Role of RNase MRP in viral RNA degradation and RNA recombination.

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

The combined effect of environmental and host factors on the emergence of viral RNA recombinants.

Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.

A host Ca2+/Mn2+ ion pump is a factor in the emergence of viral RNA recombinants.

Viruses change rapidly due to genetic mutations, and viral RNA recombination in RNA viruses can lead to the emergence of drug-resistant or highly virulent strains. Here, we report that host Pmr1p, an ion pump that controls Ca2+/Mn2+ influx into the Golgi from the cytosol, affects the frequency of viral RNA recombination and the efficiency of replication. Inactivation of PMR1 leads to an approximately 160-fold increase in RNA recombination of Tomato bushy stunt virus (TBSV) in yeast, a model host. Expression of separation-of-function mutants of Pmr1p reveals that the ability of Pmr1p to control the Mn2+ concentration in the cytosol is a key factor in viral RNA recombination. Indeed, a high Mn2+ concentration in a cell-free TBSV replication system increases the recombination frequency, and knockdown of Ca2+/Mn2+ exporters in plants increases virus replication and RNA recombination. Thus, a conserved host protein could affect the adaptive evolution of RNA viruses.

Silencing of Nicotiana benthamiana Xrn4p exoribonuclease promotes tombusvirus RNA accumulation and recombination.

The cytosolic 5'-to-3' exoribonuclease Xrn1p plays a major role in recombination and degradation of Tomato bushy stunt tombusvirus (TBSV) replicon (rep)RNA in yeast, a model host (Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., and 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.). To test if the plant cytosolic 5'-to-3' exoribonuclease Xrn4p, similar to the yeast Xrn1p, could also affect TBSV recombination, in this paper, we silenced XRN4 in Nicotiana benthamiana, an experimental host. The accumulation of tombusvirus genomic RNA and repRNA increased by 50% and 220%, respectively, in XRN4-silenced N. benthamiana. We also observed up to 125-fold increase in the emergence of new recombinants and partly degraded viral RNAs in the silenced plants. Using a cell-free assay based on a yeast extract, which supports authentic replication and recombination of TBSV, we demonstrate that the purified recombinant Xrn1p efficiently inhibited the accumulation of recombinants and partly degraded viral RNAs. Altogether, the data from a plant host and cell-free system confirm a central role for the plant cytosolic 5'-to-3' exoribonuclease in TBSV replication, recombination and viral RNA degradation.

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

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.

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins.

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.