The Ty1 Retrotransposon Restriction Factor p22 Targets Gag

Abstract

A novel form of copy number control (CNC) helps maintain a low number of Ty1 retrovirus-like transposons in the Saccharomyces genome. Ty1 produces an alternative transcript that encodes p22, a trans-dominant negative inhibitor of Ty1 retrotransposition whose sequence is identical to the C-terminal half of Gag. The level of p22 increases with copy number and inhibits normal Ty1 virus-like particle (VLP) assembly and maturation through interactions with full length Gag. A forward genetic screen for CNC-resistant (CNCR) mutations in Ty1 identified missense mutations in GAG that restore retrotransposition in the presence of p22. Some of these mutations map within a predicted UBN2 domain found throughout the Ty1/copia family of long terminal repeat retrotransposons, and others cluster within a central region of Gag that is referred to as the CNCR domain. We generated multiple alignments of yeast Ty1-like Gag proteins and found that some Gag proteins, including those of the related Ty2 elements, contain non-Ty1 residues at multiple CNCR sites. Interestingly, the Ty2-917 element is resistant to p22 and does not undergo a Ty1-like form of CNC. Substitutions conferring CNCR map within predicted helices in Ty1 Gag that overlap with conserved sequence in Ty1/copia, suggesting that p22 disturbs a central function of the capsid during VLP assembly. When hydrophobic residues within predicted helices in Gag are mutated, Gag level remains unaffected in most cases yet VLP assembly and maturation is abnormal. Gag CNCR mutations do not alter binding to p22 as determined by co-immunoprecipitation analyses, but instead, exclude p22 from Ty1 VLPs. These findings suggest that the CNCR alleles enhance retrotransposition in the presence of p22 by allowing productive Gag-Gag interactions during VLP assembly. Our work also expands the strategies used by retroviruses for developing resistance to Gag-like restriction factors to now include retrotransposons.

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces

“…Our results show that p22/p18 inhibits several steps in the process of retrotransposition prior to reverse transcription (Nishida, et al 2015, Saha, et al 2015, Tucker, et al 2015), which clarifies and greatly extends earlier work (Garfinkel, et al 2003, Matsuda and Garfinkel 2009) (Fig. 3).…”

“…The relative amount of p18 verses p22 is correlated with the level of PR produced from Ty1 (Tucker, et al 2015). When PR is expressed from the full ensemble of elements present in a genome or from pGTy1 induction p18 is the predominant form, whereas in low copy strains or an spt3 mutant only p22 is detected.…”

“…The functional organization of Ty1 Gag is not well understood (Fig, 4), therefore, mapping the regions responsible for Gag:p22/p18 interaction and CNC has revealed more information about GAG -encoded proteins (Nishida, et al 2015, Saha, et al 2015, Tucker, et al 2015). GST pulldown and co-immunoprecipitation analyses reveal interactions between p22/p18 and Gag.…”

Characterizing the functions of Ty1 Gag and the Gag-derived restriction factor p22/p18

“…14 A recent study from the Garfinkel lab found that mutations in GAG confer resistance to the p22 restriction factor. 24 Most of the mutations mapped within the Gag region important for VLP assembly but they did not interfere with p22 binding to Gag. 24 Thus, productive Gag/Ty1 RNA interactions were rescued from p22 restriction in those mutants.…”

“…24 Most of the mutations mapped within the Gag region important for VLP assembly but they did not interfere with p22 binding to Gag. 24 Thus, productive Gag/Ty1 RNA interactions were rescued from p22 restriction in those mutants. Other characterized resistant mutants contained substitutions in the NAC region important for RNA binding but their NAC functions remain to be studied.…”