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Viruses May 2024In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of... (Review)
Review
In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.
Topics: Reverse Genetics; Caliciviridae; Genome, Viral; Animals; Humans; Virus Replication
PubMed: 38932159
DOI: 10.3390/v16060866 -
Viruses May 2024The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon...
The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon gene (STING) is an essential component of the host defense mechanisms against respiratory viral infections. Although the role of the cGAS/STING signaling axis in the innate immune response to DNA viruses has been thoroughly characterized, mounting evidence shows that it also plays a key role in the prevention of RNA virus infections. In this study, we investigated the role of STING activation during Influenza virus (IFV) infection. In both mouse bone marrow-derived macrophages and monocytic cell line THP-1 differentiated with PMA, we found that dimeric amidobenzimidazole (diABZI), a STING agonist, had substantial anti-IFV activity against multiple strains of IFV, including A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. On the other hand, a pharmacological antagonist of STING (H-151) or the loss of STING in human macrophages leads to enhanced viral replication but suppressed IFN expression. Furthermore, diABZI was antiviral against IFV in primary air-liquid interface cultures of nasal epithelial cells. Our data suggest that STING agonists may serve as promising therapeutic antiviral agents to combat IFV.
Topics: Animals; Humans; Immunity, Innate; Mice; Antiviral Agents; Macrophages; Membrane Proteins; THP-1 Cells; Virus Replication; Influenza, Human; Dogs; Mice, Inbred C57BL; Orthomyxoviridae Infections; Orthomyxoviridae; Benzimidazoles; Signal Transduction
PubMed: 38932148
DOI: 10.3390/v16060855 -
Viruses May 2024Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing... (Review)
Review
Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.
Topics: Humans; Genome, Viral; Virus Replication; DNA, Viral; Host-Pathogen Interactions; DNA Viruses; Animals; Viral Proteins; Herpesviridae; Vaccinia virus
PubMed: 38932138
DOI: 10.3390/v16060845 -
Viruses May 2024Cytokinins (CKs) are a group of N-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates....
Cytokinins (CKs) are a group of N-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, -isopentenyladenine (iP), -isopentenyladenosine (iPR), -isopentenyladenosine-5'monophosphate (iPMP), and 2-methylthiol--isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations.
Topics: Virus Replication; Animals; Antiviral Agents; Ranavirus; Viral Plaque Assay; Cytokinins; Cell Line
PubMed: 38932119
DOI: 10.3390/v16060826 -
Pharmaceuticals (Basel, Switzerland) Jun 2024G-quadruplexes (G4s) are guanine-rich non-canonical secondary structures of nucleic acids that were identified in vitro almost half a century ago. Starting from the...
G-quadruplexes (G4s) are guanine-rich non-canonical secondary structures of nucleic acids that were identified in vitro almost half a century ago. Starting from the early 1980s, these structures were also observed in eukaryotic cells, first at the telomeric level and later in regulatory regions of cancer-related genes, in regulatory RNAs and within specific cell compartments such as lysosomes, mitochondria, and ribosomes. Because of the involvement of these structures in a large number of biological processes and in the pathogenesis of several diseases, including cancer, the interest in G4 targeting has exponentially increased in the last few years, and a great number of novel G4 ligands have been developed. Notably, G4 ligands represent a large family of heterogeneous molecules that can exert their functions by recognizing, binding, and stabilizing G4 structures in multiple ways. Regarding anti-cancer activity, the efficacy of G4 ligands was originally attributed to the capability of these molecules to inhibit the activity of telomerase, an enzyme that elongates telomeres and promotes endless replication in cancer cells. Thereafter, novel mechanisms through which G4 ligands exert their antitumoral activities have been defined, including the induction of DNA damage, control of gene expression, and regulation of metabolic pathways, among others. Here, we provided a perspective on the structure and function of G4 ligands with particular emphasis on their potential role as antitumoral agents. In particular, we critically examined the problems associated with the clinical translation of these molecules, trying to highlight the main aspects that should be taken into account during the phases of drug design and development. Indeed, taking advantage of the successes and failures, and the more recent technological progresses in the field, it would be possible to hypothesize the development of these molecules in the future that would represent a valid option for those cancers still missing effective therapies.
