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RETROTRANSPOSONS AS MODELS FOR THE REPLICATION OF RETROVIRUSES
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Henry L. Levin, PhD, Head, Section on Eukaryotic Transposable Elements Angela Atwood-Moore, BA, Senior Research Assistant |
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| The medical importance of retroviruses such as HIV-1 has intensified the need to understand the molecular details of reverse transcription and integration. As a result of their similarity to retroviruses, long terminal repeat (LTR)-retrotransposons in simple eukaryotes are a powerful model, with information about LTR-retrotransposons possibly leading to the identification of new antiviral strategies or targets that could be used to combat the spread of HIV-1. The retrotransposon studied in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. One of our objectives is to map the residues of reverse transcriptase (RT) that mediate individual steps of reverse transcription. Another important objective of our research is to identify the mechanisms responsible for the insertion preference of Tf1 at pol II promoters. Our work is largely motivated by similar preferences that have been reported for the integration of HIV-1 and murine leukemia virus. Our third objective centers on the possibility that the nuclear import of viruses such as HIV-1 could be the target of antiviral therapies. We have chosen as an important goal the study of Nup124p, a nuclear pore factor that is specifically required for the import of Tf1. The primer grip in the RNase H of RT, a conserved domain that contributes specifically to removal of the plus-strand primer from the 5' end of the cDNA Atwood-Moore, Ejebe, Levin; in collaboration with LeGrice The reverse transcription of retroviruses and LTR-retrotransposons unfolds as a complex sequence of reactions that produce several critical intermediate products, including the initial minus-strand product of cDNA called a strong stop, the extended minus strand, the plus-strand primer (PPT), the plus-strand strong stop, and, ultimately, the full-length double-stranded cDNA. The synthesis of these intermediates requires both the DNA polymerization and RNase H activities of RT. The RNase H domain must degrade the RNA once it is used as template, recognize and preserve the PPT, and then remove the PPT after it has primed plus-strand synthesis. Although much is known about the amino acids that catalyze the DNA synthesis and RNA degradation, much less is known about the residues and structures required for the recognition and removal of the PPT. To identify which residues of RT mediate the interactions with the PPT, we used random mutagenesis of Tf1 RT and screened for mutations that allowed the synthesis of cDNA intermediates but inhibited integration. We reasoned that the mutants would likely be defective in the late steps of reverse transcription such as those requiring interactions with the PPT. We generated a collection of 3,000 strains that were unable to support transposition and screened them with a genetic assay that detects intermediates of reverse transcription. We found 35 strains that, despite their defect in transposition, exhibited wild-type levels of homologous recombination. Immunoblot analysis demonstrated that the mutations did not affect levels of RT and integrase (IN). Surprisingly, DNA blots showed that many of the mutants produced what appeared to be normal levels of full-length, double-stranded cDNA. The remaining elements produced an interesting array of incomplete products. Our experiments focused on a cluster of mutations in RNase H that included a region with five mutations in a five-amino acid segment. Interestingly, the transposons with mutations in this region produced normal levels of full-length double-stranded cDNA. We demonstrated the significance of this domain by a crystallographic study of HIV RT showing that the domain is a "primer grip" that interacts directly with the nucleotides of the PPT adjacent to the position cleaved by RNase H to create and then remove the plus-strand primer. Both the position and the sequence of the primer grip residues in HIV corresponded to the cluster of mutations we identified in the RNase H of Tf1. The specific residues corresponded to HIV RT residues 475, 497, 501, 502, 503, and 505. These observations led us to propose that the mutations in the RNase H of Tf1 that result in full-length cDNA are defective for transposition because the recognition of the PPT was altered. A defect of a few nucleotides in the cleavage of the PPT from the 5' end of the plus strand would be "templated" into altered sequences at the 3' ends of the minus strand, producing a drastic impact on the ability of IN to catalyze strand transfer.
To test the hypothesis that the cluster of mutants in RNase H altered the cleavages of the PPT, we characterized the sequences of the 3' end of the minus strand cDNA. We used RNA ligase to attach an oligo to the 3' ends of the cDNA to amplify by PCR a product containing the junction of the 3' end. We inserted the product into a vector and 150 to 200 independent junctions and sequenced them for each mutant RT. In the 3' ends of the cDNA produced by wild-type Tf1-neo, just 35 percent corresponded to the full-length species. However, the mutations showed a significant drop in the full-length species and correlated with an increase in 3' ends that retained the PPT sequence. Thus, our screen for mutations in RT that make cDNA products identified the primer grip of RNase H as contributing to the removal of the PPT RNA from the end of the LTR. Surprisingly, this ability to process the PPT is specific and not required for the other steps in the pathway of reverse transcription.
