The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its Nterminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that "cuts" the transposon from a plasmid and "pastes" it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.

By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. (3) are responsible for the observed permissive and restrictive phenotypes. Retroviral replication can also be restricted by the action of the host APOBEC3G͞F proteins (for review, see ref. 4). Species differences for infection by human immunodeficiency virus type 1 (HIV-1) (5) have been exploited to clone a dominant restriction factor TRIM5␣ (6). Species differences in the TRIM5␣ sequence determine that it acts as a restriction factor for HIV-1 in simian cells, and it restricts N-tropic MLV in human cells (4). Another example is cyclin T1, which partners with HIV Tat (7), but a single amino acid difference in the murine protein renders it incompetent for Tat-mediated transactivation (8).We set out to identify further host-cell proteins that may be involved in the early life cycle of retroviruses. Therefore, we mutagenized hamster V79-4 cells and selected clones that were resistant to infection by MLV and HIV-1 viral vectors. Here we report on the isolation and characterization of one clone that is refractory to infection by MLV, feline immunodeficiency virus (FIV), and HIV-1 viral vectors. The block is postentry and before reverse transcription at uncoating of the virus. The block can be reversed pharmacologically with the proteasome (protease) inhibitor MG-132. Furthermore, the mutant can be complemented by a cDNA coding for a protein of unknown function, which we have termed a modulator of retrovirus infection (MRI). ResultsMutant 67-1 Cells Are Refractory to Infection by MLV, HIV-1, and FIV Viral Vectors. We initially mutagenized hamster V79-4 cells with the frameshift mutagen ICR-191 (an acridine half-mustard), and we multiply infected this population with an MLV-based retroviral vector that transduces the toxic gene barnase. Because these vectors recapitulate the early steps of the retroviral life cycle, we reasoned that cells that survive infection are either mutant in a cellular protein that is required for infection, or they simply escaped

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