An interchangeable prion-like domain is required for Ty1 retrotransposition

Significance Retrovirus-like retrotransposons help shape the genome evolution of their hosts and replicate within cytoplasmic particles. How their building blocks associate and assemble within the cell is poorly understood. Here, we report a prion-like domain (PrLD) in the budding yeast retrotransposon Ty1 Gag protein that builds virus-like particles. The PrLD has similar sequence properties to prions and disordered protein domains that can drive the formation of assemblies that range from liquid to solid. We demonstrate that the Ty1 PrLD can function as a prion and that certain prion sequences can replace the PrLD and support Ty1 transposition. This interchangeable system is a useful platform to study disordered sequences in living cells.

Retrotransposons are pervasive across diverse eukaryotes and influence genome evolution and affect host fitness. The budding yeast Saccharomyces cerevisiae contains Ty1-5 long terminal repeat (LTR)-retrotransposons, with Ty1 as the most abundant element in many laboratory strains (1,2). LTR-retrotransposons are the evolutionary progenitors of retroviruses; Ty1 elements share many structural hallmarks with retroviral genomic RNA and undergo an analogous replication cycle but lack an extracellular phase. Ty1 is transcribed from LTR-to-LTR and contains two partially overlapping open reading frames (ORFs): GAG and POL. Ty1 RNA serves as a template for protein synthesis and reverse transcription. Translation of Ty1 POL requires a programmed +1 frameshift near the C terminus of GAG, resulting in a large Gag-Pol precursor (p199) (3). Like retroviral RNA, Ty1 RNA is specifically packaged into virus-like particles (VLPs) where RNA is present in a dimeric form (3,4). Proteolytic protein maturation occurs within VLPs by a protease (PR) encoded within GAG and POL. Ty1 PR cleaves the Gag-p49 precursor near the C terminus to generate p45, the capsid protein, and Gag-Pol-p199 to form mature PR, integrase (IN), and reverse transcriptase (RT) (3). Reverse transcription occurs within mature VLPs and, like HIV-1, requires a complex formed between RT and IN (3,5). Ty1 preferentially integrates upstream of genes actively transcribed by RNA Polymerase III (Pol III) due to interactions between IN and Pol III subunits (3,(6)(7)(8).
Ty1 Gag performs the same functions as retroviral capsid and nucleocapsid. Amino acids 159 to 355 encode N-terminal domain (NTD) and C-terminal domain (CTD) capsid folds, assembling VLPs (9), and C-terminal sequences of Gag display nucleic acid chaperone (NAC) activity (10,11). Sequences in the Ty1 RNA encoding the Gag protein are required for packing, dimerization, and reverse transcription (3). The N-terminal region of Gag has unknown function, and it is not known whether it is required for transposition.
While several steps of retrotransposon life cycles have been investigated, it is not well understood how their RNA genomes and protein machinery associate within the cellular milieu to facilitate VLP assembly and replication. Retroviral particle assembly often occurs in subcellular domains, referred to as "viral factories" or "viral inclusions" (12,13). The sites of Ty1 VLP assembly are cytoplasmic foci termed retrosomes, or T-bodies, which contain Ty1 RNA, Gag, Gag-Pol, and perhaps additional cellular proteins (14)(15)(16). What drives the biogenesis of retrosomes is not understood. Mounting evidence suggests liquid-liquid phase separation (LLPS) underlies many examples of membraneless compartments (17,18). Aggregation-prone proteins that drive LLPS have overlapping properties with prions, and both are implicated in age-related disease (19)(20)(21)(22)(23)(24)(25)(26). Spontaneous demixing in these systems is often facilitated by intrinsically disordered domains, multivalent proteins, and scaffolding around nucleic acids. Indeed, prion-like and LLPS mechanisms provide intriguing models for retroelement assembly steps. Ty1 retrosomes contain Ty1 RNA and Gag oligomers associated with the RNA. Several viruses utilize LLPS in replication and assembly, including rabies virus (27), influenza A (28), herpes simplex virus 1 (29), measles virus (30), HIV-1 (31), and SARS-CoV-2 (32). Also, the human retrotransposon Long INterspersed Element-1 (LINE-1) has been reported to phase separate in vitro (33). Here, we present evidence that the Ty1 Gag protein contains a prion-like domain (PrLD) required for VLP assembly and transposition, raising the possibility that Ty1 Gag facilitates prion-like or phase-separating behaviors within retrosomes.

