Identification of essential host factors affecting tombusvirus RNA replication based on the yeast Tet promoters Hughes Collection

Abstract

To identify essential host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model plant virus, we screened 800 yeast genes present in the yeast Tet promoters Hughes Collection. In total, we have identified 30 new host genes whose down-regulation either increased or decreased the accumulation of a TBSV replicon RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, protein metabolism/transport, or other cellular processes. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts/functions of p33 and p92(pol) viral replication proteins, (ii) the standard 10 to 20:1 ratio between p33 and p92(pol) in the viral replicase, (iii) the activity of the tombusvirus replicase, and (iv) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous genetic screening of yeast (Panavas et al., Proc. Natl. Acad. Sci. USA 102:7326-7331, 2005) led to the identification of 126 host genes (out of approximately 5,600 genes that represent approximately 95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA.

FIG. 1.

Schematic representation of launching replication of TBSV DI RNA replicon (repRNA) in yeast strains based on the yTHC collection. Tombusvirus p33 and p92^pol replication proteins are expressed constitutively from the ADH1 promoter, whereas DI-AU-FP repRNA (or the wild-type DI-72 RNA; not shown) is expressed from the regulatable GAL1 promoter. Replication of repRNA takes place in the cytoplasm (on peroxisomal membrane surfaces). The expression of a particular host gene occurs in the absence of doxycycline (−Dox) (as shown in panel A), whereas the expression of the particular host gene is switched off in the presence of doxycycline (+Dox) (as shown in panel B). Replication of DI-AU-FP repRNA with four noncontiguous regions (RI to RIV, also present in DI-72 RNA [not shown]) derived from TBSV genomic RNA and the artificial AU-FP region is shown via a minus-stranded intermediate RNA (42).

FIG. 2.

Representative group of yTHC yeast strains supporting a decreased level of TBSV repRNA accumulation in the presence of doxycycline. Panels A and B include different sets of host genes as shown. Rows A1 and B1 show Northern blot analysis of total RNA extracts from the shown yeast strains was performed with a radiolabeled RNA complementary to RIII/IV. Four independent samples are shown for each strain; two samples were grown without (−Dox) and two with (+Dox) doxycycline to illustrate the reproducibility of repRNA accumulation. An arrow points at the DI-AU-FP replicon RNA. Rows A2 and B2 show Northern blot analysis of DI-72 repRNA accumulation. The probe was complementary to RIII/IV. Rows A3 and B3 show ethidium bromide-stained agarose gel to demonstrate DI-72 repRNA accumulation in selected yeast strains. Rows A4 and B4 show ethidium bromide-stained agarose gel with rRNA as a control for yeast growth. Rows A5 and B5 show Northern blot analysis of DI-72 RNA transcripts in the absence of replication from total RNA extracts obtained from selected yTHC yeast strains coexpressing p33, but lacking p92^pol. Rows A6 and B6 show Western analysis of p33 replication protein in total protein samples using anti-His-tagged antibody. Rows A7 and B7 show Western analysis of p92^pol and rows A8 and B8 show Western analysis of p33 replication proteins in membrane-enriched fractions as described in Materials and Methods. Note that the left sample is shown for “no doxycycline” and the right sample for “plus doxycycline” in each experiment.

FIG. 3.

Identification of yTHC yeast strains supporting increased level of repRNA accumulation in the presence of doxycycline. See further details in the legend to Fig. 2.

FIG. 4.

Down-regulation of expression of selected essential yeast genes inhibits the activity of CNV replicase in vitro. (A) (Top row) In vitro activity of CNV replicase present in membrane-enriched preparations. Each replicase preparation, obtained from the yeast shown coexpressing p33, p92^pol, and DI-72⁺ RNA, was tested with the copurified endogenous template. Strains were grown in the absence (−DOX) or presence (+DOX) of doxycycline, as indicated. ³²P-labeled RNA products from the above preparations were analyzed on denaturing 5% PAGE-8 M urea gels. For quantification, we measured the intensity of ³²P-labeled RNA products by using a PhosphorImager. Activity of the CNV replicase obtained from the parental yeast strain in the absence of doxycycline corresponds to 100%. Each experiment was performed three times. (Bottom row) A Western blot shows the accumulation level of p33 in the membrane-enriched preparations. (B) Effect of down-regulation of selected essential genes on asymmetrical RNA synthesis by the CNV replicase obtained from the shown yeast strains. Unlabeled T7 RNA polymerase transcripts of DI-72⁺ (marked as “+” or “∥”) and DI-72⁻ (marked as “=”) (400 ng each), respectively, were blotted on the membrane. The blotted RNAs were then hybridized with denatured ³²P-labeled RNA probes, which were generated by the CNV replicase in vitro in a runoff experiment on the endogenous templates present in the enriched membrane fractions obtained from selected yTHC strain grown in the absence (left row) or presence (right row) of doxycycline. The ratio between plus- and minus-stranded RNAs in the in vitro replicase assay was calculated based on PhosphorImager analyses from three separate experiments. For example, the value of 13.7 means that the CNV replicase produced 13.7-fold more plus-stranded RNA than minus-stranded RNA in vitro using the endogenous template.

FIG. 5.

Down-regulation of NOG1, ARP9, and PRP5 expression alters the in vitro activity of the CNV replicase. See further details in the legend to Fig. 4.