Genomic RNA Research Articles

First complete genome sequence of tulip mild mottle mosaic virus (Ophiovirus tulipae)

Tulip mild mottle mosaic disease, caused by tulip mild mottle mosaic virus (TMMMV, species Ophiovirus tulipae), was first reported in Japan in 1979. TMMMV has a negative-sense ssRNA genome and is closely related to ophioviruses such as Mirafiori lettuce big vein virus (MLBVV, Ophiovirus mirafioriense). However, its complete nucleotide sequence has not yet been reported. Therefore, in this study, we determined the complete nucleotide sequences of all four TMMMV segments. The 5′-terminal tetranucleotide sequences were highly conserved, and the five bases at the 5′ end were identical in all segments. Nucleotide sequence comparisons of the four segments showed the highest sequence identity to those of MLBVV (70–76% for the genomic RNA sequences and 81–87% for the amino acid sequences of each ORF, except for ORF2). The amino acid sequences of the coat proteins of TMMMV and MLBVV were found to be 83.1% identical, which is below the 85% threshold for assignment to the same species, indicating that these viruses belong to different species in the same genus.

Ultra-Sensitive Aptamer-Based Diagnostic Systems for Rapid Detection of All SARS-CoV-2 Variants

The emergence of numerous SARS-CoV-2 variants, characterized by mutations in the viral RNA genome and target proteins, has presented challenges for accurate COVID-19 diagnosis. To address this, we developed universal aptamer probes capable of binding to the spike proteins of SARS-CoV-2 variants, including highly mutated strains like Omicron. These aptamers were identified through protein-based SELEX using spike proteins from three key variants (D614G-substituted Wuhan-Hu-1, Delta, and Omicron) and virus-based SELEX, known as viro-SELEX. Leveraging these universal aptamers, we created a highly sensitive lateral flow assay (LFA) and an ultra-sensitive molecular diagnostic platform that integrates a novel rapid PCR technique, enabling fast and reliable detection across all SARS-CoV-2 variants.

G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication

Future pandemic threats may be caused by novel coronaviruses and influenza A viruses. Here we show that when directly added to a cell culture, 12mer guanine RNA (G12) and its phosphorothioate-linked derivatives (G12(S)), rapidly entered cytoplasm and suppressed the propagation of human coronaviruses and influenza A viruses to between 1/100 and nearly 1/1000 of normal virus infectivity without cellular toxicity and induction of innate immunity. Moreover, G12(S) alleviated the weight loss caused by coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with left-handed parallel-stranded G-quadruplex, and inhibit the replication process by impeding interaction between viral nucleoproteins and viral RNA in the cytoplasm. Unlike previous antiviral strategies that target the G-quadruplexes of the viral genome, we now show that excess exogenous G-quadruplex-forming small RNA displaces genomic RNA from ribonucleoprotein, effectively inhibiting viral replication. The approach has the potential to facilitate the creation of versatile middle-molecule antivirals featuring lipid nanoparticle-free delivery.

Genomic, Evolutionary, and Pathogenic Characterization of a New Polerovirus in Traditional Chinese Medicine Viola philippica

Viola philippica, a medicinal herbaceous plant documented in the Chinese Pharmacopoeia, is a promising candidate for research into plant-derived pharmaceuticals. However, the study of newly emerging viruses that threaten the cultivation of V. philippica remains limited. In this study, V. philippica plants exhibiting symptoms such as leaf yellowing, mottled leaves, and vein chlorosis were collected and subjected to RNA sequencing to identify potential viral pathogens. A novel polerovirus, named Viola Philippica Polerovirus (VPPV), was identified in V. philippica. VPPV possesses a linear, positive-sense, single-stranded RNA genome consisting of 5535 nucleotides (nt) and encodes seven highly overlapping open reading frames (ORFs). Two potential recombination events were identified within ORF2, ORF3a, and ORF3, providing insights into the genetic diversity and evolution history of this novel polerovirus. An infectious cDNA clone of VPPV was successfully constructed and shown to infect Nicotiana benthamiana. Using a PVX-based heterologous expression system, the VPPV P0 protein was shown to trigger a systemic hypersensitive response (HR)-like reaction in N. benthamiana, indicating that P0 functions as the main pathogenicity determinant. These findings contributed to the detection and understanding of pathogenic mechanisms and control strategies for VPPV in V. philippica.

