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PubMed1995

Stability of replicative form and fitness among RNA variants transcribed by Qbeta replicase.

Stability in the replicative form of RNA molecules transcribed by Qbeta replicase was demonstrated to provide a sequence-dependent indicator of their fitness. This follows from the finding that replication rates reported for 17 RNA species (genome length, 77-370 nucleotides) correlate with the self-interaction free energy of these self-annealed strands. Formation of double-stranded molecules during replication conversely decreased with self-interaction free energy. H-bond formation between self-complementary segments of folded RNA molecules plainly produces a potential energy barrier opposing the transition to a double-stranded, non-replicating form. Melting point temperature and resistance of RNA synthesis to elevated salt levels among three variants also increased with strand configuration free energy. Genome-based estimates of fitness in other self-replicating RNA species were therefore possible. Once a link between the kinetic parameters of replication and base sequence of these RNA species is adequately established, estimates of fitness can be dissociated from survival states following evolution, and Darwin's fundamental precept, 'survival of the fittest,' could be appraised as an experimentally testable hypothesis.

Allolevivirus↗

PubMed1988

Specificity of cleavage in replicative-form DNA of bovine herpesvirus 1.

The linear double-stranded DNA genome of herpesvirus as it is present in infectious virions needs to be circularized after infection of host cells and before DNA replication. Replicative-form genomes have to be cleaved into linear unit-length molecules during virion maturation and are most probably the substrate for inversion of the short segment relative to the long segment of the bovine herpesvirus 1 (BHV-1) genome. Those regions of the BHV-1 genome which are functionally involved in these processes have been analyzed at the molecular level by cloning and sequencing the genomic termini, the fusion of both termini from replicative-form molecules, and the junction between the short and the long genome segment. On the basis of the simple genome arrangement of BHV-1, it was inferable that the cleavage of replicative-form genomes by a hypothetical BHV-1 terminase activity may be specified by a sequence at the left end of UL (An element), which is located proximal to a reiterated beta element that makes up the cleavage site itself. The relationship of those elements in BHV-1 and the comparison to similar regions of other herpesviruses indicate consensus sequence elements which are functionally important for cleavage and isomerization of viral DNA during maturation of virions.

Animals↗

PubMed1982

DNA sequence of the 5' terminus containing the replication origin of parvovirus replicative form DNA.

The nucleotide sequence of the 5' terminus of the parvovirus H-1 was determined. There are two orientations of the 242-base-pair terminal palindrome in native replicative form DNA, one inverted with respect to the other. Adjacent to the terminal palindrome is an AT-rich region that is noncoding and contains a 55-base-pair tandem repeat. The addition mutant of H-1, DI-1, was also sequenced in this region and shown to have three copies of the tandem repeat sequence. Similarly, the related parvovirus H-3 contains only one copy of this repeat sequence. This region contains the replication origin for parvovirus replicative form DNA replication. Some of the implications of these results are discussed.

Base Sequence↗

PubMed1988

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.

Animals↗

PubMed1986

Novel dimeric configurations from bacteriophage G4 replicative form DNA.

The oligomeric fraction of the replicative form of phage G4 was prepared by sedimentation on three successive CsCl velocity gradients followed by resolution on CsCl-propidium diiodide equilibrium gradients and subfractions through the equilibrium gradients were examined by electron microscopy. The most frequent dimer species were the circular dimer, the singly linked catenane and the figure 8; these occurred in a ratio of 10:3:1. The high enrichment for dimers and other oligomers made possible the observation and the determination of the frequency of occurrence of a number of minor species, some of them of novel configuration. These are (a) dimers similar to figure 8s except containing long, apparently four-stranded junctions common to the two halves (theta forms); (2) dimers similar to those in (1) except that the long junctions separate the two halves (dumbbell forms); (3) multiply catenated dimers with apparent right-handed intertwines; and (4) dimers containing a knot. Theta forms cleaved by EcoRI were shown to be stable under conditions in which EcoRI-treated figure 8s were resolved by branch migration.

Coliphages↗

PubMed1972

Initiation of DNA synthesis: synthesis of phiX174 replicative form requires RNA synthesis resistant to rifampicin.

