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PubMed2026

A noncontiguous code for RNA-guided DNA recognition at the origin of CRISPR-Cas.

CRISPR-Cas provides RNA-mediated adaptive immunity, but how its first RNA-guided effector arose is unclear. In this study, we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein ancestral to the earliest CRISPR-Cas effectors and VIPR RNAs (vrRNAs) comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that pair with target nucleic acids through contiguous complementarity, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NN dinucleotides collectively specify a gapped target sequence. Natural vrRNA targets suggest that VIPR systems act against competing phages, and we demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that adaptive immunity originated from ancient warfare between viruses, revealing a previously unidentified logic for encoding information in sequence.

CRISPR-Cas Systems↗

PubMed2026

Endosymbiotic theory of aging revisited: Age-related leakage of mitochondrial dsDNA/RNA stimulates cytosolic nucleic acid sensors which remodel the immune network and promote the aging process.

About 1.5-2 billion years ago, an endosymbiosis between aerobic α-proteobacteria and anaerobic archaeal cells generated mitochondria, i.e., organelles capable of producing oxidative energy. The bacterial genome was fundamentally reduced and a circular mitochondrial genome evolved containing mainly the genes coding for the subunits of the electron transport chain. Before the symbiotic event, there existed a virus-host co-evolution which involved the development of sensors for detecting dangerous viral DNA/RNA molecules. Endosymbiosis supplied eukaryotic cells not only with an oxidative powerhouse to allow the evolution of more complex multicellular organisms but it also meant that cells now housed an organelle which was able to generate reactive oxygen species (ROS) and to leak mitochondrial DNA (mtDNA) and double-stranded RNA (dsRNA) into the cytoplasm. There is now abundant evidence that during aging and age-related diseases mitochondria are prone to release both mtDNA and dsRNA. In the cytoplasm, mtDNA/dsRNA molecules activate a number of cytosolic nucleic acid sensors leading to the secretion of type-1 interferons (IFN) and many other cytokines which promote an age-related proinflammatory state. Currently, it is known that mtDNA can activate the cGAS-STING pathway, AIM2 inflammasomes, IFI16 receptors, and ZBP1 sensors and in addition mitochondrial dsRNA stimulates RIG-1/MDA5 signaling. Interestingly, there is abundant evidence that all these receptors are drivers of cellular senescence and inflammaging. For decades, there has been mounting evidence that mitochondria have a crucial role in the aging process. We will examine this question from the perspective of evolution and propose that mitochondrial evolution created an endogenic source for the leakage of dangerous mtDNA/dsRNA which subsequently stimulated cytosolic DNA/RNA sensors, an evolutionarily conserved viral defence mechanism. It seems that these two evolutionary events provided not only the basis for the inevitable process of aging but also ensuring the death of parental organisms.

Aging↗

PubMed2026

Differential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutants.

Recognition of double-stranded RNA (dsRNA) triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus mutants engage distinct RNA-sensing pathways and promote differential assembly of cytoplasmic RNP granules. Infection with splicing-defective ∆E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proximity labeling proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While ∆VA-induced granules were PKR-dependent, ∆E4 mutants induced RLB-like granules independently of PKR and RNase L. In cells lacking these sensors, granule assembly during ∆E4 infection coincided with translational arrest independent of eIF2α phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.

RNA, Double-Stranded↗

PubMed2003

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

Animals↗

PubMed2025

Impact of RNA extraction on respiratory microbiome analysis using third-generation sequencing.

BACKGROUND: The respiratory microbiome, which comprises bacteria, fungi, and viruses, plays a crucial role in respiratory health and disease. However, its study is limited by the low microbial biomass in respiratory samples and the dominance of host RNA. Metatranscriptomics offers comprehensive insights into active microbial communities and their interactions with the host but requires optimized RNA extraction protocols for robust and unbiased analysis. This study evaluated two RNA extraction kits—one employing chemical lysis (CL) and another combining chemical and mechanical lysis (CML)—to determine their effectiveness for metatranscriptomic analysis of respiratory samples. RESULTS: The CML protocol significantly increased double-stranded DNA (dsDNA) library yields, leading to higher sequencing read counts for both sample types (p < 0.0001). The read length was unaffected by the lysis protocol for the BAL and NPS samples. Taxonomic profiling revealed that CML enhanced the detection of robust microorganisms, such as gram-positive bacteria and fungi, without compromising viral detection. CONCLUSIONS: The CML protocol demonstrated superior recovery of genetic material, particularly for fungi and gram-positive bacteria, making it better suited for comprehensive metatranscriptomic analyses. These findings underscore the need for tailored RNA extraction strategies on the basis of sample type and research objectives. Optimized metatranscriptomic protocols are pivotal for advancing our understanding of the respiratory microbiome and its role in health and disease.

