Biology subjects

Huber, R. G.

Publications and source records attributed to Huber, R. G..

11 recordsLinked to original sources

bioRxiv2026

Layered phage therapy exploits capsule-loss escape and altered antibiotic susceptibility in CG23 hypervirulent Klebsiella pneumoniae

The rise of drug-resistant Klebsiella pneumoniae infections is prompting a re-evaluation of phage therapy as an alternative treatment. Phage therapy has achieved successful outcomes, yet important limitations persist, including the rapid emergence of phage resistance and the time required to source suitable phages. Combining multiple phages into a phage cocktail based on genetic diversity, morphology or distinct infection mechanisms is a common strategy to mitigate resistance. Phage pairing based on these criteria neither mitigates nor delays the emergence of resistance, using CG23 hypervirulent Klebsiella pneumoniae (hvKp) EC0675 as a model. In CG23 Kp strains, resistance arises through disruption of the capsular biosynthetic pathway, leading to capsule impairment and global cross-resistance to all capsule-active phages. Combining phages active against encapsulated wild-type bacteria with those active against capsule-deficient escape mutants can overcome this constraint, improving bacterial suppression in vitro and reducing bacterial burdens across multiple organs in a mouse systemic infection model. In parallel, phage-driven capsule loss increased susceptibility of hvKp EC0675 to selected antibiotics targeting the bacterial envelope or cell wall in vitro. These findings highlight the contribution of capsule-associated resistance to phage cross-resistance in the CG23 Kp strains examined, and support further evaluation of phage combinations targeting both wild-type bacteria and resistant derivatives, including their use alongside selected antibiotics, informing future clinical translation.

microbiology↗

bioRxiv2025

HomRank: Homogeneous RNA Ranking for 3D Structure Evaluation

Leveraging rigorously curated RNA sequences and a novel ranking-based evaluation paradigm, we propose an improved pipeline for RNA 3D structure assessment. To enhance generalization, we construct a dataset comprising non-redundant single-chain RNA sequences from the PDB and apply unsupervised clustering to minimize data leakage. Based on this, we retrain the previous state-of-the-art method, ARES, to develop ARES+, which improves the top-1 success rate by 21% on the RNA Puzzles benchmark. To further boost near-native identification, we introduce HomRank, a homogeneous RNA ranking method that directly optimizes the selection of near-native conformations from candidate sets, resembling expert evaluation strategies. HomRank achieves 95% and 100% top-1 and top-5 success rates, respectively, significantly outperforming ARES+. These results demonstrate that carefully designed datasets and the expert-like selection paradigm can substantially improve the accuracy and robustness of RNA 3D structure assessment, offering a promising direction for deep learning-based RNA evaluation and near-native conformation selection.

bioinformatics↗

bioRxiv2025

Dengue virus capsid protein interaction with nucleic acids

Dengue virus (DENV) transmission has greatly increased in the last decade, in part due to the geographical expansion of its vector, the Aedes spp. mosquitoes. These arthropods are now found even in temperate climates, including Europe, where outbreaks have already occurred. A better understanding of the virus life cycle is essential, as it may enable the development of specific treatments or therapies which are currently lacking. However, recent breakthroughs concerning the biologically relevant viral capsid (C) protein structure and function are encouraging. It is now clear the C protein binds both to host lipid droplets and to the viral genome, a set of interactions that is crucial for viral encapsidation and replication. Previously, we studied DENV C protein interaction with lipid droplets, at the molecular level. Here, we examine how the association of DENV C protein with the viral genome occurs. Using DENV C protein and single-stranded DNA (ssDNA) sequences analogous to previously identified important regions of the viral genome, we biophysically characterize their interactions. A decrease in fluorescence intensity and lifetime, an increase in both anisotropy and hydrodynamic diameter, as well as changes in the proteins secondary structure, were observed upon binding to ssDNA. These changes are consistent with molecular condensation and raise the possibility of liquid-liquid phase separation involvement in DENV C-nucleic acid complex formation. These findings may be useful for the development of future therapeutic strategies based on nucleic acids, not only for DENV but also for other related viruses.

biophysics↗

bioRxiv2025

A conserved long-range RNA interaction in SARS-CoV-2 recruits ADAR1 to enhance virus proliferation

