Search Results (124)

Background: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing. Methods: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA–crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon. Results: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65–24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations. Conclusions: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes. Full article

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging. Full article

The genus Bacillus is widely recognized for its metabolic versatility, enabling it to colonize extreme environments, including sites contaminated with metals. In this study, we report the genome of B. velezensis strain HM1, isolated from sulfur-rich mine tailings from silver mining activities in southwestern Mexico. Isolation was performed by heat treatment followed by selective cultivation in a medium enriched with mine tailings extract (metals and sulfates), resulting in a single dominant morphotype corresponding to strain HM1. Whole-genome sequencing was carried out using the Illumina NovaSeq platform (2 × 250 bp). The assembled genome of strain HM1 has a size of 4,044,128 bp, distributed across 20 contigs, with an N50 of 700,388 bp and an L50 of 3, and an average coverage of 66.8×. The GC content was 46.31%, with an estimated completeness of 99.81% and contamination of 0.01%. Genome analyses indicate that the assembly corresponds to a single chromosome, with no evidence of plasmid replicons. Genome annotation identified 3950 coding sequences (CDSs), 83 tRNAs, 11 rRNAs, 26 ncRNAs, and 4 sORFs. Phylogenomic analysis, together with genomic similarity metrics (ANI > 98.6%, AAI > 98.8%, dDDH > 87%), confirms its classification as Bacillus velezensis. Functionally, the genome encodes multiple genes involved in resistance to metals and metalloids (including ABC transporters, efflux pumps, and biotransformation enzymes), as well as a complete pathway for sulfate assimilation. Collectively, these genomic features reveal a broad repertoire of adaptive strategies employed by strain HM1 to thrive in metal-contaminated environments. Full article

Viral internal ribosome entry sites (IRESs) are specialized RNA structures that facilitate cap-independent translation as a strategy to usurp the host translational machinery. The Type 6 IRESs are the most streamlined mechanism to date, as they adopt a three pseudoknot RNA structure to initiate factorless translation initiation by directly recruiting the ribosome and drive translation. The Halastavi árva virus (HalV) IRES represents the most minimalistic subclass identified to date, whereby the IRES lacks specific pseudoknot domains that bind to the 40S subunit but instead recruits pre-assembled 80S ribosomes via a mechanism that is not fully understood. Here, we examined cellular conditions that can support HalV IRES translation. We demonstrated that the HalV IRES is translationally active in insect Sf21 lysates and Drosophila S2 cells, but inactive in mammalian RRL and wheat germ extract. Cells treated with heat shock or serum starvation suppressed HalV IRES activity, whereas virus infection robustly enhanced HalV IRES-mediated translation. Finally, the HalV IRES can support viral translation and replication using a heterologous viral replicon. These findings highlight the context-specific cellular conditions that allow ribosome assembly and translation by a factorless minimalist IRES. Full article

Hepatitis C Virus (HCV) is a plus-strand RNA virus that replicates its genome via a minus-strand intermediate, which in turn is the template for the synthesis of progeny plus-strand genomes. In order to characterize sequence elements in the HCV 5′-untranslated region (5′UTR) that are possibly involved in the regulation of minus-strand RNA synthesis starting at the genome’s 3′end, we used a replicon system in which a possible function of these sequences is uncoupled from other functions like translation regulation. For the specific detection by RT-qPCR of minus strands newly synthesized in the cells from the transfected replicon RNAs, we carefully eliminated the contaminating DNA and transfected RNA and avoided self-priming caused by hairpin formation. We found that the absence of any HCV sequences at the 5′end does not allow genome replication. Stem-loop I-II sequences only allow extremely low-level replication, whereas the presence of stem-loops I-III or the complete 5′UTR allows efficient replication. The mutation of sequences required for the binding of translation initiation factor 3 (eIF3) and the ribosomal 40S subunit in the 5′UTR of the plus strand severely impairs minus-strand synthesis. This suggests that eIF3 and the 40S subunit are involved in plus-strand 5′-3′-end communication and the regulation of minus-strand synthesis. Full article

With the ongoing emergence of SARS-CoV-2 variants, continued surveillance of antiviral susceptibility remains critical for detecting resistance that could compromise treatment efficacy. This study evaluated the activity of 2 SARS-CoV-2 RNA-dependent RNA polymerase (Nsp12) inhibitors against emerging Omicron variants: remdesivir (RDV), an approved antiviral for the treatment of COVID-19, and obeldesivir (ODV), an oral prodrug that shares the same parent nucleoside as RDV. Both RDV and ODV were shown to retain antiviral activity against the Omicron subvariants BA.2.86.1, JN.1.7, KP.2, KP.3.1.1, KP.3.3, LP.8.1, NB.1.8.1, XBB.2, XEC, and XFG compared with wild-type reference strains. Only 1 new lineage-defining Nsp12 substitution, D284Y (detected in NB.1.8.1), was observed. Phenotypic analysis demonstrated that a replicon containing this substitution remained susceptible to both RDV and ODV. These findings are consistent with previous studies showing that RDV and ODV retain potent activity against previously identified Omicron variants, support the continued clinical use of RDV against circulating SARS-CoV-2 variants, and reinforce the potential of ODV as an oral antiviral therapeutic. 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

