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Molecular Cell Jun 2024The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral...
The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.
Topics: Nucleotidyltransferases; Humans; Membrane Proteins; Signal Transduction; Micronuclei, Chromosome-Defective; Nucleotides, Cyclic; Phosphorylation; Chromosome Segregation; DNA Replication; Protein Serine-Threonine Kinases; Interferon Regulatory Factor-3; Immunity, Innate; DNA Damage; HEK293 Cells; Animals; Radiation, Ionizing; HeLa Cells
PubMed: 38749421
DOI: 10.1016/j.molcel.2024.04.017 -
Emerging Microbes & Infections Dec 2024Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we...
Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral -elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.
Topics: Lassa virus; Reverse Genetics; Humans; Animals; Antiviral Agents; Chlorocebus aethiops; Cell Line; Virus Replication; Lassa Fever; Ribavirin; Vero Cells; Containment of Biohazards; Genome, Viral; Virion
PubMed: 38747061
DOI: 10.1080/22221751.2024.2356149 -
Research Square May 2024Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver miniaturized dystrophin (micro-dystrophin or µDys), which...
Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver miniaturized dystrophin (micro-dystrophin or µDys), which does not provide full protection for striated muscles as it lacks many important functional domains within full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We rationally split FL-dystrophin into three fragments (N, M, and C) linked to two orthogonal pairs of split intein, allowing efficient, unidirectional assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mice. Dystrophin-glycoprotein complex components are also restored in the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective ERK signaling in heart. The FL-dystrophin gene therapy therefore promises to offer superior protection for DMD.
PubMed: 38746161
DOI: 10.21203/rs.3.rs-3867299/v1 -
Methods in Molecular Biology (Clifton,... 2024Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid...
Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.
Topics: RNA, Viral; Morbillivirus; Animals; Blotting, Northern; Virus Replication; Polymerase Chain Reaction; RNA, Small Interfering; Genome, Viral; RNA, Double-Stranded; Humans
PubMed: 38743363
DOI: 10.1007/978-1-0716-3870-5_6 -
Methods in Molecular Biology (Clifton,... 2024RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource...
RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.
Topics: Reverse Genetics; Defective Viruses; Animals; Virus Replication; Genome, Viral; Morbillivirus; Humans; Virion; Vero Cells; Chlorocebus aethiops; RNA, Viral
PubMed: 38743362
DOI: 10.1007/978-1-0716-3870-5_5 -
PLoS Pathogens May 2024Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely...
Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/βR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.
Topics: Animals; Mice; Hepatocytes; Receptor-Interacting Protein Serine-Threonine Kinases; Cytokines; Hepatitis Delta Virus; Hepatitis D; Cell Death; Mice, Inbred C57BL; Apoptosis; Mice, Knockout; Humans; Tumor Necrosis Factor-alpha; Disease Models, Animal
PubMed: 38739648
DOI: 10.1371/journal.ppat.1011749 -
The Journal of Dermatological Treatment Dec 2024Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in , the gene encoding type VII collagen (COL7), is... (Review)
Review
BACKGROUND/PURPOSE
Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in , the gene encoding type VII collagen (COL7), is characterized by skin blistering, scarring, and extracutaneous manifestations that markedly reduce patient quality-of-life. Beremagene geperpavec-svdt ('B-VEC') is a gene therapy employing a non-integrating, replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding two copies of full-length human to restore COL7 protein after topical administration to DEB wounds. B-VEC was approved in the United States in 2023 as the first topical gene therapy and the first approved treatment for DEB. However, few providers have experience with use of this gene therapy.
METHODS
Data was obtained through literature review and the experience of providers who participated in the B-VEC clinical study or initiated treatment after B-VEC approval.
RESULTS
This review discusses the burden of disease, describes the clinical trial outcomes of B-VEC, and provides physician and patient/caregiver recommendations as a practical guide for the real-world use of B-VEC, which can be administered in-office or at the patient's home.
CONCLUSIONS
By continuing to optimize the practical aspects of B-VEC administration, the focus will continue to shift to patient-centric considerations and improved patient outcomes.
Topics: Humans; Genetic Therapy; Epidermolysis Bullosa Dystrophica; Collagen Type VII; Genetic Vectors; Herpesvirus 1, Human; Treatment Outcome; Quality of Life
PubMed: 38724041
DOI: 10.1080/09546634.2024.2350232 -
Frontiers in Immunology 2024There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two...