Role of the Psi Packaging Signal and Dimerization Initiation Sequence in the Organization of Rous Sarcoma Virus Gag-gRNA Co-Condensates

Retroviral genome selection and virion assembly remain promising targets for novel therapeutic intervention. Recent studies have demonstrated that the Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type-1 (HIV-1) undergo nuclear trafficking, colocalize with nascent genomic viral RNA (gRNA) at transcription sites, may interact with host transcription factors, and display biophysical properties characteristic of biomolecular condensates. In the present work, we utilized a controlled in vitro condensate assay and advanced imaging approaches to investigate the effects of interactions between RSV Gag condensates and viral and nonviral RNAs on condensate abundance and organization. We observed that the psi (Ψ) packaging signal and the dimerization initiation sequence (DIS) had stabilizing effects on RSV Gag condensates, while RNAs lacking these features promoted or antagonized condensation, depending on local protein concentration and condensate architecture. An RNA containing Ψ, DIS, and the dimerization linkage structure (DLS) that is capable of stable dimer formation was observed to act as a bridge between RSV Gag condensates. These observations suggest additional, condensate-related roles for Gag-Ψ binding, gRNA dimerization, and Gag dimerization/multimerization in gRNA selection and packaging, representing a significant step forward in our understanding of how these interactions collectively facilitate efficient genome packaging.

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

Coronaviruses (CoVs) encode non-structural proteins (nsp's) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3'-5' exoribonuclease in non-structural protein 14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14-ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-nsp10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between wild type-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-nsp10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-nsp10 interface for viral inhibition and attenuation.IMPORTANCECoronavirus replication requires assembly of a replication transcription complex composed of nsp's, including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic cofactor nsp10, but the determinants and importance of the nsp14-nsp10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-nsp10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non-active-site targets for virus inhibition and attenuation.

Unveiling the ghost: machine learning's impact on the landscape of virology.

The complexity and speed of evolution in viruses with RNA genomes makes predictive identification of variants with epidemic or pandemic potential challenging. In recent years, machine learning has become an increasingly capable technology for addressing this challenge, as advances in methods and computational power have dramatically improved the performance of models and led to their widespread adoption across industries and disciplines. Nascent applications of machine learning technology to virus research have now expanded, providing new tools for handling large-scale datasets and leading to a reshaping of existing workflows for phenotype prediction, phylogenetic analysis, drug discovery and more. This review explores how machine learning has been applied to and has impacted the study of viruses, before addressing the strengths and limitations of its techniques and finally highlighting the next steps that are needed for the technology to reach its full potential in this challenging and ever-relevant research area.

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Precursor of H-Type II Histo-Blood Group Antigen and Subterminal Sialic Acids on Gangliosides Are Significantly Implicated in Cell Entry and Infection by a Porcine P[11] Rotavirus

Rotaviruses, non-enveloped viruses with a double-stranded RNA genome, are the leading etiological pathogen of acute gastroenteritis in young children and animals. The P[11] genotype of rotaviruses exhibits a tropism for neonates. In the present study, a binding assay using synthetic oligosaccharides demonstrated that the VP8* protein of P[11] porcine rotavirus (PRV) strain 4555 binds to lacto-N-neotetraose (LNnT) with the sequence Galβ1,4-GlcNAcβ1,3-Galβ1,4-Glc, one of the core parts of histo-blood group antigen (HBGA) and milk glycans. However, infections were significantly inhibited by blocking of endogenous monosialoganglioside (GM) GM1a with cholera toxin B subunit and preincubation of the virus with exogenous GM1a, suggesting that GM1a is involved in the infection of P[11] PRV 4555. In addition to GM1a, preincubation of the virus with exogenous disialogangliosides (GD) GD1a, GD1b, and trisialoganglioside (GT) GT1b also prevented infection. In contrast, exogenous ganglioside GM3 only inhibited infections at an early time point, and exogenous asyalosphingolipids GA1 and LacCer did not show any inhibitory effect on infections. This indicates that P[11] PRV 4555 preferentially utilizes gangliosides containing subterminal sialic acids. Further experiments revealed that P[11] PRV 4555 infections were prevented by preincubation of the virus with Neu5Ac and Neu5Gc. These results confirmed that sialic acids are essential for P[11] PRV 4555 cell entry, despite the classification as NA-resistant strain. Overall, our results proved that P[11] rotavirus not only binds to the Gal-GlcNAc motif but also utilizes gangliosides containing subterminal sialic acids.

