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PubMed1976

Homogenization-resistant and -susceptible components of tobacco mosaic virus replicative form RNA.

When prepared from tissue frozen with liquid nitrogen, tobacco mosaic virus replicative form RNA (TMV RF) was uniform in size but when prepared by high-speed homogenization, or when TMV RF prepared with liquid nitrogen was homogenized, 80 to 90% of the RF broke into relatively discrete pieces. The unbroken RF was not fragmented by additional homogenization. The TMV RF components susceptible and resistant to breakage, respectively, were synthesized with similar kinetics in relation to length of labelling period, but the slightly more resistant component was synthesized during the early infection period. Both components were produced by different strains of TMV but leaves infected with cowpea chlorotic mottle or southern bean mosaic viruses yielded only RF resistant to breakage. TMV replicative intermediate RNA was also broken by homogenization. The occurrence of the two RF components may be of significance in the replication of RNA viruses.

Nucleic Acid Denaturation↗
PubMed1989

A novel replicative form DNA of Aleutian disease virus: the covalently closed linear DNA of the parvoviruses.

The analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50% of the 5'-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3'-terminal hairpin. This finding together with the presence of nicks in the 3' palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated 'monomer covalently closed linear RF DNA' (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.

Aleutian Mink Disease Virus↗
PubMed1980

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.

Bacterial Proteins↗
PubMed1975

Degradation of the viral strand of phiX174 parental replicative-form DNA in a rep- host.

A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled phiX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 mug/ml, a concentration sufficient to block RF leads to SS (single strand) synthesis but not RF leads to RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150 mug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand.

Chloramphenicol↗
PubMed1977

Discontinuous replication of replicative form DNA from bacteriophage phiX174.

Bacteriophage phiX174 DNA has been labeled with short pulses of [3H]thymidine during synthesis of replicative form molecules in infected Escherichia coli HF4704 cells. The replicating phiX174 DNA was isolated and analyzed by sedimentation in an alkaline sucrose gradient. During a brief pulse (5 sec at 30 degrees), the radioactivity incorporated into the complementary strand was found in chains much shorter than one genome length. Of the radioactivity incorporated into the viral strand, two-thirds was in the short pieces and the rest was in chains of one genome length or longer. RNA attachment to the 5' end of both strand components of the nascent short pieces was shown by the appearance of spleen exonuclease-digestable nascent molecules after alkali treatment. These observations suggest that the viral as well as the complementary strand is synthesized by the discontinuous mechanism with RNA primers during replication of duplex phiX174 DNA.

Centrifugation, Density Gradient↗
PubMed1981

Induction of single strand scission in bacteriophage phi X174 replicative form I DNA by mitomycin C.

The action of mitomycin C on double-stranded replicative form I DNA (RF I DNA; supercoiled, covalently closed, circular duplex DNA) of bacteriophage phi X174 was investigated using the technique of agarose gel electrophoresis. Mitomycin C reduced with sodium hydrosulfite (sodium dithionite, Na2S2O4) caused single strand scission in phi X174 RF I DNA in the presence of Cu2+. Cu2+ was essential for this DNA cleave action, and other transition metal ions such as Fe2+, Fe3+, Mn2+, Co2+ and Zn2+ were of no effect. This DNA strand scission was inhibited by catalase (EC 1.11.1.6) and various radical scavengers. This DNA strand scission was caused by free oxygen radicals generated during autoxidation of reduced mitomycin C in the presence of Cu2+.

Bacteriophage phi X 174↗
PubMed1993

The monomer covalently closed linear replicative form DNA of Aleutian disease parvovirus is infectious after transfection into permissive cells.

The recently described monomer covalently closed linear replicative form DNA (Mccl RF DNA) of Aleutian disease parvovirus (ADV) is an infectious intermediate of the viral DNA replication cycle. Transfection of highly purified Mccl RF DNA into susceptible feline kidney cells (CCC clone 81 cells) resulted in viral DNA replication, expression of viral proteins and synthesis of infectious progeny virus. Mccl RF DNAs generated under permissive (32 degrees C) or non-permissive (37 degrees C) conditions were shown to be biologically indistinguishable. The accumulation of the Mccl RF DNA form at the non-permissive temperature in vitro strongly resembles that in bone marrow cells of naturally infected mink and may reflect one mechanism contributing to virus persistence of ADV in vivo.

