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PubMed1989

Characterization of woodchuck hepatitis virus DNA and RNA in the hepatocellular carcinomas of woodchucks.

Integration and transcription of woodchuck hepatitis virus DNA were studied by Southern and Northern blot analysis in 26 hepatocellular carcinomas and in adjacent nontumor tissue of woodchucks (Marmota monax). All liver tissue chronically infected with woodchuck hepatitis virus contained various amounts of episomal and replicative forms of woodchuck hepatitis virus DNA: episomal and replicative forms of woodchuck hepatitis virus DNA without integration were found in six tumors, episomal and integrated woodchuck hepatitis virus DNA was observed in 18 tumors and exclusively integrated woodchuck hepatitis virus DNA was found in two tumors. In most tumors and in all of the liver tissues chronically infected with woodchuck hepatitis virus, two major woodchuck hepatitis virus RNA species (3.7 and 2.1 kilobases) were detected. In tumors of two other animals (HW76 and HW89) with integrated woodchuck hepatitis virus DNA, only single major transcripts of 3.5 and 2.5 kilobases, respectively, were detected. Hybridization with subcloned woodchuck hepatitis virus DNA probes showed that both aberrant transcripts lacked the C gene and a part of the pre-S1 gene; characterization of corresponding integrated woodchuck hepatitis virus DNA sequences revealed that the C gene was deleted in one tumor, although not in the other. In agreement with the nucleic acid data, we found expression of core protein by Western blotting only in chronically infected liver tissue of these animals, but not in the corresponding tumors. Deletion of the C gene in mRNA may be due to deletion of this gene in the integrated sequences or due to transcriptional regulation.

Animals↗

PubMed1977

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.

Cell-Free System↗

PubMed1969

Sindbis virus-induced viral ribonucleic acid polymerase.

A cytoplasmic structure containing the viral ribonucleic acid (RNA) polymerase has been isolated by sucrose density centrifugation from cells infected with Sindbis virus. Uninfected cells did not contain any such structure. Preliminary experiments indicated that the structure may be associated with membranes. This structure incorporated (3)H-guanosine triphosphate in vitro in the absence of added template. The RNA synthesized in vitro by the enzyme consisted of single-stranded 40S RNA, the ribonuclease-resistant replicative form, and possibly the replicative intermediate form of viral RNA. The products formed in vitro by the enzyme are identical in sedimentation rates to those formed in the infected cells in vivo.

Animals↗

PubMed1976

The role of Escherichia coli dna A gene and its integrative suppression in M13 coliphage DNA synthesis.

An F+ derivative of Escherichia coli E508 thermosensitive in dna A function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508 (ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also, but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dna A function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dna A mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dna A function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.

Coliphages↗

PubMed1986

Hepatitis B virus DNA in mononuclear cells and analysis of cell subsets for the presence of replicative intermediates of viral DNA.

To determine whether peripheral blood mononuclear cells (PBMCs) contain replicating forms of hepatitis B virus (HBV) DNA and to define which cell subset may be permissive for viral replication, we analyzed the PBMC DNA from 14 carriers positive for hepatitis B surface antigen (HBsAg) by Southern blot hybridization. HBV-related DNA, which was present exclusively in an extrachromosomal state, was found in the PBMCs of all five hepatitis B e antigen (HBeAg)-positive and three of nine HBeAg-negative carriers. Serum-associated HBV DNA was detected only in those HBsAg carriers whose PBMCs contained HBV DNA forms resembling replicative intermediates (1.0-3.2 kilobase pairs in the EcoRI digests). Analysis of PBMC subsets revealed that replicating forms of the HBV genome were present primarily in monocytes. Low levels of hybridization also were detected in B cells, whereas the T cell fraction (which contained natural killer cells) appeared to be devoid of these replicating forms.

DNA Replication↗

PubMed1990

Proteins of viral and cellular origin bind to the Aleutian disease virus (ADV) DNA 3'-terminal hairpin: presentation of a scheme for encapsidation of ADV DNA.

We have observed the binding of viral and cellular proteins to the Aleutian disease virus (ADV) 3' terminus of replicative-form DNA. Gel retardation assays showed specific band shifts produced by whole-cell extracts from either ADV-infected or uninfected cells, as well as band reduction produced by ADV capsids. In all cases, binding was confined to the turnaround, T-shaped terminal form; no binding to the extended conformation of replicative-form DNA was detected. This indicates the importance of the T-shaped secondary structure in protein recognition. We have previously reported the binding of a 3'-terminal ADV DNA restriction fragment to the ADV capsid protein VP1 (K. Willwand and O.-R. Kaaden, Virology 166:52-57, 1988). Here we show that the region between nucleotides 14 and 102 on the ADV genome is required for binding. It is suggested that the VP1-DNA interaction mediates the binding of ADV DNA to empty viral capsids and that this is followed by displacement synthesis and packaging of ADV progeny DNA. A scheme for the possible mechanism of this process is presented.