Microbiota↗

PubMed1975

Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H.

DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.

Centrifugation, Zonal↗

PubMed1976

Naturally-occurring double-stranded RNA and immune responses. IV. Influence of molecular size on antigenicity and adjuvant activity.

The immunological properties of a naturally-occurring double-stranded ribonucleic acid (ds-RNA), obtained from a mycophage of Penicillium chrysogenum, have been studied in relation to molecular size. Materials of reduced size, as reflected by molecular weight measurements, produced by ultrasonication of native ds-RNA, exhibited progressively lowered ability to induce an anti-ds-RNA response in mice. Adjuvant and immunosuppressive activities were of similar magnitude in both high and low molecular weight fractions. Evidence was also obtained of increased toxicity in materials of reduced size.

Adjuvants, Immunologic↗

PubMed1977

RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms.

The alpha, beta2, and alphabeta forms of the RNA-dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. 1. The Km values for dTTP and dGTP for enzyme forms alpha, beta2, and alphabeta were 77, 39, and 74, and 6.8, 3.1, and 6.1 micronM, respectively. 2. The affinity of 70 S Rous sarcoma virus RNA for enzyme form alphabeta was about twice that for the other two forms. 3. The relative specific activities of the three enzyme forms on synthetic primer-templates such as poly(rA)-poly(dT) were almost the same. The viral 70 S RNA-dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms alpha:beta2:alphabeta. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA-dependent activity of enzyme forms alphabeta and beta2 by a factor of 3, and that of enzyme form alpha by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. 4. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer-template were about 4,500 nucleotides long. About one-third of the total amount of polynucleotides polymerized by the alphabeta enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. 5. All three enzyme forms were capable of transcribing single-stranded into double-stranded DNA. 6. The 3-propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 microng/ml, inhibited enzyme forms beta2 and alphabeta by over 99.5 and 96%, respectively, but enzyme form alpha by only about 60%. 7. The beta2 and alphabeta forms of the enzyme were processive DNA polymerases, the alpha form a nonprocessive polymerase. 8. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the alpha form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the alphabeta enzyme form performs significantly better than either of the other two enzyme forms.

Avian Sarcoma Viruses↗

PubMed1976

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.

Avian Myeloblastosis Virus↗

PubMed2025

Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.

The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.

CRISPR-Cas Systems↗

PubMed2026

The halophilic archaeon Halogranum roseipondis sp. nov. is susceptible to a virus carrying an exceptionally high number of viral tRNA genes.

UNLABELLED: Archaea constitute a diverse group of organisms, many of which inhabit extreme environments, such as haloarchaea that dominate hypersaline ecosystems, like solar salterns. Sampling of solar salterns and other hypersaline environments has resulted in numerous haloarchaeal isolates, including 3 classified and 27 uncharacterized Halogranum species. However, no complete genome has so far been reported for any member of this genus. Here, we present the first comprehensive study of Halogranum sp. SS5-1 isolated from a solar saltern in Samut Sakhon, Thailand. Hgn. SS5-1 is a pleomorphic, aerobic heterotroph that thrives in high salinity and moderate temperature and is capable of hydrolyzing starch. Its genome consists of a 3.6 Mbp chromosome and seven additional plasmids. Based on our phylogenetic analyses, which establish Hgn. SS5-1 as a distinct species, we propose that it will be classified as the novel species Halogranum roseipondis sp. nov. SS5-1T. Additionally, we report that Hgn. roseipondis sp. nov. SS5-1T is infected by Hagravirus capitaneum (HGTV-1), the only virus known to infect a Halogranum host. HGTV-1 exhibits a unique head-tailed morphology and encodes the largest archaeal virus double-stranded DNA genome known to date, including 34 tRNA-encoding genes. Codon usage analysis of the viral genome suggests partial alignment with host preferences, yet the abundance of viral tRNA genes hints at broader roles, potentially including roles in translation and host regulation. This study establishes Hgn. roseipondis and HGTV-1 as a novel virus-host system, opening avenues to explore infection dynamics and the roles of virus-encoded tRNA in archaea. IMPORTANCE: Archaea that thrive in high-salinity environments are key players in geochemical cycles and important contributors to ecosystem productivity. Despite their ecological significance and importance for the development of novel methodologies in synthetic biology, haloarchaea remain poorly studied. Further exploration of haloarchaea is required to obtain valuable information on the evolution of cellular complexity and the molecular mechanisms that allow cells to thrive in harsh environmental conditions. Here, we present the characterization of a novel archaeon, Halogranum roseipondis sp. SS5-1T, alongside the infection cycle of its associated virus, Hagravirus capitaneum. This tailed myovirus carries an extraordinary set of 34 viral tRNA genes, a feature that opens intriguing questions about virus-host interactions and translational control. Our findings lay the groundwork for future investigations into the expression and function of viral tRNAs in an archaeal model system, thereby opening a new frontier for studying archaeal translation and virus-driven modulation of host cellular processes.