Long-range RNA-RNA pairing impacts the genome structure and function of SARS-CoV-2 variants. To understand the structure and function relationships of different SARS-CoV-2 variants that have emerged during the COVID-19 pandemic, we performed high-throughput structure probing and modelling of the genomic structures of the wildtype (WT), Alpha, Beta, Delta and Omicron variants of SARS-CoV-2. We observed that genomes of SARS-CoV-2 variants are generally structurally conserved, and that single-nucleotide variations and interactions with RNA binding proteins can impact RNA structures across the viruses. Importantly, using proximity ligation sequencing, we identified many conserved ultra-long-range RNA-RNA interactions, including one that spans more than 17 kb in both the WT virus and the Omicron variant. We showed that mutations that disrupt this 17 kb long-range interaction reduce viral fitness at later stages of its infection cycle, while compensatory mutations partially restore virus fitness. Additionally, we showed that this ultra-long-range RNA-RNA interaction structure binds directly to ADAR1 to alter the RNA editing levels on the viral genome. These studies deepen our understanding of RNA structures in the SARS-CoV-2 genome and their ability to interact with host factors to facilitate virus infectivity.

genomics↗

bioRxiv2025

Evaluating Molecular Docking Programs for RNA-Targeted Ligand Screening: Influence of Binding Modes and Ligand Types

Structured RNAs are critical regulators of gene expression and represent promising targets for drug discovery. Despite this potential, structure-based virtual screening approaches--in particular, molecular docking--have been underutilized for RNA targets, largely due to the intrinsic conformational flexibility of RNA and the limited docking methods specifically optimized for RNA. In our study, we evaluated the efficacy of three widely used docking programs for RNA-ligand docking screens. The performance of these programs -- Glide, Gold, and rDock, were evaluated with the dataset of 25 diverse RNA targets with 432 small molecule RNA binders. We found that the performance was similar across docking methods, but consistent differences emerged among RNA classes. We further explored consensus scoring across both receptor conformations and docking methods, finding that selecting the average scores across docking programs and RNA structures yielded stable performance when compared to individual methods. Finally, clustering analysis of RNA binders revealed distinct scoring function preferences for different chemotypes of RNA binders, underscoring the potential for tailored scoring strategies in RNA-targeted drug discovery. Together, this work provides a comprehensive evaluation of the performance of available docking methods in different cases of RNA-ligand docking and offers practical guidance for improving virtual screening against structured RNAs.

bioinformatics↗

bioRxiv2021

Genome-wide RNA structure changes during human neurogenesis drive gene regulatory networks

The distribution, dynamics and function of RNA structures in human development is under- explored. Here, we systematically assayed RNA structural dynamics and its relationship with gene expression, translation and decay during human neurogenesis. We observed that the human ESC transcriptome is globally more structurally accessible than that of differentiated cells; and undergo extensive RNA structure changes, particularly in the 3UTR. Additionally, RNA structure changes during differentiation is associated with translation and decay. We also identified stage-specific regulation as RBP and miRNA binding, as well as splicing is associated with structure changes during early and late differentiation, respectively. Further, RBPs serve as a major factor in structure remodelling and co-regulates additional RBPs and miRNAs through structure. We demonstrated an example of this by showing that PUM2-induced structure changes on LIN28A enable miR-30 binding. This study deepens our understanding of the wide-spread and complex role of RNA-based gene regulation during human development.

molecular biology↗

bioRxiv2021

Comprehensive mapping of SARS-CoV-2 interactions in vivo reveals functional virus-host interactions

