Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

Abstract

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

Figure 1.

Mutational analysis of Ty1i RNA initiation codons AUG1 and AUG2. (A) Map of the Ty1 and pGTy1 mutant derivatives used to assess translation of Ty1i RNA from two closely spaced initiation codons AUG1 and AUG2. pGPOLΔ lacks most of POL (22), but contains GAG, including sequences required for transcribing Ty1i RNA and cleavage of p22 to p18 by Ty1 protease (PR). (B) pGPOLΔ (URA3, 2μ) plasmids were introduced into a Ty1-less Saccharomyces paradoxus strain containing a single chromosomal Ty1his3-AI element. Cells propagated on glucose are repressed for transcription of Ty1 mRNA from GAL1 promoter, but allow synthesis of Ty1i RNA and p22/p18. Ty1his3-AI mobility analyses using this assay are shown in Table 1. (C) Total cell protein isolated after growth in SC-Ura medium for 2 days at 22°C was immunoblotted with p18 antiserum (anti-p18). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.

Figure 2.

Functional organization of GAG and coexpression of subgenomic segments with pGTy1his3-AI. (A) At the top is the mature Gag (p45) coding sequence with selected ATG codons highlighted (green) and below are segments expressed ectopically from the pYES2 GAL1 promoter. At the bottom, XtalPred (http://ffas.burnham.org/XtalPred-cgi/xtal.pl) was used to predict Gag disordered (yellow) and α-helical (red) regions. Also shown is an invariant tryptophan residue (W184) found in Pseudoviridae Gag proteins (19), the position of the nucleic acid chaperone region (21), a C-terminal disordered region (C-DR), and the Ty1 protease (PR) cleavage site (H401-N402). An ATG codon was added adjacent to P173 for expression of the CTR and sCTR. (B) Two plasmids, pGTy1his3-AI (TRP1, CEN) and GAG segment under pYES2 (URA3, 2 μ) were induced from the GAL1 promoter in a Ty1-less Saccharomyces paradoxus strain to determine whether different Gag proteins inhibited Ty1his3-AI mobility (Table 2). (C) Total cell protein from induced cultures was immunoblotted with p18 antiserum (anti-p18) or TY-tag (anti-TY-tag). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.

Figure 3.

In vitro translation of Ty1 RNAs in wheat germ extract (WGE). In vitro transcribed RNA AUG1AUG2, GCG1AUG2 and AUG1GCG2 were translated in WGE in the presence of ³⁵S-methionine followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis electrophoresis and autoradiography. Quantitation of the translation products normalized to the level of Renilla Luciferase is shown below the gel (SD ≤ 0.09).

Figure 4.

Nucleic acid aggregation and chaperone activities of Ty1 Gag-derived proteins. Percent of (A) Ty1 mini RNA (560-nt) and (B) TAR RNA and DNA aggregated by each protein at 24°C. Aggregation assays were performed as a function of protein concentration as indicated. Proteins concentrations correspond to 1:28; 1:14; 1:9; 1:7 and 1:5.6 protein to nt ratios, respectively. The graphs represent averaged data from three independent experiments. The error bars represent standard deviations. (C) Schematic of the DNA/RNA strand annealing reaction. The annealing assays were performed using TAR(−) DNA/TAR RNA substrates as a function of protein concentration (0; 0.1; 0.2; 0.3; 0.4; 0.6 μM). Protein concentrations correspond to 1:7.8; 1:4; 1:2.6; 1:2 and 1:1.3 protein to nt ratios. (D) A representative electrophoretic analysis of TAR(−) DNA/TAR RNA annealing. Lane 1 contains a control sample (denoted C) lacking protein and lanes 2–6, 7–11, 12–16 and 17–21 contain increasing amounts of CTR, sCTR, AUG2p18 and AUG1p18, respectively, as described above. (E) The graphs represent averaged data from three independent annealing experiments for each protein. The error bars represent standard deviations.

Figure 5.

