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.     

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.     

Proteolytic processing of Ty3 proteins is required for transposition.

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Development of a rapid and convenient method for the quantification of HIV-1 budding

In cells, the expression of Gag protein, one of the major structural proteins of retroviruses, is sufficient for budding virus-like particles (VLPs) from the cell surface. We have previously reported that spheroplasts of Saccharomyces cerevisiae expressing HIV-1 Gag proteins from an episomal plasmid constitutively released a large amount of VLPs into culture media; however, commercially available ELISA kits which detect mature capsid of HIV-1 could not detect uncleaved 55-kDa Gag proteins released from budding yeast. We therefore developed a method to quantitate VLP levels released from budding yeast by using fusion protein from HIV-1 Gag and Firefly Luciferase. This system is useful for screening cellular factor(s) involved in retrovirus budding from S. cerevisiae.     

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.     

Structural and dynamics studies of the D54A mutant of human T cell leukemia virus-1 capsid protein

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.