-
Emerging Microbes & Infections Dec 2023N-methyladenosine (mA) is one of the most abundant modifications of cellular RNA, where it serves various functions. mA methylation of many viral RNA species has also...
N-methyladenosine (mA) is one of the most abundant modifications of cellular RNA, where it serves various functions. mA methylation of many viral RNA species has also been described; however, little is known about the mA epitranscriptome of haemorrhagic fever-causing viruses like Ebola virus (EBOV). Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the mA methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of mA methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for mA that is conserved among diverse haemorrhagic fever-causing viruses (i.e. EBOV, JUNV and CCHFV), making METTL3 a promising target for broadly-acting antivirals.
Topics: Humans; Hemorrhagic Fever Virus, Crimean-Congo; Ebolavirus; Hemorrhagic Fever, Ebola; RNA, Viral; Dengue Virus; Nucleoproteins; Methyltransferases
PubMed: 37306620
DOI: 10.1080/22221751.2023.2223732 -
Viruses Jan 2024Henipaviruses are a genus of emerging pathogens that includes the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of disease. Henipaviruses... (Review)
Review
Henipaviruses are a genus of emerging pathogens that includes the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of disease. Henipaviruses rely on two surface glycoproteins, known as the attachment and fusion proteins, to facilitate entry into host cells. As new and divergent members of the genus have been discovered and structurally characterized, key differences and similarities have been noted. This review surveys the available structural information on glycoproteins, complementing this with information from related biophysical and structural studies of the broader family of which Henipaviruses are members. The process of viral entry is a primary focus for vaccine and drug development, and this review aims to identify critical knowledge gaps in our understanding of the mechanisms that drive fusion.
Topics: Humans; Henipavirus; Nipah Virus; Henipavirus Infections; Glycoproteins; Hendra Virus
PubMed: 38399971
DOI: 10.3390/v16020195 -
Nature Communications Oct 2023Arthritogenic alphaviruses are positive-strand RNA viruses that cause debilitating musculoskeletal diseases affecting millions worldwide. A recent discovery identified...
Arthritogenic alphaviruses are positive-strand RNA viruses that cause debilitating musculoskeletal diseases affecting millions worldwide. A recent discovery identified the four-and-a-half-LIM domain protein 1 splice variant A (FHL1A) as a crucial host factor interacting with the hypervariable domain (HVD) of chikungunya virus (CHIKV) nonstructural protein 3 (nsP3). Here, we show that acute and chronic chikungunya disease in humans correlates with elevated levels of FHL1. We generated FHL1 mice, which when infected with CHIKV or o'nyong-nyong virus (ONNV) displayed reduced arthritis and myositis, fewer immune infiltrates, and reduced proinflammatory cytokine/chemokine outputs, compared to infected wild-type (WT) mice. Interestingly, disease signs were comparable in FHL1 and WT mice infected with arthritogenic alphaviruses Ross River virus (RRV) or Mayaro virus (MAYV). This aligns with pull-down assay data, which showed the ability of CHIKV and ONNV nsP3 to interact with FHL1, while RRV and MAYV nsP3s did not. We engineered a CHIKV mutant unable to bind FHL1 (CHIKV-ΔFHL1), which was avirulent in vivo. Following inoculation with CHIKV-ΔFHL1, mice were protected from disease upon challenge with CHIKV and ONNV, and viraemia was significantly reduced in RRV- and MAYV-challenged mice. Targeting FHL1-binding as an approach to vaccine design could lead to breakthroughs in mitigating alphaviral disease.
Topics: Animals; Humans; Mice; Arthritis; Chikungunya Fever; Chikungunya virus; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Muscle Proteins; O'nyong-nyong Virus; Vaccines
PubMed: 37884534
DOI: 10.1038/s41467-023-42330-2 -
Viruses Sep 2023Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea... (Review)
Review
Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination.
Topics: Animals; Swine; Coronavirus; Reverse Genetics; Coronavirus Infections; Porcine epidemic diarrhea virus; RNA; Diarrhea; Swine Diseases
PubMed: 37896780
DOI: 10.3390/v15102003 -
Viruses Sep 2023Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the genus in the family. CSFV causes a severe... (Review)
Review
Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the genus in the family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.
