Search Results (149)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense RNA virus, and its genome includes a highly conserved 5′ untranslated region (5′UTR). This region contains the so-called ‘leader sequence’, a crucial genomic region responsible for the viral replication and the synthesis of all subgenomic RNAs (sgRNAs). It has been demonstrated that targeting highly conserved genomic regions is essential for developing broad-spectrum antiviral therapies that resist viral mutation and evasion. Hypothesis: Given the high level of nucleotide homology between SARS-CoV and SARS-CoV-2, particularly in essential regions like the 5′UTR, the identification of a perfect sequence alignment across SARS-CoV-2 variants within this conserved region would provide a robust, mutation-resistant target for novel RNA-based drugs, such as small interfering RNAs (siRNAs) or microRNAs (miRNAs). Materials and Methods: Sequence alignment was performed across the different SARS-CoV-2 strains (i.e., the different variants that have appeared so far) to identify conserved genomic areas, leading to the selection of potential target sites for antiviral molecules. Specifically, computational analyses were utilized to map available binding sites for human miRNAs within the SARS-CoV-2 5′UTR. Results: Comparative alignments revealed that the leader sequence/5′UTR region is highly stable and conserved in all the considered SARS-CoV-2 sequences, representing a common therapeutic target across different variants and strains. Discussion: The perfect alignment observed in the 5′UTR confirms that this region is a highly critical target, less prone to mutations in all the considered variants. This property makes the region ideal for therapeutic intervention using non-coding RNAs. If endogenous miRNAs were found to bind this region (e.g., miR-638, miR-3150b-3p, etc.) and promote viral replication similarly to mechanisms observed in viruses like hepatitis C virus (HCV), their activity could be inhibited using chemically modified antisense analogs, such as locked nucleic acid (LNA) oligonucleotides. Full article

Whole-genome duplication (WGD) is a powerful evolutionary force, yet the mechanisms by which neopolyploids achieve transcriptomic stability and phenotypic success remain poorly understood. This study investigated the phenotypic and transcriptomic consequences of ploidy changes in the flatworm Macrostomum lignano, a “successful” neopolyploid model. We exploited two established sublines derived from the inbred DV1 line: the euploid DV1_8 (hidden tetraploid, SSL₁L₂) and the aneuploid DV1_10 (hidden hexaploid, SSL₁L₁L₂L₂). By integrating whole-genome sequencing (WGS)-informed normalization with RNA-seq analysis, we differentiated true regulatory shifts from gene-dosage effects. We revealed that while most genes scale linearly with ploidy, 1308 genes exhibited a nonlinear aneuploidy-induced transcriptional response. The remarkable trans-acting effects were observed across subgenome S encoded by disomic small chromosomes. Differentially expressed genes (DEGs) were enriched in pathways essential for homeostasis and growth: mTOR signaling, ubiquitin-mediated proteolysis, and the Hippo/Wnt pathways. Phenotypes of the DV1_10 worms exhibited increased body size, enhanced cell proliferation, and higher viability in comparison to the DV1_8 worms (60.25% vs. 21.5%). These findings suggest that M. lignano possesses mechanisms for dosage compensation to mitigate the deleterious effects of aneuploidy. Ultimately, this study demonstrates how genomic plasticity and rewiring of the transcriptome may facilitate the evolutionary success of animal neopolyploids. Full article

The concentration of extracellular vesicles (EVs) in the peripheral blood of COVID-19 patients is increased. Nevertheless, their potential role in the transmission of infection remains unclear. This study was performed to determine whether EVs produced by the sub-genomic replicon system developed in Baby Hamster Kidney (BHK-21) cells could transfer SARS-CoV-2 replicon RNA, leading to the establishment of a viral replication system in human cells. Purified EVs from the SARS-CoV-2 sub-genomic replicon cell line BHK-21 were cultured with a naive human cell line. The success of EV-mediated transfer of SARS-CoV-2 replicon RNA and its productive replication was assessed using G-418 selection, a luciferase assay, immunostaining, and Western blot. We found that the A549 cell line cultured with EVs isolated from SARS-CoV-2 BHK-21 replicon cells developed G-418-resistant cell colonies. SARS-COV-2 RNA replication in A549 cells was confirmed by nano luciferase, Nsp1 protein. SARS-CoV-2 RNA replication causes massive morphological changes. Treatment of cells with the FDA-approved Paxlovid demonstrated a dose-dependent inhibition of viral replication. We isolated two human epithelial cell lines (gastrointestinal and neuroblastoma) and one vascular endothelial cell line that stably support high-level replication of SARS-CoV-2 sub-genomic RNA. Viral elimination did not revert the abnormal cellular shape, vesicle accumulation, syncytia formation, or EV release. Our study’s findings highlight the potential implications of EV-mediated transfer of replicon RNA to permissive cells. The replicon model is a valuable tool for studying virus-induced reversible and irreversible cellular reprogramming, as well as for testing novel therapeutic strategies for SARS-CoV-2. Full article