Results
Bioinformatic Analyses Reveal a PrLD in Ty1 Gag. Ty1 Gag contains several protein features, including capsid and NAC domains (9,11). The N-terminal region of the protein, meanwhile, is predicted to be unstructured and does not have previously reported function. We analyzed Ty1 Gag (Fig. 1A) and Gag-Pol (SI Appendix, Fig. S1) using several bioinformatic tools designed to predict protein disorder, amyloidogenic secondary structures, and amino acid composition similarity to known yeast prions (34)(35)(36)(37). For comparison, we ran the well-studied yeast prions Sup35 and Ure2, the mouse prion protein PrP, and Alzheimer's disease-associated human Aβ 1-42 through the same bioinformatic analyses ( Fig. 1 B-E). Ty1 Gag contains a 71-amino acid domain with strikingly similar amino acid composition to yeast prions in its disordered N terminus, comparable to Sup35 and Ure2. This Gag PrLD is predicted to be unstructured by AlphaFold (38), and no published structures of the region are available, similar to canonical prions (9,(39)(40)(41)(42) Fig. S2). Given the computational predictions and the requirement for Gag in forming Ty1 retrosomes, we further investigated prionogenic properties of the Gag PrLD, which we define as amino acid residues 66 to 136. Prionogenic Properties of the Gag PrLD . We used a wellcharacterized Sup35-based in vivo reporter system to assess the ability of the Gag PrLD to promote prionogenesis in a yeast strain harboring a mutant allele of the adenine biosynthesis gene, ade1-14, which contains a premature stop codon ( Fig. 2A) (43)(44)(45). Soluble Sup35 functions as a translation termination factor, resulting in a truncated nonfunctional Ade1 protein. Yeast fails to grow on media lacking adenine and appears red due to the buildup of a metabolic intermediate. However, formation of a prion state (termed [PSI + ]) aggregates Sup35 away from the ribosome, allowing for translational read-through. This can be detected by adenine prototrophy and yeast colonies appearing white. Fusion of the PrLD of interest to the Sup35 N or NM domains promotes prion nucleation and has previously been used to study mammalian PrP and Aβ (45). Expression of Sup35NM-Gag PrLD fusion under the CUP1 copper-inducible promoter stimulates prionogenesis, as detected by increased papillation on SC-Ade when compared to the reporter alone (Fig. 2B). Protein expression and Ade + growth is copper responsive; however, we found that the Sup35N reporter construct displays a high background growth upon induction (SI Appendix, Fig. S3 A-E). We next biochemically monitored prion aggregation using semidenaturing detergent-agarose gel electrophoresis (SDD-AGE) (46). Gag PrLD fusions formed large, slow-migrating, copper-inducible aggregates with both Sup35N (SI Appendix, Fig. S2F) and Sup35NM ( Fig. 2C) above reporter alone. Finally, we verified prion nucleation specifically, as opposed to colony growth due to accumulating suppressor mutations, by curing colonies of the prion after passaging cells on guanidine hydrochloride (GdHCl) (47,48). Representative cells are shown for the naïve [psi − ], induced [PSI + ], and cured states for Sup35NM fusions to Gag PrLD or positive control Aβ, both HA-tagged ( Fig. 2D) and untagged (SI Appendix, Fig. S2G). A large fraction of Sup35NM-Gag PrLD Ade + colonies were curable by GdHCl.