Halobacteriaceae↗

PubMed2026

Megamimivirus double-stranded DNA linear genomes flanked by highly diverse terminal inverted repeats.

UNLABELLED: Giant viruses have fundamentally expanded our understanding of virology by challenging the conventional boundaries of both virion size and genome complexity. However, the scarcity of isolates has left many of their unique biological features unexplored. Here, we report the isolation and characterization of four new giant virus species belonging to the subfamily Megamimivirinae, sampled from distinct environments across China. Among these, Megavirus daqingense is the first giant virus isolated from an oil reservoir; it exhibits virion stability under high salinity, chloroform exposure, and elevated temperatures, suggesting fitness adaptations to subsurface conditions. Using a hybrid sequencing approach that integrates short- and long-read technologies, we assembled complete linear genomes for all four isolates, each flanked by long terminal inverted repeats (TIRs). Comparative genomic and synteny analyses identified 29 distinct TIRs from 46 megamimivirus genomes. Gene content within these TIRs was highly diverse, with no orthologous proteins conserved across all repeats. Furthermore, TIR genes experienced weaker purifying selection than those in non-TIR regions (i.e., the genomic regions excluding the TIRs), consistent with their role as drivers of genome plasticity. Notably, we discovered for the first time that identical tRNA genes are shared between TIRs and non-TIR regions of eukaryotic viruses. Collectively, our work provides insights into the structural and evolutionary complexity of megamimiviruses, revealing TIRs as reservoirs of genetic diversity and hotspots for gene transfer, thereby playing a pivotal role in shaping the dynamic architecture of giant virus genomes. IMPORTANCE: Terminal inverted repeats (TIRs) are critical structural elements at the termini of linear genomes essential for fundamental processes such as recombination, replication, and integration across diverse organisms. However, the inherent limitations of short-read sequencing technologies have left the complete structure, diversity, and evolutionary significance of long TIRs in giant viruses unexplored. In this study, we leverage hybrid sequencing and comparative genomic analyses to unveil the complexity of TIRs across the subfamily Megamimivirinae. We demonstrate that TIRs are dynamic genomic hotspots characterized by remarkable gene diversity and unexpected conservation of specific tRNA genes. These findings establish TIRs as key drivers of genome plasticity, serving as hotspots for horizontal gene transfer and genetic innovation. By resolving the long-hidden terminal structures of megamimivirus genomes, this work provides a foundational framework for understanding how TIRs shape the evolution of giant viruses and, more broadly, advances our understanding of genome architecture in large DNA viruses.

Megavirus↗

PubMed2026

Molecular characterization of a novel partitivirus harboring an additional third dsRNA segment from Trichoderma harzianum.

We report the complete genome sequence of a novel partitivirus identified from Trichoderma harzianum NFCF092 strain, designated Trichoderma harzianum partitivirus 4 (ThPV4). Unlike canonical members of the family Partitiviridae, which possess a bipartite genome consisting of two double-stranded RNA (dsRNA) segments encoding an RNA-dependent RNA polymerase (RdRP) and a capsid protein (CP), ThPV4 harbors a third dsRNA segment encoding a protein of unknown function. The complete genome consists of dsRNA1 (1,950 bp; encoding the RdRP), dsRNA2 (1,772 bp; encoding the CP), and dsRNA3 (1,629 bp; encoding a protein with unknown function). Sequence analysis shows that each segment possesses a single open reading frame (ORF). The deduced amino acid sequence of the RdRP shows the highest similarity (90.5% identity) to that of Trichoderma gamsii alphapartitivirus 1. Phylogenetic analyses based on the RdRP indicate that ThPV4 clusters within the genus Alphapartitivirus of the family Partitiviridae. To our knowledge. ThPV4 is the first member of the genus Alphapartitivirus identified from T. harzianum to possess an additional, conserved third dsRNA segment.

