T-body formation precedes virus-like particle maturation in S. cerevisiae

Abstract

T-bodies are localized S. cerevisiae RNPs containing Ty1 retroviral components and speculated to play a role in the assembly of virus-like particles (VLPs). Mapping requirements for T-body formation, we demonstrate that ectopic expression of immature TyA1/Gag (Gag-p49), a structural component of the Ty1 capsid, is sufficient for T-body formation both under normal conditions as well as in a strain background devoid of endogenous Gag. Moreover, T-bodies are readily formed when Ty1 transposition is blocked. Thus, T-bodies represent an early stage in the Ty1 life cycle, preceding VLP maturation.

Figure 1

Validation of the tagTy1 construct as a tool to study T-body biology. (A) Schematic representations of the PgalTy1H3 his3AI (tagTy1) and the endogenous Ty1 (endTy1) elements showing regions recognized by the Ty1-INT (dark grey stippled box) and Ty1-h (light grey stippled box) probes. Other features highlighted are the Ty1 LTRs (grey triangles); the GAL1 promoter driving transcription of tagTy1 (black box); the his3AI tag region (red box) and the Ty1 RNAs (thin arrows). (B) Ty1 northern analysis of total RNA harvested from wt cells transformed with ptagTy1 plasmid and grown at 25°C in AA-Ura media with different carbon sources as indicated. rRNA 25S was probed as an internal standard. RNAs were visualized using 5′end radiolabeled Ty1-INT, Ty1-h and rRNA probes, respectively. (C) Cells grown in glucose (repression) or galactose (induction) were fixed and processed for tagTy1 specific RNA-FISH using a Cy3-labeled Ty1-h probe. The chromatin-rich parts of yeast nuclei were visualized by DAPI staining and overlaid with the Cy3 signal. The fraction of T-body containing cells is indicated. (D) Cells grown in galactose-containing medium and fixed as in (C) were co-hybridized with FITC-labeled Ty1-h and Cy3-labeled Ty1-INT probes (upper parts). As a control for fluorescence bleeding, single FITC-labeled Ty1-h hybridized cells are shown in the lower parts. The letter coloring indicates whether the probe was labeled with Cy3 (red) or FITC (green). The fraction of cells containing overlapping Cy3 and FITC signals is indicated.

Figure 2

Gag-p49 expression is sufficient for T-body formation. (A) Schematic representation of investigated constructs, indicating the EcoNI [E], SphI [S] and BstEII [B] restriction sites, the TYA DNA region (dark grey solid bar) and the grey Gag variants (grey stippled bar). The Gag variants predicted to be expressed by each of the constructs are indicated. Ribosomal frameshifting (FS). (B) Northern analysis of total RNA harvested from cells transformed with an empty vector, ptagTy1, ptagTYA or ptagTYA C-terminal deletions (tagTYAΔEB and -ΔSB) as well as Klenow treated restriction digests (tagTYA-E* and -S*) as indicated. Cell growth and northern hybridization conditions were as in Figure 1B. (C) RNA-FISH and Gag-IF analysis of cells transformed with an empty vector, ptagTy1, ptagTYA or ptagTYA-S* using Cy3-labeled Ty1-h or Ty1-INT probes, or the BB2 anti-Gag primary antibody followed by an anti-mouse FITC conjugated secondary antibody. The fractions of cells containing cytoplasmic foci defined by either RNA or protein detection are indicated. (D) western blotting analysis of extracts from cells grown as in (B) using the BB2 anti-Gag antibody. The bands corresponding to Gag-49 and Gag-p45 are indicated. Two differentially loaded gels were run. An anti-Nop1p antibody was used to estimate the relative protein concentrations of the extracts. Cell growth, fixation and image treatment were as in Figure 1C.

Figure 3

Endogenous Ty1 components are not required for Gag-p49 induced T-body formation. (A) MFA1 and mfa1Δ cells were grown to exponential phase in rich media at 25°C and processed for RNA-FISH and IF using a Cy3-labeled Ty1-INT probe and the BB2 anti-Gag antibody, respectively. The fractions of cells containing cytoplasmic foci detected by each reagent are indicated. (B) Western blotting analysis of protein extract prepared from MFA1 and mfa1Δ transformed with an empty vector, ptagTy1 or ptagTyA performed as in Figure 2D. (C) Cells from (B) were fixed and processed for RNA-FISH (Cy3-labeled Ty1-h probe) and IF (BB2 anti-Gag antibody). The fractions of cells containing cytoplasmic foci detected by each reagent are indicated.

Figure 4

T-body formation occurs in the transposition incompetent xrn1Δ strain. XRN1 and xrn1Δ cells transformed with an empty vector or ptagTy1 were grown exponentially in AA-Ura⁺ galactose medium and processed for RNA-FISH (Cy3 labeled Ty1-INT) and IF (BB2 anti-Gag antibody). The fractions of cells containing cytoplasmic foci detected by either of the reagents are indicated.