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.

Introduction

Ty1 is an endogenous S. cerevisiae LTR-retrotransposon belonging to the pseudoviridae family. Ty1 RNA and Gag protein locally concentrate in a cytoplasmic region, the Ty1- or T-body, with a putative role as Ty1 VLP assembly site.^1,2 Ty1 retrotransposons contain two partially overlapping ORFs, TYA1 and TYB1, that encode for TyA1/Gag and TyB1/Pol proteins (hereafter denoted Gag and Pol), but lack a functional homolog of the Env protein, essential for infectivity.^3,4 While Gag can be readily translated as an immature Gag-p49 form, Pol synthesis requires a ribosomal frameshift, creating a template for the production of a Gag-Pol fusion protein that is further processed by proteolysis.^5,6 Similar to retroviruses, Ty1 genomic RNA, Gag and unprocessed Gag-Pol assemble to form immature VLPs.⁷ VLP maturation, including the proteolytic conversion of Gag-p49 into Gag-p45 and the precise cleavage of Gag-Pol, both achieved by a protease function contained in Pol, is a prerequisite for Ty1 RNA reverse transcription and transposition.^8,9

In the haploid genome of most S. cerevisiae laboratory strains, Ty1 elements are present in ∼30 copies responsible for an impressive 8–10% of steady state total cellular mRNA. This level is significantly reduced in diploid cells due to the mating type dependent transcriptional regulation of Ty1. Individual Ty1 copies vary with respect to sequence, expression level and transposition activity,^3,10,11 making precise analysis of Ty1 biology rather cumbersome. In this paper, we use the PgalTy1H3 his3AI element¹² as a model system to show that an undisrupted TYA ORF is both necessary and sufficient to form T-bodies. Our data support a role of the T-body in the initial assembly of immature VLPs during the transposition cycle.

Results

To be able to follow the distribution of specific Ty1 RNA variants, we employed wild type (wt) S. cerevisiae strains of the BY background transformed with the galactose-inducible Ty1H3his3AI element (hereafter denoted tagTy1), widely used as a tool to study the biology of Ty1 and other viruses^12,13 (Fig. 1A). Northern analysis, using the 5′ end labeled Ty1-h probe, specifically targeting the “tag” sequence, or the Ty1-INT probe,¹ targeting both the tagTy1 and the endogenous Ty1 (endTy1) RNA, revealed the expected transcripts (Fig. 1B). In addition, RNA-FISH analysis using fluorophore-labeled Ty-h or Ty1-INT probes demonstrated that tagTy1 RNAs form high-intensity single cytoplasmic foci that overlay, and appear with a frequency similar to, endogenous Ty1 foci (Fig. 1C and D). We conclude that the tagTy1 system provides a good model to study T-body biology.

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.

The T-body formation ability of the GAL1 promoter regulated tagTy1 construct demonstrated the dispensability of the R region of Ty1 5′UTR for this specialized RNA localization. To further delineate region(s) of the Ty1 RNA necessary for its localization in T-bodies, different deletions of the tagTy1 element were constructed and analyzed. Deletion of the TYB region, ptagTy1ΔBN construct, exhibited robust steady state RNA levels and a strong T-body signal (data not shown). Further deletion of the 3′UTR of this TYB less construct resulted in “tagTYA” (Fig. 2A). Northern, RNA-FISH and IF analysis showed that RNA and Gag from this construct are present at high levels and readily forms T-bodies (Fig. 2B and C). As previously shown for endogenous Gag and Ty1 RNA,¹ signals obtained using the Gag-antibody and Ty1-h FISH probe fully overlap (data not shown). We conclude that neither Pol nor the 5′- or 3′-UTRs of Ty1 are needed for T-body formation, hence, TYA is sufficient for this specialized localization of Ty1 RNA.

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.

Specialized RNA localization may rely on the association of trafficking proteins with target RNA sequences, sometimes termed “zip codes”.^14,15 To determine if the T-body stimulatory information of TYA is exclusively embedded in the RNA or whether it also requires synthesis of Gag, we analyzed four tagTYA variants with different alterations of the Gag ORF (Fig. 2A). Neither the tagTYAΔEB and tagTYAΔSB constructs, deletions of 1,060 and 575 nts respectively from the 3′end of the TYA ORF, nor the tagTYA-E* and agTYA-S* variants, insertion of one nt and deletion of 4 nts respectively, are capable of forming T-bodies (Fig. 2C and data not show). We conclude that an intact TYA ORF is necessary for T-body formation and that there is unlikely to be simple Gag-independent zip-codes in the TYA RNA.

