A supramolecular assembly mediates lentiviral DNA integration

Published on Jan 6, 2017in Science41.063
· DOI :10.1126/science.aah7002
A. Ballandras-Colas5
Estimated H-index: 5
(Francis Crick Institute),
D.P. Maskell5
Estimated H-index: 5
(Francis Crick Institute)
+ 11 AuthorsPeter Cherepanov38
Estimated H-index: 38
(Imperial College London)
Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo–electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.
  • References (49)
  • Citations (33)
📖 Papers frequently viewed together
55 Citations
42 Citations
7 Authors (Zhiqi Yin, ..., Hideki Aihara)
39 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1Dario Oliveira Passos (Salk Institute for Biological Studies)H-Index: 4
#2Min Li (NIH: National Institutes of Health)H-Index: 10
Last. Dmitry Lyumkis (Salk Institute for Biological Studies)H-Index: 18
view all 10 authors...
55 CitationsSource
#1Paul LesbatsH-Index: 3
#2Alan EngelmanH-Index: 65
Last. Peter CherepanovH-Index: 38
view all 3 authors...
The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retrovira...
50 CitationsSource
#1Shawn Q. Zheng (UCSF: University of California, San Francisco)H-Index: 12
#2Eugene Palovcak (UCSF: University of California, San Francisco)H-Index: 5
Last. David A. Agard (UCSF: University of California, San Francisco)H-Index: 84
view all 5 authors...
Correction of electron beam-induced sample motion is one of the major factors contributing to the recent resolution breakthroughs in cryo-electron microscopy. Improving the accuracy and efficiency of motion correction can lead to further resolution improvement. Based on observations that the electron beam induces doming of the thin vitreous ice layer, we developed an algorithm to correct anisotropic image motion at the single pixel level across the whole frame, suitable for both single particle ...
56 CitationsSource
#1James M. Bell (BCM: Baylor College of Medicine)H-Index: 2
#2Muyuan Chen (BCM: Baylor College of Medicine)H-Index: 7
Last. Steven J. Ludtke (BCM: Baylor College of Medicine)H-Index: 51
view all 4 authors...
EMAN2.1 is a complete image processing suite for quantitative analysis of grayscale images, with a primary focus on transmission electron microscopy, with complete workflows for performing high resolution single particle reconstruction, 2-D and 3-D heterogeneity analysis, random conical tilt reconstruction and subtomogram averaging, among other tasks. In this manuscript we provide the first detailed description of the high resolution single particle analysis pipeline and the philosophy behind it...
51 CitationsSource
#1Erik Serrao (Harvard University)H-Index: 12
#2Peter Cherepanov (Francis Crick Institute)H-Index: 38
Last. Alan Engelman (Harvard University)H-Index: 65
view all 3 authors...
Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restr...
12 CitationsSource
#1A. Ballandras-Colas (Harvard University)H-Index: 5
#2M. R. Brown (Salk Institute for Biological Studies)H-Index: 11
Last. Alan Engelman (Harvard University)H-Index: 65
view all 8 authors...
42 CitationsSource
#1Zhiqi YinH-Index: 4
#2Ke ShiH-Index: 16
Last. Hideki AiharaH-Index: 19
view all 7 authors...
39 CitationsSource
#1Alexis Rohou (HHMI: Howard Hughes Medical Institute)H-Index: 11
#2Nikolaus Grigorieff (HHMI: Howard Hughes Medical Institute)H-Index: 56
Abstract CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope’s contrast transfer function (CTF) to an image’s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new...
835 CitationsSource
#1D.P. MaskellH-Index: 5
#2Ludovic RenaultH-Index: 13
Last. Peter CherepanovH-Index: 38
view all 10 authors...
Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1, 2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome–nucleosome interface involving...
78 CitationsSource
#1Sjors H.W. Scheres (LMB: Laboratory of Molecular Biology)H-Index: 49
The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. Wit...
