Virus Research

RNA viruses are common in tephritid fruit flies including the Queensland fruit fly, Australias most significant horticultural pest. For many their transmission, tissue tropism and load across host development remain unexplored. Yet these factors are important for host biology, ecology and pest management. We investigated Bactrocera tryoni orbivirus (OV), Bactrocera tryoni xinmovirus (XV), Bactrocera tryoni toti-like virus (TLV) and Bactrocera tryoni iflavirus species 2 (IVsp.2) that commonly coinfect B. tryoni laboratory populations. OV and XV transmission was vertical within and on eggs, while TLV transmission was vertical within eggs. IVsp.2 was not detected in eggs but was present in adults; however, IVsp.2 was horizontally transmitted, with viral load increasing with cohabitation time with infected flies. Horizontal transmission was not observed for the other viruses. OV had a similar load across all tissues, while XV was consistently more abundant in ovaries. TLV had a high viral load in the brain whereas IVsp.2 was abundant in the thorax, foregut and midgut. Besides differences in eggs, the viruses were detected in all other developmental stages, but viral load patterns differed: viral load remained constant for TLV, fluctuated for OV and XV, and was low in pre-adult stages and high in adults for IVsp.2. Our findings demonstrate distinct transmission strategies and tissue tropism among the viruses, providing new insights into their epidemiology and role in host biology. Furthermore, contrary to prevailing views that viruses are generally horizontally transmitted, most known RNA viruses of B. tryoni are vertically transmitted affecting the evolution of host-virus interactions.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

The yerba mate psyllid (Gyropsylla spegazziniana) poses a significant threat to yerba mate crops, causing extensive economic losses. While some ecological aspects as well as control strategies have been studied, its associated viral diversity remains unexplored. Here, by generating the first RNA high-throughput analysis (HTS) of this pest, we explored the G. spegazziniana virome, revealing novel and diverse RNA viruses. We characterized five new viral members belonging to distinct families, with evolutionary cues of beny-like viruses (Benyviridae), picorna-like viruses (Picornaviridae), and sobemo-like viruses (Solemoviridae); which were tentatively named Gyropsylla spegazziniana beny-like virus 1 (GSBlV1), Gyropsylla spegazziniana picorna-like virus 1 (GSPlV1), and Gyropsylla spegazziniana sobemo-like virus 1-3 (GSSlV1-3), respectively. Phylogenetic analysis of the bi-segmented and highly divergent sobemo-like viruses showed a distinctive evolutionary trajectory of its encoding proteins at the periphery of recently reported invertebrate Sobelivirales. The beny-like virus belonged to a cluster of insect-associated beny-like viruse; while the picorna-like virus clustered together with psyllid-associated picorna-like viruses. These results highlight the existence of a complex virome within G. spegazziniana and establish the basis for future studies investigating the ecological roles, evolutionary dynamics, and potential biocontrol applications of these viruses in the G. spegazziniana -yerba mate eco-systems.

Climate change and ecosystem collapse promote geographic expansion of vector-borne diseases, as witnessed by the recent incursions into Spain of the virus responsible for Crimean-Congo hemorrhagic fever (CCHFV). CCHFV is maintained in a tick-vertebrate cycle, principally involving ticks of the genus Hyalomma. Faced with the spread of Hyalomma ticks, and therefore the threat of a natural introduction of CCHFV into Western Europe, appropriate surveillance tools and control measures need to be implemented. It is both within and by the tick that CCHFV is maintained and spread in the environment. Despite prolonged portage of the virus, the tick is not overtly affected by CHFV infection. One of the prerequisites in conceiving control strategies is to understand the molecular mechanisms that intimately link the virus to its arthropod host. Despite the central role of the tick in the biology of CCHFV, these mechanisms are ill-defined, owing in part to the constraints associated with handling CCHFV-infected ticks in biosafety level 4 containment. In this study, we established the network of interactions between the S segment of the RNA genome Hazara virus (HAZV), a BSL-2 model of CCHFV, and Hyalomma proteins using ChIRP-MS technique. We identified 166 tick proteins, 21 of which have been described as RNA-binding proteins. Gene ontology and pathway enrichment analyses revealed that the S segment RNA interacts predominantly with mitochondrial proteins that belong to various mitochondrial metabolic pathways.