Background/Objectives: Since its emergence, COVID-19—caused by the novel coronavirus SARS-CoV-2—has affected millions globally and led to over 1.2 million deaths in the United States alone. This global impact, coupled with the emergence of five new human coronaviruses over the past two decades, underscores the urgency of understanding its pathogenic mechanisms at the molecular level—not only for managing the current pandemic but also preparing for future outbreaks. Small non-coding RNAs (sncRNAs) critically regulate host and viral gene expression, including antiviral responses. Among the molecular regulators implicated in antiviral defense, the microRNA-processing enzyme Drosha has emerged as a particularly intriguing factor. In addition to its canonical role, Drosha also exerts a non-canonical, interferon-independent antiviral function against several RNA viruses. Methods: To investigate this, we employed q/RT-PCR, Western blot, and immunocytochemistry/immunofluorescence in an immortalized normal human lung/bronchial epithelial cell line (NuLi-1), as well as a human colorectal carcinoma Drosha CRISPR knockout cell line. Results: In this study, we observed a striking shift in Drosha isoform expression following infection with multiple SARS-CoV-2 variants. This shift was absent following treatment with the viral mimetic poly (I:C) or infection with other RNA viruses, including the non-severe coronaviruses HCoV-OC43 and HCoV-229E. We also identified a distinct alteration in Drosha’s cellular localization post SARS-CoV-2 infection. Moreover, Drosha ablation led to reduced expression of SARS-CoV-2 genomic and sub-genomic targets. Conclusions: Together, these observations not only elucidate a novel aspect of Drosha’s antiviral role but also advance our understanding of SARS-CoV-2 host–pathogen interactions, highlighting potential therapeutic avenues for future human coronavirus infections. Full article

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that enhances transcriptomic and proteomic diversity by generating multiple mRNA isoforms from a single gene. In plants, AS plays a central role in modulating growth, development, and stress responses. We summarize the prevalence and functional roles of AS in plant development and stress adaptation, highlighting mechanisms that link AS to hormone signaling, RNA surveillance, and epigenetic regulation. Polyploid crops, with their duplicated genomes, exhibit expanded AS complexity, contributing to phenotypic plasticity, stress tolerance, and adaptive evolution. Thus, this review synthesizes current knowledge on AS in plants, with a focus on three economically important polyploid crops—Brassica napus, Gossypium hirsutum, and Triticum aestivum. We also discuss how subgenome interactions shape diversity in polyploids and influence trait variation. Despite significant advances enabled by high-throughput sequencing, mechanistic studies that directly link specific AS events to phenotypic outcomes remain limited. Understanding how polyploidy reprograms AS and how isoform variation contributes to stress adaptation will be critical for harnessing AS in crop improvement. Full article