Enzymatic browning, driven by polyphenol oxidase (PPO), remains a major postharvest challenge for potato (Solanum tuberosum L.), reducing product quality, shelf life, and consumer acceptance. To mitigate this trait in the elite tetraploid cultivar ‘Yagana-INIA’, we applied a geminivirus-derived CRISPR–Cas9 system to edit the StPPO genes most highly expressed in tubers, StPPO1 and particularly StPPO2. A paired-gRNA strategy generated a double-cut deletion in StPPO1, while StPPO2 editing required a complementary single-gRNA screening workflow. High-resolution fragment analysis and sequencing identified three StPPO2-edited lines, including one that lacked GFP, Cas9, and Rep/RepA sequences, confirming a transgene-free editing outcome. Edited tubers exhibited visibly reduced browning relative to wild type, and biochemical assays showed decreased PPO activity consistent with targeted disruption of StPPO2. Amplicon sequencing verified monoallelic editing at the gRNA2 site in the non-transgenic line. These results demonstrate the utility of a replicon-based CRISPR system for achieving targeted, transgene-free edits in tetraploid potato and identify a non-GM StPPO2-edited line with improved postharvest quality under Chile’s regulatory framework. Full article

Alphaviruses pose a growing global health threat, with Chikungunya virus (CHIKV) epidemics ongoing. Although several CHIKV vaccine candidates have progressed to late-stage clinical evaluation, none have yet achieved licensure or widespread availability. The CHIKV nonstructural proteins nsP2 and nsP4 encode essential enzymatic activities that represent key targets for antiviral development, yet the biochemical basis of nsP4 RNA-dependent RNA polymerase (RdRp) activity remains poorly understood. Here, we identify a minimal, functional precursor form of nsP4 derived from the nsP3–nsP4 polyprotein (P34) that is active in a cell-based RNA replicon system. Using synthetic, capped mRNAs, we show that cleavage of P34 by the nsP2 protease is required for robust reporter expression, and that a truncated form retaining only the C-terminal 50 residues of nsP3 (CT50-P34) supports near-wild-type replication. Unexpectedly, ubiquitin–nsP4 fusions failed to substitute for P34, likely reflecting the transient expression supported by our RNA-based system. We propose that precursor forms of nsP4 interact with the nsP1 dodecamer at the site of genome replication, where cleavage activates the RdRp and localization within the nsP1 dodecamer maintains nsP4 in its active conformation. Dissociation from the nsP1 dodecamer triggers a conformational switch to an inactive state. Together, these findings establish a tractable framework for interrogation of the assembly, activation, and regulation of the alphavirus polymerase. Full article

Background/Objectives: Despite extensive research on hepatitis B virus (HBV), its post-transcriptional regulatory mechanisms remain incompletely characterized, particularly regarding epitranscriptomic modifications. This study aims to systematically profile the transcriptomic complexity and RNA modification landscape of HBV in hepatocellular carcinoma models. Methods: We transfected PLC/PRF/5 and Huh7 cells with the HBV 1.3-mer WT replicon plasmid, followed by qPCR measurement of viral load. Total nucleic acids extracted from transfected cells underwent nanopore direct RNA sequencing. The complete HBV transcriptome was then analyzed in two established hepatocellular carcinoma cell lines (PLC/PRF/5 and Huh7), with alternative splicing, polyadenylation, and RNA modifications identified through comprehensive bioinformatics analysis. Results: Our analysis revealed substantial transcriptomic diversity, identifying 34 distinct splice variants—including 14 previously unreported isoforms—with cell-type-specific expression patterns. Additionally, we detected 30 high-confidence RNA modification sites across HBV transcripts, with 93% (28 sites) conserved between both cellular environments. Notably, we observed significant intercellular heterogeneity in poly(A) tail length distributions. Conclusions: A comparison of the post-transcriptional processing modifications of HBV in PLC/PRF/5 and Huh7 cells reveals that the former may be better able to mimic the immune evasion mechanisms of chronic HBV infection. In contrast, the longer poly(A) tails present in Huh7 cells facilitate efficient replication, rendering these cells more amenable to the study of HBV transcription and replication mechanisms. These findings comprehensively elucidate the post-transcriptional regulatory mechanisms of hepatitis B virus in different hepatocellular carcinoma cell lines, establishing a critical benchmark for selecting appropriate experimental models in virology research. The identified transcriptomic features may provide new insights for developing antiviral strategies targeting the viral epigenome. Full article