Conversion of single-stranded DNA of phage varphiX174 to the double-stranded replicative form in Escherichia coli uses enzymes essential for initiation and replication of the host chromosome. These enzymes can now be purified by the assay that this phage system provides. The varphiX174 conversion is distinct from that of M13. The reaction requires different host enzymes and is resistant to rifampicin and streptolydigin, inhibitors of RNA polymerase. However, RNA synthesis is essential for varphiX174 DNA synthesis: the reaction is inhibited by low concentrations of actinomycin D, all four ribonucleoside triphosphates are required, and an average of one phosphodiester bond links DNA to RNA in the isolated double-stranded circles. Thus, we presume that, as in the case of M13, synthesis of a short RNA chain primes the synthesis of a replicative form by DNA polymerase. Initiation of DNA synthesis by RNA priming is a mechanism of wide significance.

Anti-Bacterial Agents↗

PubMed1981

Involvement of host cell gene products in conversion of bacteriophage S13 single-stranded DNa to duplex replicative form DNA in vitro.

The single-stranded circular DNA of bacteriophage S13 was converted to the duplex replicative form DNA by soluble extracts from Escherichia coli strain H560 (polA, endA) in vitro. The maximal conversion required four deoxyribonucleoside triphosphates, Mg2+, exogenous S13 DNA and ATP, but not CTP, UTP, or GTP. The conversion was blocked by N-ethylmaleinimide but not by rifampicin. The product was identified as a gapped duplex replicative form DNA. Using extracts from some thermosensitive mutants of E. coli defective in DNA replication, we found that dnaB and dnaC gene products are involved in the conversion stage of single-stranded DNA to duplex DNA in vitro.

Coliphages↗

PubMed1972

Conversion of phiX174 and fd single-stranded DNA to replicative forms in extracts of Escherichia coli.

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.

Adenine Nucleotides↗

PubMed1981

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA.

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.

Bacteriophage phi X 174↗

PubMed1992

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR.

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Animals↗

PubMed1980

Replication of bacteriophage M13. XV. Location of the specific nick in M13 replicative form II accumulated in Escherichia coli polAex1.

M13 replicative form II (RFII) DNA was prepared from Escherichia coli RS5052 (polAex1) cells in the late stage of infection, and the DNA sequence at the discontinuity was examined. The data presented here suggest that the single discontinuity in the late stage of infection RFII maps at the same position as the gene II protein nicking site on fd RFI which was determined in vitro (Meyer et al., Nature (London) 278:365-367, 1979) and has a 5' terminal nucleotide sequence identical to that at the nick produced by gene II protein in vitro. The discontinuity in the in vivo RFII appears to be a single break in the phosphodiester backbone, leaving a 3' OH terminus. RFII molecules containing a gap, i.e., missing nucleotides at the site of discontinuity, were not detected.

Base Sequence↗

PubMed1986

The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas.

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis. A partial restriction map has been established. The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector. The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient. After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation. The cloned SpV4 RF DNA was demonstrated to be infectious by transfection.

Bacteriophages↗

PubMed1985

Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication.

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogeneous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.

Animals↗

PubMed2025

Dengue Virus Replicative-Form dsRNA Is Recognized by Both RIG-I and MDA5 to Activate Innate Immunity.

RIG-I like receptors (RLRs) are a family of cytosolic RNA sensors that sense RNA virus infection to activate innate immune response. It is generally believed that different RNA viruses are recognized by either RIG-I or MDA5, two important RLR members, depending on the nature of pathogen-associated molecular patterns (PAMPs) that are generated by RNA virus replication. Dengue virus (DENV) is an important RNA virus causing serious human diseases. Despite extensive investigations, the molecular basis of the DENV PAMP recognized by the host RLR has been poorly defined. Here, we demonstrated that the DENV infection-induced interferon response is dependent upon both RIG-I and MDA5, with RIG-I playing a predominant role. Next we purified the DENV PAMP RNA from the DENV-infected cells, and demonstrated that the purified DENV PAMP is viral full-length double-stranded RNA bearing 5'ppp modifications, likely representing the viral replicative-form RNA. Finally, we confirmed the nature of the DENV PAMP by reconstituting the viral replicative-form RNA from in vitro synthesized DENV genomic RNA. In conclusion, our work not only defined the molecular basis of the RLR-PAMP interaction during DENV infection, but also revealed the previously underappreciated recognition of a distinct moiety of the same PAMP by different RLRs in innate immunity against RNA viruses.