The Gag PrLD Is Required for Ty1 Transposition. Given that the Gag PrLD promotes prionogenesis of a Sup35-based reporter, we investigated its functional role in Ty1 transposition. In a Saccharomyces paradoxus strain lacking genomic Ty1 elements (49,50), we first deleted the PrLD from Gag in a Ty1 element provided on a plasmid and tagged with the robust and sensitive his3-AI retrotranscript indicator gene (51) (Fig. 3A). This marker contains a mutant his3 gene split by an antisense artificial intron (AI) that is inserted at the 3′ untranslated region of Ty1 in the opposite transcriptional orientation. The AI is in the correct orientation to be spliced only in Ty1his3-AI RNA; cDNA reverse transcribed from this product results in a functional HIS3 allele. Insertion into the genome, either by integration or recombination, allows cells to grow on media lacking histidine. The frequency of His + prototrophy is a direct measure of Ty1his3-AI retrotransposition or cDNA recombination, collectively known as retromobility. Deletion of the Gag PrLD in a complete Ty1his3-AI element overexpressed under the GAL1 promoter completely abolished retromobility (SI Appendix, Fig. S4A), despite retaining similar Gag protein levels (SI Appendix, Fig. S4B). However, the PrLD region of Gag contains cis-acting RNA signals required for efficient reverse transcription (52,53). To distinguish between a functional role in retrotransposition of the PrLD in the Gag protein versus the role of the RNA sequences that encode for the PrLD, we used a two-plasmid system to separate Ty1 RNA and protein functions (Fig. 3B). A helper-Ty1 encodes a functional mRNA, providing protein products, but lacks a 3′ LTR thus disrupting cis-acting signals required for reverse transcription. Mini-Ty1his3-AI lacks complete ORFs but contains cis-acting signals for dimerization, packaging, and reverse transcription of mini-Ty1his3-AI RNA (53,54). Retromobility is monitored through the his3-AI reporter.
In the two-plasmid assay, deletion of the Gag PrLD also inhibits retromobility ( Fig. 3 C and D), despite producing similar levels of Gag protein (Fig. 3E), confirming a critical contribution from the PrLD in the Gag protein to retromobility.

Ty1 Mobility of Gag Chimeras Containing Foreign PrLDs.
To better understand the nature of the PrLD's contribution to retromobility, we asked whether the Gag PrLD sequence is uniquely capable of facilitating retromobility. Since the Gag PrLD has prionogenic properties and sequence similarity to prions, we created chimeric Ty1 Gags in which the PrLD is replaced with prion domains from well-studied prions and aggregating proteins (Fig. 3A). We chose the yeast prions Sup35 and Ure2, the mouse prion protein PrP, and Alzheimer's disease-associated human Aβ 1-42 using domains predicted computationally ( Fig. 1) (44,45,55). Chimeric Ty1 elements on the helper-Ty1 plasmid were coexpressed with mini-Ty1his3-AI, and the level of Ty1 mobility was determined.
Remarkably, substitution of the Gag PrLD with the prion domain from yeast Sup35 or mouse PrP supported Ty1 retromobility in qualitative (Fig. 3C) and quantitative retromobility assays (Fig. 3D). Gag Sup35N retromobility is not significantly different from wild type (WT), whereas Gag PrP is an order of magnitude lower, although still readily detectable on a qualitative plate assay. Replacing the PrLD sequence disrupts RNA signals, (D) Quantitative mobility assay of galactose-induced cells. Each bar represents the mean of at least eight independent measurements, displayed as points, and the error bar ± the SD. Error bars are omitted for PrLDΔ, Ure2, and Aβ chimeras that did not transpose; one retromobility event was observed in one replicate of PrLDΔ. Adjusted retromobility frequency is indicated above the bars. For Ure2 and Aβ, frequencies are indicated as less than the calculated frequency if one retromobility event had been observed. Significance is calculated from a two-sided Student's t test compared with WT (n.s. not significant, ***P < 0.001. Exact P-values are provided in SI Appendix, Table S1). (E) Protein extracts prepared from galactose-induced cells expressing the indicated Gag constructs in the two-plasmid system were immunoblotted for the protein indicated on left. Polypeptide precursors are bracketed and mature RT and IN sizes are noted on right. Pgk1 serves as a loading control. Migration of molecular weight standards is shown alongside the immunoblots. A representative image of at least three replicates is shown.