Aleutian Mink Disease Virus↗
PubMed1988

Terminal sequences of the replicative form of RNA of the Japanese encephalitis virus.

The 5' and 3' terminal sequences of the replicative form (RF) of RNA of a flavivirus, the Japanese encephalitis (JE) virus, strain Ja0ArS982, have been determined by in vitro labelling and mobility shift analysis. The plus strand sequence was 5'AGAAGUUUAUCUGUGUGAA...UCUOH3', while the minus strand sequence was 5'AGAUCCUGUGUUCUUCCUCA...UCUOH3'. These sequences were similar to those of West Nile (WN) virus being identical in 12 nucleotides at the 5'terminal of the minus strand, and in the 5'terminal dinucleotides, 5'AG3'. Somewhat more internal hexanucleotides 5'CUGUGU3' are conserved among 3 flaviviruses, the JE, WN, and yellow fever viruses.

Base Sequence↗
PubMed1975

dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form.

The protein responsible for the initiation of conversion of single-stranded phage G4 DNA to the duplex replicative form has been purified approximately 3000-fold and identified with Escherichia coli dnaG gene product. The protein is a rifampicin-resistant RNA polymerase of approximately 64,000 daltons. It catalyzes the incorporation of the four ribonucleoside triphosphates into an oligoribonucleotide, using as template the single-stranded DNA coated with the DNA unwinding protein of E. coli. An RNA transcript of a unique region of the chromosome can serve as a primer by covalent extension by DNA polymerase III holoenzyme to form a nearly full-length linear complementary strand. A similar role for the dnaG protein in the initiation of nascent (Okazaki) fragments in replication of the host chromosome is discussed.

Bacterial Proteins↗
PubMed1977

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.

DNA Replication↗
PubMed1994

Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA.

The alphavirus mutant Sindbis virus HR ts4, which has been assigned to the A complementation group, possessed a selective defect in negative-strand synthesis that was similar although not identical to that observed for the B complementation group mutant ts11 (Y.-F. Wang, S. G. Sawicki, and D. L. Sawicki, J. Virol. 65:985-988, 1991). The causal mutation was identified as a change of a C to a U residue at nucleotide 4903 in the nsP3 open reading frame that predicted a change of Ala-268 to Val. Thus, both nsP3 and nsP1 play a role selectively in the transcription of negative strands early in infection. The assignment of the mutation carried by an A complementation group mutant of Sindbis virus HR to nsP3 was unexpected, as mutations in other A complementation group mutants studied to date mapped to nsP2. Another mutant with a conditionally lethal mutation, ts7 of the G complementation group, also possessed a causal mutation resulting from a single-residue change in nsP3. Negative-strand synthesis ceased more slowly after a shift to the nonpermissive temperature in ts7-than in ts4-infected cells, and ts7 complemented ts11, but ts4 did not. However, the nsP3 of both ts4 and ts7 allowed reactivation of negative-strand synthesis by stable replication complexes containing nsP4 from ts24. Therefore, mutations in nsP3 affected only early events in replication and probably prevent the formation and/or function of the initial replication complex that synthesizes its negative-strand template. Because neither ts4 nor ts7 complemented 10A complementation group mutants, the genes for nsP2 and nsP3 function initially as a single cistron. We interpret these findings and present a model to suggest that the initial alphavirus replication complex is formed from tightly associated nsP2 and nsP3, perhaps in the form of P23, and proteolytically processed and trans-active nsP4 and nsP1.

Alanine↗
PubMed2002

Frequent detection of the replicative form of TT virus DNA in peripheral blood mononuclear cells and bone marrow cells in cancer patients.

