Genomic RNA Research Articles (Page 1)

Detection of cross-reactivity of antibodies to the N proteins of feline morbillivirus and canine distemper virus in Japanese cat plasma samples.

Preparation of monoclonal antibodies for the VP1 protein of bovine enterovirus and precise mapping of linear B-cell epitopes.

Background: Mitoxantrone (MX) is regularly used to treat several cancers. Despite its long history in the clinic, recent studies continue to unveil novel protein targets. These targets may contribute to the cytotoxic effects of the drug, as well as potential non-canonical antitumor activity. A better understanding of MX’s cellular targets is required to fully comprehend the molecular consequences of treatment and to interpret MX sensitivity in homologous recombination (HR)-deficient cancer. Methods: Here, we evaluated MX activity in HR-deficient UWB1.289 (BRCA1−) ovarian cancer cells and surveyed the binding profile of MX using TMT-labeled quantitative proteomics and chemoproteomics. Results: Mass spectrometry (MS) analysis of cellular extracts from MX-treated BRCA1−UWB1.289 cells revealed unique downregulation of pathways instrumental in maintaining genomic stability, including single-strand annealing. Moreover, the BRCA1− cells exhibited a significant upregulation of proteins involved in ribosome biogenesis and RNA processing. Additional MS analyses following affinity-purification using a biotinylated-mitoxantrone probe corroborated these findings, which showed considerable targeting of proteins involved in genome maintenance and RNA processing. Conclusions: Our results suggest that an interplay of both canonical and non-canonical MX-antitumor activity overwhelms the BRCA1− UWB1.289 cells. Furthermore, this study characterizes the target landscape of MX, providing insights into off-target effects and MX action in HR-deficient cancer.

Potato mop-top virus (PMTV), vectored by soil-borne protist Spongospora subterranea f. sp. subterranea (Sss) is increasingly becoming a threat to potato production in the USA. Major means of spread is through infected tubers, vector, and contaminated farm equipment, and the virus is known to be mechanically poorly transmitted. Using infectious cDNA clones of PMTV, in vitro produced viral RNA transcripts were used to mechanically inoculate two potato cultivars differing in their sensitivity to PMTV, as well as model plants Nicotiana benthamiana, and N. tabacum. Infection was measured using primers to each of the three genomic RNAs by real-time PCR. The movement of all three genomic RNAs and their relative accumulation were studied over several time points. Overall, 100% of the inoculated plants showed infection. All three RNAs of PMTV were detected in younger, uninoculated leaves at 7, 14, and 21 dpi, suggesting successful infection followed by long distance movement. Potato cv. Clearwater Russet had higher levels of PMTV compared to Castle Russet, which was in agreement with their response to PMTV under natural field conditions. Findings suggest that the use of infectious RNA transcripts in combination with mechanical inoculation has the potential to study PMTV-potato interactions at the omics level.

Type-1 diabetes (T1D) pathogenesis is either initiated or accelerated by human enteroviruses, particularly Coxsackie viruses. Because the structural makeup of the virus's viral protein 2 C (P2-C) and the glutamic acid decarboxylase (GAD65) autoantigen in human beta-cells is similar, Coxsackie B viruses are one of the primary causes of type 1 diabetes. The study identified the molecular presence of the Coxsackie B virus and ascertained its seroprevalence in patients with type 1 diabetes mellitus. From March 2023 to January 2024, a hospital-based cross-sectional study was conducted in three hospitals in Jigawa State: Dutse General Hospital, Hadejia General Hospital, and Ringim General Hospital. Ninety blood samples from diabetes patients who gave their consent were collected from the research facilities. Blood sugar levels exceeding 200 mg/dL or 11.1mmol/L were used to diagnose type-1 diabetes mellitus (T1DM) in patients based on clinical characteristics. IgM indirect ELISA detection against the GAD65 autoantibody in the patients' serum was used to confirm the diagnosis of T1DM. The virus was also detected by IgM detection and molecular technique (RT-PCR). Out of 90 blood samples, 52% of all samples of T1D patients were positive for Anti-GAD65. 11 were positive for the CBV IgM antibody, indicating a prevalence of 12.2% of T1D patients across all patient groups. The RT-PCR results indicated the presence of the RNA genome of Coxsackie B virus in the serum of some T1DM patients, with a prevalence of 2.2% (2/90), as indicated by the detection of the VP1 gene. The findings demonstrated that in Jigawa state, CBV is not the most likely causative agent of T1D; rather, risk variables for the disease include sex, age, educational attainment, parental status, and the mechanism of disease acquisition and residency. However, other factors may be taken into consideration in future research. To lower the prevalence or eradicate the disease, it may be advisable to conduct more studies on the various causative agents of T1D and to provide early treatment for affected individuals. Additionally, relevant agencies should establish public awareness campaigns on the impacts of the disease.

