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. 2025 Oct 9;21(10):e1011898.
doi: 10.1371/journal.pgen.1011898. eCollection 2025 Oct.

Probing the molecular determinants of Ty1 retrotransposon restriction specificity in yeast

Sean L Beckwith  1   2 , Matthew A Cottee  3 , J Adam Hannon-Hatfield  1 , Abigail C Newman  1 , Emma C Walker  1 , Justin R Romero  2 , Jonathan P Stoye  4 , Ian A Taylor  3 , David J Garfinkel  1
Affiliations

Probing the molecular determinants of Ty1 retrotransposon restriction specificity in yeast

Sean L Beckwith et al. PLoS Genet. .

Abstract

The evolutionary history of retrotransposons and their hosts shapes the dynamics of transposition and restriction. The Pseudoviridae of yeast includes multiple Ty1 LTR-retrotransposon subfamilies. Saccharomyces cerevisiae prevents uncontrolled retrotransposition of Ty1 subfamilies using distinct mechanisms: canonical Ty1 is inhibited by a self-encoded restriction factor, p22/p18, whereas Ty1' is inhibited by an endogenized restriction factor, Drt2. The minimal inhibitory fragment of both restriction factors (p18m and Drt2m) is a conserved C-terminal capsid domain. Here, we use biophysical and genetic approaches to demonstrate that p18m and Drt2m are highly specific to their subfamilies. Although the crystal structures of p18m and Drt2m are similar, three divergent residues found in a conserved hydrophobic interface direct restriction specificity. By mutating these three residues, we re-target each restriction factor to the opposite transposon. Our work highlights how a common lattice-poisoning mechanism of restriction evolved from independent evolutionary trajectories in closely related retrotransposon subfamilies. These data raise the possibility that similar capsid-capsid interactions may exist in other transposons/viruses and that highly specific inhibitors could be engineered to target capsid interfaces.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Subfamily specific restriction of Ty1 elements.
(A) Schematic contrasting the two restriction mechanisms evolved for Ty1c and Ty1’: the self-encoded p22 protein restricts Ty1c whereas an endogenized gene at a fixed locus, DRT2, expresses an inhibitory protein against Ty1’. (B) Schematic illustrating the transposon expression plasmid and the chromosomal DRT2 restriction factor. pTy1chis3-AI is on a URA3 marked vector and pTy1’his3-AI is on a TRP1 marked vector. Each transposon is expressed under its native promoter and contains the his3-AI retromobility indicator gene; histidine prototrophy requires retromobility. (C) Quantitative mobility assay. Each bar represents the mean of the four independent measurements displayed as points. The error bar center represents the mean of the four measurements and the error bar extent ± the standard deviation. Significance is calculated from a two-sided Student’s t-test compared with wildtype (n.s not significant, *** p < 0.001. Exact p-values are provided in S1 Table). Significant fold-change in restriction compared to wildtype is indicated above the bars. (D) Sequence alignment of p18m and Drt2m restriction factors. Numbering is according to the equivalent position in Ty1c Gag. Positions of α-helices observed in crystal structures are indicated by the green bars above the alignment. Divergent residues are indicated by boxes; thick red boxes indicate Dimer-1 interface residues where p18m differs from Drt2m. Gray shaded residues indicate residues where Dimer-2 suppression serine mutations have been made. (E) Bar representation of Gag and CA, residue numbers correspond to the Gag sequence. The CA-NTD is colored dark blue and CA-CTD is orange. (F) p18-type restriction factors, residue numbers correspond to the Gag sequence. p18m is colored orange like Ty1c CA-CTD and Drt2m is light blue. (E & F) VTF and AVL indicate the tri-point mutations that convert the Ty1c CA or p18m to a Drt2m interface (AVL to VTF) and the Drt2m to a Ty1c interface (VTF to AVL).
Fig 2
Fig 2. Crystal structures of p18m and Drt2m restriction factors.
Crystal structures of (A) p18m and (B) Drt2m(SS) dimers. The protein backbone is shown in cartoon representation, p18m in orange and Drt2m in pale blue. Helical secondary structures in each monomer are labelled sequentially from N- to C-terminus. (C) 3D structural superposition of p18m and Drt2m dimers. Coloring and orientation are the same as in A and B. Structures were aligned using 155 backbone Cα atoms, yielding an RMSD of 0.7 Å. (D) p18m and (E) Drt2m Dimer-1 interfaces. The view is perpendicular to (upper) and into (lower) the interface of a single monomer. The protein mainchain is shown in gray cartoon. Residues that make apolar and salt bridge interactions at the interface are shown in stick representation. Specificity determining residues in p18m (A266, V270 and L312) and Drt2m (V266, T270 and F312) are highlighted with bold labelling and shown in light brown and green respectively. (F & G) View of the Dimer-1 interface of (F) p18m and (G) Drt2m. View and cartoon coloring is the same is A and B. The variable interfacial residues (A/V)266, (V/T)270 and (L/F)312 together with R315 on helices α1 and α3 are shown in stick representation. The T270-R315 hydrogen bonding interaction at the Drt2m interface is indicate by the purple dashes. Red crosses indicate a lack of hydrogen bonding in p18m interface.