which is reflected in the single plasmid assay, in which Gag Sup35N and Gag PrP chimeras have dramatically reduced retromobility (SI Appendix, Fig. S4A), despite producing similar Gag protein levels (SI Appendix, Fig. S4B), highlighting the importance of separating protein and RNA function with the two-plasmid assay. Retromobility measured as the frequency of His + prototrophs formed from his3-AI tagged elements includes both new chromosomal integrations likely created via retrotransposition, and recombination of the spliced cDNA with homologous sequences present on the mini-Ty1his3-AI plasmid or solo LTRs present in the genome. To assess whether the chimeras support retrotransposition or merely recombination, we distinguished the two by, first, monitoring histidine prototrophy after segregating the helper and mini-Ty1his3-AI plasmids (SI Appendix, Fig. S4C). In our strain background with the WT two-plasmid system, 4% of retromobility events were due to recombination with either of the plasmids. The Gag Sup35N and Gag PrP chimeras had modestly increased recombination events, although only Gag Sup35N reached statistical significance (P = 0.024) (SI Appendix, Fig. S4D). Secondly, we measured retromobility in a rad52 mutant, thereby blocking Ty1 cDNA recombination (56,57). RAD52-dependent recombination contributes to total retromobility of Gag Sup35N and Gag PrP , but each also still support RAD52-independent retromobility (SI Appendix, Fig. S4E). Approximately one half of Gag Sup35N retromobility events, and one-third of Gag PrP events, are RAD52-independent. WT Gag had no statistically significant decrease in retromobility in a rad52 mutant. We conclude that the Gag chimeras support de novo retrotransposition (56,58).

Effect of Gag PrLD Chimeras on Ty1 Protein Level and Maturation.
The result that the Gag PrLD can be replaced by foreign prion sequences indicates its function is not unique to the PrLD sequence and may be the same as provided in aggregation-prone proteins. However, not all the disordered domains tested in Gag chimeras supported transposition. Ty1 chimeras containing the domains from yeast Ure2 or human Aβ did not transpose (Fig. 3  C and D). All the chimeric Gags were expressed at similar levels ( Fig. 3E), arguing against different transposition phenotypes due to effects on protein stability from the foreign prion domains. The substituted prion domains are of various sizes, and Gag chimeras had predicted electrophoretic mobilities. Gag proteolytically matures from p49 to p45 and is subject to posttranslational modifications, often resulting in multiple bands observed by western blot (3). To determine whether the Gag chimeras affected protein maturation, we assessed the relative levels of mature RT and IN by western blotting with antibodies specific to each protein.
Deletion of the PrLD results in dramatically reduced mature RT and IN levels (Fig. 3E). The Gag Sup35N chimera transposed as well as WT and produced equivalent levels of mature RT and IN. The transposition-deficient chimeras, Gag Ure2 and Gag Aβ , have very reduced levels, comparable to Gag PrLD Δ. Interestingly, Gag PrP supports transposition, although reduced from WT, and has low levels of mature RT and IN. These results raise the possibility that Gag chimeras can block PR function and production of mature RT and IN that are essential for Ty1 mobility. (59), which are believed to be assembled in retrosomes (14)(15)(16). Gag fused to GFP has been used as a reporter for retrosome assembly and location (60), therefore we examined formation of cytoplasmic foci of WT, mutant, and chimeric Gag-GFP in the Ty-less background. WT Gag-GFP fusions formed discrete cytoplasmic foci, as previously reported using this construct, but deleting the PrLD resulted in diffuse localization throughout the cytoplasm (Fig. 4). We found that a 24-h galactose induction, shorter than 48-h induction used above, was ideal for live-cell microscopy and GFP-detection as yeast cultures are in log-phase growth (SI Appendix, Fig. S5). Twenty four-hour-induced Gag Sup35N formed similarly discrete foci patterns as WT Gag, whereas Gag Ure2 had diffuse localization similar to Gag PrLD Δ. Gag PrP supports transposition and predominately formed foci similar to WT, but also had a modest fraction of cells containing a visually distinct fluorescent morphology that appears as a single, large, very bright focus. Ty1 Gag Aβ does not transpose, yet formed foci and an even larger fraction of cells contained these single, large foci. Forming Gag-GFP foci correlates with a requirement for transposition but, as Gag Aβ shows, is not sufficient.

Protein (GFP) Affect Aggregation and Localization. Proteolytic maturation of RT and IN via PR occurs within VLPs
In addition, we investigated the structures formed by Gag-GFP chimeras in fixed yeast cells by thin-section transmission electron microscopy (TEM) (SI Appendix, Fig. S6) using methods similar to those used for detecting Ty1 VLPs (16). WT Gag-GFP produced electron-dense structures that appear similar to VLPs but look incomplete or incorrectly assembled, lacking a circular shell with a hollow interior. Gag PrP -GFP also produced clusters of structures reminiscent of VLPs, but in this case appearing denser and lacking a hollow interior. Gag PrLD Δ-GFP and Gag Ure2 -GFP did not form any VLP-like structures detectable in micrographs. The Gag Sup35N -GFP strain produced tubular or filamentous structures,  Table S2.
also not resembling proper VLPs. And strikingly, the Gag Aβ -GFP strain formed large densities in defined regions of the cell, instead of clusters of particles or filaments across the cytoplasm, perhaps corresponding to the single large foci seen by fluorescent microscopy. These results suggest that Gag-GFP can reveal severe assembly defects as evidenced by Gag PrLD Δ-GFP and Gag Ure2 -GFP but GFP may confer aberrant VLP assembly properties when WT or chimeric Gag-GFP fusions are produced in cells.