Ginseng rusty root symptoms (GRSs) compromise the yield and quality of Panax ginseng. While transcriptomic analyses have demonstrated extensive remodeling of stress signaling networks, the post-transcriptional defense circuitry remains obscure. We profiled alternative splicing (AS) in three phloem tissues, the healthy phloem (AG), the non-reddened phloem neighboring lesions (BG), and the reddened lesion core (CG), to delineate AS reprogramming during GRS progression. The frequency of AS was sharply elevated in CG, with intron retention predominating. Extensive gains and losses of splice events indicate large-scale rewiring of the splice network. Overlapping differentially alternative spliced genes (DAGs) identified in both CG vs AG and CG vs BG contrasts were significantly enriched for RNA–spliceosome assembly and stress–response pathways, revealing a conserved post-transcriptional response associated with lesion formation. Integrative analysis of differentially expressed genes uncovered 671 loci under dual regulation; functional classification categorized these genes in receptor-like kinase signaling and chromatin-remodeling modules, underscoring the synergy between AS and transcriptional control. Moreover, the B subgenome disproportionately contributed stress-responsive transcripts in diseased tissue, suggesting an adaptive, subgenome-biased strategy. These findings demonstrate that dynamic AS remodeling and subgenome expression bias jointly orchestrate ginseng defense against GRS and provide a framework for breeding disease-resilient crops. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, remains a major global health threat. The virus enters host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Several small-molecule antiviral drugs, including molnupiravir, favipiravir, remdesivir, and nirmatrelvir have been shown to inhibit SARS-CoV-2 replication and are approved for treating SARS-CoV-2 infections. Nirmatrelvir inhibits the viral main protease (M^pro), a key enzyme for processing polyproteins in viral replication. In contrast, molnupiravir, favipiravir, and remdesivir are prodrugs that target RNA-dependent RNA polymerase (RdRp), which is crucial for genome replication and subgenomic RNA production. However, undergoing extensive metabolism profoundly impacts their therapeutic effects. Carboxylesterases (CES) are a family of enzymes that play an essential role in the metabolism of many drugs, especially prodrugs that require activation through hydrolysis. Molnupiravir is activated by carboxylesterase-2 (CES2), while remdesivir is hydrolytically activated by CES1 but inhibits CES2. Nirmatrelvir and remdesivir are oxidized by the same cytochrome P450 (CYP) enzyme. Additionally, various transporters are involved in the uptake or efflux of these drugs and/or their metabolites. It is well established that drug-metabolizing enzymes and transporters are differentially expressed depending on the cell type, and these genes exhibit significant polymorphisms. In this review, we examine how CES-related cellular and genetic factors influence the therapeutic activities of these widely used COVID-19 medications. This article highlights implications for improving product design, targeted inhibition, and personalized medicine by exploring genetic variations and their impact on drug metabolism and efficacy. Full article

With the emergence of the SARS-CoV-2 pandemic the process of coronavirus replication has been under increasing scrutiny. During the replication of their genomic RNA, coronaviruses produce a range of other RNAs in addition to the negative-sense replicative intermediates of the genome, which includes a set of subgenomic RNAs. These subgenomic RNAs are nested within the sequence of the complete genome and can be both replicated further and act as templates for protein production. Alongside these functional products of discontinuous replication, coronaviruses produce defective viral genomes that can potentially impact both the virus and infected host cells. These interactions can arise from the ability of these defective viral genomes to impact the production of new infectious virions, through either competition with the wild-type genome for replication or by stimulating an antiviral response. Examining the behaviour of defective viral genomes can also help to elucidate the functional elements of the genome involved in the processes of replication and packaging. This review covers the process of intracellular replication by coronaviruses describing the mechanisms by which the different RNA species are produced. Of particular focus are factors involved in discontinuous replication that produces defective viral genomes, and the behaviour of coronavirus defective viral genomes. Full article

The possible origin of four cultivated species of the genus Avena of different ploidy and different subgenome composition (A. strigosa, A. abyssinica, A. byzantina, and A. sativa) from possible wild species was investigated. The region of the internal transcribed spacer ITS1 and the 5.8S rRNA gene in the cultivated species was studied with next-generation sequencing (NGS), and the patterns of occurrence and distribution of the ribotypes were compared among them and with those of the wild species. According to these data diploid, A. strigosa is more closely related to the diploid A. hirtula than to polyploid oats, and it could have evolved independently of polyploid cultivated species. The tetraploid Avena abyssinica could be a cultivated derivative of A. vaviloviana. Two hexaploid cultivated species, A. byzantina and A. sativa, could have a different origin; A. sativa could be the cultivated form of A. fatua, whereas A. byzantina could originate independently. It was found that the oat species with the A and C subgenomes, even with strong morphological and karyological differences, could intercross and pass the further stages of introgression producing a new stable combination of genomes. Our data show that almost all species of Avena could form an introgressive interspecies complex. Full article

