Screening of the Yeast yTHC Collection Identifies Essential Host Factors Affecting Tombusvirus RNA Recombination

Abstract

RNA recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing ϳ80% of all genes of yeast, led to the identification of 11 host genes affecting RNA recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV recombination and evolution.RNA viruses are successful pathogens because they are capable of rapid evolution that helps them to overcome host resistance and other antiviral strategies (13,14,17,27,54,55,64). RNA recombination, the joining of two noncontiguous RNA segments together, is an especially powerful tool for viruses to create new resistance-breaking or drug-resistant strains and/or viruses (27,64). Accordingly, the generation of novel recombinant RNAs (recRNAs) has been described for many human, animal, and plant viruses as well as RNA bacteriophages (1,4,5,11,16,21,23,24,27,32,42,59,64,65).Progress in our understanding of viral RNA recombination has been slowed down by the difficulty of detection of new recRNAs, the adverse selection pressure on some recRNAs, and the poor predictability of recombination events. Development of powerful model RNA recombination systems, however, has revealed many unique features of viral RNA recombination. For example, sequencing of numerous recRNAs in Brome mosaic virus (BMV) (3,33,36,53), Turnip crinkle virus (TCV) (6,7,37,38,40), and tombusviruses (61, 62) established that recombination does not occur randomly within the viral RNA genome but rather, there are recombination "hot spots". These include AU-rich sequences (31, 34, 58), inter-or intramolecular secondary structures (19,35,62), and cis-acting RNA elements with high affinity toward the viral replicase (8,10,40). Mutagenesis of the replicase proteins has led to altered recombination frequencies or altered the sites of recombination (15,30,47), suggesting that many recombination events are due to template switching (replicase jumping) by the viral replicase (22,27,39...

An attempt to identify recombinants between two sobemoviruses in doubly infected oat plants

“…Screening the recombination efficiency of TBSV using a yeast single-knockout library revealed that host genes involved in RNA degradation were suppressing the generation of new viral RNA recombinants. In contrast, genes contributing to the intracellular transport of proteins were identified as viral RNA recombination accelerators (Serviene et al, 2005;Serviene et al, 2006). In this study, approximately one quarter of the CfMV and RGMoV genomes were monitored with highly sensitive and specific RT-PCR for the generation of recombinant molecules in co-inoculated oat plants.…”

A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis

“…Because of their limited genetic complement, viruses need to recruit numerous host factors to be able to replicate and move inside the infected plant. This is exemplified by genome-wide screens performed in yeast, in which over 100 genes with diverse functions and involved in a variety of cellular processes turned out to have an effect on the replication and recombination of Tomato bushy stunt virus (TBSV; Panavas et al, 2005;Serviene et al, 2005Serviene et al, , 2006. TBSV belongs to the Tombusviridae family, whose members, like potyviruses, contain linear single-stranded (+) RNA genomes.…”

Genome 3′-end repair in dengue virus type 2

“…Tomato bushy stunt virus recombination in a model host, yeast Saccharomyces cerevisiae, is enhanced 10-to 50-fold in host XRN1 5-39 exoribonuclease-negative mutant cells (Serviene et al 2006). Various housekeeping RNases that are abundantly distributed in intracellular compartments such as lysosomes, endoplastic reticulum, and ribosomes also could potentially act on the 39 termini of viral RNAs (Irie 1999).…”