The Ty1 Gag PrLDΔ and Gag Chimeras Affect VLP Assembly.
To evaluate VLP assembly in the chimeras using the two-plasmid system, we examined Gag sedimentation profiles of yeast lysate run through a 7 to 47% continuous sucrose gradient, as previously reported (9,50,61). WT VLPs accumulated in more dense sucrose fractions near the bottom half of the gradient, with peak fractions indicated by a bar (Fig. 5). Gag PrLD Δ appears unable to assemble complete VLPs, as Gag in these mutants accumulated in less dense sucrose fractions near the top of the gradient. The transpositioncompetent chimeras Gag Sup35N and Gag PrP had similar sedimentation profiles as WT, whereas transposition-deficient Gag Ure2 accumulated near the top of the gradient like Gag PrLD Δ. Gag Aβ does not support retrotransposition, but peaked in similar fractions as WT, although somewhat more broadly distributed across the gradient.
To further examine the VLPs assembled by each chimera, we visualized thin sections of fixed yeast cells by TEM. Cells overexpressing the WT two-plasmid Ty1 system produced large clusters of VLPs (Fig. 6). VLPs are characteristically round with an electron-dense shell and their interior appears hollow in micrographs. Importantly, these particles were not observed in the parental yeast strain expressing empty vectors. Ty1 VLPs are heterogeneously sized and are approximately 30 to 80 nm in diameter, based on previous measurements of purified particles (62,63). In thin-section TEM, particles may be in different Z-planes when sectioned, therefore masking the diameter of a roughly spherical particle, and preventing quantitative particle-size data collection from thin-section TEM. With this limitation in mind, we measured particle diameters from several cells in multiple micrographs to estimate an approximate size, and found WT particles ranging from 40 to 80 nm, with a median diameter of 59 nm (SI Appendix, Fig. S7), largely agreeing with previous reports of purified particles.
We did not observe any cells producing VLPs in the Gag PrLD Δ mutant, in agreement with Gag PrLD Δ-GFP imaging and sucrose sedimentation profiles. Taken together, these data lead us to conclude that the PrLD is required for Ty1 VLP assembly. The transposition-deficient Gag Ure2 chimera also did not assemble VLPs as monitored by thin-section TEM, again agreeing with sucrose sedimentation results. The two transposition-competent chimeras, Gag Sup35N and Gag PrP , assembled VLPs similar in size and appearance to WT. These chimeras also produced large numbers of particles in each cell, although consistently appearing somewhat more dispersed throughout the cell than WT particle clusters. Interestingly, Gag Aβ does not support retrotransposition, but has a similar sucrose sedimentation profile as WT, suggesting it may assemble particles that are defective for transposition. In thin-section TEM, we observed particles in cells expressing Gag Aβ that are visually distinct from WT. The most striking difference is that these particles do not have the characteristic hollow center and instead appear electron-dense throughout. They are smaller than WT with a median diameter of 42 nm (SI Appendix, Fig. S7), and, like the Gag Sup35N and Gag PrP chimeras, are produced in large numbers of particles but are dispersed throughout the cell. Together, these results illustrate the robust and flexible nature of VLP assembly. However, our data also underscore the requirement for PrLD functionality as yeast and mammalian Gag-prionogenic chimeras form VLPs in vivo whereas the Gag PrLD Δmutant does not.

Discussion
The data presented here permit several conclusions about prionogenic domains, the functional organization of Ty1 Gag, and VLP assembly. Our results show that Ty1 Gag contains a PrLD that is required for VLP assembly and retrotransposition. The Gag PrLD has intrinsic prionogenic properties as demonstrated by a cellbased Sup35 reporter, and its function in Ty1 transposition can be replaced by certain yeast and mammalian prion domains. Our findings also raise interesting questions about sequence constraints of PrLDs and how widespread PrLD functions are across retroelements. Furthermore, our work suggests Ty1 retromobility is an effective in vivo screening platform to study intrinsically disordered domains.
