RETROTRANSPOSONS AS MODELS FOR THE REPLICATION OF RETROVIRUSES
, Head, Section on Eukaryotic Transposable Elements
Angela Atwood-Moore, BA, Senior Research Assistant
Tracy Ripmaster, PhD, Research Assistant
Atreyi Chatterjee, PhD, Visiting Fellow
Hirotaka Ebina, PhD, Visiting Fellow
Yabin Guo, PhD, Visiting Fellow
Young-Eun Leem, PhD, Visiting Fellow
Anasua Majumdar, PhD, Visiting Fellow
Adam Evertts, BA, Postbaccalaureate Fellow
Robert Judson, BA, Postbaccalaureate Fellow
Ryan Rampersaud, BA, Postbaccalaureate Fellow
GPY/F is a highly conserved domain of integrase that mediates multimerization
The C-terminal domains in the integrases (INs) of LTR retrotransposons and retroviruses are not well conserved. However, close examination of C-termini did identify one motif that exists in a wide variety of INs. This module, termed the GPY/F motif, is present in the INs of a diverse set of LTR retrotransposons in the Metaviridae family (formally Ty3/gypsy) and in the gamma class of retroviruses. The function of the motif has not been studied. As a recombinant protein, the IN of Tf1 is highly soluble and possesses robust catalytic activity. These properties motivated us to consider the IN of Tf1 as a potential model for studying the function of the GPY/F domain.
In solution, the INs of HIV-1, MLV, and avian sarcoma virus (ASV) form a dimer-tetramer equilibrium. To measure the ability of Tf1 IN to multimerize, we tested full-length IN for interactions with the individual portions of the protein. Using a precipitation procedure and recombinant proteins, we found that the N-terminal domain, the central core, and the 71–amino acid GPY/F fragment all bound to the full-length IN. To test directly for stable multimers, we performed gel filtration with superdex 200. IN eluted as a single peak with an observed molecular weight of 126.5 kDa, a size consistent with a dimer. We observed that the central core by itself also forms a stable dimer. The ability of the central core and the full-length proteins to dimerize was typical of other INs.
To investigate the contribution of the C-terminal domain to the multimerization of IN, we subjected the GPY/F fragment to gel filtration with superdex 75. The elution profile had three major peaks, reflecting sizes of three species as monomer, dimer, and trimer. To test whether the GPF residues that define the motif contributed to multimerization, we generated single amino acid substitutions. Substitutions G364A and P365A completely disrupted multimerization of the GPY/F fragment, indicating that the GPY/F residues play a central role in promoting multimerization. In separate experiments to test for multimers, we subjected the GPY/F fragment to the chemical cross-linker dithiobis-(succinimidyl) propionate. The results indicated that the protein was in an equilibrium of monomers, dimers, trimers, and tetramers. We propose a model wherein full-length IN in its synaptic complex with the donor and target DNA forms a tetramer. Such a model is supported by the recent finding of Robert Craigie and colleagues (NIDDK) that HIV-1 IN does form a tetramer when purified in a synaptic complex. Our finding that the GPY/F fragment forms trimers and tetramers suggests that the GPY/F motif plays an important role in tetramerizing IN in the synaptic complex.
The C-terminal domains of INs are known to bind to DNA without sequence specificity. To map which sections of Tf1 IN interact with DNA, we assayed each of the individual domains for DNA binding. We mixed labeled oligonucleotides with the individual domains and cross-linked the mixtures with UV. The full-length IN, core, and GPY/F fragment demonstrated substantial DNA binding activity that corresponds well to what has been described for other INs. In additional experiments, the G364A and P365A substitutions in the GPF residues of the GPY/F fragment did not reduce DNA binding, indicating that other sequences in the GPY/F fragment mediated the DNA binding activity and that the GPY/F residues appear to be specific for promoting multimerization. Our finding that the G364A and P365A mutations did not reduce DNA binding also indicated that multimerization is not required for DNA binding.
By generating recombinant IN with the substitution G364A, we tested the GPY/F residues’ contribution to catalysis. The mutation significantly reduced strand transfer activity, as is consistent with the model that tetramer formation is required for strand transfer activity. The widespread conservation of the GPY/F motif suggests that, in other INs, the motif may also promote multimerization and strand-transfer activity.
Gao X, Hou Y, Ebina H, Levin H, Voytas D. Retrotransposons and heterochromatin: the role of integrase chromodomains in target site specificity. Genome Res 2007, in press.
Hizi A, Levin H. The integrase of the long terminal repeat-retrotransposon Tf1 has a chromodomain that modulates integrase activities. J Biol Chem 2005;80:39086-94.
