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PubMed1979

A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection.

The longest DNA molecules synthesized by endogenous reverse transcription in detergent-permeabilized Moloney murine sarcoma virus (Mo-MSV) virions (clone G8-124) are double-stranded DNA molecules of 5,8 kilobase pairs (kbp). This DNA species has been purified by sedimentation of total in vitro synthesized Mo-MSV DNA through neutral sucrose gradients. A physical map of the positions of the cleavage sites for a series of restriction endonucleases has been derived for this 5.8 kbp DNA. Mo-MSV DNA synthesized in vitro was found to induce morphological transformation of NIH-3T3 mouse fibroblasts upon transfection. The foci had a morphology indistinguishable from that of Mo-MSV-induced foci, and the induced transformed phenotype was stable. The 5.8 kbp double-stranded DNA (dsDNA) purified by agarose gel electrophoresis also induced focal transformation. Furthermore, gel-purified, restriction endonuclease-generated fragments of 5.8 kbp dsDNA containing the region from 2.8--4.9 kbp on the physical map of Mo-MSV DNA were able to induce foci. In contrast, endonuclease-generated DNA fragments lacking this region on the map were unable to transform cells upon transfection. When transformants derived by transfection with 5.8 kbp dsDNA were infected with Moloney murine leukemia virus (Mo-MLV) helper virus, Mo-MSV was rescued from a small portion of these cells, suggesting the establishment of the complete viral genome in these cells. One Mo-MSV DNA fragment, spanning 2.8--4.9 kbp on the physical map, was generated by cleavage of 5.8 kbp DNA with endonucleases Hind III + Sal I and currently represents our maximum estimate for the size of the transforming region of the Mo-MSV genome. This fragment includes the Mo-MSV sequences which are found in the DNA of uninfected mouse cells.

Base Sequence↗
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↗
PubMed1975

Poliovirus-induced infectious double-stranded RNA: Effect of RNA-degrading enzymes.

The infectivity of replicative form RNA (RF-RNA) isolated from poliovirus-infected HeLa cells is completely resistant to the action of T-1 RNase but decreases after exposure to RNase A in the presence of 0.3 M NaCl. Under these conditions neither enzyme produces single-stranded nicks in RF-RNA. Three endonuclease-free exonuleases (RNase II, polynucleotide phosphorylase and spleen phosphodiesterase) rapidly destroy the infectivity of single-stranded RNA, but do not alter the infectivity of RF-RNA. It is concluded that RF-RNA does not contain single-stranded ends essential for infectivity. Indirect evidence suggests that all or most of the poly A region at the 3' end of the plus strand of infectious RF-RNA is base-paired to a poly U region at the 5 end of the minus strand.

Base Sequence↗
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↗
PubMed1977

Mouse mammary tumor virus DNA in infected rat cells: characterization of unintegrated forms.

Mouse mammary tumor virus (MMTV) DNA in chronically infected rat hepatoma cells is maintained in both the integrated and unintegrated state. Fractionation of DNA by the procedure of Hirt (1967) as well as by sedimentation through alkaline sucrose suggests that about two thirds of the viral DNA is associated with high molecular weight cell DNA. The remainder of the viral DNA is unintegrated and is present primarily as linear or open circular duplexes consisting of a genome-length strand complementary to the viral RNA ("minus" strand) and "plus" strands of subgenomic length. Approximately 20% of the unintegrated MMTV DNA is present as double-stranded, covently closed circles (form I) with a molecular weight of 6 X 10(6) daltons. Form I viral DNA is found primarily in the nucleus, whereas the open forms are both nuclear and cytoplasmic.

Cell Line↗
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↗
PubMed1978

Antibodies to synthetic polyribonucleotides in spouses of patients with inflammatory bowel disease.

Serum from patients with inflammatory bowel disease (I.B.D.) and their unaffected spouses bound significantly more synthetic single-stranded (poly.rA) and double-stranded (poly.rA.poly.rU) R.N.A. but not KB cell D.N.A. than did serum from age-matched controls and their spouses. Binding activity resided in Ig fractions and was predominantly of IgM class. A significant corelation was observed between the amount of double-stranded but not single-stranded R.N.A. bound by serum from patients and from their respective spouses. These findings provide indirect support for the presence of R.N.A. viruses in patients with I.B.D. and the transmission of such agents to their close personal contacts.

Antibodies, Viral↗
PubMed1977

Isolation and characterization of DNA-DNA and DNA-RNA.

A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.

Cell Line↗
PubMed1978

Effect of a polynucleotide interferon inducer of fungal origin on experimental rhinovirus infection in humans.

A double-stranded RNA of fungal origin (BRL 5907) was given intranasally to volunteers. Apart from mild local irritancy of the higher dosage, the compound was well tolerated. A double-blind placebo-controlled trial of a three-day course (5 mg per day) of BRL 5907 against challenge with rhinovirus type 4 showed that treatment was associated with a delay in onset of symptoms and a reduction in shedding of virus, but the differences were not statistically significant. Low titers of interferon were found in nasal washings.

Adolescent↗
PubMed1979

New procedure for the production of influenza virus-specific double-stranded DNA's.

A novel technique is described for the production of pure, full-length influenza virus ds DNA's corresponding to each segment of the influenza virus genome, and suitable for molecular cloning and restriction endonuclease mapping. The method involves the synthesis of DNA complementary to both virion (negative strand) and messenger (positive strand) RNA, gel purification and annealing. By avoiding the use of SI nuclease, which often removes the terminal regions of DNA duplexes, the method allows transcription of the total sequence information of influenza virion and messenger RNA's into a ds DNA form.

Avian Myeloblastosis Virus↗
PubMed1978

Inverted complementary terminal sequences in single-stranded RNAs and snap-back RNAs from vesicular stomatitis defective interfering particles.

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

Base Sequence↗
PubMed1975

Infectious viral DNA in Rous sarcoma virus-transformed nonproducer and producer animal cells.

Nonproducer and producer RSV-transformed cells and producer nontransforming virus-infected cells harbor viral DNA specifying the respective avian tumor virus. In nonproducer Rous sarcoma cells, the residing viral DNA is linear, double-stranded and covalently linked to the chromosomal DNA. Both double-stranded and single-stranded forms of RSV DNA transfect chicken cells. The progeny virus is indistinguishable from the DNA parent with respect to the morphological, biological and antigenic properties. Unlike the DNA extracted from nonproducer RSV-transformed mammalian cells, that extracted from producer RSV-transformed chicken cells gives rise, in transfection experiments, to both sarcoma virus and its nontransforming derivative. The DNA from nontransforming virus-infected chicken cells generates only nontransforming viruses. The frequency of nontransforming virus recovery is different from that of sarcoma virus recovery, the latter being a nonlinear function of the amount of transfecting DNA, while the former may suggest a linear relationship. On the other hand, the end-point dilution of transfecting DNA for sarcoma virus recovery is approximately the same as that for nontransforming virus recovery. The following is assumed: Sarcoma viruses and nontransforming derivatives are recovered from two different species of viral DNA which carry or lack, respectively, the transforming genetic material. The size of double-stranded viral DNA is substantially smaller than 20 times 10(6) daltons. In transfection experiments, the sarcoma virus DNA is first integrated into the host cell genome before being expressed, while the nontransforming viral DNA apparently bypasses the integration step. The latter DNA generates the progeny virus when taken up, carried, and transcribed in a permissive cell.

Animals↗
PubMed1975

Reaction of poliovirus RNAs with antibodies to double-stranded RNA demonstrated by an immunochemical binding assay.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.

Animals↗
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↗
PubMed1977

Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.

Centrifugation, Density Gradient↗