RNA binding properties of CTR, AUG2p18 and AUG1p18 proteins. Data plots of the filter-binding assay performed in different concentrations of NaCl (10–500 mM) for Ty1 mini RNA and (A) CTR, (B) AUG2p18 and (C) AUG1p18. The lines correspond to the best fit of the data. (D) Data plot of the dissociation constant measured for CTR, AUG2p18 and AUG1p18 as a function of NaCl concentration. The error bars represent standard deviations. (E) 2D structure model of +1–362 region (35) of Ty1 mini RNA with the positions protected from hydroxyl radical cleavage in the presence of the Ty1 Gag derived proteins marked (red). (F) Reactivity plots of protein free Ty1 mini RNA (black) in comparison with RNA probed in the presence of CTR (red) and p18 (green). Regions showing consistent increased reactivity over several nucleotides are boxed.

Figure 6.

Protein-mediated annealing of tRNA_i^Met with Ty1 RNA. (A) A representative electrophoretic analysis in the presence of increasing concentrations (0; 0.75; 1.5; 3; 6 μM) of CTR, AUG2p18 or AUG1p18. Proteins concentrations correspond to 1:8; 1:4; 1:2 and 1:1 protein to nt ratios. Lanes 1, 6 and 11 (denoted C) represent control samples that lack protein and lanes 2–5, 7–10 and 12–15 contain increasing amounts of CTR, AUG2p18 and AUG1p18, respectively, as described above. (B) The graph representing the averaged percent of annealed tRNA_i^Met from three independent experiments for CTR, AUG2p18, AUG1p18 or sCTR protein. The error bars represent standard deviations. (C) A representative electrophoretic analysis performed in the presence of CTR (constant concentration of 1.5 μM) and increasing concentrations of sAUG2 (0.75, 1.5, 3, 6 μM). Lane 1 (denoted C) is a control sample lacking protein and lanes 2–7 contain increasing amounts of sAUG2 relative to CTR protein, as described above. (D) The graph representing the averaged percent of annealed tRNA_i^Met in the presence of both proteins. Respective molar protein ratios are indicated.

Figure 7.

GST-CTR interacts with p18 or deleted CD-R p18. Protein extracts (Input) from a Ty1-less strain induced for expression of GST [pEG(KT); pGST], GST-CTR (pBDG1496; pGST-CTR) and (A) p18 (pAUG1p18) or (B) p18 lacking the C-DR (pBDG1612; psAUG1) were incubated with glutathione-coated resin. Bound proteins (Pull-down) were analyzed by immunoblotting with GST and p18 antisera to detect, GST, GST-CTR, p18 or deleted C-DR p18 after extensive washing with lysis buffer. A putative p18 degradation product of ∼12.5 kDa is noted in panel A (*; also refer to Supplementary Figure S7).

Figure 8.

Impact of p22/p18 or p18 on RNA dimerization and packaging. (A) A representative electrophoretic analysis in the presence of increasing concentrations of CTR, AUG2p18 or AUG1p18. Proteins concentrations correspond to 1:20; 1:10; 1:7 and 1:5 protein to nt ratios. Lanes denoted C represent control samples that lack protein. (B) The graph representing the averaged percent of Ty1 mini RNA dimer from three independent experiments. The error bars represent standard deviations. (C) Nuclease protection of Ty1 mRNA in cells coexpressing pGTy1his3-AI alone and pAUG1p22 or pAUG1p18. Equal aliquots of whole-cell extracts from galactose-induced cells were incubated with (+) or without (−) the nuclease benzonase. RNA extracted from these samples was analyzed by Northern blotting using a ³²P-labeled riboprobe specific for Ty1 mRNA. Protection from benzonase is expressed as a ratio of treated to untreated Ty1 mRNA. ACT1 was used as a control to confirm RNA degradation in the nuclease treated samples. (D) Total protein extracts used in (C) was immunoblotted with p18 antiserum (anti-p18). Histidyl tRNA synthetase (anti-Hts1) served as a loading control.