Defective interfering particles (DIPs) derived from the influenza A virus (IAV) are a promising antiviral agent due to their strong antiviral efficacy demonstrated in various animal models. OP7 is an unconventional IAV DIP with multiple point mutations in the viral RNA (vRNA) of genome segment 7, as opposed to the large internal genomic deletions typically found in conventional IAV DIPs. Further, OP7 showed an even higher interfering efficacy than conventional DIPs. However, the inhibitory effect of OP7 on standard virus (STV) replication has primarily been investigated in Madin-Darby Canine Kidney (MDCK) cells, which lack a functional myxovirus resistance (Mx)-mediated antiviral activity against IAV. In this study, we examined the antiviral activity and mechanism of antiviral action of OP7 in an interferon (IFN)-competent human lung carcinoma cell line (Calu-3) in vitro. We performed STV and OP7 co-infection experiments using a variety of infection conditions and measured the time-resolved dynamics in viral titer, vRNA, protein level, and host cell gene expression. We observed that OP7 co-infection results in enhanced type I IFN responses and markedly reduced infectious virus release, even at low doses. Additionally, we found that at a high STV multiplicity of infection (MOI), the replication interference of OP7, suppressing the replication of STV vRNA, appears to be the dominant mechanism of its antiviral action. At a low MOI, however, IFN induction seems to be more important. Furthermore, we examined the efficacious co-infection time window for potential prophylactic and therapeutic antiviral treatment. We observed an antiviral effect exerted by OP7 infection for up to seven days before STV infection and up to 24 hours after STV infection. Together, these findings demonstrate that OP7 is a potent antiviral DIP. Therefore, this work supports the further development of OP7 as a therapeutic and prophylactic antiviral agent.

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

Chronic co-infections by HBV and its satellite virus HDV are associated with a high risk of progression to cirrhosis and liver cancer, and therapeutic options for achieving a cure are still unsatisfactory. HBs is the main surface glycoprotein of both viruses, and is also massively secreted by infected hepatocytes in the form of empty subviral particles which suppress the host immune responses. This makes HBs an attractive target to develop therapeutic strategies. Here, we took advantage of the known interaction between the Large form HDV antigen (HDAg-L) and the small form of HBs (S-HBs) to develop a non-infectious, minimalistic reporter assay for the assembly and secretion of HDV particles. By screening the existing pharmacopeia for drugs that could interfere with S-HBs and HDAg-L co-secretion, we found that ritonavir and other Cytochrome P450 inhibitors affect the biogenesis of HBs and impair the production of infectious HDV virions. Mechanistically, we established that these drugs induce oxidative stress which dysregulates disulfide bond formation in the endoplasmic reticulum. As a consequence, the production of HBs, which depends on a dense network of disulfide bonds, is markedly affected as evidenced by an abnormal glycosylation profile, altered antigenic properties, and a poor expression of the largest form of HBs (L-HBs) which is essential to virus entry into target cells. This is associated with induction of the unfolded protein response, with the upregulation of CHOP/DDIT3 and key enzymes involved in the synthesis of the reducing metabolite glutathione (PHGDH, SHMT2, MTHFD2). Overall, our results indicate that alterations in redox homeostasis significantly impact HBs biogenesis, and reveal a druggable pathway that could be exploited to eliminate HDV in chronically infected patients. IMPACT AND IMPLICATIONSMore effective therapies are still needed to achieve a functional cure in patients chronically co-infected by HBV and HDV. In this study, we discovered that ritonavir, along with other cytochrome P450 inhibitors, can affect the production of infectious HDV particles in human hepatocyte cultures. Mechanistically, ritonavir induces oxidative stress and the unfolded protein response in the endoplasmic reticulum, thereby altering the biogenesis of HBs, the surface glycoprotein of both viruses. This work highlights the potential benefit and mechanism of action of ritonavir and related molecules in the treatment of co-infected patients.