Advancements in Antiviral Therapeutics: A Comprehensive Review of Hepatitis C Virus and Novel Flavone Leads

This review explores the fundamental characteristics and implications of viruses, focusing on their classification, structure, and the specific case of Hepatitis C Virus (HCV). Viruses, nonliving biological entities reliant on host cells for replication, have been historically pivotal in understanding infectious diseases. The classification of viruses has evolved significantly, leading to a hierarchical system that categorizes them by order, family, genus, and species based on genomic characteristics and structural features. HCV, a member of the Flaviviridae family, presents a significant global health concern, affecting over 150 million people and causing approximately 500,000 deaths annually. The virus is characterized by its enveloped structure and single-stranded RNA genome, which encodes a polyprotein that is cleaved into functional proteins essential for viral replication and assembly. The epidemiology of HCV reveals regional prevalence variations, with higher rates observed in developing countries. Notably, the virus exhibits considerable genetic diversity, categorized into seven genotypes, each demonstrating different responses to treatment. The natural history of HCV infection is complex, often remaining asymptomatic for extended periods, which complicates diagnosis and treatment initiation. Chronic infections can lead to severe complications, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Understanding the mechanisms of HCV entry, replication, and immune evasion is crucial for developing effective antiviral therapies and preventive measures. This review aims to provide a comprehensive overview of the current knowledge surrounding HCV, highlighting the need for ongoing research to better understand its biology and to improve therapeutic strategies against this pervasive virus.

Structure of the Nipah virus polymerase complex

Nipah virus is a highly virulent zoonotic paramyxovirus causing severe respiratory and neurological disease. Despite its lethality, there is no approved treatment for Nipah virus infection. The viral polymerase complex, composed of the polymerase (L) and phosphoprotein (P), replicates and transcribes the viral RNA genome. Here, we describe structures of the Nipah virus L-P polymerase complex and the L-protein’s Connecting Domain (CD). The cryo-electron microscopy L-P complex structure reveals the organization of the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L-protein, and shows how the P-protein, which forms a tetramer, interacts with the RdRp-domain of the L-protein. The crystal structure of the CD-domain alone reveals binding of three Mg ions. Modelling of this domain onto an AlphaFold 3 model of an RNA-L-P complex suggests a catalytic role for one Mg ion in mRNA capping. These findings offer insights into the structural details of the L-P polymerase complex and the molecular interactions between L-protein and P-protein, shedding light on the mechanisms of the replication machinery. This work will underpin efforts to develop antiviral drugs that target the polymerase complex of Nipah virus.

Molecular Diagnostics for Infectious Diseases in Low or MiddleIncome Countries

Infectious diseases remain a leading cause of morbidity and mortality in Low and MiddleIncome Countries (LMICs). Indisputably, molecular diagnostics is the key to a rapid and reliable identification of an infectious agent in clinical specimens. Unlike traditional techniques, such as microscopy and cell culture, molecular diagnostics can detect extremely low levels of pathogens within a short time and can provide additional information about the pathogen by looking at the genome. The term molecular diagnostics encompasses various laboratory techniques that directly target the DNA or RNA genome of pathogen, the proteome of a pathogen, or in an indirect manner, the immune reaction of the infected system to a specific pathogen. In a particular setting, molecular diagnostics can replace, support or confirm a clinical diagnosis and is specifically helpful for differential diagnosis in infectious diseases, that cross-present a high similarity in clinical symptoms

Barley stripe mosaic virus-induced gene silencing for functional validation of abiotic stress in barley.