Background: Effective treatment of cardiac diseases requires a deep understanding of disease-relevant cellular mechanisms that drive pathology in the human heart. Traditional cell lines often lack the complexity and genetic context necessary to capture these mechanisms. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes provide a physiologically relevant and genetically diverse platform to model human cardiac disease biology. Evotec’s iPSC platform enables detailed study of disease processes critical for identifying therapeutic targets and developing novel treatments. Methods&Results: We developed a robust, scalable human iPSC-based cardiomyocyte platform integrating a genetically diverse bank of well-characterized iPSC lines and optimized cardiac differentiation protocols. The platform enables the generation of multiple human-relevant cardiac cell types and model systems, including cardiomyocytes, cardiac fibroblasts, 2D co-cultures of these cell types, 3D cardiac spheroids, and engineered heart tissue (EHT). Genome editing technologies, including CRISPR/Cas9 and RNA interference, are routinely applied at both iPSC and cardiomyocyte stages. These tools enable the introduction of disease-relevant mutations to model specific cardiac pathologies, as well as the validation and exploration of novel target gene functions within a human cellular context. To assess disease mechanisms and potential target effects, we use multi-electrode arrays (MEA) for electrophysiology, xCELLigence and FlexCyte for contractility, and calcium transient assays. This enables high-throughput, quantitative, and physiologically relevant phenotyping. Pharmacological testing with well-characterized compounds confirms the platform’s sensitivity to cardiac functional changes. Conclusion: Evotec’s iPSC cardiomyocyte platform, combined with the Evotec Heart Atlas, offers a human-relevant system for cardiac disease modeling, drug target validation, and mechanistic insights. Together, these platforms accelerate cardiovascular therapy discovery and increase clinical success through improved human and disease relevance.

RNA can fold to form complex three-dimensional tertiary structures, with large roles in RNA biology. However, accurate discovery of motifs that form true tertiary structures remains an unsolved challenge. Here we demonstrate that at rare but distinctive sites, RNA tertiary folding creates electronegative pockets that undergo selective chemistry with a small positively-charged chemical probe, trimethyloxonium (TMO). Sites preferentially reactive with the TMO cation, relative to uncharged dimethyl sulfate (DMS), termed T-sites, reflect preferential interaction of TMO at punctate electronegative pockets created by juxtaposition of a reactive nucleobase with non-bridging oxygen atoms in the RNA backbone. T-sites map specifically to centers of higher-order structural interactions and functional cores in diverse RNAs, including in specific states in conformational ensembles. Probing the 10,723-nt dengue virus RNA genome revealed exactly three T-sites, each of which is embedded within a complex RNA structure with direct roles in viral replication. T-site chemistry creates broad opportunities for high-confidence discovery and analysis of functional RNA tertiary structures across long and complex RNAs, including transcriptome-wide.

Transcription is regulated at multiple levels, including initiation, elongation, and termination. Whereas much research has focused on the initiation of transcription, regulation of elongation plays an important role not only in transcription dynamics but also in cotranscriptional RNA processing and genome stability. Despite advances in high-throughput approaches for global quantification of RNA polymerase II (RNAPII) speed, RNAPII elongation rate studies have been limited to a relatively small number of long genes or to velocity estimates inferred indirectly from RNAPII occupancy data. Here, we present DRB/TTchem-seq2, a modified version of the DRB/TTchem-seq method, to directly measure gene-specific elongation rates of more than 3000 genes. By combining short time point sampling after synchronized RNAPII release into the gene body and a new computational framework to track the distance traveled by RNAPII, we greatly increase the number of genes for which it is possible to obtain elongation rates. Our direct RNAPII elongation rate quantification reveals that elongation rates vary not only among genes but also within genes. Additionally, we describe how specific histone modifications and elongation factor occupancy correlate with subclasses of genes based on their elongation rates. Together, we present a robust and powerful method for RNAPII transcription elongation rate measurement.