Sudan virus (SUDV) is a member of the family Filoviridae, which comprises highly pathogenic viruses associated with unusually high case fatality rates. The development of medical countermeasures against filoviruses, including antivirals, vaccines, and therapeutic antibodies, requires preclinical evaluation in suitable animal models. C57BL/6J IFNAR-/- mice, which lack the type I interferon (IFN-/{beta}) receptor, have been reported to be susceptible to filovirus infections, although their impaired innate immune response may represent a potential limitation of the model. Here, we show that IFNAR-/- mice constitute a suitable model for SUDV infection. Following infection, animals developed a clear clinical disease characterized by significant weight loss and pronounced changes in behaviour and appearance. Mice reached the predefined clinical endpoint 3-5 days post infection. Post mortem analysis of terminal samples revealed high viral loads and viral genome copies in all tested organs as well as in serum, indicating widespread systemic dissemination. Importantly, infection was associated with a marked increase in several key chemokines and cytokines linked to systemic inflammation, consistent with the development of a cytokine storm-like response. Together, these findings demonstrate that SUDV infection in IFNAR-/- mice induces systemic viral dissemination and a pronounced inflammatory response, supporting the suitability of this model for investigating filovirus pathogenesis and infection-associated immune dysregulation.

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

Borealpox virus (BRPV; formerly Alaskapox) is an orthopoxvirus that has caused seven reported human infections in Alaska since 2015, including a fatal case in 2023. The natural reservoir of BRPV is unknown, although previous investigations have raised the possibility of wild small mammals transmitting the virus to humans, either through direct contact or via domestic cats and dogs. To understand which species may be involved in the maintenance and/or spillover of BRPV in Alaska, we trapped and sampled wild small mammals (including voles, shrews, and squirrels) in 2021 and 2024 near reported human case locations in Fairbanks and the Kenai Peninsula, respectively. We found evidence of previous exposure to orthopoxviruses in five species (including the House Mouse, Mus musculus) and detected BRPV DNA as well as viable virus in Northern Red-backed Voles (Clethrionomys rutilus). Further, screening of tissues from historical museum specimens revealed BRPV DNA in C. rutilus specimens collected in Denali National Park and Preserve in 1998 and 1999, 17 years before the first reported human case of BRPV. Phylogenomic analysis of all human and animal BRPV isolates strongly supports the hypothesis of local human infections through multiple spillover events. These findings suggest C. rutilus as a possible reservoir species for BRPV and indicate that BRPV has been present in Alaskan wild small-mammal populations for at least 25 years. Our study highlights the potential of museum collections to elucidate the temporal, spatial, and host ranges of emerging pathogens. Further museum- and field-based sampling will clarify the true geographic range of BRPV, which is closely related to Old World orthopoxviruses and may be circulating beyond North America.

BackgroundThere is currently no approved antiviral therapy against measles virus (MeV). Repurposing available compounds with broad antiviral activity may rapidly identify candidate drugs for clinical evaluation. Here we evaluated the antiviral activity of the clinically approved drugs azelastine hydrochloride and zafirlukast as well as the flavonoids quercetin and isoquercetin against MeV in preventative and therapeutic in vitro studies. MethodsCompounds were tested for antiviral activity against MeV in preventative (prophylactic and virucidal) and therapeutic (steady-state and persistent) assays in Vero/hSLAM cells. Viral loads and cell viability were measured 48h post-infection, and dose-response curves were used to calculate EC50 values. Flavonoids were also tested in the presence of 1 mM ascorbic acid. ResultsAzelastine hydrochloride did not show evidence of antiviral activity against MeV under these conditions, whereas zafirlukast, quercetin, and isoquercetin showed therapeutic activity against MeV. The addition of ascorbic acid enhanced the therapeutic potency of quercetin to 4.2-4.8 {micro}M and of isoquercetin to 10.7-10.9 {micro}M. Antiviral activity was dose-dependent when administered post-infection. ConclusionAmong the four compounds tested, quercetin showed the most potent therapeutic antiviral activity against MeV in vitro. Isoquercetin and zafirkulast also showed therapeutic activity. These findings support further evaluation of quercetin, isoquercetin, and zafirlukast as candidate antiviral drugs for MeV and highlight the utility of in vitro platforms for rapid antiviral drug screening.

