Abstract
Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. To define some of these functions, the cell cycle dependence of transposition of the yeast retroviruslike element Ty3 was examined. Ty3 is unique among retroviruslike elements because of the specificity of its integration, which occurs upstream of genes transcribed by RNA polymerase III. A physical assay for Ty3 transposition which takes advantage of this position-specific integration was developed. The assay uses PCR to amplify a product of Ty3 integration into a target plasmid that carries a modified tRNA gene. By using the GAL1 upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated. This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone alpha-factor. The restriction of transposition was not due to changes in transcription of either Ty3 or tRNA genes or to aspects of the mating pheromone response unrelated to cell cycle arrest. The block of the Ty3 life cycle was reversed when cells were released from G1 arrest. Examination of Ty3 intermediates during G1 arrest indicated that Ty3 viruslike particles were present but that reverse transcription of the Ty3 genomic RNA into double-stranded DNA had not occurred. In G1, the Ty3 life cycle is blocked after particle assembly but before the completion of reverse transcription.
Highlights
Host cell cycle genes provide important functions to retroviruses and retroviruslike elements
The common steps of replication and integration for these elements, referred to collectively as retroviruslike elements, are (i) transcription of an integrated DNA copy of the element into RNA, (ii) translation of the RNA followed by assembly of particles composed of the RNA as well as the proteins encoded by the element, (iii) reverse transcription of the RNA into DNA, and (iv) integration of this DNA copy into the host cell genome
To study the effect of cell cycle arrest on Ty3 transposition it was necessary to (i) efficiently arrest cells at a particular point in the cell cycle, (ii) control expression of the Ty3 element so that it could be activated during cell cycle arrest, and (iii) assay Ty3 transposition during cell cycle arrest
Summary
Host cell cycle genes provide important functions to retroviruses and retroviruslike elements. By using the GALl upstream activating sequence to regulate expression of Ty3, transposition was detected within one generation of cell growth after Ty3 transcription was initiated This physical assay was used to show that Ty3 did not transpose when yeast cells were arrested in G1 during treatment with the mating pheromone aL-factor. In cases in which productive infection of quiescent cells is impeded [16, 17, 22, 59, 62], reverse transcription of genomic RNA is deficient. RSV cannot productively infect cells arrested in S phase [25] Reverse transcription of both MLV and RSV genomic RNAs occurs during these cell cycle arrests, but integration does not. MLV DNA is excluded from the nucleus until the cell has completed M phase [48], but for RSV it is not known how integration is affected
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