SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction.

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

The National Cryo-EM Facility at Frederick National Laboratory: Operational Procedures, Methods and Outputs.

The National Cryo-Electron Microscopy Facility (NCEF) at the National Cancer Institute was launched in May of 2017 to provide free and rapid access to high resolution cryo-EM data collection to United States researchers working on problems of broad general relevance to cancer biology. The decision about suitability of projects for data collection is made on a first-come, first-served basis by NCEF staff, and is based solely on the quality of the screening images provided without need for a scientific proposal. Here, we provide an overview of the operation of the facility, typical data collection procedures and some insights that have emerged from the structures reported from data collected at the facility.

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.

Ultrastructural analysis of hepatitis C virus particles.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.

Probing the early temporal and spatial interaction of the Sindbis virus capsid and E2 proteins with reverse genetics.

A 7-Å cryoelectron microscopy-based reconstruction of Sindbis virus (SINV) was recently generated. Fitting the crystal structure of the SINV capsid protein (Cp) into the density map revealed that the F2-G2 loop of the Cp was shifted away from cytoplasmic domain of E2 (cdE2) in the 7-Å reconstruction relative to its position in the Cp crystal structure. Furthermore, the reconstruction demonstrated that residue E395 in region I of the cytoplasmic domain of the E2 envelope protein (cdE2-RI) and K252 of Cp, part of the Cp F2-G2 loop, formed a putative salt bridge in the virion. We generated amino acid substitutions at residues K250 and K252 of the SINV Cp and explored the resulting phenotypes. In the context of cells infected with wild-type or mutant virus, reversing the charge of these two residues resulted in the appearance of Cp aggregates around cytopathic vacuole type I (CPV-I) structures, the absence of nucleocapsid (NC) formation, and a lack of virus particle release in the infected mammalian cell. However, expressing the same Cp mutants in the cell without the envelope proteins or expressing and purifying the mutants from an Escherichia coli expression system and assembling in vitro yielded NC assembly in all cases. In addition, second-site mutations within cdE2 restored NC assembly but not release of infectious particles. Our data suggest an early temporal and spatial interaction between cdE2-RI and the Cp F2-G2 loop that, when ablated, leads to the absence of NC assembly. This interaction also appears to be important for budding of virus particles.

Interactions of the cytoplasmic domain of Sindbis virus E2 with nucleocapsid cores promote alphavirus budding.

Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.

Patient-specific risk of undetected malignant disease after investigation for haematuria, based on a 4-year follow-up.

OBJECTIVES: • To estimate the diagnostic accuracy of a guidelines-based haematuria clinic protocol by measuring the incidence of undetected malignancy during a follow-up period. • To estimate an individual's post-test risk of having undetected malignancy using the protocol likelihood ratio and the population prevalence of disease. METHODS: • Data were collected prospectively on a cohort of 4020 consecutive patients who were referred to a 'one-stop' haematuria clinic between 1998 and 2003. • All patients had a plain radiograph taken and underwent ultrasonography and flexible cystoscopy as a part of 'first-line' investigation. • Intravenous urography was performed where indicated after abnormal first-line tests or in patients with persistent haematuria where no abnormality had been detected. • Records of the initial 687 participants from the first year of the study were reviewed 4 years after the original consultation. Missed diagnoses of urinary tract malignancy were recorded and sensitivities, likelihood ratios and the post-test probability of missing all disease and upper tract malignancy were calculated. RESULTS: • As previously reported, the overall prevalence of malignant disease was 12.1% (18.9% for macroscopic haematuria compared with 4.8% for microscopic haematuria). • The records of the first year's cohort of patients (N = 687) were analysed 4 years after their original consultation and 10 potentially 'missed' tumours were identified. • The sensitivity of the protocol was 90.9% for the detection of all urinary tract malignancy (95% CI, 82.4 to 95.5) and 71% for upper tract tumours alone (95% CI, 45.4-88.3). The latter improves to 78.6% (95% CI, 52.4-92.4) with the addition of further upper tract testing. • The probability of missing malignant disease overall was 1.7% (95% CI, 0.95-3.04) but this rose sharply to >4% for males over 60 with macroscopic haematuria. • For those with non-visible haematuria, the percentage probability of missed malignant disease was less than 1%. CONCLUSIONS: • The haematuria clinic protocol described is robust but it is not infallible. • The risk of missing malignant disease in the higher risk groups identified in the study is much greater than previous studies would suggest. • If additional upper tract testing or interval follow-up were to be recommended, it could be rationally targeted at these groups, given the measurable risk shown here.

Influence of pr-M cleavage on the heterogeneity of extracellular dengue virus particles.

During dengue virus replication, an incomplete cleavage of the envelope glycoprotein prM, generates a mixture of mature (prM-less) and prM-containing, immature extracellular particles. In this study, sequential immunoprecipitation and cryoelectron microscopy revealed a third type of extracellular particles, the partially mature particles, as the major prM-containing particles in a dengue serotype 2 virus. Changes in the proportion of viral particles in the pr-M junction mutants exhibiting altered levels of prM cleavage suggest that the partially mature particles may represent an intermediate subpopulation in the virus maturation pathway. These findings are consistent with a model suggesting the progressive mode of prM cleavage.