Role of Mitochondrial Membrane Spherules in Flock House Virus Replication.

UNLABELLED: Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCE: It is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

Structural insights into the stabilization of the human immunodeficiency virus type 1 capsid protein by the cyclophilin-binding domain and implications on the virus cycle.

During infection, human immunodeficiency virus type 1 (HIV-1) interacts with the cellular host factor cyclophilin A (CypA) through residues 85-93 of the N-terminal domain of HIV-1's capsid protein (CA). The role of the CA:CypA interaction is still unclear. Previous studies showed that a CypA-binding loop mutant, Δ87-97, has increased ability to assemble in vitro. We used this mutant to infer whether the CypA-binding region has an overall effect on CA stability, as measured by pressure and chemical perturbation. We built a SAXS-based envelope model for the dimer of both WT and Δ87-97. A new conformational arrangement of the dimers is described, showing the structural plasticity that CA can adopt. In protein folding studies, the deletion of the loop drastically reduces CA stability, as assayed by high hydrostatic pressure and urea. We hypothesize that the deletion promotes a rearrangement of helix 4, which may enhance the heterotypic interaction between the N- and C-terminal domains of CA dimers. In addition, we propose that the cyclophilin-binding loop may modulate capsid assembly during infection, either in the cytoplasm or near the nucleus by binding to the nuclear protein Nup385.

A fast cross-validation method for alignment of electron tomography images based on Beer-Lambert law.

In electron tomography, accurate alignment of tilt series is an essential step in attaining high-resolution 3D reconstructions. Nevertheless, quantitative assessment of alignment quality has remained a challenging issue, even though many alignment methods have been reported. Here, we report a fast and accurate method, tomoAlignEval, based on the Beer-Lambert law, for the evaluation of alignment quality. Our method is able to globally estimate the alignment accuracy by measuring the goodness of log-linear relationship of the beam intensity attenuations at different tilt angles. Extensive tests with experimental data demonstrated its robust performance with stained and cryo samples. Our method is not only significantly faster but also more sensitive than measurements of tomogram resolution using Fourier shell correlation method (FSCe/o). From these tests, we also conclude that while current alignment methods are sufficiently accurate for stained samples, inaccurate alignments remain a major limitation for high resolution cryo-electron tomography.

Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer-Lambert law.

Cryo-electron tomography (cryo-ET) is an emerging technique that can elucidate the architecture of macromolecular complexes and cellular ultrastructure in a near-native state. Some important sample parameters, such as thickness and tilt, are needed for 3-D reconstruction. However, these parameters can currently only be determined using trial 3-D reconstructions. Accurate electron mean free path plays a significant role in modeling image formation process essential for simulation of electron microscopy images and model-based iterative 3-D reconstruction methods; however, their values are voltage and sample dependent and have only been experimentally measured for a limited number of sample conditions. Here, we report a computational method, tomoThickness, based on the Beer-Lambert law, to simultaneously determine the sample thickness, tilt and electron inelastic mean free path by solving an overdetermined nonlinear least square optimization problem utilizing the strong constraints of tilt relationships. The method has been extensively tested with both stained and cryo datasets. The fitted electron mean free paths are consistent with reported experimental measurements. The accurate thickness estimation eliminates the need for a generous assignment of Z-dimension size of the tomogram. Interestingly, we have also found that nearly all samples are a few degrees tilted relative to the electron beam. Compensation of the intrinsic sample tilt can result in horizontal structure and reduced Z-dimension of tomograms. Our fast, pre-reconstruction method can thus provide important sample parameters that can help improve performance of tomographic reconstruction of a wide range of samples.

Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells.

UNLABELLED: During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. IMPORTANCE: Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

TxBR montage reconstruction for large field electron tomography.

Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.

Contribution of the Golgi apparatus in morphogenesis of a virus-induced cytopathic vacuolar system.

The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of a peroxidase-tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here, we propose a model for the conversion of the GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our results have implications for alphavirus-induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles.

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.

A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV).

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Šresolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Synthesis of biotin-tagged chemical cross-linkers and their applications for mass spectrometry.

Chemical cross-linking combined with mass spectrometry (MS) has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine-reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them to enrich cross-linked peptides. The reaction of biotin-tagged cross-linkers with purified HIV-1 CA resulted in the formation of hanging and intramolecular cross-links. The peptides modified with biotinylated cross-linkers were effectively enriched and recovered using a streptavidin-coated plate and MS-friendly buffers. The enrichment of modified peptides and removal of the dominantly unmodified peptides simplify mass spectra and their analyses. The combination of the high mass accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS and the tandem mass spectrometric (MS/MS) capability of the linear ion trap allows us to unambiguously identify the cross-linking sites and additional modification, such as oxidation.

Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange.

To characterize the intersubunit interactions underlying assembly and maturation in HIV-1, we determined the amide hydrogen exchange protection pattern of capsid protein in the immature virion and the mature virion using mass spectrometry. Alterations in protection upon maturation provide evidence for the maturation-induced formation of an interaction between the N- and C-terminal domains in half of the capsid molecules, indicating that only half of the capsid protein is assembled into the conical core.

Diverse Morphology and Structural Features of Old and New World Hantaviruses.

To further understanding of the structure and morphology of the Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus.

Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy.

Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are approximately 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle.

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications.

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.

High-sensitivity mass spectrometry for imaging subunit interactions: hydrogen/deuterium exchange.

In recent years, advances in mass spectrometry have provided unprecedented knowledge of protein expression within cells. It has become apparent that many proteins function as macromolecular complexes. Structural genomics programs are determining the fold of these proteins at an increasing rate and electron microscopic tomography potentially provides a means to determine the location of these complexes within the cell. A complete understanding of the molecular mechanism of these proteins requires detailed information on the interactions and dynamics within the complex. Recent advances in mass spectrometry now make it possible to use hydrogen/deuterium exchange to detect intersubunit interfaces and dynamics within supramolecular complexes.

3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.

Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding.

Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry.

The pleomorphic nature of the immature and mature HIV-1 virions has made it difficult to characterize intersubunit interactions using traditional approaches. While the structures of isolated domains are known, the challenge is to identify intersubunit interactions and thereby pack these domains into supramolecular structures. Using high-resolution mass spectrometry, we have measured the amide hydrogen exchange protection factors for the soluble capsid protein (CA) and CA assembled in vitro. Comparison of the protection factors as well as chemical crosslinking experiments has led to a map of the subunit/subunit interfaces in the assembled tubes. This analysis provides direct biochemical evidence for the homotypic N domain and C domain interactions proposed from cryo-electron microscopy image reconstruction of CA tubes. Most significantly, we have identified a previously unrecognized intersubunit N domain-C domain interaction. The detection of this interaction reconciles previously discrepant biophysical and genetic data.

Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis.

For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange is determined by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form.

Characterization of a direct detection device imaging camera for transmission electron microscopy.

The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography.

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.