Haag LE, Lin J, Levin H. Evidence for the packaging of multiple copies of Tf1 mRNA into particles and the trans priming of reverse transcription. J Virol 2000;74:7164-7170. Levin H. The retrotransposons of Schizosaccharomyces pombe. In: Egal R, ed. The molecular biology of Schizosaccharomyces pombe. Heidelberg: Springer, 2003; in press. Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses Teysset, Kim, Dang, Levin To determine whether the 27 kDa protein encoded at the N-terminus of the Tf1 ORF is the functional equivalent of a retroviral Gag, we generated deletions of 10 amino acids in each of the four regions of hydrophylicity. We tested the transposition activities of Tf1 elements with the deletions, referred to as Tf1-delta-A, Tf1-delta-B, Tf1-delta-C, and Tf1-delta-D, by using a genetic assay that is based on the resistance to G418 resulting from the insertion of a neo-marked copy of Tf1. The transposition assays showed that Tf1-delta-B and Tf1-delta-C exhibited no transposition at all; Tf1-delta-A and Tf1-delta-D generated very low levels.
To test whether the deletions in Gag caused defects in the production of cDNA, we used DNA blot analysis, which showed that Tf1-delta-A generated a wild-type level of cDNA. Tf1-delta-D produced significantly lower levels of cDNA while repeated examination of Tf1-delta-B and Tf1-delta-C detected no cDNA bands. The data indicated that the reduced levels of transposition observed for Tf1-delta-B, Tf1-delta-C, and Tf1-delta-D were attributable to significantly reduced levels of reverse transcription. On the other hand, the reduced transposition caused by Tf1-delta-A likely reduced transport of Tf1 into the nucleus. We base our conclusion on our work that identified a nuclear localization signal within the sequence deleted by Tf1-delta-A. To investigate whether the transposition defects were attributable to decreased stability of Tf1 proteins, we performed immunoblot analysis with antibodies raised against the Tf1 Gag, IN, and RT. Although the deletions had little impact on the levels of RT and IN, Tf1-delta-B and Tf1-delta-C produced very low levels of Gag. Thus, the Gag protein was required for transposition.
Previous work established that Gag, RT, IN, and cDNA assemble into large macromolecular complexes that cosediment in sucrose gradients. We tested whether Tf1-assembled spherical virus-like particles (VLPs) can be observed with electron microscopy and found that particles with typical structures were easily detectable in the cytoplasm of cells expressing the Tf1-WT element. They appeared as clusters of well-defined particles or aggregates. The maximum diameter of these intracytoplasmic particles was 50 nm. We used the same techniques of electron microscopy to determine whether the forms of Tf1 with deletions in Gag produced spherical particles and found no particles or dense aggregates in the strains with Tf1-delta-C. Given that the Gag proteins were unstable in Tf1-delta-C, the results indicate that Gag was required for particle formation. In the case of Tf1-delta-A, we found dense aggregates in the cytoplasm and for Tf1-delta-D, with the size and number of particles similar to wild-type Tf1.
Many Gag proteins of retroviruses and LTR-retrotransposons are able to form particles in the absence of any other retroelement proteins. We tested whether the expression of the Tf1 Gag was sufficient for the formation of spherical particles. Tf1-PRfs, the version of Tf1 with the frame shift at the beginning of PR, does not express RT or IN. Electron microscopy revealed that a large percentage of cells expressing Tf1-PRfs contained particles and dense aggregates, indicating that Tf1 Gag is sufficient for the assembly of spherical particles. One principal role of Gag proteins is to assemble into particles that package the RNA of the retroelement. We therefore tested whether Tf1 Gag was required for packaging the Tf1 RNA. Our approach followed a previously developed assay that detects the amount of Tf1 RNA protected from degradation by the nuclease Benzonase. The strains expressing Tf1-delta-B and Tf1-delta-C were unable to protect RNA from degradation, corresponding to the reduced levels of Gag and the lack of particles detected by electron microscopy. Thus, Gag was required for packaging the Tf1 mRNA.