Mpox virus (MPXV) gained increased attention following the declaration of two Public Health Emergencies of International Concern (PHEICs) in 2022 and 2024. The rapid spread of MPXV and the increase in human-to-human transmission highlighted the need for improved diagnostic tools for characterizing infection patterns and transmission dynamics. While PCR is effective for detecting active infections, serological approaches can help identify previous or asymptomatic infections and support retrospective surveillance. However, many serological assays developed during recent outbreaks have not been evaluated in endemic settings such as the Democratic Republic of the Congo (DRC). This study aims to define antigen-specific serological cutoff values to differentiate MPXV-seroreactive individuals from those with other orthopoxvirus (OPXV) exposure or different vaccination histories, specifically for use in the DRC. Here, we analyzed 134 individuals, divided into six distinct cohorts with different exposures. Serum samples were tested using Mesoscale Discovery (MSD) to screen for five MPXV and vaccinia virus (VACV) orthologous antigens: A29L/A27L, A35R/A33R, B6R/B5R, E8L/D8L, and M1R/L1R. Receiver operating characteristic (ROC) analysis identified the best-performing antigens and established seroreactivity cutoff values. A binary composite rule was also evaluated to improve the classification of these results. We identified three MPXV antigens, E8L (cut-off=12.33 AU/mL), A35R (cut-off=5.22 AU/mL), and B6R (cut-off=9.77 AU/mL), that showed the strongest discriminatory performance in the dataset. Collectively, these three antigens form a significant panel that demonstrated clear separation between our mpox survivor cohort and other OPXV-exposed individuals.

Hantavirids, specifically the members within the genus Orthohantavirus, represent a significant global public health threat, with bat-associated lineages challenging traditional reservoir paradigms. To investigate the genetic diversity of hantavirids in Southeast Asia, we conducted an expanded surveillance program in Lao PDR from May 2023 to October 2025 in bat populations and wild animals from local wet markets. Using molecular screening and deep sequencing to characterize hantavirids from bat populations and wild animals from local wet markets, we identified 20 positive samples across four bat species, recovering coding-complete genomes for multiple novel variants. Phylogenetic analysis confirmed that these viruses form a monophyletic group within Mobatvirus, resolving into two major subclades. The first subclade clustered with Quezon and Robina viruses found in fruit-eating bats. The second subclade further split into two lineages corresponding to Thakrong and Xuan Son viruses, which are associated with trident and leaf-nosed bats, respectively. Despite the strong host specificity observed, the detection of these viruses in a wet market, a critical interface for human-wildlife contact, indicates a potential zoonotic risk. These findings significantly expand the known diversity of mobatviruses in Laos and highlight the urgent need for serological surveillance in at-risk human populations to assess the potential for spillover.

Dengue virus (DENV) is a major burden to global public health, affecting hundreds of millions annually. Children represent the major proportion of global dengue cases, ranging from asymptomatic or subclinical presentation to dengue fever (DF) and severe dengue hemorrhagic fever or shock syndrome (DHF/DSS). The factors that distinguish this range of disease severity are still poorly understood. To identify biomarkers of severity, we analyzed the plasma proteome of acute DENV infected children including both subclinical and hospitalized cases. Proteins associated with the acute-phase response, innate immune and lysosomal activation, and components of the coagulation cascade showed marked differences between hospitalized and subclinical cases during early infection. Longitudinal profiling demonstrated that endothelial dysfunction emerges early, with PTX3 showing the strongest and most rapid upregulation in hospitalized patients, supporting its potential role as a marker of imminent vascular involvement. When comparing severe (DHF/DSS) and classical DF hospitalized cases, CLEC11A displayed the highest fold change at hospital admittance. We used machine-learning analysis to predict disease severity at the acute phase of infection, distinguishing subclinical from hospitalized cases and patients that develop classical dengue fever or severe disease based on the identified complement regulators and inflammatory markers. The panel of identified plasma proteins shed light on the mechanisms of dengue related disease progression and may provide a handle to predict disease severity based on blood markers present during the acute phase of infection.