The tagTYA construct lacks almost completely the Pol region, which is only represented by a truncated fragment of the N-terminal protease. This suggests that immature Gag can stimulate T-body formation. Western blotting analysis, using the monoclonal BB2 anti-Gag antibody¹⁶ showed, in addition to a strong increase in Gag levels in cells expressing the tagTy1 and tagTYA constructs (Fig. 2D; note loading 1/10^th the amount of extract (middle part) the Gag signal is still saturated), a shift in the predominant band from Gag-p45 to Gag-p49 in cells expressing tagTYA (Fig. 2D). The additional band of high molecular weight is in agreement with ribosomal frameshifting allowing partial translation of the tag region (see Fig. 2A). Thus, immature Gag-p49 stimulates T-body formation.

To minimize the possibility of interference effects in trans by endogenous Ty1 components on our assays, we tested the effect of expression of tagTy1 and tagTYA in a strain background with only low expression of endogenous Ty1. For this purpose, we used the mfa1Δ mutant, identified in our lab as a T-body deficient strain (Fig. 3A upper row) with decreased Ty1 RNA levels (data not shown). Low level signals of Gag in both IF (Fig. 3A lower row) and western (Fig. 3B) analysis complemented these data. However, despite these low levels of endogenous Gag, expression of both tagTy1 and tagTYA constructs (see Fig. 3B for expression levels) readily induced T-body formation (Fig. 3C). Thus, expression of endogenous Ty1 is not required for Gag-p49 induced T-body formation.

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.

The ability of Gag-p49 by itself to stimulate T-body formation indicates that Ty1 focal accumulation precedes transpositional competence, i.e., VLP maturation. To test our hypothesis using an approach alternative to Gag-p49 induction, we examined the ability of tagTy1 to form T-bodies in the xrn1Δ strain. We chose this background due to the strong inhibition of Ty1 transposition in the absence of the Xrn1p exonuclease,¹⁷ probably due to the accumulation of Ty1 antisense RNAs that inhibit VLP maturation.^17,18 Consistent with a previous report in reference ², we observed significantly decreased T-body counts in xrn1Δ cells transformed with an empty vector (Fig. 4). In contrast, xrn1Δ cells growing under the same conditions but transformed with the tagTy1 system are T-body formation proficient (Fig. 4) and deficient in transposition (data not shown). Thus, T-body formation does not require VLP transpositional competence.

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.

Discussion

Given the high level of regulation of the Ty1 transposition cycle,³ it seems reasonable that Ty1 transposition could also be regulated at the level of VLP assembly. Creation of a favorable viral microenvironment, gathering Ty1 elements in a spatially restricted volume of the cell, may facilitate VLP assembly. We and others previously proposed that T-bodies may be involved in such a process.^1,2 Here we present evidence demonstrating that T-body formation precedes formation of mature VLPs and transposition.

Our results showing that Ty1 Gag is sufficient for RNA localization are in agreement with other studies showing that Gag proteins from the Ty3 and the Rous sarcoma viruses also play central roles in the sub-cellular localization of these viral RNAs.^19,20 This suggests that Gag-mediated T-body formation may play a central role in the initial assembly of VLPs. First, Ty1 features required for VLP- and T-body-formation are similar. Indeed, Gag on its own is capable of assembling VLPs⁷ and expression of a complete TYA in E. coli leads to VLP formation.^21,22 In addition, mutations resulting in C-terminal truncation of Gag impair T-body formation (this paper) as well as the intrinsic ability of Gag to multimerize into spheroidal particles.^16,22 Second, T-body formation does not require transpositional competence: (1) T-bodies are independent of the proteolytic processing of Gag-p49 and (2) are still formed in a transposition-deficient context. Interestingly, HIV-1 regulates viral assembly by local accumulation of viral components and the host factor Staufen 1 into SHRNPs outside of P-bodies and stress granules.²³ Thus, the assembly of immature Ty1 VLPs resembles the behavior of HIV-1, where Gag and HIV-1 RNA accumulation in the plasma membrane occurs prior to virion assembly.^24,25

