Automatic tracking of viral and intracellular structures displayed as spots with varying sizes in fluorescence microscopy images is an important task to quantify cellular processes. We propose a ...novel probabilistic tracking approach for multiple particle tracking based on multi-detector and multi-scale data fusion as well as Bayesian smoothing. The approach integrates results from multiple detectors using a novel intensity-based covariance intersection method which takes into account information about the image intensities, positions, and uncertainties. The method ensures a consistent estimate of multiple fused particle detections and does not require an optimization step. Our probabilistic tracking approach performs data fusion of detections from classical and deep learning methods as well as exploits single-scale and multi-scale detections. In addition, we use Bayesian smoothing to fuse information of predictions from both past and future time points. We evaluated our approach using image data of the Particle Tracking Challenge and achieved state-of-the-art results or outperformed previous methods. Our method was also assessed on challenging live cell fluorescence microscopy image data of viral and cellular proteins expressed in hepatitis C virus-infected cells and chromatin structures in non-infected cells, acquired at different spatial–temporal resolutions. We found that the proposed approach outperforms existing methods.
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•Probabilistic particle tracking by multi-detector fusion and Bayesian smoothing.•Detections from multiple detectors fused by intensity-based covariance intersection.•Multi-detector fusion exploits image intensities, positions, and uncertainties.•Information from future time points integrated by Bayesian smoothing.•Quantitative evaluation using microscopy images of viral and chromatin structures.
An estimated 170 million persons worldwide are infected with hepatitis C virus (HCV), a major cause of chronic liver disease. Despite increasing knowledge of genome structure and individual viral ...proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. A full-length consensus genome was cloned from viral RNA isolated from an infected human liver and used to construct subgenomic selectable replicons. Upon transfection into a human hepatoma cell line, these RNAs were found to replicate to high levels, permitting metabolic radiolabeling of viral RNA and proteins. This work defines the structure of HCV replicons functional in cell culture and provides the basis for a long-sought cellular system that should allow detailed molecular studies of HCV and the development of antiviral drugs.
•Probabilistic particle tracking by multi-sensor data fusion and Bayesian smoothing.•Integration of multiple measurements from separate measurement processes.•Past and future information exploited by ...smoothing and covariance intersection.•Motion information employed in the cost function for correspondence finding.•Improved results for microscopy image data of different types of viruses.
Automatic tracking of viral structures displayed as small spots in fluorescence microscopy images is an important task to determine quantitative information about cellular processes. We introduce a novel probabilistic approach for tracking multiple particles based on multi-sensor data fusion and Bayesian smoothing methods. The approach exploits multiple measurements as in a particle filter, both detection-based measurements and prediction-based measurements from a Kalman filter using probabilistic data association with elliptical sampling. Compared to previous probabilistic tracking methods, our approach exploits separate uncertainties for the detection-based and prediction-based measurements, and integrates them by a sequential multi-sensor data fusion method. In addition, information from both past and future time points is taken into account by a Bayesian smoothing method in conjunction with the covariance intersection algorithm for data fusion. Also, motion information based on displacements is used to improve correspondence finding. Our approach has been evaluated on data of the Particle Tracking Challenge and yielded state-of-the-art results or outperformed previous approaches. We also applied our approach to challenging time-lapse fluorescence microscopy data of human immunodeficiency virus type 1 and hepatitis C virus proteins acquired with different types of microscopes and spatial-temporal resolutions. It turned out, that our approach outperforms existing methods.
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane ...anchor domain of NS5A as determined by NMR spectroscopy. An α-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic α-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.
The hepatitis C virus (HCV) is a major causative agent of transfusion‐acquired and sporadic non‐A, non‐B hepatitis worldwide. Infections most often persist and lead, in ≈ 50% of all patients, to ...chronic liver disease. As is characteristic for a member of the family Flaviviridae, HCV has a plus‐strand RNA genome encoding a polyprotein, which is cleaved co‐ and post‐translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin‐like serine proteinase located in the N‐terminal domain of non‐structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54‐residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X‐ray crystal structure and current attempts towards development of efficient inhibitors.
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