Faculty Bibliography

Overexpression of dominant-negative mutants of various viral proteins can result in 'intracellular immunization'. Here we describe a new approach to interfering with viral replication in which a nuclease is fused to a capsid component so that the nuclease is encapsidated inside the virion where it can inactivate viral nucleic acid. We used Ty1, a yeast retrotransposon whose transposition closely parallels retroviral replication mechanisms and serves as an easily manipulated model for the retroviral infection process. We constructed fusion genes consisting of the region encoding the N-terminal portion of the TYA/TYB open reading frames of retrotransposon Ty1 and either of two different nuclease genes. Ty1-nuclease fusion proteins are targeted to Ty1 virus-like particles, and are active in degrading nucleic acids. A Ty1-barnase fusion protein causes 98-99% reduction in the efficiency of Ty1 transposition in vivo, presumably by degrading encapsidated Ty1 RNA. This strategy, referred to as capsid-targeted viral inactivation, may be useful for interfering with the replication of retroviruses and other viruses.

Haploid yeast strains bearing approximately double the normal number of Ty1 elements have been constructed using marked GAL/Ty1 fusion plasmids. The strains maintain their high transposon copy number and overall genome structure in the absence of selection. The strains bearing extra Ty1 copies are surprisingly similar phenotypically to the parental strain. The results suggest that the limit to transposon copy number, if any, has not been reached. When these strains are crossed by wild-type strains (i.e., bearing the normal complement of Ty1 elements) or by strains of opposite mating type also bearing excess Ty1 elements, normal to very slightly reduced spore viability is observed, indicating that increasing the extent of transposon homology scattered around the genome does not result in significant increases in frequency of ectopic reciprocal recombination. The results suggest that yeast cells have evolved mechanisms for coping with excess transposon copies in the genome.

Recent developments in the area of the transposition mechanisms used by retrotransposons and related retroviral pathways are discussed. In particular, advances in the areas of retrotransposon gene expression, virus-like particle assembly, reverse transcription, and integration are reviewed.

Genetic screening of a yeast genomic library in a high-copy-number vector identified the normally single copy tRNA(CCU Arg) gene as one of the genes and reduces Ty1 transposition frequency when overexpressed. Immunoblot analyses of Ty1-encoded proteins indicate an inverse correlation between the copy number of the tRNA gene and the production of the TYB protein. Thus, Ty1 transposition frequency is apparently regulated by the level of tRNA(CCU Arg) in yeast cells.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.