Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.     

Ty3 nucleocapsid controls localization of particle assembly

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX₂CX₄HX₄C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.     

The TY3 Gag3 spacer controls intracellular condensation and uncoating

Cells expressing the yeast retrotransposon Ty3 form concentrated foci of Ty3 proteins and RNA within which virus-like particle (VLP) assembly occurs. Gag3, the major structural protein of the Ty3 retrotransposon, is composed of capsid (CA), spacer (SP), and nucleocapsid (NC) domains analogous to retroviral domains. Unlike the known SP domains of retroviruses, Ty3 SP is highly acidic. The current studies investigated the role of this domain. Although deletion of Ty3 SP dramatically reduced retrotransposition, significant Gag3 processing and cDNA synthesis occurred. Mutations that interfered with cleavage at the SP-NC junction disrupted CA-SP processing, cDNA synthesis, and electron-dense core formation. Mutations that interfered with cleavage of CA-SP allowed cleavage of the SP-NC junction, production of electron-dense cores, and cDNA synthesis but blocked retrotransposition. A mutant in which acidic residues of SP were replaced with alanine failed to form both Gag3 foci and VLPs. We propose a speculative "spring" model for Gag3 during assembly. In the first phase during concentration of Gag3 into foci, intramolecular interactions between negatively charged SP and positively charged NC domains of Gag3 limit multimerization. In the second phase, the NC domain binds RNA, and the bound form is stabilized by intermolecular interactions with the SP domain. These interactions promote CA domain multimerization. In the third phase, a negatively charged SP domain destabilizes the remaining CA-SP shell for cDNA release.     

Ty1 Gag Enhances the Stability and Nuclear Export of Ty1 mRNA

Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus‐like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1‐less strain expressing galactose‐inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense‐mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.     

SIRE-1, A putative plant retrovirus is closely related to a legume TY1-copia retrotransposon family

SIRE-1 is a potential soybean retrovirus which has a gene order similar to Ty1-copia retrotransposons but also contains an envelope-like open reading frame (ORF), which is characteristic of retroviruses. PCR and Southern analysis reveals that SIRE-1 is closely related to a legume-wide family of envelope-lacking Ty1-copia group retrotransposons which suggests that SIRE-1 was formed by the recent acquisition of an envelope gene by a Ty1-copia retrotransposon.     

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

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.     

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag–GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.     

Frameshift Signal Transplantation and the Unambiguous Analysis of Mutations in the Yeast Retrotransposon Ty1 Gag-Pol Overlap Region

The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in cis by "transplanting" the frameshift signal to a position upstream of its native location. These "frameshift transplant" mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.     

Isolation of two novel complete Ty1<Emphasis Type="Italic">-copia</Emphasis> retrotransposons from apple and demonstration of use of derived S-SAP markers for distinguishing bud sports of <Emphasis Type="BoldItalic">Malus domestica</Emphasis> cv. Fuji

Retrotransposon-based molecular markers are a powerful tool for mapping and diversity studies. The scarcity of retrotransposon long terminal repeat (LTR) sequences limits the application of retrotransposon-based molecular marker systems. Here, we isolated two novel complete Ty1-copia retrotransposons (CTcrm1 and CTcrm2) in apple using a genome walking strategy. The CTcrm retrotransposons are nearly 5 kb long, and they have all the features of Ty1-copia retrotransposons. The differences in gene organization and nucleotide sequence length between the CTcrm retrotransposons and other reported complete retrotransposons in apple showed that CTcrm1 and CTcrm2 are the first two distinct complete Ty1-copia retrotransposons in the apple genome. To investigate the potential utility of the two retrotransposons as molecular markers, primers complementary to the CTcrm LTRs were designed to develop sequence-specific amplification polymorphism markers for discriminating bud sports of Fuji apple. Multiple polymorphisms corresponding to CTcrm1 and CTcrm2 were detected and could easily be used to discriminate bud sports from their Fuji progenitor, as well as from each other.     

The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB) is counteracted by Vif and requires its Zinc-binding domain

Background

DSB, the 3-O-(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium.

Results

Wild-type Vif (Vif^wt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H¹⁰⁸C¹¹⁴C¹³³H¹³⁹ coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vif^wt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone.

Conclusion

The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vif^wt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.