The fact that T-bodies do not seem to be required for VLP maturation and cDNA synthesis “per se”, along with the Gag self-assembly properties to form VLPs in an heterologous prokaryote host, raises the question of the functionality of T-bodies in S. cerevisiae. Although this is still an open question, we favor the idea that the kinetics of VLP formation is stimulated by locally concentrating Ty1 elements. In agreement with this, while the transposition level of endogenous Ty1 elements are very low,²⁶ ectopic expression of Ty1 stimulates T-body formation as well as VLP accumulation and transposition efficiency.^2,12,26 Alternatively, or in addition to this, VLP assembly into T-bodies may restrict the actions of host VLP destabilizing factors, such as Fus3p.²⁷ In this scenario, the exit of mature VLPs from the T-bodies, or the dissolution of T-bodies altogether, may be required for the release of the preintegration complex (Int plus cDNA) from the VLP shell, mediated by the action of host VLP destabilizing factors.

Materials and Methods

Strains and yeast manipulations.

Unless otherwise indicated, media, growth conditions and yeast manipulations were performed as previously described in reference ¹. Experiments were done with cells grown exponentially at 25°C in rich AA minimal media lacking uracil and supplemented with 2% galactose as carbon source. All strains used are direct derivatives of the BY series of the yeast knock out collection (S288C background).

Plasmids.

Plasmids pRS426,²⁸ and pPgalTy1H3 his3AI¹² have been described previously. Plasmids were propagated in E. coli DH5α in rich LB media supplemented with ampicillin. DNA extractions and manipulations were done using standard techniques. Blunt ends were created by the Klenow enzyme when required. ptagTy1ΔBN were made by digesting ptagTy1(=pPgalTy1H3 his3AI) with BstEII and NheI followed by Klenow treament and religation. ptagTy1ΔBNΔCA (ptagTyA) was made by digesting PtagTy1ΔBN with ClaI and NarI followed by Klenow treament and religation. PtagTyAΔEB and PtagTyAΔSB were made, respectively, by cutting ptagTyA with EcoNI and BstEII or with SphI and BstEII, by Klenow treament and religation. PtagTyA-E* and PtagTYA-S* were made respectively by cutting ptagTyA with EcoNI or SphI followed by Klenow treament and religation.

RNA-FISH and IF analyses.

Unless otherwise indicated, RNA-FISH and IF analyses were performed as previously described in reference ¹. RNA from tagTy1 and its deletion variants was visualized using 60 ng of fluorophore labeled Ty1-h probe (5′-CGG CTG GTC GCT AAT CGT TGA GTG CAT TGG TGA CTT ACA CAT AGA CGA C); T represents aminomodified C6 dT residues amenable for Cy3, Cy5 or FITC conjugation. Similar results were obtained with the Ty1-hB probe (5′-CGT ACG CAG TTG TCG AAC TTG GTT TGC AAA GGG AGA AAG TAG GAG ATC T). RNA co-localization experiments were performed using combinations of Cy3, Cy5 and FITC labeled probes as indicated in figure legends (controls experiments using individually labeled probes were done in parallel to discern putative unspecific signal due to fluorescence bleeding).

Imaging.

Image acquisition and treatment was done as previously described in reference ¹. Images were acquired with a BX51 (Olympus) or an Axiovert 200M (Zeiss) microscope equipped with a Coolsnap HQ camera (Ropers Scientific), and artificially colored and overlayed using Adobe Photoshop. All images in a single part were modified in a similar way. Cell counting was done by selecting random fields under the DAPI filter followed by image acquisition using the different filters. A minimum of 100 cells per experiment and per strain were counted.

Northern and western blotting analysis.

RNA extractions and northern blotting were done as described in reference ¹. Protein extraction was done starting with frozen pellets of cells grown in the same conditions as for northern and microscopic experiments, and using the NaOH protein extraction method.²⁹ Briefly, frozen pellets from 2 ml of cells at O.D._{600 nm} of 0.5 were resuspended in 250 µl of 0.1 M NaOH, incubated at room temperature for 3 min, centrifuged, resuspended in 300 µl of loading buffer and incubated for 10 min at 95°C. Due to the extreme abundance of Gag, different dilutions were loaded in parallel 10% PAGE-SDS gels (typically 5 µl of undiluted sample for the Nop1p loading control; and 5 µl of a 50- or 500-fold dilution for Gag).

Abbreviations

VLP

virus-like particle

FISH

fluorescence in situ hybridization

IF

immunofluorescence