The TT virus (TTV), a member of a family of human viruses related to the circoviridae viruses, was associated initially with acute and chronic liver diseases. TTV consists of a single-stranded, circular DNA genome of 3.8 kilobases (kb) and at least three open reading frames (ORFs). The objective of the present study was to determine whether or not TTV replicated in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs). DNA was extracted from the PBMCs or BMCs of 153 cancer patients and from the PBMCs of 50 healthy blood donors (the controls). By using a single round of polymerase chain reaction (PCR), TTV was detected in 98.6% (141 of 143) of the PBMCs and in 90% (9 of 10) of the BMCs from cancer patients. TTV DNA was detected in significantly fewer control subjects at 86% (43 of 50; P < 0.05). Strand-specific PCR (SSPCR) targeting the ORF2 of the common genotypes of TTV was developed specifically to detect TTV positive or negative strand DNA and to examine TTV replication. TTV positive strand DNA, which may be an intermediate of viral replication, was detected in 55.3% (78 of 141) of the TTV-infected PBMCs of the cancer patients and in 7% (3 of 43) of the controls (P < 0.001). The replicative form of TTV was also detectable in 55.6% (5 of 9) of the TTV-infected BMCs. The existence of double-strand (positive and negative strands) TTV DNA in PBMCs and BMCs of the cancer patients was also supported by the finding that TTV DNA extracted from these cells was resistant to S1 nuclease. Using in situ hybridization, TTV DNA was also demonstrated to be present in the nucleus of PBMCs. It is concluded that replicative intermediate forms of TTV DNA are present in both PBMCs and BMCs, indicating that blood cells may be a site of TTV replication.

Bone Marrow Cells↗
PubMed1987

The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31.

The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix. Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome. A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31. The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C. They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure. Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state. Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable. Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.

Bacteriophage phi X 174↗
PubMed1993

Digitaria streak geminivirus replicative forms are abundant in S-phase nuclei of infected cells.

We have analyzed the replication of Digitaria streak geminivirus (DSV) using flow cytometry. Nuclei from infected plants were sorted on the basis of their DNA content in the different phases of the cell cycle and viral DNA forms in them were analyzed. DSV replicative forms were much more abundant (up to 20 times in one experiment) in S-phase nuclei than in G0-G1 and G2 nuclei, while single-stranded viral DNA did not show such dramatic increase during any phase. DSV replicative forms constitute almost 90% of total viral DNA forms in S-phase nuclei. DSV replication seems to be synchronized with host DNA replication and might be primarily under control of host factors becoming available only at the G1/S boundary.

Blotting, Southern↗
PubMed1984

DNA methylation inhibits the transfecting activity of replicative- form phi X174 DNA.

Replacement of virtually all the cytosine residues with 5-methylcytosine residues in the complementary strand of the replicative form (RF) of phi X174 DNA caused a 300- to 500-fold loss in its transfecting activity. Similar results were obtained with analogously methylated M13 RF. Transfection experiments with phi X RF hemimethylated in only part of the molecule, as assessed by analysis with restriction endonucleases, indicated that gene A of phi X, which needs to be nicked at a specific site by the gene A protein for RF replication, was not the main target for this inhibition by DNA methylation. We propose that the loss of transfecting activity was due to hemimethylation of the phi X RF interfering with the processively catalyzed movement of the replication fork.

5-Methylcytosine↗
PubMed1976

DNA synthesis in vitro dependent upon phiX174 replicative form I DNA.

Extracts of Escherichia coli strains infected with bacteriophage phiX174 catalyze DNA synthesis dependent on double-stranded, circular phiX174 replicative form I (phiX RFI) by a semiconservative process. The reaction required Mg++, ATP, all four dNTP, and exogenous phiX RFI DNA as template and yielded phiX RFI and phiX RFII. The reaction was inhibited by nalidixic acid and novobiocin but not by rifampicin. DNA synthesis required the phiX174 gene A product and E. coli gene products dnaB, dnaC(D), dnaG, and rep.

Bacterial Proteins↗