The barley stripe mosaic virus (BSMV) uses its genomic RNA components (alpha, beta, and gamma) as an efficient method for studying gene functions. It is a newly developed method that utilizes gene transcript suppression to determine the role of plant genes. BSMV derived from virus induced gene silencing (VIGS) is capable of infecting various key farming crops like barley, wheat, rice, corn, and oats. Nevertheless, the growing acceptance and enhancement of BSMV-VIGS will benefit all kinds of plants. Abiotic stresses such as drought and salt are highly affecting plant growth, development, and production. BSMV-induced temporal gene knockdown is performed during particular stressful situations to determine their specific function. The quick physiological and biochemical changes aid in confirming the role of the target genes. VIGS has a significant role to improve crop genetics and breeding, despite having certain restrictions. Thus, exploring the possible solution and addressing these difficulties will enhance the technology in the continuous advancement of plant manufacturing. BSMV-mediated VIGS has become popular in functional genomics; gene function can be determined without permanent transformation. In general, BSMV-mediated VIGS will be very helpful in the ongoing effort to develop resilient crops.

Diversity and evolution of viroids and viroid-like agents with circular RNA genomes revealed by metatranscriptome mining.

Viroids, the agents of several plant diseases, are the smallest and simplest known replicators that consist of covalently closed circular (ccc) RNA molecules between 200 and 400 nucleotides in size. Viroids encode no proteins and rely on host RNA polymerases for replication, but some contain ribozymes involved in replication intermediate processing. Although other viroid-like agents with cccRNAs genomes, such as satellite RNAs, ribozyviruses and retrozymes, have been discovered, until recently, the spread of these agents in the biosphere appeared narrow, and their actual diversity and evolution remained poorly understood. Extensive, targeted metatranscriptome mining dramatically expanded the known diversity of cccRNAs genomes. These searches identified numerous, diverse viroid-like cccRNAs, many found in environments devoid of plant and animal material, suggesting replication in unicellular eukaryotic and/or prokaryotic hosts. Several cccRNAs are targeted by CRISPR systems, supporting their association with bacteria. In addition to small cccRNAs in the viroid size range, a broad variety of ribozyviruses and novel viruses with cccRNAs genomes, with genomes reaching nearly 5 kilobases, were discovered. Thus, metatranscriptome mining shows that the diversity of viroid-like cccRNAs genomes is far greater than previously suspected, prompting reassessment of the relevance of these replicators for understanding the primordial RNA world.

The protein structurome of Orthornavirae and its dark matter.

Advanced methods for protein structure prediction, such as AlphaFold2, greatly expand our capability to identify protein domains and infer their likely functions and evolutionary relationships. This is particularly pertinent for proteins encoded by viruses that are known to evolve rapidly and as a result often cannot be adequately characterized by analysis of the protein sequences. We performed an exhaustive structure prediction and comparative analysis for uncharacterized proteins and domains ("dark matter") encoded by viruses with RNA genomes. The results show the dark matter of RNA virus proteome consists mostly of disordered and all-α-helical domains that cannot be readily assigned a specific function and that likely mediate various interactions between viral proteins and between viral and host proteins. The great majority of globular proteins and domains of RNA viruses are already known although we identified several unexpected domains represented in individual viral families.

Formalin and 2.5% Glutaraldehyde/2% Paraformaldehyde in 0.1 M Cacodylate Buffer Inactivation Protocols to Ensure the Proper Fixation of Positive Sense RNA Viruses and Genomic Material Prior to Removal from Containment.

Recommendations released by the CDC in 2023 address the need to demonstrate that the RNA genome of positive-strand RNA viruses is inactivated in addition to viral particles. This recommendation is in response to the similarities between host mRNA and the viral genome that allow the viral RNA to be used as a template by host replication mechanisms to produce infectious viruses; therefore, there is concern that through artificial introduction into host cells, active positive-strand RNA genomes can be utilized to produce infectious viruses out of a containment facility. Utilizing 10% formalin for 7 days or 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer (glut/PFA) for 2 days to fix eastern equine encephalitis virus (EEEV)-infected non-human primate (NHP) brain tissue was found to effectively inactivate EEEV particles and genomic RNA. The methods assessed in this paper outline an effective means to validate both genomic RNA and viral particle inactivation.

