-
Nature Reviews. Microbiology Jan 2022A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell... (Review)
Review
A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell chromosome. Integrase is the viral enzyme responsible for the catalytic steps involved in this process, and integrase strand transfer inhibitors are widely used to treat people living with HIV. Over the past decade, a series of X-ray crystallography and cryogenic electron microscopy studies have revealed the structural basis of retroviral DNA integration. A variable number of integrase molecules congregate on viral DNA ends to assemble a conserved intasome core machine that facilitates integration. The structures additionally informed on the modes of integrase inhibitor action and the means by which HIV acquires drug resistance. Recent years have witnessed the development of allosteric integrase inhibitors, a highly promising class of small molecules that antagonize viral morphogenesis. In this Review, we explore recent insights into the organization and mechanism of the retroviral integration machinery and highlight open questions as well as new directions in the field.
Topics: Crystallography, X-Ray; DNA, Viral; HIV Integrase; HIV-1; Humans; Integrases; Models, Molecular; Protein Conformation; Retroviridae; Virus Integration
PubMed: 34244677
DOI: 10.1038/s41579-021-00586-9 -
Microbiology Spectrum Feb 2015The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the... (Review)
Review
The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.
Topics: Integrases; Models, Biological; Molecular Biology; Mutagenesis, Insertional; Recombination, Genetic
PubMed: 26104563
DOI: 10.1128/microbiolspec.MDNA3-0014-2014 -
Nature Biotechnology Dec 2017Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate...
Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.
Topics: Amino Acids; Animals; Click Chemistry; Female; Gene Expression Regulation; Integrases; Male; Methionine-tRNA Ligase; Mice; Mice, Transgenic; Neurons; Proteome; Proteomics
PubMed: 29106408
DOI: 10.1038/nbt.4016 -
Nature Nov 2019The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles...
The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.
Topics: Animals; Axons; Cerebral Cortex; Female; Integrases; Male; Mice; Mice, Inbred C57BL; Neural Pathways; Thalamus
PubMed: 31666704
DOI: 10.1038/s41586-019-1716-z -
Nature Jun 2023CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for...
CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Cryoelectron Microscopy; DNA; Exonucleases; Integrases; Biocatalysis; Genome, Bacterial
PubMed: 37316664
DOI: 10.1038/s41586-023-06178-2 -
Cell May 2015Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV...
Most cancer cells release heterogeneous populations of extracellular vesicles (EVs) containing proteins, lipids, and nucleic acids. In vitro experiments showed that EV uptake can lead to transfer of functional mRNA and altered cellular behavior. However, similar in vivo experiments remain challenging because cells that take up EVs cannot be discriminated from non-EV-receiving cells. Here, we used the Cre-LoxP system to directly identify tumor cells that take up EVs in vivo. We show that EVs released by malignant tumor cells are taken up by less malignant tumor cells located within the same and within distant tumors and that these EVs carry mRNAs involved in migration and metastasis. By intravital imaging, we show that the less malignant tumor cells that take up EVs display enhanced migratory behavior and metastatic capacity. We postulate that tumor cells locally and systemically share molecules carried by EVs in vivo and that this affects cellular behavior.
Topics: Animals; Cell Line, Tumor; Humans; Integrases; Mice; Neoplasm Metastasis; Neoplastic Cells, Circulating; Transport Vesicles
PubMed: 26000481
DOI: 10.1016/j.cell.2015.04.042 -
Nature Biotechnology Feb 2016Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is...
Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.
Topics: Animals; Dependovirus; Female; Genetic Engineering; Genetic Vectors; HEK293 Cells; Humans; Integrases; Mice; Transfection
PubMed: 26829320
DOI: 10.1038/nbt.3440 -
Current Opinion in Structural Biology Dec 2017Retroviral DNA integration takes place in the context of the intasome nucleoprotein complex. X-ray crystal structures of functional spumaviral intasomes were previously... (Review)
Review
Retroviral DNA integration takes place in the context of the intasome nucleoprotein complex. X-ray crystal structures of functional spumaviral intasomes were previously revealed to harbor a homotetramer of integrase, and it was generally believed that integrase tetramers catalyzed the integration of other retroviruses. The elucidation of new structures from four different retroviruses over the past year has however revealed this is not the case. The number of integrase molecules required to construct the conserved intasome core structure differs between viral species. While four subunits suffice for spumaviruses, α- and β-retroviruses require eight and the lentiviruses use up to sixteen. Herein we described these alternative architectures, highlighting both evolutionary and structural constraints that result in the different integrase-DNA stoichiometries across Retroviridae.
Topics: DNA, Viral; Integrases; Models, Molecular; Multiprotein Complexes; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Retroviridae; Structure-Activity Relationship; Virus Integration
PubMed: 28458055
DOI: 10.1016/j.sbi.2017.04.005 -
Genesis (New York, N.Y. : 2000) Aug 2016DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling...
DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs, and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic β-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology, we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions. genesis 54:439-446, 2016. © 2016 Wiley Periodicals, Inc.
Topics: Animals; Cells, Cultured; Gene Targeting; Integrases; Ligands; Mice; Mice, Inbred C57BL; Protein Binding; Receptors, G-Protein-Coupled
PubMed: 27194399
DOI: 10.1002/dvg.22949 -
Neuron Mar 2015An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current...
An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.
Topics: Animals; Gene Targeting; Hippocampus; Integrases; Mice; Mice, Transgenic; Neurons; Optogenetics; Organ Culture Techniques; Visual Cortex
PubMed: 25741722
DOI: 10.1016/j.neuron.2015.02.022