Deoxycholic acid liganded HBs contributes to HBV maturation

Understanding the underlying mechanism of HBV maturation and subviral particle production is critical to control HBV infection and develop new antiviral strategies. Here, we demonstrate that deoxycholic acid (DCA) plays a central role in HBV production. HBV infection increased DCA levels, whereas elimination of DCA-producing microbiome decreased HBV viral load. DCA can bind to HBs antigen via LXXLL motif at TM1 and TM2 region to regulate HBs-HBc interaction and the production of mature HBV. Plasma DCA levels from patients undergoing antiviral therapy were significantly higher in those with positive HBV viral load. These results suggest that intestinal DCA-producing microbiome can affect the efficiency of antiviral therapy and provide a potential novel strategy for HBV antiviral therapy.

Verification of an algorithm for actin tail detection of subviral particles by comparison with expert validated data

The locomotion of subviral particles of Marburg virus has been shown to be primarily actin based. For this work, a virologist selected 14 subviral particles that show actin tails in fluorescence image sequences. Using the tracked coordinates, examination areas around these subviral particles are defined. The brightness of within the examination area behind the subviral particle is analysed. In addition, the speed of the particle in each frame is calculated to investigate potential correlations between actin activity and particle speed. The results show that actin tracks can be automatically detected and analysed. First hints of a correlation between subviral particle movement and actin activity could be gathered with the presented actin tail quantifier.

Estimation of accuracy loss by training a deep-learning-based cell organelle recognition software using full dataset and a reduced dataset containing only subviral particle distribution information

In collaboration with the Institute of Virology, Philipps University, Marburg, a deep-learning-based method that recognizes and classifies cell organelles based on the distribution of subviral particles in fluorescence microscopy images of virus-infected cells has been further developed. In this work a method to recognize cell organelles by means of partial image information is extended. The focus is on investigating loss of accuracy by only providing information about subviral particles and not all cell organelles to an adopted Mask-R convolutional neural network. Our results show that the subviral particle distribution holds information about the cell morphology, thus making it possible to use it for cell organelle-labelling.

Parameter estimation of support vector machine with radial basis function kernel using grid search with leave-p-out cross validation for classification of motion patterns of subviral particles

The classification of subviral particle motion in fluorescence microscopy video sequences is relevant to drug development. This work introduces a method for estimating parameters for support vector machines (SVMs) with radial basis function (RBF) kernels using grid search with leave-pout cross-validation for classification of subviral particle motion patterns. RBF-SVM was trained and tested with a large number of combinations of expert-evaluated training and test data sets for different RBF-SVM parameters using grid search. For each subtest, the mean and standard deviation of the accuracy of the RBF-SVM were calculated. The RBF-SVM parameters are selected according to the optimal accuracy. For the optimal parameters, the accuracy is 89% +- 13% for N = 100. Using the introduced computer intensive machine learning parameter adjustment method, an RBF-SVM has been successfully trained to classify the motion patterns of subviral particles into chaotic, moderate and linear movements.

Role of Small Envelope Protein in Sustaining the Intracellular and Extracellular Levels of Hepatitis B Virus Large and Middle Envelope Proteins

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion.

Noncrystallographic symmetry-constrained map obtained by direct density optimization

Noncrystallographic symmetry (NCS) averaging following molecular-replacement phasing is generally the major technique used to solve a structure with several molecules in one asymmetric unit, such as a spherical icosahedral viral particle. As an alternative method to NCS averaging, a new approach to optimize or to refine the electron density directly under NCS constraints is proposed. This method has the same effect as the conventional NCS-averaging method but does not include the process of Fourier synthesis to generate the electron density from amplitudes and the corresponding phases. It has great merit for the solution of structures with limited data that are either twinned or incomplete at low resolution. This method was applied to the case of the T = 1 shell-domain subviral particle of Penaeus vannamei nodavirus with data affected by twinning using the REFMAC5 refinement software.

Selection and Characterization of a Reovirus Mutant with Increased Thermostability

ABSTRACTThe environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual-layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) σ3 is degraded to generate the infectious subviral particle, and (ii) μ1 facilitates membrane penetration and subsequent core delivery. μ1-σ3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (μ1 M258I and σ3 S344P) within heat-resistant strains. σ3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability.IMPORTANCENonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, these findings show capsid function is balanced for optimal replication and for spread to a new host.

Selection and characterization of a reovirus mutant with improved thermostability

ABSTRACTThe environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol and sodium dodecyl sulfate) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) σ3 is degraded to generate the infectious subviral particle and (ii) μ1 facilitates membrane penetration and subsequent core delivery. μ1-σ3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (μ1 M258I and σ3 S344P) within heat resistant strains. σ3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability.SIGNIFICANCENonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, capsid function is balanced for optimal replication and for spread to a new host.