Intracellular Ebola virus nucleocapsid assembly revealed by in situ cryo-electron tomography.

Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus to illuminate assembly intermediates, as well as a 9 Å map of the complete intracellular assembly. This structure reveals a previously unresolved third and outer layer of NP complexed with VP35. The intrinsically disordered region, together with the C-terminal domain of this outer layer of NP, provides the constant width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity.

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: 'associated' (73° of DNA bend), 'half-wrapped' (107°) and 'fully-wrapped' (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Amino Acid Composition in Various Types of Nucleic Acid-Binding Proteins.

Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments.

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.

TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation.

Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.

Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein.

The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N- and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.

The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein.

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.

The nucleoid-associated protein Dan organizes chromosomal DNA through rigid nucleoprotein filament formation in E. coli during anoxia.

Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan-DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation.

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.

Exploiting radiation damage to map proteins in nucleoprotein complexes: the internal structure of bacteriophage T7.

In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique ("bubblegram imaging") by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 s) intervals between exposures of 17 electrons/Å(2) each, bubbling starts in the third exposure, with 1-4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation.

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers.

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures.

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy.

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.

Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2.

The human breast cancer susceptibility gene BRCA2 is required for the regulation of RAD51-mediated homologous recombinational repair. BRCA2 interacts with RAD51 monomers, as well as nucleoprotein filaments, primarily though the conserved BRC motifs. The unrelated C-terminal region of BRCA2 also interacts with RAD51. Here we show that the BRCA2 C terminus interacts directly with RAD51 filaments, but not monomers, by binding an interface created by two adjacent RAD51 protomers. These interactions stabilize filaments so that they cannot be dissociated by association with BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. We suggest that interactions of the BRCA2 C-terminal region with RAD51 may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair.

Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats.

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes [corrected] of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.