Search

Abstract: In many studies of HIV replication, it is useful to quantify the number of HIV proviruses in cells against a background of unintegrated forms of the HIV DNA. A popular method for doing so involves quantitative PCR using one primer complementary to the HIV long terminal repeat (LTR), and a second primer complementary to a cellular Alu repeat, so that PCR product only forms from templates where a provirus is integrated in the human genome near an Alu repeat. However, several recent studies have identified conditions that alter distributions of HIV integration sites relative to genes. Because Alu repeats are enriched in gene rich regions, this raises the question of whether altered integration site distributions might confound provirus abundance measurements using the Alu-PCR method. Here modified versions of the HIV tethering protein LEDGF/p75 were used to retarget HIV integration outside of transcription units, and show that this has a negligible effect on Alu-PCR quantitation of proviral abundance. Thus altered integration targeting, at least to the degree achieved here, is not a major concern when using the Alu-PCR assay. [Copyright &y& Elsevier]

Mobile elements rely on cellular processes to replicate, and therefore, mobile element proteins frequently interact with a variety of cellular factors. The integrase (IN) encoded by the retrotransposon Ty5 interacts with the heterochromatin protein Sir4, and this interaction determines Ty5's preference to integrate into heterochromatin. We explored the hypothesis that Ty5's targeting mechanism arose by mimicking an interaction between Sir4 and another cellular protein(s). Mutational analyses defined the requirements for the IN-Sir4 interaction, providing criteria to screen for cellular analogues. Esc1, a protein associated with the inner nuclear membrane, interacted with the same domain of Sir4 as IN, and 75% of mutations that disrupted IN-Sir4 interactions also abrogated Esc1-Sir4 interactions. A small motif critical for recognizing Sir4 was identified in Esc1. The functional equivalency of this motif and the Sir4-interacting domain of IN was demonstrated by swapping these motifs and showing that the chimeric IN and Esc1 proteins effectively target integration and partition DNA, respectively. We conclude that Ty5 targets integration by imitating the Esc1-Sir4 interaction and suggest molecular mimicry as a general mechanism that enables mobile elements to interface with cellular processes. [ABSTRACT FROM AUTHOR]

Summary: The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5''s mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock''s hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge. [Copyright &y& Elsevier]