Phylogeny↗

PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗

PubMed1976

Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA.

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.

Animals↗

PubMed1977

In vitro transcription of the avian oncornavirus genome by the RNA-directed DNA polymerase: effect of actinomycin D on the extent of transcription.

The minus-strand component of double-stranded DNA synthesized by the avian oncornavirus RNA-directed DNA polymerase in vitro represents as much of the nucleotide sequences of the viral genome as single-stranded DNA synthesized in the presence of actinomycin D. Since actinomycin D inhibits doublestranded DNA formation, we propose that the principal effect of the drug on RNA-directed DNA synthesis is the reduction in synthesis of plus-strand DNA, thereby allowing single-stranded minus-strand DNA to accumulate. This effect, rather than increasing the extent of genome transcription, is responsible for the more uniform nature of the DNA transcripts synthesized in the presence of actinomycin D.

Avian Sarcoma Viruses↗

PubMed1977

Structure of hepatitis B Dane particle DNA and nature of the endogenous DNA polymerase reaction.

The circular DNA of hepatitis B Dane particles, which serves as the primer/template for an endogenous DNA polymerase, was analyzed by electrophoresis before and after a polymerase reaction and after digestion by restriction endonuclease or single-strand-specific endonuclease S1. The unreacted molecules extracted from the particles were electrophoretically heterogeneous, and treatment with S1 nuclease produced double-stranded linear DNA ranging in length from 1,700 to 2,800 base pairs (bp). After an endogenous DNA polymerase reaction, two discrete species of DNA molecules were found: a circular form and a linear form 3,200 bp long. The reaction resulted in a population of molecules with an elongated and more homogeneous double-stranded region. These results suggest that the circular molecules in Dane particles have single-stranded regions of varying lengths that are made double stranded during the DNA polymerase reaction. The endogenous DNA polymerase was found to initiate apparently at random in a region spanning more than a third of the molecule. Analysis of restriction endonuclease cleavage fragments of the fully elongated DNA revealed that although the molecules were of a uniform length, they were somewhat heterogeneous in sequence. The sum of the sizes of the 10 major endonuclease Hae III-generated fragments, detected by ethidium bromide, was 3,880 bp. Two additional fragments (B and G) detected by autoradiography after an endogenous DNA polymerase reaction with (32)P-labeled deoxynucleoside triphosphates made the total 4,910 bp.

DNA Restriction Enzymes↗

PubMed2026

Quercetin, a flavonoid, suppresses viral proliferation by interfering with the ubiquitin transfer from E1 to E2 enzymes.

Quercetin is recognized for diverse pharmacological activities. However, the mechanism underlying its broad-antiviral effects has not been elucidated. Herein, we identified quercetin as a potent inhibitor of both double-stranded DNA virus Bombyx mori nucleopolyhedrovirus (BmNPV) and single-stranded RNA virus porcine reproductive and respiratory syndrome virus (PRRSV). Surface plasmon resonance (SPR) revealed that quercetin targets host ubiquitin-activating enzyme 1 (Uba1) homologs. Uba1 knockdown reduced viral proliferation and enhanced the antiviral effect of quercetin, whereas Uba1 overexpression functioned oppositely. Quercetin bound Uba1 homologs with high affinity. Notably, mutation of two binding residues, Q977 and G978, significantly disrupted the binding between BmUba1 and quercetin, and abolished quercetin's antiviral activity. Quercetin obstructed the transfer of ubiquitin from Uba1 to the E2 enzyme Ubc6, impairing the ubiquitination process. Similarly, quercetin inhibited PRRSV proliferation via targeting Uba1 in mammals. These findings elucidate the molecular mechanism underlying the pharmacological effects of quercetin, providing a theoretical basis for the development of novel antiviral agents against both DNA and RNA viruses.

Quercetin↗