Bridging predictions with experimental reality: In vitro evaluation of phenolic natural products as SARS-CoV-2 exoribonuclease inhibitors.

Duck hepatitis A virus 3CD and 3D proteins are key facilitators of hnRNP K expression and cytoplasmic entry, and hijack hnRNP K to facilitate viral translation and replication.

In addition to their role in canonical autophagy, autophagy proteins (ATG) contribute to various cellular processes, including phagocytosis, membrane remodeling, and vesicle secretion. Several viruses also exploit components of the autophagy pathway for their own replication. Here, we explore the role of ATG proteins in HIV-1 assembly. Postulating that host proteins crucial for virion assembly are present at the assembly site and can be incorporated within virions, we analyze the proteome of HIV-1 preparations using mass spectrometry. We identify an enrichment of macroautophagy-related terms, notably 3 of the 6 ATG8 (LC3/GABARAP) proteins. Functional studies reveal that GABARAP proteins are critical for the production of infectious virions. Knockout of GABARAP proteins reduces the packaging of viral genomic RNA (gRNA) into particles, impairing virion infectivity. GABARAPL1 associates with gRNA and interacts with Gag in an RNA-dependent manner. Additionally, GABARAP knockout increases cellular Gag:gRNA complexes and decreases gRNA association with membranes, suggesting that GABARAP proteins regulate gRNA fate during HIV-1 assembly by facilitating its packaging. This study uncovers a novel role for GABARAP proteins in HIV-1 genome packaging.

Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging highly pathogenic bunyavirus, poses a significant public health threat with a case fatality rate of up to 30%. The viral nucleocapsid protein (NP) encapsidates the genomic RNA to form a ribonucleoprotein (RNP) complex, which is critical for transcription and replication. However, the molecular mechanism underlying SFTSV RNA encapsidation remains poorly understood, largely due to the lack of structural information on the NP-RNA complex. Here, we report a cryo-electron microscopy structure of the SFTSV NP in complex with single-stranded RNA. The structure reveals a pentameric NP assembly that sequesters RNA along the inner surface of the oligomeric ring in a sequence-independent manner. Strikingly, all the RNA bases face the protein, rendering them inaccessible for transcription and replication. Each NP subunit accommodates four nucleotides within an evolutionarily conserved hydrophobic cleft, with an additional two to three nucleotides bound at the inter-subunit interface. The functional importance of the NP-RNA interactions is further corroborated by a minigenome-based assay. This work provides structural insight into RNA encapsidation by SFTSV NP and offers a foundation for the rational design of antiviral therapeutics targeting this essential viral protein.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus that causes severe hemorrhagic fever, leukopenia, thrombocytopenia, and multi-organ failure, with a case fatality rate of up to 30%. No licensed vaccines or specific antiviral therapies are currently available. The viral nucleocapsid protein (NP) is essential for viral transcription and replication, forming a ribonucleoprotein complex (RNP) by encapsidating viral genomic RNA. However, the structural basis of RNA recognition and encapsidation by SFTSV NP remains poorly understood. In this study, we determined a cryo-electron microscopy structure of the SFTSV NP-RNA complex. Structural comparisons and evolutionary conservation analysis of NPs across the family Phenuiviridae uncovered a conserved RNA-binding mode among phenuiviruses, suggesting a shared RNA encapsidation mechanism among related viruses. Our findings provide critical structural insights into SFTSV RNA encapsidation and will aid future efforts to develop antivirals against SFTSV and related pathogenic viruses.

The nuclear transcription factor NFYA3_0 promotes MTA-mediated m6A modification of Potato virus Y genomic RNA to confer antiviral resistance in Nicotiana benthamiana.