During viral infection, viral replication perturbs endoplasmic reticulum (ER) homeostasis and triggers the unfolded protein response (UPR). XBP1s, a transcription factor generated by one branch of the UPR, is known to potentiate both innate and adaptive immunity, but its role in antiviral responses remains incompletely understood beyond its ability to augment type I interferon (IFN) mRNA induction. Here, we show that XBP1s positively regulates the RIG-I-like receptors (RLRs), ribonuclease L (RNase L), and protein kinase R (PKR) pathways, indicating that it enhances all three major antiviral response pathways. We further show that RNase L activation rapidly decreases XBP1 mRNA levels in an RNase activity-dependent manner, leading to a prompt reduction in XBP1s expression. Consistent with this, RNase L deletion significantly increased both thapsigargin-mediated XBP1s induction and XBP1s expression following Japan encephalitis virus infection. Poly(I:C)-induced IFNB mRNA expression was significantly enhanced in RNase L-knockout cells. This enhancement was completely abolished by RNase L reconstitution. XBP1 knockdown also significantly attenuated IFNB mRNA expression in RNase L-knockout cells. These findings suggest a negative-feedback loop in which RNase L suppresses XBP1s, thereby fine-tuning antiviral responsiveness during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/713401v1_ufig1.gif" ALT="Figure 1000"> View larger version (19K): org.highwire.dtl.DTLVardef@112d312org.highwire.dtl.DTLVardef@df79a9org.highwire.dtl.DTLVardef@1ac571borg.highwire.dtl.DTLVardef@18ac610_HPS_FORMAT_FIGEXP M_FIG C_FIG

The co-circulation and rapid expansion of the genus Orthoflavivirus, including dengue virus (DENV), Zika (ZIKV), and Japanese encephalitis virus (JEV), pose significant global health challenges. Developing inclusive pan-genus molecular diagnostics is hindered by high nucleotide divergence (>25%-30%) and the computational limitations of traditional multiple sequence alignment in detecting conserved motifs across large datasets. To overcome these limitations, we developed a systematic alignment-free design pipeline that uses rigorous k-mer analysis and compacted De Bruijn graphs. We analyzed 11,846 RefSeq viral genomes to identify phylogenetically conserved, functionally relevant signatures within the Orthoflavivirus genus as a case study. The pipeline identified a conserved 600-bp region within the non-structural protein 5 gene, facilitating the design of a broad-spectrum TaqMan RT-qPCR assay. Analytical validation against standard reference strains demonstrated a limit of detection of 1-10 copies/{micro}L for DENV1-4, ZIKV, and JEV, with no cross-reactivity against non-target pathogens. In a clinical evaluation of archived samples, the assay achieved 97.33% overall accuracy. It demonstrated 100% sensitivity and specificity for DENV serotypes, yielding significantly earlier cycle threshold (Ct) values compared to a standard commercial kit, while ZIKV detection showed 100% specificity with 71.43% sensitivity. This study validates an alignment-free, k-mer guided approach for uncovering conserved diagnostic targets in highly variable viral genera. The resulting assay offers a robust tool for frontline surveillance, and the computational framework provides a scalable solution for future pandemic preparedness.

Toll-like receptors (TLRs) are vital components of the innate immune system, recognizing both exogenous pathogens signals (PAMPs) and internal stress signals (DAMPs). TLR2 is unique among the human (Homo sapiens) TLR family members, as it contains a large cavity for binding hydrophobic ligands, such as lipoteichoic acid (LTA) and di/triacyl lipopeptides (Pam2/3CSK4). This study analyzed the structural phylogeny of cavity presence in the TLR2 lineage in vertebrates (vTLR) enabled by AI protein structure predictions and explored the potential convergent evolution of similar features in invertebrates (iTLRs). Analysis of AI models of TLR2s shows that this cavity is consistently present in TRL2 orthologs across jawed vertebrates (Gnathostomata). In jawless vertebrates (Cyclostomatha), these cavities were found in lamprey (Petromyzon marinus) TLR2 model, but only in some extant hagfish (Myxini), suggesting an ancestral origin in basal vertebrates followed by lineage-specific losses. TLR2 paralogs were found in several species, with a similar central cavity but potentially different ligand specificities. In silico ligand docking showed Pam2CSK4 binds to this cavity in all TLRs and paralogs consistently, demonstrating the conserved function of the ligand-binding pocket in gram-positive bacteria recognition across TLR2 branches. Changes in the TLR2 cavity size and shape in some vertebrate groups show the evolution of this DAMP recognition mechanism adapted to its respective pathogens. iTLRs form a separate phylogenetic branch with distinct structural features, but in literature some are considered to be TLR2 orthologs. Indeed, TLRs from some species of Helobdella and Ciona, contain a cavity with some similarity to that in the vTLR2 lineage. However, detailed structural comparisons of their location in the LRR domain and the structural details of the models suggest that their cavities have developed independently from that in TLR2s. Smaller cavities are present in other branches of the LRR family, but show different locations, shapes, and features, indicating that the binding of small ligands in the internal cavities within the LRR domains evolved multiple times in the LRR domain family history.

