Host co-factors of the retrovirus-like transposon Ty1

Abstract

Background: Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event.

Results: To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ.

Conclusion: Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 cDNA include characterized RNA metabolism and modification genes, consistent with their having roles in early steps in retrotransposition such as expression, nuclear export, translation, localization, or packaging of Ty1 RNA. Our results suggest that Bud21, Hcr1, Loc1, and Puf6 promote efficient synthesis or stability of Ty1 Gag.

Figure 1

Modified synthetic genetic array screen for rhfΔ mutants. (A) Schematic of the Ty1his3AI element that is used to assay the frequency of retrotransposition. Ty1 long terminal repeats are represented as tripartite black and white boxes that flank internal region of the element. The internal region has two partially overlapping ORFs, gag and pol (green rectangles). As illustrated, the gag ORF begins in the 5′ LTR. The his3AI retrotransposition indicator gene is inserted in non-coding DNA between the end of pol and the beginning of the 3′ LTR. The his3AI gene consists of a HIS3 gene (red rectangles) in the opposite orientation to gag and pol. HIS3 is interrupted by insertion of an artificial intron (AI; blue rectangle) in an unspliceable orientation; however, AI is spliced from the Ty1his3AI transcript. When splicing occurs and the transcript is reverse transcribed, a Ty1HIS3 cDNA is produced, which, when integrated into the genome, confers a His⁺ phenotype. (B) Schematic of the genetic manipulations used to generate haploid progeny containing the Ty1his3AI element, the rtt101Δ or med1Δ mutation, and an orfΔ mutation. The query strain containing the rtt101Δ or med1Δ and the Ty1his3AI-MET15 allele was mated to each orfΔ::kanMX strain in the yeast ORF deletion library. Following induction of transposition by growth of cells in YEPD broth at 20°, progeny were plated on YEPD agar containing G418 to assess growth (not shown) and SC -His agar to measure retrotransposition. (C) The results of the Ty1his3AI retrotransposition assay on one SC -His plate of rtt101Δ: LEU2orfΔ:kanMX progeny. Cells that sustained a retrotransposition event give rise to His⁺ papillae, which were counted at each address. Addresses that are blank lack progeny because of synthetic lethality or slow growth (green circles). Addresses with ≤5 His⁺ papillae (red circles) harbor progeny with reduced retrotransposition. The parental rtt101Δ strain (blue circle) was plated in an empty address prior to induction of retrotransposition.

Figure 2

Reproducibility of results of Ty1 his3AI retrotransposition assay across 10 trials. Progeny of 94 orfΔ::kanMX strains on one plate and the rtt101Δ query strain were isolated 10 independent times, and retrotransposition was measured in all 940 isolates. The fraction of trials at each address that yielded a ≥5-fold reduction in His⁺ papillae formation relative to the rtt101Δ query strain is plotted on the x-axis. The percentage of addresses within each category is plotted on the y-axis.

Figure 3

Frequency of distribution of suppressors of rtt101Δ hypertransposition, suppressors of med1Δ hypertransposition, RHF genes, and all yeast genes in Gene Ontology molecular function categories. The histogram indicates the percent of the total number of genes in each gene set that are found in each GO molecular function category. The GO categories were assigned using yeast GO slim mapper ( www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl).

Figure 4

Levels of Ty1 retromobility, RNA, and Gag: GFP protein in six rhfΔ mutants with defects in ribosome biogenesis. (A) The frequency of His⁺ prototroph formation (retrotransposition) in wild-type strain JC3807 (WT) and congenic rhfΔ derivatives harboring a chromosomal Ty1his3AI element. The frequency reported for the dbp7Δ strain is the maximum possible frequency determined as if one His ⁺ colony had formed in each independent culture tested. Error bars: standard error. (B) Northern blot analyses of Ty1 RNA (top panel) and PYK1 RNA (bottom panel) in each strain, using ³²P-labeled riboprobes. The ratio of ³²P activity in the Ty1 band to ³²P activity in the PYK1 band was determined by phosphorimaging. Ty1/PYK1 RNA ratios for each strain normalized to that of the wild-type strain are provided below each lane. (C) The average level of Ty1 RNA in total RNA from three biological replicates of each strain relative to the wild-type strain was determined by qPCR analysis (left panel). The spt3Δ strain is a negative control. The average level of RNA derived from the Ty1(gag::GFP)-3566 chromosomal element in total RNA from three biological replicates of the wild type strain and the congenic bud21Δ derivative was measured by qPCR analysis. Error bars: standard error. (D) Western blot analyses of total cell lysate with anti-VLP antibody, which recognizes unprocessed p49-Gag and processed p45-Gag (top panel), and anti-alpha-tubulin antibody (bottom panel) as a loading control. (E) Histogram of the mean Gag:GFP fusion protein activity, as measured by flow cytometry, in rhfΔ strains relative to the wild-type strain. Each strain harbors the chromosomal Ty1(gag::GFP)-3566 element. Error bars: standard error.