Self-amplifying RNA (saRNA) is a promising platform for the production of vaccines, anti-tumor therapeutics, and gene therapy solutions. One of the advantages of the saRNA platform is the ability to use small doses of the therapeutic while maintaining prolonged expression of the target protein. However, the presence of auxiliary sequences encoding non-structural alphavirus proteins, which facilitate the replication of saRNA in cells, necessitates a thorough assessment of the biosafety of this platform. In our review, we focus on saRNA functions in the context of its interaction with the innate immune system. Firstly, an analysis is conducted of the side effects of candidate saRNA therapeutics, as observed in preclinical and clinical trials. Then, the mechanisms underlying the function of saRNA products derived from various alphavirus genomes in cell systems are discussed, as well as the reasons for their reactogenicity. The key approaches to optimizing the saRNA platform, which are aimed at reducing the activation of the innate immune response and cytopathic effects, are described. To summarize, this review enables us to systematize our knowledge on the advantages and disadvantages of saRNA, as well as potential approaches to improving this platform in order to develop more effective and safer therapeutics. Full article

Background/Objectives: A number of different vaccines against feline leukemia virus (FeLV) are available; however, there is continuous debate regarding the efficacy advantages of adjuvanted vaccines versus the potential safety advantages of non-adjuvanted vaccines. Methods: For this reason, we developed a non-adjuvanted vaccine based on a replicon RNA particle (RP) expressing the FeLV gp85 envelope protein, which possesses the safety of a non-adjuvanted vaccine while consistently providing high efficacy. Results: In two efficacy studies, a high-level of protection against virulent FeLV challenge was demonstrated with two doses given 3 weeks apart based on the prevention of FeLV p27 antigenemia. Furthermore, in both studies, we compared this novel vaccine against a non-adjuvanted, canarypox-vectored FeLV vaccine, demonstrating that none of the cats that received two doses of the RP-FeLV vaccine developed persistent antigenemia post-challenge. In comparison, of cats receiving the canarypox-vectored FeLV vaccine, three of seven (43%) became persistently antigenemic in one study, and three of ten (30%) became persistently antigenemic in the other study. In a field safety study using two commercial serials, safety of the RP-FeLV vaccine was demonstrated in over 800 cats receiving two doses of the vaccine. Conclusions: These data suggest that the RP-FeLV vaccine offers advantages over some current FeLV vaccines by combining the safety profile of a non-adjuvanted vaccine with the induction of a robust immune response demonstrated by some adjuvanted vaccines. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. The development of antiviral drugs for COVID-19 has been hampered by the requirement of a biosafety level 3 (BSL3) laboratory for experiments related to SARS-CoV-2, and by the lack of easy and precise methods for quantification of infection. Here, we developed a SARS-CoV-2 viral vector composed of all four SARS-CoV-2 structural proteins constitutively expressed in lentivirally transduced cells, combined with an RNA replicon deleted for SARS-CoV-2 structural protein genes S, M, and E, and expressing a luciferase–GFP fusion protein. We show that, after concentrating viral stocks by ultracentrifugation, the SARS-CoV-2 viral vector is able to infect two human cell lines expressing receptors ACE2 and TMPRSS2. Both luciferase activity and GFP fluorescence were detected, and transduction was remdesivir-sensitive. We also show that this vector is inhibited by three type I interferon (IFN-I) subtypes. Although improvements are needed to increase infectious titers, this vector system may prove useful for antiviral drug screening and SARS-CoV-2-related investigations. Full article

Viral replicons are efficient tools to understand the mechanisms of viral replication and screen antiviral drugs. In this study, a viral-cDNA-based replicon of Japanese encephalitis virus (JEV), which is the causative agent of Japanese encephalitis, was constructed by replacing the viral structural proteins with a green fluorescent protein (JEV-GFP replicon). The resulting JEV-GFP replicon was used as a tool to screen antiviral drugs targeting JEV nonstructural proteins, and the five compounds JNJ-A07, HZ-1157, NITD-2, quinine, and NITD008 were obtained, which significantly inhibited the replication of the JEV-GFP replicon and JEV in vitro, and the properties of these five compounds were also analyzed. The CC₅₀, EC₅₀, and SI indices of these five compounds were analyzed. In addition, the JEV-GFP replicon was used as a tool to identify the residues of viral nonstructural proteins involved in RNA replication, and the cysteine residue at position 4 of nonstructural protein 1 was found to be essential for JEV RNA replication. These data suggested that the noninfectious JEV-GFP replicon could be used as tool for different purposes, such as antiviral drug screening and gene function studies. Full article

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article