Prion Properties of the Ty1 Gag PrLD . We have examined prionogenic properties of the Ty1 Gag PrLD using a cell-based assay in which Gag PrLD is fused to the N and NM domains of Sup35 (45). Nonsense read-through is measured by auxotrophic growth and colony color, aggregate formation is monitored biochemically with SDD-AGE, and curability is assessed after GdHCl treatment. Further characterization of the Ty1 Gag PrLD will include analyzing fusions to other Sup35 regions, measuring binding of the amyloidsensitive dye thioflavin-T, and determining non-Mendelian inheritance (64, 65).

RNA-Contributions to Gag PrLD Function.
To determine proteinlevel effects of mutations in the Ty1 Gag PrLD from mutations of cis-acting RNA sequences, we separated RNA and protein function in a two-plasmid system (53,54). Retromobility is lower in the two-plasmid system (Fig. 3C) than a single-plasmid expressing the intact transposon (SI Appendix, Fig. S4A). Gag Sup35N restored retromobility in the two-plasmid system but had a severe retromobility defect in the single-plasmid assay. This is likely due to disruption of a functional pseudoknot in the RNA region that also encodes for the PrLD (52, 53). pnas.org Sequence Requirements of the Ty1 Gag PrLD . We replaced the Gag PrLD with exogenous prion domains based on computational predictions and functional analyses. We chose the entirety of Aβ 1-42 and the complete N-terminal domain of Sup35 2-123 . We introduced the highest scoring 60 amino acid stretch predicted by prion-like amino acid composition (PLAAC), Ure2  , which is within the established prion domain reported as the first 89 amino acids (44,55). The infectious PrP 27-30 isoform contains about 142 amino acids and spans 90 to 230 (66), but shorter truncations still display prion phenotypes (67)(68)(69), and PrP 90-159 is sufficient to induce prionogenesis in yeast (45). PrP 121-231 is soluble, and its structure has been determined using solution NMR (41,70). We introduced PrP 90-159 as a Ty1 Gag chimera based on prior success in yeast. It will be interesting to examine other regions of PrP for function when present in Gag.
Although the sequence features constraining Ty1 PrLD function are not well defined, both the transposition-competent Gag chimeras (Sup35 and PrP) are from proteins with oligopeptide repeats associated with prionogenesis (71,72). However, the PrP sequence introduced as a Ty1 Gag chimera does not contain these repeats. Moreover, the Ty1 Gag PrLD does not have equivalent repeats of 8 to 10 amino acids. Instead, like other reported prion domains, the Gag PrLD is Q/N-rich and is depleted of charged residues. Additionally, a large number of prolines in the Gag PrLD likely prevents secondary structure formation and contrasts with the highly alpha-helical folding of the Gag capsid domain (9,61). Further investigation will be required to understand the sequence parameters, such as length, amino acid composition, charge, or oligorepeats, that govern function of the Gag PrLD .

Gag Chimeras Reveal Varied Properties across the Ty1 Life
Cycle. Importantly, the Gag Sup35N chimera restored Ty1 mobility, produced VLPs with WT morphology and sedimentation profiles, as well as mature IN and RT. Gag PrP supported retromobility, although less well than WT or Gag Sup35N . Whereas Gag PrP produces VLPs with WT morphology by TEM (Fig. 6) and similar sedimentation profiles (Fig. 5), Gag PrP accumulates low levels of mature RT and IN (Fig. 3E). This could indicate an incompatibility of Gag PrP as a substrate for PR. Another possibility is that Gag PrP VLPs inefficiently incorporate Gag-Pol or are partially defective in ways not detectable by TEM or sedimentation. The reduced retromobility of Gag PrP may be explained by impaired RT and IN protein maturation. Meanwhile, Gag Aβ produces morphologically altered particles that do not support retrotransposition or Pol maturation. These particles lack the characteristic hollow center observed in TEM of WT VLPs (Fig. 6) and are noticeably smaller in diameter (SI Appendix, Fig. S7). These observations highlight the robust nature of VLP assembly but reveal that simply assembling particles is not sufficient for transposition or protein maturation. It will be informative to measure packaging of the mini-Ty1 RNA into chimeric VLPs as productive reverse transcription requires both mature enzymes, a correctly folded RNA substrate, and the tRNA Met primer to be present in VLPs. Our sedimentation and TEM results build upon previously published sedimentation experiments (9,50,61), and strengthen the value of sedimentation as a proxy for VLP assembly. Nonetheless, the value of TEM is exemplified by the Gag Aβ chimera, which sediments similarly to WT but TEM reveals aberrant particle morphology.
