Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses

Abstract

Viruses are devastating pathogens of humans, animals, and plants. To further our understanding of how viruses use the resources of infected cells, we systematically tested the yeast single-gene-knockout library for the effect of each host gene on the replication of tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins, and other compounds and in protein targeting/transport. Comparison with published genome-wide screens reveals that the replication of TBSV and brome mosaic virus (BMV), which belongs to a different supergroup among plus-strand RNA viruses, is affected by vastly different yeast genes. Moreover, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related alpha-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This finding suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may suggest connections between chronic diseases and prior virus infection.

Fig. 1.

A schematic presentation of expression of tombusvirus replicase genes and an RNA replicon (DI-72 RNA) in the YKO library. p33 and p92 replicase proteins are expressed constitutively from separate plasmids by using the ADH1 promoter (PADH1), whereas the precursor for DI-72 RNA is under the control of inducible GAL1 promoter (PGAL1). 6× His, 6× His-tag at the amino terminus of p33 and p92. The self-cleaving tobacco ringspot virus satellite ribozyme (Rz sat) produces an authentic 3′end in DI-72 RNA after the transcription of DI-72 precursor from pYC/DI-72 plasmid.

Fig. 2.

Comparison of viral RNA and replicase protein levels in selected YKO strains with the parental yeast strain. (A) Ethidium-bromide-stained agarose gel of total RNA obtained from selected YKO strains showing a reduced level of DI-72 RNA accumulation. The ribosomal rRNA and DI-72 RNA bands are indicated with arrows. Each strain was transformed with three plasmids (see Fig. 1). (B and C) Northern blot analysis of total RNA extracts for DI-72 RNA (B) with the 3′ end of DI-72 as a probe and for DI-72 RNA transcript (C) with a probe for the 5′ plasmid-borne leader sequence, which is deleted in the replicating DI-72 RNA (13). (D and E) Western analysis of p33 and p92 replicase proteins in total protein samples with anti-His-tag antibody.

Fig. 3.

Increased viral RNA replication in six YKO strains. Northern blot and Western analyses were performed as described in the legend to Fig. 2.

Fig. 4.

Comparison of relative abundance of yeast genes with known functions within selected functional categories known to enhance the cytotoxicity of α-synuclein (35) or mutated huntingtin and affect tombusvirus replication. We included those genes from the three genome-wide screens, which matched or were known to have similar functions. The data for comparison are taken from ref. and Table 1. YKO, the relative abundance of genes with given functions among nonessential genes in the YKO collection. The standard error is shown.