Diseases caused by retroviruses such as AIDS and leukemia have intensified the need to understand the mechanisms of retrovirus replication. Our primary objectives are to understand how reverse transcription of viral mRNA occurs and how the cDNA products are integrated into the genome of infected cells. Owing to their similarity to retroviruses, LTR-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. During the synthesis of cDNA, reverse transcriptase (RT) generates a series of highly specific intermediates. We identified residues of Tf1 RT that recognize specific intermediates of cDNA by screening large numbers of RT mutants. A combination of genetic assays and physical analyses identified a set of 35 mutations that inhibited integration without reducing reverse transcription. Our experiments focused on a cluster of mutations in RNase H that included a region with five single amino acid substitutions in a five?amino acid segment. Surprisingly, these mutations in RNase H did not reduce the levels of full-length double stranded cDNA. Crystallographic studies by Dr. Edward Arnold and colleagues indicated that the corresponding residues of HIV-1 RT interact directly with the PPT. This observation led us to test whether the mutations in the RNase H of Tf1 were defective for either the recognition or processing of the PPT. A defect in the position of the PPT cleavage would alter the sequences at the 3' end of the minus strand and as a result have a drastic impact on the ability of IN to catalyze strand transfer. The sequence from the 3? ends of the minus strand cDNA from Tf1 particles was determined using ligation mediated PCR. The mutations clearly increased the levels of cDNA that retained the PPT at the 3? end. RNase H cleaves on either side of the PPT during the selection of the primer. In addition, RNase H cleaves the PPT to remove the primer after reverse transcription of the plus strand is initiated. Alterations in either the selection or removal of the PPT could result in cDNA that retains PPT sequence at the 3? end. A defect in the selection of the PPT would result in changes in the DNA sequence at the 3? and 5? ends of the cDNA while a reduction in PPT removal would not alter the DNA sequence at the 5? end of the cDNA. To distinguish between these two possibilities we analyzed the 5? ends of the cDNA by primer extension. Our results provided strong evidence that the mutations in RNase H specifically inhibited the removal of the PPT RNA from the 5? end of the plus strand. As a result, our data identified a cluster of conserved amino acids in RNase H that have the specific function of removing the PPT. The IN and cDNA of retroviruses form a preintegration complex (PIC) that must access the nucleus to perform integration. Because HIV-1 infects nondividing cells, the PIC must enter the nucleus through the nuclear pore complex. In an effort to model the import of HIV-1 into the nucleus, we examined the import of Tf1 in S. pombe. The Gag and cDNA of Tf1 enter the nucleus only after cells reach the stationary phase of growth. Previous studies identified a nuclear localization signal (NLS in the N-terminus of Gag that is required for transposition. Mutations in the NLS cause a severe defect in the nuclear localization of Gag and the cDNA. In separate experiments, we found that a factor of the nuclear pore, Nup124p, had a specific activity required for nuclear import of Tf1. Mutations in nup124 cause a significant defect in the import of Tf1 Gag and, surprisingly, do not reduce the import of other proteins. The results of two-hybrid analyses and precipitation studies revealed an interaction between the N-terminus of Nup124p and the Gag of Tf1. We proposed that the binding of Gag to Nup124p could mediate the nuclear import of Tf1. We further explored the function of Nup124p in the import of Tf1 Gag by studying the import of large proteins consisting of sections of Gag fused to GFP and lacZ. Interestingly, Nup124p was required for import of the first 50 amino acids of Gag fused to GFP-lacZ. The requirement for Nup124p was mapped to residues 10 through 30 of Gag. To understand how the Gag of Tf1 is imported into the nucleus and the role of Nup124p in this process, we introduced five independent mutations in Gag residues 10 through 30. The Gags of mutants A1, A2, and A3 were not imported into the nucleus. These residues are adjacent to the NLS and may contribute to its activity. We tested whether the mutants A1, A2, and A3 altered the interaction between Gag and Nup124p. The results of precipitation experiments showed that the mutations did not reduce binding with Nup124p. In addition, the interaction with Nup124p was mapped to 50 amino acids at the C-terminus of Gag. Surprisingly, the localization of Gag with mutations A4 and A5 occurred in the nucleus at both the log and stationary phase of cell growth and, the import of Gag with the A4 and A5 mutations did not depend on Nup124p. By subjecting cell extracts to gradient sedimentation we found Gag-A4 and Gag-A5 were defective for particle formation. Electron micrographs confirmed these results. Therefore, the ability of Gag to enter the nucleus corresponds with the loss in particle structure, suggesting that, at least in stationary-phase cells, the import of Tf1 into the nucleus is impeded by its particle structure and that Nup124p is required to overcome this block. Our analysis of Tf1 insertions resulting from the induction of transposition under laboratory conditions revealed a strong preference for pol II promoters. To define the determinants of the target sites we developed an in vivo assay for integration using a plasmid that contained ade6 as the target. Deletions in the 405 nucleotide region upstream of ade6 revealed that just the center third was required for efficient integration. The positions of insertion were determined by isolating kanamycin resistant plasmids and sequencing the DNA flanking Tf1. We isolated 50 separate insertions in the intact target plasmid and found ninety-five percent occurred within 300 nucleotides of the ade6 ORF. All of these insertions occurred within the same central third of the upstream region that was found to be necessary for efficient integration. In addition, the insertions clustered into four sites separated by 30 nucleotides each. To explore the significance of these four clusters, we scanned the intergenic region for transcription factors using micrococcyl nuclease. The clusters of integration corresponded closely with sites of micrococcyl supersensitivity. This indicated that Tf1 integration in the promoter of ade6 may be positioned by transcription factors. Together these data indicate that elements of the ade6 promoter are necessary and sufficient for recognition by Tf1. Additional experiments using fbp1 as a target support the conclusion that pol II promoters are the determinants of Tf1 integration. Over 85% of the insertions in the plasmid with fbp1 occurred in the promoter region.
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