Single molecule methods have become prevalent tools in elucidating molecular processes across various life science fields over the past three decades, driving breakthroughs in understanding their underlying molecular mechanisms. In our study, we employed two single-molecule methods, Forster Resonance Energy Transfer (smFRET) and high-resolution optical tweezers, to investigate the transcription of Mycobacterium tuberculosis RNA polymerase (MtbRNAP) from initiation through to termination. We aim to provide a set of comprehensive biophysical tools to deepen our current understanding of MtbRNAP and its transcription factors. These experimental assays represent an important step towards unraveling the molecular dynamics and interactions that support transcription in Mycobacterium tuberculosis.

Cell cycle manipulation is critical to oncogenesis, including cancers associated with oncogenic gammaherpesviruses, Epstein-Barr Virus and Kaposis Sarcoma-associated Herpesvirus. Infection with these viruses can result in various cancers, including lymphomas and carcinomas. In healthy individuals, gammaherpesvirus infections result in lifelong latent infections with occasional reactivation. The cell cycle plays a critical role in infection, particularly in reactivation from quiescent latency to lytic virus replication. A number of cyclin-dependent kinase (CDK) inhibitors are clinically available but with little investigation thus far for virus-associated cancers. Using the mouse gammaherpesvirus model, we assessed the impact of CDK inhibitors on virus reactivation. First, we tested chemical inducers of reactivation, and found that optimal reactivation occurred with a combination of PMA and sodium butyrate. Application of optimal reactivation triggers demonstrated distinct stage-specific outcomes of reactivation, distinguished using flow cytometry to measure expression of GFP (early reactivation) and vRCA, a late viral protein (late reactivation). Following chemical induction of reactivation, we used flow cytometry to demonstrate that the early effects of induction were unaffected by CDK inhibitors. However, all broad spectrum CDK inhibitors tested, Dinaciclib, Alvocidib, and Seliciclib, decreased both reactivation from latency and primary lytic replication. In contrast, the impact of targeted CDK 4/6 inhibitors, Palbociclib, Ribociclib, and Abemaciclib, was more nuanced, with decreased reactivation when given concurrently, but increased reactivation when administered prior to induction. These findings were consistent for both murine gammaherpesvirus and Epstein-Barr Virus. Overall, our data indicate that CDK inhibitors may be useful for targeted treatment of gammaherpesvirus-associated cancers, but optimal use of targeted CDK 4/6 inhibitors requires careful consideration of cell state and order of therapies.

Coat protein (CP) tertiary structures and their capsid organization of spherical viruses are highly conserved within each virus family. While AlphaFold successfully predicts the tertiary structures of individual CPs, their association to form proper quaternary assemblies cannot be easily accomplished. Here, we report a generalized methodology and associated web-based utility (https://foldavirus.org) that combines AlphaFold predictions of CPs with the knowledge on corresponding icosahedral architectures (e.g., T=1, 3, 4...) based on the known structures from the same virus family to generate associated capsids. The resulting assemblies are subjected to Amber energy minimization to relieve any steric clashes at the inter-subunit interfaces. Significantly, the capsid models are validated by calculating robust Mahalanobis distance using the residue annotations categorized as interface, core and surface amino acids with respect to those observed in the experimentally determined analogous structures. Given the amino acid sequence of CP(s), we successfully generated capsids up to T=9 icosahedral symmetry, including those of Picornaviruses that display pseudo-T=3 symmetry comprising different CPs. As the number of currently available CP sequences are 3-4 orders of magnitude larger than the experimentally determined 3D-structures, this approach bridges the huge gap that exists between the corresponding sequence and structure space.