Fig 3
Fig 3. Sedimentation equilibrium analysis of p18m and Drt2m self-association.
(A and B) Upper panels are the multi-speed sedimentation equilibrium profiles determined from interference (left) and absorbance (right) data collected on (A) p18m(F323S) at 45 µM and (B) Drt2m(SSS) at 44 µM. Data was recorded at the speeds indicated. Data points in each panel are colored according to the speed. The solid lines represent the global best fit to the data using a monomer-dimer-tetramer model (p18m(F323S); KD(1-2) = 0.71 µM, KD(2-4) = 30.5 µM, reduced χ2 = 0.407) and (Drt2m(SSS); KD(1-2) = 0.48 µM, KD(2-4) = 277 µM; reduced χ2 = 0.190). The lower panels are the residuals to the fit, the color of points corresponds to that of the fitted profile in the upper panels, see also S3 Table.
Fig 4
Fig 4. SEC-MALLS analysis of restriction factor specificity.
SEC-MALLS analysis of (A-D) Ty1c CA(F323S) and (E-H) Ty1c CA-VTF(F323S) interaction with p18m(F323S), p18m-VTF(F323S), Drt2m(SSS) and Drt2m-AVL(SSS) restriction factors. In each panel, the differential refractive index (dRI) is plotted against column retention time for sample loadings of 50 µM Ty1c CA(F323S) or Ty1c CA-VTF(F323S) (green), 50 µM p18m(F323S), p18m-VTF(F323S), Drt2m(SSS) or Drt2m-AVL(SSS) (blue) and 50 µM equimolar mixtures (red) as indicated. The molar mass, determined at 1-second intervals throughout the elution of peaks, is plotted as points. The molecular masses of p18m or Drt2m homodimers, p18m-Ty1c CA or Drt2m-Ty1c CA heterodimers and the Ty1c CA or Ty1c CA-VTF homodimers are indicated with the gray dashed lines.
Fig 5
Fig 5. Restriction factor specificity in vivo.
(A) Schematic illustrating the two plasmids used to co-express the transposon and restriction factor (RF). The expression of each is driven by a galactose-inducible promoter from the GAL1 gene. The transposon is marked with the his3-AI retromobility indicator gene; histidine prototrophy requires retromobility. (B) Western blot of restriction factor and Gag expression. Protein extracts of galactose-induced cells were immunoblotted with an anti-hexa-histidine antibody to detect restriction factors, anti-TY tag to detect Ty1c Gag, and anti-p18’ to detect Ty1’ Gag. Pgk1 serves as a loading control. Migration of molecular weight standards is shown alongside each immunoblot. A representative image of at least 3 replicates is shown; original images of entire-gel immunoblots are provided in the Supporting Information. (C) Quantitative mobility assay of galactose-induced cells. Each bar represents the mean of the four independent measurements displayed as points. The error bar center represents the mean of the four measurements and the error bar extent ± the standard deviation. Fold restriction is plotted compared to empty vector. Error bars are omitted for Ty1’-AVL with Drt2m-AVL in which no retromobility events were observed; instead, the value graphed represents the theoretical maximum fold restriction if one retromobility event had been observed. Significance is calculated from a two-sided Student’s t-test compared with empty (n.s not significant. Exact p-values are provided in S1 Table). Bar color denotes homotypic or heterotypic interactions at Dimer-1. The cartoon below illustrates the Dimer-1 residues present in the restriction factor and Gag in each bar.
Fig 6
Fig 6. Restriction factor disruption of particle assembly.
Protein extracts from galactose-induced yeast cells (Input) were fractionated over a 7-47% (w/v) continuous sucrose gradient and immunoblotted with anti-TY tag for Gag and anti-hexahistidine for restriction factors. The bars below Gag blots denote peak Gag fractions containing more than 1/9 of the Gag signal across the gradient, as determined by densitometric analysis. A representative image of at least 3 replicates is shown. Migration of molecular weight standards is shown alongside the immunoblots. Images of the whole gel immunoblots are provided in the Supporting Information. The cartoon to the right illustrates the restriction factor and Gag present in each strain with the color denoting the Dimer-1 residues of each.
See this image and copyright information in PMC

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