Both hepatitis B virus (HBV), an hepadnavirus with a DNA genome, and hepatitis A virus (HAV), a picornavirus, require the TRAMP-like host ZCCHC14-TENT4 complex for efficient replication. However, whereas HBV requires the nucleotidyltransferase activity of TENT4 to extend and stabilize the 3′ poly(A) tails of mRNA transcribed from its genome, the role played by TENT4 in HAV replication is uncertain. HAV proteins are synthesized directly from its genomic RNA, which possesses a 3′ poly(A) tail, with its length and composition presumably maintained by 3D^pol-catalyzed RNA transcription during its replicative cycle. Using nanopore long-read sequencing of RNA from infected cells, we confirm here that the length of the HAV 3′ poly(A) tail is not altered by treating infected cells with RG7834, a small molecule TENT4 inhibitor with potent anti-HAV activity. Despite this, TENT4 catalytic activity is essential for HAV replication. Surprisingly, nanopore sequencing revealed a low abundance of HAV subgenomic RNAs (hsRNAs) that extend from the 5′ end of the genome to a site within the 5′ untranslated RNA (5′UTR) immediately downstream of a stem-loop to which the ZCCHC14-TENT4 complex is recruited. These hsRNAs are polyadenylated, and their abundance is sharply reduced by RG7834 treatment, implying they are likely products of TENT4. Similar subgenomic RNAs were not identified in poliovirus-infected cells. hsRNAs are present not only in HAV-infected cell culture but also in the liver of HAV-infected mice, where they represent 1–3% of all HAV transcripts, suggesting their physiological relevance. However, transfecting exogenous hsRNA into TENT4-depleted cells failed to rescue HAV replication, leaving the functional role of hsRNA unresolved. These findings reveal a novel picornaviral subgenomic RNA species while highlighting mechanistic differences in the manner in which HAV and HBV exploit the host ZCCHC4-TENT4 complex for their replication. Full article

The current clinical management of SARS-CoV-2 disease control and immunity may be not optimal anymore. Reverse transcription polymerase chain reaction (RT-PCR) of genomic viral RNA is broadly used for diagnosis, even though the virus may still be detectable when it is already non-infectious. Regarding serology, commercial assays mostly still rely on ancestral spike detection despite significant changes in the genetic sequence of the current circulating variants. We followed a group of 105 non-vaccinated individuals, measuring their viral shedding until negativity and antibody response up to six months. The mean viral detection period until a negative RT-PCR result was 2.2 weeks when using subgenomic RNA-E as a detection target, and 5.2 weeks when using genomic RNA as a detection target. Our neutralising antibody results suggest that, when challenged against a variant different from the variant of first exposure, commercial immunoassays are suboptimal at predicting the neutralising capacity of sera. Additionally, anti-Alpha and anti-Delta antibodies showed very low cross-reactivity between variants. This study provides insights into viral shedding and immune response in pre-Omicron variants like Alpha and Delta, which have been understudied in the published literature. These conclusions point to potential improvements in the clinical management of SARS-CoV-2 cases in order to organise vaccination campaigns and select monoclonal antibody treatments. Full article

Feline infectious peritonitis (FIP) is a complex immune-mediated disease caused by feline coronavirus (FCoV). Despite advancements in understanding its pathogenesis, challenges persist in elucidating viral factors related to virion composition and replication. This study examined FCoV-infected cats with and without FIP for potential associations with or variation in expression ratios for different viral genes. We analyzed tissue samples with FIP lesions from 46 cats with FIP and mesenteric lymph nodes from 10 FCoV-infected cats (7b RT-qPCR positive) without FIP with three RT-qPCR assays, targeting (sub)-genomic RNAs of the RNA-dependent RNA polymerase (RdRp) and envelope (E) genes. In cats with FIP, the RdRp mRNA assay yielded the highest copy numbers, followed by the combined RdRp gRNA and mRNA assay; the E mRNA assay yielded the lowest copy numbers. In cats without FIP, significantly fewer viral RNA copies were detected regardless of the assay. Viral gene expression was not detected in six and was observed only at low levels in one or more assays in four samples. The observed correlation between assays and the intragroup correlation assay indicate consistent transcription of both the structural E protein and RdRp genes within FIP lesions in cats with FIP but not in cats with systemic FCoV infection alone. Full article