206 CitationsSource
Cited By33
#2Pierre CappyH-Index: 1
Last. Marc RuffH-Index: 23
view all 12 authors...
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 39-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal st...
#1Chang-Ying Chiang (UCM: University of California, Merced)
#2Gloria Denise Ligunas (UCM: University of California, Merced)
Last. Chih-Wen Ni (UCM: University of California, Merced)
view all 4 authors...
Abstract Efficient transgene delivery is critical for genetic manipulation and therapeutic intervention of target cells. Two well-characterized integrative systems have been described that rely on viral and non-viral vectors. However, use of viral vectors for gene therapy has been associated with several safety concerns. Here, we report a virus-free method for stable transgenesis based on the reaction of retroviral integrase. We constructed a gateway cloning compatible vector containing two trun...
#1Lorena de Pablo-Maiso (CSIC: Spanish National Research Council)H-Index: 1
#2Irache Echeverría (CSIC: Spanish National Research Council)
Last. Ramsés Reina (CSIC: Spanish National Research Council)H-Index: 16
view all 8 authors...
Small ruminant lentiviruses (SRLVs) are widely spread in the ovine and caprine populations, causing an incurable disease affecting animal health and production. Vaccine development is hindered owing to the high genetic heterogeneity of lentiviruses and the selection of T-cell and antibody escape mutants, requiring antigen delivery optimization. Sendai virus (SeV) is a respiratory paramyxovirus in mice that has been recognized as a potent inducer of innate immune responses in several species, inc...
#1Dario Oliveira Passos (Salk Institute for Biological Studies)H-Index: 4
#2Min Li (NIH: National Institutes of Health)H-Index: 10
Last. Stephen H. HughesH-Index: 65
view all 13 authors...
The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretrovirals, integrase (IN) strand transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of HIV i...
2 CitationsSource
#1Min Li (NIH: National Institutes of Health)H-Index: 12
#1Min LiH-Index: 10
Last. Robert CraigieH-Index: 52
view all 6 authors...
Abstract The low solubility and aggregation properties of HIV-1 integrase are major obstacles for biochemical and structural studies. Lens epithelium-derived growth factor (LEDGF) is a cellular factor that binds integrase and tethers preintegration complexes to chromatin prior to integration. LEDGF also stimulates HIV-1 integrase DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of LEDGF ...
1 CitationsSource
#1Xuemin Chen (LMB: Laboratory of Molecular Biology)H-Index: 1
#2Yanxiang Cui (UCLA: University of California, Los Angeles)H-Index: 1
Last. Wei Yang (LMB: Laboratory of Molecular Biology)H-Index: 13
view all 6 authors...
The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA-strand transfer complex of mouse RAG at 3.1-A resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination,...
1 CitationsSource
#1N. Cook (Francis Crick Institute)H-Index: 8
#2Wen Li (Harvard University)
Last. Peter Cherepanov (Imperial College London)H-Index: 38
view all 10 authors...
Despite worldwide prescription, the mechanistic basis for superiority of second-generation HIV integrase (IN) strand transfer inhibitors (INSTIs) is poorly understood. We use single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near atomic resolution. Q148H/G140S amino acid substitutions in IN that pervade clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical sc...
2 CitationsSource
#1Jennifer Elliott (WashU: Washington University in St. Louis)H-Index: 3
Last. Sebla B. KutluayH-Index: 13
view all 10 authors...
A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization a...
#1Chang LiuH-Index: 1
#2Yang Yang (Yale University)H-Index: 10
view all 3 authors...
Transposons have had a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1–RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joi...
3 CitationsSource
#1George Ghanim (University of California, Berkeley)
#2Elizabeth H. Kellogg (California Institute for Quantitative Biosciences)H-Index: 11
Last. Donald C. Rio (University of California, Berkeley)H-Index: 41
view all 4 authors...
P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprote...
1 CitationsSource