Interferon-Induced Helicase, IFIH1↗

PubMed1977

Electron microscopy analysis of the interaction between Escherichia coli DNA-dependent RNA polymerase and the replicative form of phage fd DNA. 1. Mapping of the binding sites.

The interaction of Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6) with the replicative form of the DNA from the filamentous coliphage fd cleaved by the restriction endonuclease HindII has been studied by electron microscopy at low and high ionic strength. In the presence of ATP or GTP, and heparin, RNA polymerase binds to fd replicative-form DNA at a few specific sites which have been mapped. The map was oriented so that transcription is from right to left. Three main GTP initiator sites are found at 15%, 82% and 94% of the genome length. One main ATP initiator site is found which cannot be mapped with the same accuracy, and which is localized between 38% and 50%. In the absence of initiator triphosphates and heparin, the binding of the enzyme to fd DNA is much more heterogeneous and therefore the mapping is more difficult. Nevertheless it seems that the preferential binding regions correspond to the specific sites mapped in the presence of GTP or ATP. The mean number of polymerase molecules bound to DNA as a function of the molecular ratio enzyme to DNA present in the mixture has been determined. From these results a binding isotherm can be obtained. The apparent equilibrium constant (K approximately 10(9) M-1) which is derived certainly represents an under-estimated value, as discussed.

Binding Sites↗

PubMed1977

Base-unpaired regions in supercoiled replicative form DNA of coliphage M13.

Superhelical covalently closed circular replicative form DNA (RF I) of coliphage M13 appears as a relaxed molecule that has a base-unpaired region in the form of a bubble (100 to 200 base pairs long) seen in electron micrographs when spread in the presence of formaldehyde and formamide or after pretreatment with glyoxal. S1 endonuclease, specific for single-stranded DNA, converts superhelical M13 RF I DNA, but not nonsuperhelical M13 RF I to a significant extent, into unit-length linear molecules by sequential nicking of two strands. The locations of S1 nuclease-susceptible sites and glyoxal-fixed base-unpaired regions were both related to the five A-T-rich regions in M13 RF DNA. While S1 nuclease does not show preference for any of these sites, glyoxal-fixed bubbles occur predominantly at the major A-T-rich region in M13 RF DNA.

Coliphages↗

PubMed1984

Porcine parvovirus DNA: characterization of the genomic and replicative form DNA of two virus isolates.

The genomic and replicative form (RF) DNA of porcine parvovirus (PPV) have been characterized. PPV isolate NADL-8 was found to have a 5000-base single-stranded genome, and a unique strand was encapsidated in virus particles. The RF DNA of isolate NADL-8 was found to be an infectious 5000-base pair (bp) molecule. Select restriction endonuclease sites were mapped along the RF DNA of PPV (NADL-8), and oriented with respect to the viral genomic DNA. The RF DNA of a second isolate of PPV, the less pathogenic, cell culture-adapted NADL-2 virus, was also analyzed. DNA preparations isolated from NADL-2 virus-infected cells contained two viral RF DNA species: a 5000-bp infectious molecule very similar if not identical to the NADL-8 virus RF DNA, and a noninfectious 4700-bp DNA molecule. The 4700-bp RF DNA molecule found in NADL-2 DNA preparations was similar in all respects to the 5000-bp RF DNA, except for a 300-bp deletion in the region encoding the viral capsid proteins. The presence of this defective variant in PPV (NADL-2) virus preparations is discussed in relation to the reduced pathogenic potential of the NADL-2 virus as compared to the NADL-8 virus.

Animals↗

PubMed1995

Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative form of viral DNA.

DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolation of plasmid DNA from Escherichia coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus.

Amino Acid Sequence↗