter dimeric RNA with 5Ј truncations (the recRNAs were missing the 5Ј RI and partly RII sequences) (4,7,42). These recRNAlike RNAs were not present when no DI-72(ϩ) repRNA was added to the assay mixture (Fig.…”

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

“…These RNA recombinants in the TBSV system are generated via the template-switching mechanism by the viral replicase using viral repRNA templates that are cleaved by cellular endo-and exoribonucleases (schematically shown in Fig. 1A) (14,15,21,22,24,50,51). In addition, yeast expressing the rpn11 -14 ts mutant as the only copy of Rpn11p led to an ϳ11-fold increase in TBSV recRNA levels at the semipermissive temperature (Fig.…”

“…For example, the cytosolic Xrn1p 5=-to-3= exoribonuclease (Xrn4 in plants) suppresses TBSV recombination via rapidly degrading TBSV RNAs (degRNAs) (Fig. 1A) cleaved by cellular endoribonucleases (22)(23)(24)(25). Thus, the degRNAs have only limited time to participate in RNA recombination in wild-type (wt) cells.…”

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

“…Empty vector pGD-35S was used as a negative control. Transformed Agrobacterium cells were selected, grown, and infiltrated into young N. benthamiana leaves as described earlier (63). Agrobacterium cultures transformed with pGD-p19 with an optical density at 600 nm (OD 600 ) of 0.5 were mixed separately with Agrobacterium cultures, transformed with pGD-35S, pGD-dnBro1, pGD-dnVps23-1, or pGD-dnVps23-2 (OD 600 , 0.5) or mixtures of cultures pGD-dnBro1 with pGD-dnVps23-1 or pGD-dnBro1 with pGDdnVps23-1 (OD 600 , 0.25 each).…”