Topics: Animals; Swine; Classical Swine Fever Virus; Pestivirus; Endocytosis; Classical Swine Fever
PubMed: 37766277
DOI: 10.3390/v15091870 -
The Journal of General Virology Sep 2023is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three...
is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three single-stranded RNA segments and encodes a nucleoprotein (NP), a glycoprotein (GP) and a large (L) protein containing RNA-directed RNA polymerase (RdRP) domains; some arenavirids encode a zinc-binding protein (Z). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family , which is available at www.ictv.global/report/arenaviridae.
Topics: Animals; Arenaviridae; Nucleoproteins; RNA; RNA-Dependent RNA Polymerase; Mammals
PubMed: 37698490
DOI: 10.1099/jgv.0.001891 -
PLoS Pathogens Sep 2023RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing...
RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing attention. Recent studies showed that the cGAS-STING pathway plays an important role in restricting RNA viruses via mitochondria DNA (mtDNA) mediated activation. However, the mechanisms of cGAS mediated innate immune evasion by RNA viruses remain unknown. Here, we report that seneca valley virus (SVV) protease 3C disrupts mtDNA mediated innate immune sensing by cleaving porcine cGAS (pcGAS) in a species-specific manner. Mechanistically, a W/Q motif within the N-terminal domain of pcGAS is a unique cleavage site recognized by SVV 3C. Three conserved catalytic residues of SVV 3C cooperatively contribute to the cleavage of pcGAS, but not human cGAS (hcGAS) or mouse cGAS (mcGAS). Additionally, upon SVV infection and poly(dA:dT) transfection, pcGAS and SVV 3C colocalizes in the cells. Furthermore, SVV 3C disrupts pcGAS-mediated DNA binding, cGAMP synthesis and interferon induction by specifically cleaving pcGAS. This work uncovers a novel mechanism by which the viral protease cleaves the DNA sensor cGAS to evade innate immune response, suggesting a new antiviral approach against picornaviruses.
Topics: Animals; Humans; Mice; DNA, Mitochondrial; Endopeptidases; Mitochondria; Peptide Hydrolases; Picornaviridae; Swine; Nucleotidyltransferases
PubMed: 37708231
DOI: 10.1371/journal.ppat.1011641 -
The Journal of Physiology Jun 2024Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These... (Review)
Review
Studies with RNA enzymes (ribozymes) and protein enzymes have identified certain structural elements that are present in some cellular mRNAs and viral RNAs. These elements do not share a primary structure and, thus, are not phylogenetically related. However, they have common (secondary/tertiary) structural folds that, according to some lines of evidence, may have an ancient and common origin. The term 'mRNA archaeology' has been coined to refer to the search for such structural/functional relics that may be informative of early evolutionary developments in the cellular and viral worlds and have lasted to the present day. Such identified RNA elements may have developed as biological signals with structural and functional relevance (as if they were buried objects with archaeological value), and coexist with the standard linear information of nucleic acid molecules that is translated into proteins. However, there is a key difference between the methods that extract information from either the primary structure of mRNA or the signals provided by secondary and tertiary structures. The former (sequence comparison and phylogenetic analysis) requires strict continuity of the material vehicle of information during evolution, whereas the archaeological method does not require such continuity. The tools of RNA archaeology (including the use of ribozymes and enzymes to investigate the reactivity of the RNA elements) establish links between the concepts of communication and language theories that have not been incorporated into knowledge of virology, as well as experimental studies on the search for functionally relevant RNA structures.
Topics: RNA, Viral; Evolution, Molecular; RNA Viruses; Humans; Animals; Archaeology
PubMed: 37818797
DOI: 10.1113/JP284416 -
EBioMedicine Jul 2023RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that...
BACKGROUND
RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice.
METHODS
We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model.
FINDINGS
We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model.
INTERPRETATION
Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.
FUNDING
Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.
Topics: Humans; Mice; Animals; CRISPR-Cas Systems; Dependovirus; COVID-19; Enterovirus; Enterovirus A, Human
PubMed: 37390772
DOI: 10.1016/j.ebiom.2023.104682 -
Nucleic Acids Research Sep 2023The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the...
The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.
Topics: Humans; Enterovirus; Genome, Viral; HeLa Cells; Nucleic Acid Conformation; Poliovirus; Protein Biosynthesis; RNA, Viral; RNA-Binding Proteins; Viral Proteins; Virus Replication
PubMed: 37486760
DOI: 10.1093/nar/gkad618