PubMed: 38931438
DOI: 10.3390/ph17060771 -
Molecules (Basel, Switzerland) Jun 2024Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome...
Hepatitis B virus (HBV) remains a global health threat. Ribonuclease H (RNase H), part of the virus polymerase protein, cleaves the pgRNA template during viral genome replication. Inhibition of RNase H activity prevents (+) DNA strand synthesis and results in the accumulation of non-functional genomes, terminating the viral replication cycle. RNase H, though promising, remains an under-explored drug target against HBV. We previously reported the identification of a series of -hydroxypyridinedione (HPD) imines that effectively inhibit the HBV RNase H. In our effort to further explore the HPD scaffold, we designed, synthesized, and evaluated 18 novel HPD oximes, as well as 4 structurally related minoxidil derivatives and 2 barbituric acid counterparts. The new analogs were docked on the RNase H active site and all proved able to coordinate the two Mg ions in the catalytic site. All of the new HPDs effectively inhibited the viral replication in cell assays exhibiting EC values in the low μM range (1.1-7.7 μM) with low cytotoxicity, resulting in selectivity indexes (SI) of up to 92, one of the highest reported to date among HBV RNase H inhibitors. Our findings expand the structure-activity relationships on the HPD scaffold, facilitating the development of even more potent anti-HBV agents.
Topics: Hepatitis B virus; Virus Replication; Antiviral Agents; Ribonuclease H; Humans; Structure-Activity Relationship; Molecular Docking Simulation; Catalytic Domain; Oximes; Molecular Structure; Hep G2 Cells; Enzyme Inhibitors
PubMed: 38931006
DOI: 10.3390/molecules29122942 -
Microorganisms May 2024In this study, we focused on evaluating the impact of BHJ04 on the growth of seedlings and its biocontrol efficacy against pine wilt disease (PWD). Additionally, the...
In this study, we focused on evaluating the impact of BHJ04 on the growth of seedlings and its biocontrol efficacy against pine wilt disease (PWD). Additionally, the colonization dynamics of BHJ04 on were examined. The growth promotion experiment showed that BHJ04 significantly promoted the growth of the branches and roots of . Pot control experiments indicated that strain BHJ04 significantly inhibited the spread of PWD. There were significant changes in the expression of several genes related to pine wood nematode defense in , including chitinase, nicotinamide synthetase, and triangular tetrapeptide-like superfamily protein isoform 9. Furthermore, our results revealed significant upregulation of genes associated with the water stress response (dehydration-responsive proteins), genetic material replication (DNA/RNA polymerase superfamily proteins), cell wall hydrolase, and detoxification (cytochrome P450 and cytochrome P450 monooxygenase superfamily genes) in the self-regulation of . Colonization experiments demonstrated that strain BHJ04 can colonize the roots, shoots, and leaves of , and the colonization amount on the leaves was the greatest, reaching 160,000 on the 15th day. However, colonization of the stems lasted longer, with the highest level of colonization observed after 45 d. This study provides a preliminary exploration of the growth-promoting and disease-preventing mechanisms of BHJ04 and its ability to colonize pines, thus providing a new biocontrol microbial resource for the biological control of plant diseases.
PubMed: 38930471
DOI: 10.3390/microorganisms12061089 -
Microorganisms May 2024Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this...
Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of 1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of 1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the Mip1 and its counterparts from and higher eukaryotes are discussed.