Long-term survival after local immunotherapy for malignant gliomas: a retrospective study with 20 years follow-up

PurposeImmunotherapy is a promising treatment for cancers but should be optimized for malignant gliomas. Because of immune privilege feature of the brain, local administration of immunotherapy may be a promising strategy for malignant glioma treatment. Identification of patients who may benefit from local immunotherapy is essential.MethodsWe retrospectively reviewed the clinicopathological characteristics and outcomes of six malignant glioma patients who received local administration of autologous cytokine-induced killer (CIK) cells through Ommaya reservoirs implanted into the tumor resection cavity. Profiles of tumor genome, transcriptome and immune microenvironment were also investigated by genomic target sequencing, RNA sequencing, electrochemiluminescence assay and immunohistochemistry (IHC) staining.ResultsFour patients died from tumor progression and the overall survival ranged from 10.0 to 33.9 months. Remarkably, two patients, including one diagnosed as diffuse hemispheric glioma H3 G34-mutant (G34-DHG, WHO grade 4) and the other diagnosed as astrocytoma (IDH1 mutation, WHO grade 3) survived more than 20 years without evidence of recurrence. The distinctive clinical feature of the two long-term survivors was tumor gross total resection (GTR) before CIK therapy. NTRK1 mutation was uniquely present and 353 genes were differentially expressed in the long-term survivors compared with the short-term survivors. These differential expression genes were highly associated with immune function. Electrochemiluminescence assay and IHC staining revealed higher expressions of cytokines and lower infiltrations of tumor-associated macrophages in the tumors of the long-term survivors.ConclusionThese findings suggest that certain patients diagnosed as malignant gliomas, including G34-DHG (WHO grade 4), can acquire long-term survival after local immunotherapy. Tumor GTR before local immunotherapy and relatively weaker immunosuppressive tumor microenvironment are the favorable factors for long-term survival. Larger, controlled studies with standardized treatment protocols, including consistent use of GTR, are warranted to further evaluate the potential benefits of locally delivered immunotherapy.

Transcription as a double-edged sword in genome maintenance.

Genome maintenance is essential for the integrity of the genetic blueprint, of which only a small fraction is transcribed in higher eukaryotes. DNA lesions occurring in the transcribed genome trigger transcription pausing and transcription-coupled DNA repair. There are two major transcription-coupled DNA repair pathways. The transcription-coupled nucleotide excision repair (TC-NER) pathway has been well studied for decades, while the transcription-coupled homologous recombination repair (TC-HR) pathway has recently gained attention. Importantly, recent studies have uncovered crucial roles of RNA transcripts in TC-HR, opening exciting directions for future research. Transcription also plays pivotal roles in regulating the stability of highly specialized genomic structures such as telomeres, centromeres, and fragile sites. Despite their positive function in genome maintenance, transcription and RNA transcripts can also be the sources of genomic instability, especially when colliding with DNA replication and forming unscheduled pathological RNA:DNA hybrids (R-loops), respectively. Pathological R-loops can result from transcriptional stress, which may be induced by transcription dysregulation. Future investigation into the interplay between transcription and DNA repair will reveal novel molecular bases for genome maintenance and transcriptional stress-associated genomic instability, providing therapeutic targets for human disease intervention.

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories.

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA.

Prolonged fecal shedding of replication-competent virus, lasting immune activation, and intestinal inflammation in a rhesus macaque after experimental SARS-CoV-2 infection.

Infection of an adult rhesus macaque with SARS-CoV-2 led to viral RNAemia in nose, throat, and lungs. The animal also presented extended fecal shedding of viral genomic and subgenomic messenger RNA and replication-competent virus for more than 3 weeks after infection. Positron emission tomography revealed increased intestinal glucose metabolism which was histologically related to inflammation of the ileum. These findings highlight the potential of the virus to cause gastrointestinal infections in macaques like this is also regularly observed in COVID-19 patients and substantiates the probability of virus transmission via the fecal-oral route. This study further adds the importance of nonhuman primates as a valuable animal model to study SARS-CoV-2 infection in humans.