Subgenomic RNAs (sgRNAs) are potential markers of active SARS-CoV-2 replication, serving as templates for the synthesis of structural and accessory proteins in infectious viral particles. This study aimed to use RT-qPCR to quantify sgRNA and negative RNA intermediates, assessing viral replication in virus samples inactivated by β-propiolactone (βPL). Inactivated viruses subjected to five blind serial passages (BSs) were amplified by RT-qPCR using primers to target the envelope (ENV) and nucleoproteins (N1 and N2) of genomic genes, subgenomic envelope RNA (sgENV), and intermediate envelope RNA (ENV-). All positive controls showed consistent viral titers across passages (10 log10 copies/mL in N1/N2 and 11 log10 copies/mL in ENV) during BSs. Inactivated viral samples for ENV and ENV- targets ranged from 11.34 log10 copies/mL in BS1 to 11.20 log10 copies/mL in BS5. The sgENV was no longer detected in the inactivated SARS-CoV-2 samples after the second passage, suggesting successful inactivation. Replication kinetics showed consistent profiles for N1/N2, ENV, and ENV- targets in the first three post-infection hours (pih) and maintained approximately 5 log10 copies/mL at 1 pih, 2 pih, and 3 pih. A sharp exponential increase in the viral titer was observed from 24 pih onwards, peaking at 11.64 log10 copies/mL at 48 pih. Transmission electron microscopy confirmed viral particles only in cells infected with active SARS-CoV-2. These results support the use of sgRNA as a reliable marker for SARS-CoV-2 replication, especially in distinguishing between active replication and non-viable particles and in the development of diagnostic and therapeutic strategies. Full article

The nucleocapsid (N) protein is the most expressed protein in later stages of SARS-CoV-2 infection with several important functions. It is translated from a subgenomic mRNA (sgmRNA) formed by template switching during transcription. A recently described translation initiation site (TIS) with a CTG codon in the leader sequence (TIS-L) is out of frame with most structural and accessory genes including the N gene and may act as a translation suppressor. We analyzed multiple sequenced samples infected by SARS-CoV-2 and found that any single variant of this virus produces multiple isoforms of the N sgmRNA. The main isoform starting at TIS-L is out of frame, but two secondary dominant isoforms (present in nearly all samples) were found to restore the reading frame and likely involved in the regulation of N protein production. Analysis of sequenced samples infected by other coronaviruses revealed that such isoforms are also produced in their transcriptomes. In SARS-CoV, they restore the reading frame for a putative TIS (also a CTG codon) in the same relative position as in SARS-CoV-2. Positions of junction breakpoints relative to stem loop 3 in the 5′-UTR suggest similar mechanisms in SARS-CoV, SARS-CoV-2, and OC43, but not in MERS-CoV. These observations may be pertinent for antisense-based antiviral strategies. Full article

Chikungunya virus (CHIKV) is an emerging, mosquito-borne arthritic alphavirus increasingly associated with severe neurological sequelae and long-term morbidity. However, there is limited understanding of the crucial host components involved in CHIKV replicase assembly complex formation, and thus virus replication and virulence-determining factors, within the central nervous system (CNS). Furthermore, the majority of CHIKV CNS studies focus on neuronal infection, even though astrocytes represent the main cerebral target. Heterogeneous ribonucleoprotein K (hnRNP K), an RNA-binding protein involved in RNA splicing, trafficking, and translation, is a regulatory component of alphavirus replicase assembly complexes, but has yet to be thoroughly studied in the context of CHIKV infection. We identified the hnRNP K CHIKV viral RNA (vRNA) binding site via sequence alignment and performed site-directed mutagenesis to generate a mutant, ΔhnRNPK-BS1, with disrupted hnRNPK–vRNA binding, as verified through RNA coimmunoprecipitation and RT-qPCR. CHIKV ΔhnRNPK-BS1 demonstrated hampered replication in both NSC-34 neuronal and C8-D1A astrocytic cultures. In astrocytes, disruption of the hnRNPK–vRNA interaction curtailed viral RNA transcription and shut down subgenomic RNA translation. Our study demonstrates that hnRNP K serves as a crucial RNA-binding host factor that regulates CHIKV replication through the modulation of subgenomic RNA translation. Full article