SARS-CoV-2 has emerged as a major threat to global public health, resulting in global societal and economic disruptions. Here, we investigate the intramolecular and intermolecular RNA interactions of wildtype (WT) and a mutant ({Delta}382) SARS-CoV-2 virus in cells using high throughput structure probing on Illumina and Nanopore platforms. We identified twelve potentially functional structural elements within the SARS-CoV-2 genome, observed that identical sequences can fold into divergent structures on different subgenomic RNAs, and that WT and {Delta}382 virus genomes can fold differently. Proximity ligation sequencing experiments identified hundreds of intramolecular and intermolecular pair-wise interactions within the virus genome and between virus and host RNAs. SARS-CoV-2 binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2-O-methylated. 2-O-methylation sites in the virus genome are enriched in the untranslated regions and are associated with increased pair-wise interactions. SARS-CoV-2 infection results in a global decrease of 2-O-methylation sites on host mRNAs, suggesting that binding to snoRNAs could be a pro-viral mechanism to sequester methylation machinery from host RNAs towards the virus genome. Collectively, these studies deepen our understanding of the molecular basis of SARS-CoV-2 pathogenicity, cellular factors important during infection and provide a platform for targeted therapy.

genomics↗

bioRxiv2020

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intend of engineering BMC-based metabolic modules for new functions in synthetic biology.

biophysics↗

bioRxiv2020

Extending the Martini coarse-grained forcefield to N-glycans

Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the forcefield (FF) parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini FF parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various n-glycan disaccharide combinations. When using standard Martini non-bonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimised the non-bonded interactions for glycans interacting with seven lectin candidates and show that scaling down the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins, carbohydrate binding proteins (CBPs) as well as their complexes, while benefiting from the efficiency of CG sampling.

biophysics↗

bioRxiv2019

Genome-wide Associations of Flavivirus Capsid Proteins

Dengue virus (DENV) and Zika virus (ZIKV) are both positive sense single-stranded RNA viruses. They are packaged within the virion with a capsid (C) protein to form the nucleocapsid. Based on cryo-electron microscopy imaging, the nucleocapsid has been described as lacking symmetry, whilst there is distinguishable separation of the C proteins from the viral RNA (vRNA) genome. Here, to elucidate the architecture of the nucleocapsid of DENV serotype 2 and ZIKV, we used a nuclease digestion assay and next-generation sequencing to map the respective vRNA genome wide association with the C protein in vitro. The C protein exhibited non-uniform binding along the vRNA, and as C protein concentration increased, the normalized read counts also increased. A saturation point of 1:100 (vRNA:C protein monomers) was found, and binding regions showed variable saturation patterns. We also observed that C protein had a preference for G-rich sequences for both viruses. Taken together, we demonstrate that the DENV 2 and ZIKV C proteins bind vRNA in a non-uniform manner with distinct patterns of association.\n\nSingificance StatementOur study demonstrates that flavivirus capsid proteins associate with the viral genome at specific sites rather than in a uniform manner as commonly expected. We estimate the number of capsid proteins binding to a single genomic RNA. We proceed to locate the capsid binding sites along the viral genomes of Dengue and Zika viruses. We characterize the binding sites in terms of affinity and analyze the nucleotide composition and sequence motifs at binding sites. We cross-reference binding sites against SHAPE reactivity data corresponding to local RNA secondary structure, which allows us to identify structural motifs of capsid binding sites. As capsid proteins are essential for viral packaging, these interactions may form attractive targets for therapeutic intervention.

genomics↗

bioRxiv2018

Structure mapping of dengue and Zika viruses reveals new functional long-range interactions

Dengue and Zika are clinically important members of the Flaviviridae family that utilizes an 11kb positive strand RNA for genome regulation. While structures have been mapped primarily in the UTRs, much remains to be learnt about how the rest of the genome folds to enable function. Here, we performed secondary structure and pair-wise interaction mapping on four dengue serotypes and four Zika strains in their native virus particles and infected cells. Comparative analysis of SHAPE reactivities across serotypes nominated potentially functional regions that are highly structured, show structure conservation, and low synonymous mutation rates, including a structure associated with ribosome pausing. Pair-wise interaction mapping by SPLASH further reveals new pair-wise interactions, in addition to the known circularization sequence. 40% of pair-wise interactions form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared pair-wise interactions between serotypes revealed macro-organization whereby interactions are preserved at their physical locations, beyond their sequence identities. In addition, structure mapping of virus genomes released in solution-as well as inside host cells-showed that other helicases, in addition to the ribosome, play a role in unwinding viral structures inside cells. Mutational experiments that disrupt in cell and in virion pair-wise interactions result in virus attenuation, demonstrating their importance during the virus life-cycle.

genomics↗