Frederic D Bushman

..Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection...

..We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences...

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..Here, we review retroviral DNA integration, with emphasis on recent genome-wide studies of targeting and on the status of efforts to modulate target-site selection...

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..These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals...

..The approaches to sequencing and bioinformatics analysis reported here should be widely useful in assessing the outcome of gene therapy trials...

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..The methods described here should allow integration site populations from human gene therapy to be deeply characterized with spatial and temporal resolution...

..Lastly, we describe possible roles for the A22 resolvase DNA-branch nuclease activity in DNA replication and repair...

..These data define the full variola virus topoisomerase recognition site and provide a new window on topoisomerase function in vivo...

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..Analysis of additional published integration site sequences also indicated that the route of entry did not affect integration site selection for other viral envelopes as well...

..e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold...

..These data show how divalent metals dictate the conformation of FPV resolvase-DNA complexes and subsequent DNA cleavage...

..Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies...

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..The pyrosequencing and bioinformatic methods described here should be useful for investigating many aspects of retroviral DNA integration...

..These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/...

..Long-term survival after lung transplantation is limited by infectious complications and by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection linked in part to microbial triggers...

..Thus, alternative enterotype states are associated with long-term diet...

..These findings provide a tractable poxvirus resolvase usable for the development of small molecule inhibitors...

..Thus these data provide an overview of the composition of the human gut virome and associate virome structure with diet...

..These findings indicate that accumulation of HERVs in the human germline is a two-step process: integration targeting biases direct initial accumulation, then purifying selection leads to loss of proviruses disrupting gene function...

..However, these two measures assign too much weight either to rare lineages or to most abundant lineages, which can lead to loss of power when the important composition change occurs in moderately abundant lineages...

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..Some 1-hydroxy-1,8-naphthyridin-2(1H)-one compounds showed anti-poxvirus activity...

..Engineered T cells are a promising form of synthetic biology for long-term delivery of protein-based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases...

..Thus, we conclude that favored integration near DNase I-hypersensitive sites does not imply that integration takes place exclusively in nucleosome-free regions...

..The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections...

..These data specify the ligands bound at the catalytically relevant IN monomer and allow more-specific modeling of the mechanism of inhibitors that also bind this surface of IN...

..Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy...

..Our objective was to analyze the pathways leading to resistance of HIV to the integrase (IN) inhibitor raltegravir (RAL)...

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..The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and C. difficile infection...

..We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells...

..These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over...

..Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well...

..However, studies of the lung are challenging due to difficulties in working with low biomass samples...

..These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing...

..Both simulations and real data applications show that ssCCA performs better than the standard sparse CCA in identifying meaningful variables when there are structures in the data...

..Thus altered integration targeting, at least to the degree achieved here, is not a major concern when using the Alu-PCR assay...

..These results provide important insights into the architecture of the retroviral intasome...

..These results have implications for therapies targeting the latent reservoir and suggest that some latent cells could be cleared by a robust immune response...

..Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases...

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..Thus, β-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control...

..Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease...

..A web-based tutorial for implementing this method is available at http://microb230.med.upenn.edu/tutorials/wangTutorial...

..Unexpectedly, this enrichment was only detected after the challenge with HIV-1(BaL), where the viral genome would form an imperfect duplex with envAS, and not HIV-1(NL4-3), where a perfectly matched duplex would form...

..A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and integration targeting...

..No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation...

..This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome...

..These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals...

..Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration...

..There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans...

..These findings provide the most detailed sequence-based analysis of HCV transmission bottlenecks to date. The analytical methods described here are broadly applicable to studies of viral diversity using deep sequencing...

..Analysis of the reaction products revealed that A22R cleaves to leave a 3' hydroxyl at the cleaved phosphodiester bond...

..It remains unclear whether specific NVP resistance mutations or combinations of mutations predict virologic failure in infants when present at low frequencies before NVP-based treatment...

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..LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells...

..The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting...

..These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication...

..CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals...

..4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase...

..Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection...

..Despite these differences, recent data hints at a common targeting mechanism-tethering of integration complexes to proteins bound at favorable sites...

..Here we describe the use of RNA interference (RNAi) in live chick embryos to block replication of an avian retrovirus. We also describe inhibition of ASLV and HIV replication in cell culture with RNAi...

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..The asymmetry of the complex is a feature not incorporated in previous structural models and potentially provides the first view of an asymmetric reaction intermediate...

..We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation...

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..Research into the mechanisms of retroviral integration site selection has shed light on the phenomena of insertional mutagenesis and viral latency...

..Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors...

..None of the cellular mutations showed a strong effect on normal integration, consistent with the idea that the cellular pathways leading to circularization are not involved in productive integration...

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..Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication...

..Lamellarin alpha 13,20-disulfate is a moderate inhibitor of both HIV-1 integrase and cancer cell lines. Lamellarin H is a more potent inhibitor of HIV-1 integrase but lacked the specificity required to be medicinally useful...

..Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer...

..2. Investigate the mechanism by which interferon treatment of target cells abrogates the early steps of HIV infection. 3. Use microarray technology to investigate the host cell response to early steps of infection. ..

..Long term, these studies, together with data on protein structures, will provide a high-resolution picture of the organization and function of preintegration complexes. ..

..abstract_text> ..

..The Specific Aims are: Aim 1. Functional studies of IN-DNA complexes Aim 2. Structural analysis of IN-DNA complexes. ..

..The integration system is a target for antiviral agents, and the proposed experiments should provide information directly useful for designing inhibitors. ..

..Ultimately, these studies taken together will provide a high-resolution picture of the organization and mechanism of action of the preintegration complex...

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..In addition, poxviruses have been implicated as possible biological warfare agents; thus poxvirus topoisomerase inhibitors might be use for treating such infections. ..

..Here we propose to apply massively parallel pyrosequencing to analyzing vector integration in samples from the historic SCID-X1 trial. ..