Systemic spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to extrapulmonary tissues has been observed following acute infections. Autopsy studies further indicate tissue-specific virus diversity, including in immune-privileged sites. Questions remain on the viral dynamics leading to the tissue tropism of SARS-CoV-2, including evolutionary trajectories and functional adaptations that could impact persistence and transmission. In this study, we characterized SARS-CoV-2 genomes from 27 distinct tissues collected from an autopsy case where the patient had a primary immune deficiency. We identified tissue-specific virus genotypes, in some instances coexisting within the same sites, with mutations primarily in the receptor-binding domain of the spike protein. Protein simulations and isolation of infectious virus indicate combinations of spike substitutions that would lead to increased protein stability and stronger binding of the virus to host cells. This highlights the importance of studying patients with weakened immune responses where potential tissue reservoirs provide an environment permissive for SARS-CoV-2 evolution and diversification.IMPORTANCEPersistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in immunocompromised individuals are considered a potential source of new viral variants. Beyond the respiratory tract, the virus can spread within days to organs like the brain, heart, and kidneys, where distinct tissue microenvironments may further drive viral evolution and the emergence of new mutations. In this study, we compared the genetic diversity of SARS-CoV-2 genomic RNA isolated from 27 distinct tissue sites collected from an individual with a weakened immune system. By linking viral population dynamics across these tissue sites, we defined the extent of compartmentalization during multi-organ spread, highlighting how non-respiratory tissues can impact SARS-CoV-2 diversification.

Nucleoside analogs are successfully used to treat viral infections. dNTP analogs are primarily DNA chain terminators, while NTP analog remdesivir can inhibit RNA synthesis by delayed chain termination or when in the template strand. Here, enzymatic assays, mass spectrometry, and cryo-EM demonstrate that SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) preferentially incorporates remdesivir triphosphate (RTP), outcompeting 10-fold excess ATP; however, successive RTP incorporations are disfavored when ATP is present. The RdRp structures demonstrate that the remdesivir:UMP base pair is resilient to translocation, reducing successive RTP incorporations. Consequently, the relative concentration of ATP-to-RTP and structural rigidity of remdesivir:UMP, would limit remdesivir occupancy to < 16% of available positions in a copied genomic RNA strand. The S759A mutant confers RTP resistance. The structures of S759A RdRp reveal that the primer 3′-end nucleotide repositioning and its altered ribose-ring conformation contribute to RTP resistance. These findings have implications for designing non-obligate nucleoside analogs with different inhibition mechanisms.

Reoviruses are non-enveloped viruses with segmented double-stranded RNA (dsRNA) genomes that are members of the Reoviridae family. These viruses may infect a wide range of hosts, including people, mammals, birds, and reptiles, and are widely known for their diversity and zoonotic potential. Bangladesh announced the first verified cases of Reovirus infection in humans in January 2025, which might be a public health concern. Five patients were confirmed to be infected with Reovirus. All exhibited mild to moderate symptoms, with full recovery observed in each case. Epidemiological analysis suggests a zoonotic origin, with potential transmission linked to exposure to raw date palm sap, a known reservoir for bat-borne viruses. The identification highlights the need for more monitoring, better diagnostic tools, and public health awareness, especially in rural regions where close animal-human interaction is more prevalent, even though the clinical results were not severe and not dangerous. Bangladesh Journal of Infectious Diseases, June 2025;12(1):181-184

Furoviruses, which are members of the Virgaviridae family, are rod-shaped particles containing bipartite and positive-sense RNA genomes. Three furoviruses, Japanese soil-borne wheat mosaic virus (JSBWMV), Chinese wheat mosaic virus (CWMV), and soil-borne wheat mosaic virus (SBWMV), infect wheat in Japan; however, differences in their pathogenicity to wheat remain unclear. In this study, the infectivity of these three furoviruses in Japanese wheat cultivars was investigated using mechanical leaf inoculation. Of the 15 Japanese wheat cultivars inoculated with these furoviruses, 4 cultivars, such as Chikugoizumi, Fukusayaka, Shirogane komugi, and Yumekaori, were resistant to JSBWMV. In contrast, no cultivars with complete resistance to CWMV or SBWMV were noticed. Infectious cDNA clones of JSBWMV and CWMV were constructed to identify the viral factors involved in this resistance. Analysis of reassortment and chimeric viruses between JSBWMV and CWMV identified cysteine-rich protein (CRP) on RNA2 as the viral factor; a chimeric virus containing CWMV CRP infected all four cultivars, whereas that containing JSBWMV CRP was resisted. Furthermore, JSBWMV could multiply in the mesophyll protoplasts of the four JSBWMV-resistant cultivars, suggesting that the resistance mechanism did not block multiplication but affected local or systemic movement of the virus. Therefore, the identification of wheat genes involved in resistance to JSBWMV will contribute to the understanding of resistance mechanisms to furoviruses and interactions between furovirus CRPs and host factors.

