Proteolytic processing of Ty3 proteins is required for transposition

Abstract

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, Tyl, 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 PI position is occupied by an amino acid with a branched beta carbon.

Ty1-Mediated Integration of Expression Cassettes: Host Strain Effects, Stability, and Product Synthesis

“…Strain BWG1-7a has been used extensively in the study of Ty1 retrotransposition and shows high frequencies of transposition . Similarly, strain TMy16 has been employed in the study of Ty3 retrotransposition (Kirchner and Sandmeyer, 1993). Strain 334 allows gratuitous induc-tion of the GAL1 promoter due to gal1 and reg1 mutations (Hovland et al, 1989).…”

The yeast Ty3 retrotransposon contains a 5'-3' bipartite primer-binding site and encodes nucleocapsid protein NCp9 functionally homologous to HIV-1 NCp7

“…transcription as described . DNA encoding Ty3 5Ј RNA under T7 promoter control was generated by PCR using pEGTy3-1 (Kirschner and Sandmeyer, 1993), a 5Ј oligonucleotide containing T7 Po, an EcoRI site and 16 nucleotides of the 5Ј end of Ty3 RNA, and a 3Ј oligonucleotide complementary to position 1106-1121 of Ty3 RNA and containing a HindIII site. DNA was cloned into pSP64 and controlled by sequencing (pTy3-CG1).…”

“…The effect of 3Ј PBS mutations on Ty3 transposition was examined using a low-copy helper plasmid and a high-copy Ty3 plasmid encoding HIS (Kirschner and Sandmeyer, 1993). The yeast strain yTM443 (derivative of TMy18: MATa,bar1::his G, GAL3 ϩ , ΔTy3) was transformed with one HIS3-marked high-copy donor plasmid (pKO254 for the wildtype plasmid) (3Ј PBS sequences of pKO254 were replaced by a SpeI site, ACTAGT, to generate plasmids pCG100 with 3ЈΔPBSa, PCG101 with 3ЈΔPBSb and pCG102 with 3ЈΔPBSab) and with one unmarked low-copy helper plasmid to supply the necessary Ty3 proteins in trans (pJK312).…”

“…In view of the ubiquitous nature of NC protein among retroviruses and of its numerous key roles in virus structure and replication, we set out to compare the functions of NC proteins from two distantly related retroelements, namely HIV-1 and the yeast Ty3 retrotransposon. Ty3, an LTR-containing retroelement, encodes the nucleocapsid protein NCp9 which possesses a single zinc finger and is required for Ty3 transposition in yeast (Kirschner and Sandmeyer, 1993;Orlinsky and Sandmeyer, 1994), implying functional homology with HIV-1 NCp7. During an in vitro comparison of HIV-1 NCp7 and Ty3 NCp9 functions in genomic RNA dimerization and reverse transcription, we discovered an unusual bipartite PBS organization for Ty3 with segments of complementarity to primer tRNA i Met located at opposite ends of the genome.…”

Ty3 requires yeast La homologous protein for wild‐type frequencies of transposition

“…Gag3p is processed into major structural proteins, capsid (CA) and nucleocapsid (NC). POL3 is expressed as a Gag3p-Pol3p fusion dependent upon a + 1 frameshift at the end of GAG3 (Kirchner et al ., 1993). This polyprotein is processed into structural as well as catalytic proteins, PR, reverse transcriptase (RT) and integrase (IN) (Hansen et al ., 1992).…”