Integration preference of Tf1 for pol II promoters
Our analysis of the genome sequence of S. pombe revealed large numbers of pre-existing Tf transposons positioned 200 bp to 400 bp from the 5¢ end of ORFs (open reading frames). Experiments based on the production of new integration events revealed that the position of Tf1 upstream of ORFs resulted from integration preference. The similarity of this pattern of integration to the preference of HIV-1 for pol II transcription units motivated us to study Tf1 integration as a model for HIV-1. To define the determinants of the target sites recognized by Tf1, we developed an in vivo assay for integration by using a plasmid that contained ade6 as the target and a plasmid with Tf1 that induced transposition. We expressed a version of Tf1 containing a neo gene that would cause cells with integration events to gain resistance to G418. When Tf1-neo was expressed, the plasmid with ade6 provided an efficient target for integration. We isolated 50 separate insertions in the intact target plasmid and found that 95 percent occurred within a 160 nt region in the ade6 promoter. To determine which sequences of ade6 were required for efficient integration, we created a series of 10 deletions within the target plasmid. The analysis revealed that the 160 nt region of the promoter was the only sequence required for efficient integration. To determine whether promoter activity was required for integration, we measured transcript levels of ade6. Deletions of sequence on either side of the 160 nt region caused 5- to 10-fold reductions in ade6 mRNA. Nevertheless, the deletions caused no reduction in integration efficiency. The results indicated that transcription was not important for target site activity.
We considered whether transcription factors themselves were directing the integration of Tf1. To identify positions where factors bind in the promoter of ade6, we used micrococcal nuclease mapping and observed a strong correlation between micrococcal-sensitive sites and the position of the prominent insertion sites, suggesting that transcription factors played a role in directing Tf1 integration. However, the factors that bind to the promoter of ade6 are not known. To test the role of transcription factors in directing Tf1, we examined the integration properties of fbp1, a promoter that binds to the transcription activator Atf1p. We found that the fbp1 promoter was a target for Tf1 insertion and that most insertions occurred 30 nt from the position where Atf1p binds. A mutation that blocks the binding of Aft1p caused a nine-fold reduction in Tf1 integration at the promoter of fbp1. We also tested Atf1p for a role in integration by measuring Tf1 activity in cells that lacked the gene for Atf1p. Target assays with these cells exhibited the same nine-fold reduction of integration in the promoter of fbp1 that we observed when the Atf1p binding site was mutated. The data indicate that Atf1p is responsible for targeting Tf1 to specific insertion sites in the fbp1 promoter. Atf1p’s role in directing integration may be to bind to promoters such as fbp1 and to recruit Tf1 IN to the binding sites by binding directly to Tf1 IN. The results of immunoprecipitation reactions demonstrated that, in vivo, Atf1p did bind to IN. The data support the model whereby Atf1p binds to the promoter of fbp1 and mediates integration by binding to IN.
Cam H, Noma K, Ebina H, Levin H, Grewal S. Host genome surveillance for retrotransposons by transposon-derived proteins. Nature 2007 [E-pub ahead of print].
Kelly F, Levin HL. The evolution of retrotransposons in Schizosaccharomyces pombe. Cytogenet Genome Res 2005;110:566-74.
Levin H. Metaviruses. In: Mahy BW, Van Regenmortel MHV, eds. Encyclopedia of Virology. Elsevier, in press.
The hermes transposon of the house fly is highly active in S. pombe
The integration of Tf1 occurs primarily into pol II promoters. Although we currently believe that such preference results from a mechanism that actively targets Tf1, it is possible that the insertion bias is attributable to greater accessibility at the promoter sequences. We are currently testing this possibility in S. pombe cells by studying the integration pattern of hermes, a “cut and paste” transposon that was isolated from the house fly. Given that hermes propagates in a host that is evolutionarily distant from S. pombe, a mechanism that would actively position insertion sites is unlikely to exist. Thus, any integration of hermes in S. pombe would probably occur at positions accessible to the transposase. In addition, unbiased activity of a transposon in S. pombe could be widely adapted as a tool for random mutagenesis. In the absence of methods for transposon mutagenesis of S. pombe, a method for insertional mutagenesis would be a significant contribution to the field.
We expressed the transposase of hermes in S. pombe by fusing its gene to the promoter of nmt1 and used three versions of the nmt1 promoter to express various levels of the transposase. Immunoblots of cell extracts demonstrated that the transposase was expressed in a stable form and in amounts that corresponded to the strength of the promoter. To measure transposition activity, we used cells that expressed both the transposase and a plasmid-encoded copy of neo flanked by the terminal inverted repeats (TIRs) of hermes. We tested the ability of the transposase to cut out neo with the TIRs and insert this DNA into the pombe genome. Once the transposase was expressed, we grew cells on medium containing 5-fluoro-orotic acid, a treatment that removes the plasmid carrying the hermes TIRs and neo. We then grew the strains on agar plates containing G418 in order to select for cells that had transposed copies of hermes. The strains expressing the transposase generated surprisingly high levels of resistance to G418. We analyzed for potential insertion events 26 independent strains that became G418-resistant. It was significant that each strain had acquired a copy of hermes as the result of a bona fide integration event. Analysis of the inserted copies revealed that 54 percent disrupted ORFs. Given that 60 percent of the pombe genome consists of coding sequence, our results indicate that the insertion of hermes did not discriminate between coding and noncoding sequences, a finding that stands in strong contrast to the integration of Tf1, for which virtually none of the inserts occurs in ORFs. The data indicate that hermes can be used as a tool for the random disruption of pombe genes.
Evertts A, Plymire C, Craig N, Levin H. The hermes transposon of Musca domestica has robust activity in Schizosaccharomyces pombe that disrupts open reading frames. Genetics 2007;177:2519-23.
COLLABORATOR
Charles Hoffman, PhD, Boston College, Chestnut Hill, MA
For further information, contact henry_levin@nih.gov.