In summary, the lack of transposition activity resulting from deletions B and C was attributable to the absence of Gag. The low levels of Gag resulted in no formation of particles, no packaging of mRNA, and no reverse transcription. The results of the electron microscopy demonstrated that the expression of Gag was sufficient for particle formation. Taken together, our results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. Teysset L, Dang V, Kim M, Levin H. An LTR-retrotransposon of Schizosaccharomyces pombe expresses a Gag-like protein that assembles into virus-like particles and mediates reverse transcription. J Virol 2003;77:5451-5463. Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements derived from the complete genome sequence of S. pombe Bowen, Levin; in collaboration with Epstein, Jordan, Wood The integration of HIV-1 cDNA shows a significant preference for actively transcribed genes. Similarly, the insertion of murine leukemia virus shows a strong preference for sites within 5kb from where pol II initiates transcription. Very little is known about how these viruses interact with the structures of chromatin and recognize their target sites. The study of the retrotransposons Ty1 and Ty3 of S. cerevisiae has demonstrated clearly that much has been learned about integration directed to the promoters of pol III-transcribed genes. However, our recent observation that the integration of Tf1 occurs specifically at pol II promoters presents an opportunity to study in S. pombe an integration mechanism that parallels that of retroviruses. The complete DNA sequence of the genome of S. pombe provides the opportunity to investigate the entire complement of transposable elements (TEs), their association with specic sequences, their chromosomal distribution, and their evolution. Through homology-based searches, we identified the sequences of Tf elements used in our analysis. Only two families of LTR retrotransposons, Tf1 and Tf2, are known to exist in S. pombe. They are closely related elements that differ only in their sequence of Gag and the LTRs. We used the nucleotide sequences of Tf1 and Tf2 to query BLAST against the entire genome of the laboratory strain of S. pombe, 972. Our analysis confirmed that there were no full-length Tf1 elements within the laboratory strain 972. There were, however, 13 full-length elements of Tf2. The full-length Tf2 elements are a homogeneous group with an average pairwise DNA sequence identity of 99.7 percent. The high level of sequence identity among the Tf2 elements indicates that the elements transposed very recently.
The solo LTRs found in the genome of S. pombe can be classified into at least three large groups as follows: (1) those that are closely related to Tf2; (2) those that are closely related to Tf1; and (3) many smaller families of LTRs that are more distantly related to Tf1 and Tf2. We derived these designations from a complete phylogenetic characterization of the LTRs by using DNA distance values from comparisons with LTRs of full-length Tf1 and Tf2 elements. The short distances of many of the terminal branches of Tf1 and Tf2 indicate that these retrotransposons represent the largest number of recently active elements within the genome.
To determine whether preferences for integration sites existed during the insertion of the 186 Tf sequences, we compared the locations of solo LTRs and full-length Tf2s with the positions of all 4,984 predicted open reading frames (ORFs) of S. pombe. We found that all insertions were located exclusively in intergenic regions of the genome, in intergenic spaces that contained pol II promoters. In addition, the LTRs were clustered within 300 nulceotides of the 5' end of ORFs. These specific positions could be the result of selective pressures, either positive or negative, that favor populations of S. pombe with each of the patterns observed. Alternatively, the patterns of the Tf sequences that we observed could be strictly the result of biochemical mechanisms of integration. Each of the biases in the position of Tf sequences described above was very similar in pattern and magnitude to the positions of insertions resulting from the induction of Tf1 transposition under laboratory conditions. These extensive similarities argue strongly that the biases in the position of Tf sequences as observed in the genome of S. pombe result from biochemical preferences of integration for specific sites. The data indicate that Tf elements recognize and insert upstream of RNA polymerase II promoters.
Bowen N, Jordan I, Epstein J, Wood V, Levin H. Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements derived from the complete genome sequence of Schizosaccharomyces pombe. Genome Res 2003;13:1984-1997. Singleton T, Levin H. An LTR-retrotransposon of fission yeast has strong preferences for specific sites of insertion. Eukaryotic Cell 2002;1:44-55. Silverstein R, Richardson B, Levin H, Allshire R, Ekwall K. A new role for the transcriptional co-repressor SIN3; regulation of centromeres. Current Biol 2003;13:68-72.
COLLABORATORS Jonathan Epstein, PhD, Unit on Biologic Computation, NICHD, Bethesda MD King Jordan, PhD, National Center for Biotechnology Information, NIH, Bethesda MD Stuart LeGrice, PhD, HIV Drug Resistance Program, NCI, Frederick MD Valerie Wood, PhD, The Sanger Centre, Cambridge, UK
For further information, contact henry_levin@nih.gov |
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