Gag-GFP fusions are used as a proxy for Ty1 retrosomes (60), although we have not formally tested for Ty1 RNA colocalization in our system. We used a previously characterized WT GFP-fusion construct that contains the mature Gag (p45) and not a full-length element (60). Here, the utility of Gag-GFP is shown by the lack of foci for the Gag PrLD Δ mutant in growing cells and the cellular mislocalization observed in Gag chimeras (Fig. 4). However, GFP is a 26 kD protein and fusion impaired proper VLP formation (SI Appendix, Fig. S6), perhaps by interfering with Gag-Gag contacts required for assembly of a complete particle structure. Examining the PrLD fused to GFP alone, without the full Gag protein, or testing a Gag truncation that lacks the NAC domain, will indicate the minimal region that promotes foci formation and whether RNA recruitment is required. Ty1 Gag contains a NAC and binds Ty1 RNA, but also binds diverse RNAs in vitro and cellular mRNAs associate with Ty1 VLPs (10,11,54,(73)(74)(75). Whether Ty1 RNA, specifically, is required to form foci or to nucleate VLP assembly, or whether there is an RNA requirement at all, requires further study. Further studies are required to understand the kinetic components and cytoplasmic localization required to form retrosomes and their progression toward VLP assembly, but dynamic transitions are evident in our Gag-GFP analyses and retrograde transport of Gag from the endoplasmic reticulum is required for protein stability (60).
Does the Ty1 Retrosome Constitute a Phase-Separated Compartment? WT cells assemble discrete VLPs that can be found throughout the cell but are often observed in a particular region of the cytoplasm, and even the WT Gag-GFP-assembled discrete structures, observed by TEM. However, the Gag Aβ -GFP strain produced large densities that may correspond to large foci observed by fluorescence microscopy. These assemblies would be consistent with LLPS compartments containing high concentrations of Gag-GFP that stall and cannot complete VLP assembly; however, we have not examined LLPS properties such as concentration dependence, droplet merging, or internal mixing (17). PrLDs can drive formation of a gradient of assemblies, from LLPS to hydrogels and amyloid-like fibers. The Ty1 Gag chimeras may exhibit a spectrum of these morphologies. The filamentous assemblies formed by Gag Sup35N -GFP are potentially similar to Sup35 amyloid fibers observed in vitro, and Gag Aβ -GFP may form liquid droplets. Sup35, while canonically known for its ability to form amyloid fibers as a prion, has more recently been appreciated to undergo LLPS upon a decrease in cytosolic pH and can mature over time into a gel-like condensate (76,77). Whereas WT Gag allows for VLP assembly to proceed and supports transposition, perhaps transiently existing in an LLPS state, chimeras may become blocked along the retrosome and VLP assembly pathway, resulting in the striking structures observed by fluorescence microscopy and TEM. Further work will be required for the rigorous characterization necessary to declare the Ty1 retrosome or other assemblies formed by Gag chimeras an LLPS compartment. Ty1 provides a promising system to unite studies of prion and LLPS pathways.

An Interchangeable Platform to Study PrLD and LLPS Domains
in Living Cells. The condensate-forming property, but not the prion-forming property, of Sup35 is conserved across 400 My from S. cerevisiae to Schizosaccharomyces pombe, emphasizing the evolutionary importance of this ancient phenotype (76). Our finding of the Ty1 PrLD raises the possibility that LLPS may be widespread among retroelements. Our preliminary computational analyses of Pseudoviridae (Ty1/copia) retroelement family members reveal predicted PrLDs in not only the closely related yeast Ty2, but also in distantly related plants in the Oryza element Retrofit and the Arabidopsis elements Evelknievel and AtRE1. The human retrotransposon LINE-1 phase separates, and retrotransposition is associated with cancer (78) and age-associated inflammation (79,80). A condensate-hardening drug was found to block human respiratory syncytial virus replication, which