The Polymycoviridae family is an expanding group of mycoviruses, generally characterized by atypical capsids and segmented double-stranded (ds) RNA genomes. Polymycoviruses have been associated with various changes in their hosts' traits, such as shifts in spore production, changes in pigmentation, and alterations in growth and virulence, either enhancing or reducing them. Here, we report the complete genome sequence of a polymycovirus identified in an entomopathogenic, fungicolous, and keratinophilic, filamentous ascomycete species Lecanicillium aphanocladii, isolated from a patient with foot dermatophytosis (Tinea pedis). The virus, denominated Lecanicillium aphanocladii polymycovirus 1 (LaPmV1), has six genomic dsRNA segments. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) sequences revealed that LaPmV1 is closely related to the previously reported Beauveria bassiana polymycovirus 4. Virus-curing studies revealed that LaPmV1 regulates pigmentation and decreases sporulation in its host while not affecting mycelial growth in vitro. LaPmV1 has also been shown to slightly increase the host's susceptibility to certain common antifungals, including itraconazole, voriconazole, and posaconazole.

Porcine deltacoronavirus (PDCoV), an enteric member of the coronavirus family, has emerged globally over the past decade, causing significant impacts on the swine industry. While studies of virus-host protein interactions provide crucial insights into viral engagement with host cells during infection, research specifically targeting PDCoV-host interaction factors remains limited. To identify host proteins involved in PDCoV replication, comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) was employed to identify host proteins interacting with the PDCoV genomic RNA. Concurrently, affinity purification mass spectrometry (AP-MS) was utilized to identify host interactors of PDCoV-encoded proteins. A total of 671 host proteins were identified in our analysis. These host interactors participate in diverse cellular processes, including extensive representation of metabolic enzymes, transcription factors, RNA-binding proteins (RBPs), and intracellular signal transduction components. Construction of a comprehensive PDCoV-host protein interaction network map revealed that SYNCRIP (heterogeneous nuclear ribonucleoprotein Q, hnRNP Q), functions as a novel host restriction factor with PDCoV. SYNCRIP interacts with the N proteins of multiple coronaviruses and competitively displaces HUWE1 to bind the PDCoV N protein, thereby blocking its ubiquitin-proteasome-mediated degradation. Furthermore, Isoforsythiaside, a small-molecule inhibitor designed to target SYNCRIP, demonstrated substantial antiviral potential both in vitro and in vivo. In summary, this study provides a comprehensive catalog of functional PDCoV viral RNA (vRNA)/viral Protein (vProtein)-host protein interactions. This resource not only informs the understanding of pan-coronavirus infection mechanisms but also nominates host cellular processes as potential targets for antiviral intervention.

We know the flu for its annual seasonal epidemics and occasional pandemics. The highest morbidity from this infection is seen in children, but the highest mortality is reserved for patients over 65 years of age with associated comorbidities. There are three known types of influenza viruses: Influenzavirus A, B, and C. All belong to the Orthomyxoviridae family and have a single-stranded, negative-polarity RNA genome. The lipid envelope contains two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which allow the influenza A virus to be subtyped. The last pandemic recorded in 2009 was caused by the AH1N1pdm (pdm: pandemic) variant. Clinical symptoms begin abruptly with fever, general malaise, headache, and respiratory symptoms ranging from mild to pneumonia. The diagnosis can be direct (detection of viral antigen, molecular diagnosis, or viral culture) or indirect (serology). In this review, we focus on viral culture. The influenza virus can be isolated in cell lines: MDCK (Madin-Darby Canine Kidney) or hCK (a cell line derived from the former, which are humanized MDCK cells). After infecting the cells, the cytopathic effect of the virus on them is observed by immunofluorescence. Viral culture demonstrates the presence of the virus, its viability, and infectivity, allowing for studies of sensitivity to antivirals. However, it has a limitation of approximately 50% sensitivity due to the meticulous nature of the cultivation procedure. The entire population is susceptible to influenza, although immunity may exist due to previous contact with identical or antigenically similar viruses or through immunization (annual vaccination). The World Health Organization (WHO) maintains a global influenza surveillance program, but the changing nature of the virus defies prediction and the onset of a pandemic is unpredictable.