PubMed: 38930434
DOI: 10.3390/microorganisms12061052 -
Life (Basel, Switzerland) May 2024Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the... (Review)
Review
Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the segregation process of replicating chromosome arms (replichores) during the cell cycle. The first part of this review discusses light microscope observations demonstrating that the nucleoid has a lower refractive index and thus, a lower density than the cytoplasm. A polymer physics explanation for this phenomenon was given by a theory discussed at length in this review. By assuming a phase separation between the nucleoid and the cytoplasm and by imposing equal osmotic pressure and chemical potential between the two phases, a minimal energy situation is obtained, in which soluble proteins are depleted from the nucleoid, thus explaining its lower density. This theory is compared to recent views on DNA compaction that are based on the exclusion of polyribosomes from the nucleoid or on the transcriptional activity of the cell. These new views prompt the question of whether they can still explain the lower refractive index or density of the nucleoid. In the second part of this review, we discuss the question of how DNA segregation occurs in in the absence of the so-called active ParABS system, which is present in the majority of bacteria. How is the entanglement of nascent chromosome arms generated at the origin in the parental DNA network of the nucleoid prevented? Microscopic observations of the position of fluorescently-labeled genetic loci have indicated that the four nascent chromosome arms synthesized in the initial replication bubble segregate to opposite halves of the sister nucleoids. This implies that extensive intermingling of daughter strands does not occur. Based on the hypothesis that leading and lagging replichores synthesized in the replication bubble fold into microdomains that do not intermingle, a passive four-excluding-arms model for segregation is proposed. This model suggests that the key for segregation already exists in the structure of the replication bubble at the very start of DNA replication; it explains the different patterns of chromosome arms as well as the segregation distances between replicated loci, as experimentally observed.
PubMed: 38929644
DOI: 10.3390/life14060660 -
Children (Basel, Switzerland) Jun 2024A ~3-kb deletion-type DNA copy number variation (CNV, esv3587290) located at intron 7 of the gene (1p13.1, MIM*610132) has been proposed as a genetic factor in lupus...
Does the esv3587290 Copy Number Variation in the Gene Differ as a Genetic Factor for Developing Nephritis in Mexican Childhood-Onset Systemic Lupus Erythematosus Patients?
A ~3-kb deletion-type DNA copy number variation (CNV, esv3587290) located at intron 7 of the gene (1p13.1, MIM*610132) has been proposed as a genetic factor in lupus nephritis (LN) development in adult systemic lupus erythematosus (SLE) patients across European-descent populations, but its replication in other ethnicities has been inconsistent and its association with LN in childhood-onset SLE (cSLE) remains unknown. Here, we performed an exploratory association study in a sample of 66 unrelated cSLE Mexican patients (11 males, 55 females; ages 7.8 to 18.6 years). Two stratified groups were compared: cSLE patients with (N = 39) or without (N = 27) LN, as diagnosed by renal biopsy (N = 17), proteinuria (N = 33), urinary protein-creatinine ratio > 0.2 (N = 34), and erythrocyturia and/or granular casts in urinary sediment (N = 16). For esv3587290 CNV genotyping, we performed an end-point PCR assay with breakpoint confirmation using Sanger sequencing. We also determined the allelic frequencies of the esv3587290 CNV in 181 deidentified ethnically matched individuals (reference group). The obtained genotypes were tested for Hardy-Weinberg equilibrium using the χ test. Associations between LN and esv3587290 CNV were tested by calculating the odds ratio (OR) and using Pearson's χ tests, with a 95% confidence interval and ≤ 0.05. The esv3587290 CNV allele (OR 0.108, 95% CI 0.034-0.33, = 0.0003) and the heterozygous genotype (OR 0.04, 95% CI 0.119-0.9811, = 0.002) showed a significant protective effect against LN development. Finally, we characterized the precise breakpoint of the esv3587290 CNV to be NG_016548.1(NM_138959.3):c.1314+1339_1315-897del in our population. This report supports the notion that a broad genetic heterogeneity underlies the susceptibility for developing LN.
PubMed: 38929291
DOI: 10.3390/children11060712