Ty3 viruslike particle morphogenesis and host interactions

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DESCRIPTION (provided by applicant):Retroviruses are the cause of AIDS and cancer and retroelements are a significant component of eukaryotic genomes. Nevertheless, there is a considerable amount which remains unknown about interactions of retroviruses and retroelements with host cells during the cytoplasmic phase of the lifecycle from assembly to arrival at the nuclear pore. We are using the Ty3 retroviruslike element in Saccharomyces cerevisiae as a model system to study this aspect of the lifecycle. Ty3 is comprised of two overlapping reading frames, GAG3 and POL3, encoding Gag3 and Gag3-Pol3 which are processed into mature capsid, nucleocapsid, protease, reverse transcriptase, and integrase. These proteins are analogous in structure and function to their retrovirus counterparts. Cells in which Ty3 is expressed produce virus like particles (VLPs) which undergo proteolytic maturation, reverse transcription, uncoating, and nuclear entry. Over past funding periods we have developed tools which will facilitate the proposed work including a set of over 100 mutants defective in Ty3 transposition, antibodies against the Ty3 proteins, and Ty3 elements tagged with fluorescent reporters. Microscopic observation of cells producing Ty3 has shown that Ty3 mRNA and proteins first appear in the peripheral cytoplasm, but are trafficked to perinuclear clusters where VLPs are observed. The proposed work has specific aims A-E:. A, B. Identification of Ty3 RNA and structural protein requirements for VLP assembly and nuclear delivery. Protein and RNA will be expressed separately and in trans to determine whether the RNA must encode Gag3 in order to be trafficked to the perinuclear region. If Gag3 is required, whether it can be supplied in trans will be determined. Gag3 residues required for assembly and trafficking of Ty3 protein and RNA will be determined. C. 130 mutants have been identified that affect Ty3 transposition. Ty3 expression and protein and cDNA intermediates will be analyzed in a high priority subset of these strains in order to identify mutations that affect assembly, processing, cDNA synthesis and nuclear delivery of the VLP. Genes that encode proteins that interact with Ty3 structural proteins will be identified by tagging Gag3 and CA with a tandem affinity tag and performing mass spectrometry on affinity-purified complexes. D, E. The spectrum of mutants and preliminary experiments support a model in which the cytoskeleton and endosomal trafficking systems move Ty3 proteins and RNAs toward a perinuclear cluster which has points of contact with the nucleus and where much of assembly and cDNA synthesis are likely to occur. Experiments are proposed to test this model.