Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing.

Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate novel folding intermediates. In an in vitro co-transcriptional assembly assay, 8 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 new intermediate structures were determined resulting from the specific inhibitions. In particular a novel intermediate class provided compelling evidence of independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S-rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA foldon docking and domain consolidation, which provides a hierarchical view of the RNP assembly process.

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

Overcoming resolution attenuation during tilted cryo-EM data collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

A common binding motif in the ET domain of BRD3 forms polymorphic structural interfaces with host and viral proteins.

The extraterminal (ET) domain of BRD3 is conserved among BET proteins (BRD2, BRD3, BRD4), interacting with multiple host and viral protein-protein networks. Solution NMR structures of complexes formed between the BRD3 ET domain and either the 79-residue murine leukemia virus integrase (IN) C-terminal domain (IN329-408) or its 22-residue IN tail peptide (IN386-407) alone reveal similar intermolecular three-stranded ß-sheet formations. 15N relaxation studies reveal a 10-residue linker region (IN379-388) tethering the SH3 domain (IN329-378) to the ET-binding motif (IN389-405):ET complex. This linker has restricted flexibility, affecting its potential range of orientations in the IN:nucleosome complex. The complex of the ET-binding peptide of the host NSD3 protein (NSD3148-184) and the BRD3 ET domain includes a similar three-stranded ß-sheet interaction, but the orientation of the ß hairpin is flipped compared with the two IN:ET complexes. These studies expand our understanding of molecular recognition polymorphism in complexes of ET-binding motifs with viral and host proteins.

Evaluating Local and Directional Resolution of Cryo-EM Density Maps.

A systematic and quantitative evaluation of cryo-EM maps is necessary to judge their quality and to capture all possible sources of error. A single value for global resolution is insufficient to accurately describe the quality of a reconstructed density. We describe the estimation and evaluation of two additional resolution measures, local and directional resolution, using methods based on the Fourier shell correlation (FSC). We apply the protocol to samples that encompass different types of pathologies a user is expected to encounter and provide analyses on how to interpret the output files and resulting maps. Implementation of these tools will facilitate density interpretation and can guide the user in adapting their experiments to improve the quality of cryo-EM maps, and by extension atomic models.

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model.

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.

Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant.

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition.

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1-44) and an HHCC zinc-finger NTD (residues 45-105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1-105 (NTR1-105 ) and 8-105 (NTR8-105 ) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1-105 packs as a dimer and NTR8-105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel ß-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three ß-strands form an extended ß-sheet with another ß-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647-656. © 2016 Wiley Periodicals, Inc.

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD ß1-ß2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD ß1-ß2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD ß1-ß2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.

Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction.

We report alterations to the murine leukemia virus (MLV) integrase (IN) protein that successfully result in decreasing its integration frequency at transcription start sites and CpG islands, thereby reducing the potential for insertional activation. The host bromo and extraterminal (BET) proteins Brd2, 3 and 4 interact with the MLV IN protein primarily through the BET protein ET domain. Using solution NMR, protein interaction studies, and next generation sequencing, we show that the C-terminal tail peptide region of MLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. The use of the unstructured tails of gammaretroviral INs to direct association with complexes at active promoters parallels that used by histones and RNA polymerase II. Viruses bearing MLV IN C-terminal truncations can provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy.

Bimodal high-affinity association of Brd4 with murine leukemia virus integrase and mononucleosomes.

The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.

BET proteins promote efficient murine leukemia virus integration at transcription start sites.

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.

Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein.

Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer.

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag.

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)(1-23), human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA(1-32), required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.

MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA.

For Moloney murine leukemia virus (M-MuLV), sustained viral infections require expression from an integrated provirus. For many applications, non-integrating retroviral vectors have been utilized to avoid the unwanted effects of integration, however, the level of expression from unintegrated DNA is significantly less than that of integrated provirus. We find that unintegrated DNA expression can be increased in the presence of HDAC inhibitors, such as TSA, when applied in combination with integrase (IN) mutations. These mutants include an active site mutation as well as catalytically active INs bearing mutations of K376 in the MuLV C-terminal domain of IN. MuLV IN K376 is homologous to K266 in HIV-1 IN, a known substrate for acetylation. The MuLV IN protein is acetylated by p300 in vitro, however, the effect of HDAC inhibitors on gene expression from unintegrated DNA is not dependent on the acetylation state of MuLV IN K376.