There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses , which is important for vaccine safety and to prevent adverse effects upon viral exposure.
Topics: Animals; Influenza Vaccines; Nanoparticles; Mice; Adjuvants, Immunologic; Mice, Inbred BALB C; Orthomyxoviridae Infections; Female; Lipids; Vaccination; Adjuvants, Vaccine; Antibodies, Viral; Disease Models, Animal; Sendai virus; Influenza, Human; Liposomes
PubMed: 38711520
DOI: 10.3389/fimmu.2024.1370564 -
Virus Research Jul 2024Cnaphalocrocis medinalis granulovirus (CnmeGV), belonging to Betabaculovirus cnamedinalis, can infect the rice pest, the rice leaf roller. In 1979, a CnmeGV isolate,... (Comparative Study)
Comparative Study
Cnaphalocrocis medinalis granulovirus (CnmeGV), belonging to Betabaculovirus cnamedinalis, can infect the rice pest, the rice leaf roller. In 1979, a CnmeGV isolate, CnmeGV-EP, was collected from Enping County, China. In 2014, we collected another CnmeGV isolate, CnmeGV-EPDH3, at the same location and obtained the complete virus genome sequence using Illumina and ONT sequencing technologies. By combining these two virus isolates, we updated the genome annotation of CnmeGV and conducted an in-depth analysis of its genome features. CnmeGV genome contains abundant tandem repeat sequences, and the repeating units in the homologous regions (hrs) exhibit overlapping and nested patterns. The genetic variations within EPDH3 population show the high stability of CnmeGV genome, and tandem repeats are the only region of high genetic variation in CnmeGV genome replication. Some defective viral genomes formed by recombination were found within the population. Comparison analysis of the two virus isolates collected from Enping showed that the proteins encoded by the CnmeGV-specific genes were less conserved relative to the baculovirus core genes. At the genomic level, there are a large number of SNPs and InDels between the two virus isolates, especially in and around the bro genes and hrs. Additionally, we discovered that CnmeGV acquired a segment of non-ORF sequence from its host, which does not provide any new proteins but rather serves as redundant genetic material integrated into the viral genome. Furthermore, we observed that the host's transposon piggyBac has inserted into some virus genes. Together, dsDNA viruses could acquire non-coding genetic material from their hosts to expand the size of their genomes. These findings provide new insights into the evolution of dsDNA viruses.
Topics: Genome, Viral; Animals; Genetic Variation; Phylogeny; China; Granulovirus; Whole Genome Sequencing; Oryza; Tandem Repeat Sequences; Plant Diseases; Recombination, Genetic
PubMed: 38710287
DOI: 10.1016/j.virusres.2024.199390 -
Homozygous variant in abolishing triokinase activities is associated with isolated immunodeficiency.Journal of Medical Genetics May 2024Triokinase and FMN cyclase (TKFC) is a bifunctional enzyme involved in fructose metabolism. Triokinase catalyses the phosphorylation of fructose-derived glyceraldehyde...
BACKGROUND
Triokinase and FMN cyclase (TKFC) is a bifunctional enzyme involved in fructose metabolism. Triokinase catalyses the phosphorylation of fructose-derived glyceraldehyde (GA) and exogenous dihydroxyacetone (DHA), while FMN cyclase generates cyclic FMN. TKFC regulates the antiviral immune response by interacting with IFIH1 (MDA5). Previously reported pathogenic variants in are associated with either a multisystemic disease or isolated hypotrichosis with loose anagen hairs.
METHODS
Whole-exome sequencing identified a homozygous novel variant in (c.1624G>A; p.Gly542Arg) in an individual with a complex primary immunodeficiency disorder. The variant was characterised using enzymatic assays and yeast studies of mutant recombinant proteins.
RESULTS
The individual presented with chronic active Epstein-Barr virus disease and multiple bacterial and viral infections. Clinical investigations revealed hypogammaglobulinaemia, near absent natural killer cells and decreased memory B cells. Enzymatic assays showed that this variant displayed defective DHA and GA kinase activity while maintaining FMN cyclase activity. An allogenic bone marrow transplantation corrected the patient's immunodeficiency.
CONCLUSION
Our report suggests that TKFC may have a role in the immunological system. The pathological features associated with this variant are possibly linked with DHA/GA kinase inactivation through a yet an unknown mechanism. This report thus adds a possible new pathway of immunometabolism to explore further.
PubMed: 38697782
DOI: 10.1136/jmg-2024-109853