Aleutian Mink Disease Virus↗

PubMed1989

Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes.

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1978

Growth of single-stranded DNA phages in replication mutants of Escherichia coli.

Host capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnam709 and dnaS707 mutants, multiplication of phiK was not restricted even at 42 degree C. In dnaM710 cells, however, growth of phiK was severely effected at 42 degree C but not at 33 degree C. Upon infection of phiK, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of phiX174 and alpha3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with lambda, microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.

Coliphages↗

PubMed1974

Inhibition of bacteriophage phi chi 174 DNA replication in dnaB mutants of Escherichia coli C.

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.

Centrifugation, Zonal↗

PubMed1982

Cloned bacteriophage phi X174 DNA sequence interferes with synthesis of the complementary strand of infecting bacteriophage phi X174.

The insertion of a particular phi X DNA sequence in the plasmid pACYC177 strongly decreased the capacity of Escherichia coli cells containing such a plasmid to propagate bacteriophage phi X174. The smallest DNA sequence tested that showed the effect was the HindII fragment R4. This fragment does not code for a complete protein. It contains the sequence specifying the C-terminal part of the gene H protein and the N-terminal part of the gene A protein, as well as the noncoding region between these genes. Analysis of cells that contain plasmids with the "reduction sequence" showed that (i) the adsorption of the phages to the host cells is normal, (ii) in a single infection cycle much less phage is formed, (iii) only 10% of the infecting viral single-stranded DNA is converted to double-stranded replicative-form DNA, and (iv) less progeny replicative form DNA is synthesized. The reduction process is phi X174 specific, since the growth of the related G4 and St-1 phages was not affected in these cells. The effect of the recombinant plasmids on infecting phage DNA shows similarity to the process of superinfection exclusion.

Bacteriophage phi X 174↗

PubMed1978

Ultrastructural studies of H-1 parvovirus replication. VI. simultaneous autoradiographic and immunochemical intranuclear localization of viral DNA synthesis and protein accumulation.

The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [(3)H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [(3)H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [(3)H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis.

Antigens, Viral↗

PubMed1986

Selective killing of simian virus 40-transformed human fibroblasts by parvovirus H-1.

A normal strain of human foreskin fibroblasts, two SV40-transformed derivatives with finite and infinite life spans, and an established line of SV40-transformed newborn human kidney cells are compared for their susceptibility to infection with parvovirus H-1. H-1 inocula, which do not detectably alter the growth of normal cells, cause a progressive degeneration of all three SV40-transformed cultures. The resistance of normal cells is not a membrane phenomenon since they adsorb and take up H-1 as efficiently as the transformants. Moreover, the fraction of infected cells supporting the synthesis and nuclear migration of H-1 proteins is similar in normal and SV40-transformed cultures. On the other hand, the enhanced H-1 sensitivity of transformed cells correlates with a 5- to 30-fold increase in their accumulation of newly synthesized parvoviral DNA, as compared with normal cultures. This stimulation of H-1 DNA replication is most pronounced for the amplification of duplex replicative forms, although the conversion of parental single-stranded DNA to replicative forms is also enhanced to a smaller extent. In addition, SV40-transformed cells support productive H-1 infection and release a burst of infectious virus, whereas no H-1 production can be detected in the normal cell strain. The latter difference was confirmed for another series of 7 normal and 16 SV40-transformed strains of human skin fibroblasts. Altogether, these results indicate that intracellular limitations on H-1 DNA replication are associated with the abortive nature of the parvoviral life cycle in normal human fibroblasts and are overcome after SV40 transformation, resulting in the selective killing of the transformants. This observation raises the possibility that oncolysis might contribute to the oncosuppressive activity displayed by parvoviruses in vivo.

Cell Survival↗

PubMed1976

Replication of semliki forest virus: polyadenylate in plus-strand RNA and polyuridylate in minus-strand RNA.

The 42S RNA from Semliki Forest virus contains a polyadenylate [poly(A)] sequence that is 80 to 90 residues long and is the 3'-terminus of the virion RNA. A poly(A) sequence of the same length was found in the plus strand of the replicative forms (RFs) and replicative intermediates (RIs) isolated 2 h after infection. In addition, both RFs and RIs contained a polyuridylate [poly(U)] sequence. No poly(U) was found in virion RNA, and thus the poly(U) sequence is in minus-strand RNA. The poly(U) from RFs was on the average 60 residues long, whereas that isolated from the RIs was 80 residues long. Poly(U) sequences isolated from RFs and RIs by digestion with RNase T1 contained 5'-phosphorylated pUp and ppUp residues, indicating that the poly(U) sequence was the 5'-terminus of the minus-strand RNA. The poly(U) sequence in RFs or RIs was free to bind to poly(A)-Sepharose only after denaturation of the RNAs, indicating that the poly(U) was hydrogen bonded to the poly(A) at the 3'-terminus of the plus-strand RNA in these molecules. When treated with 0.02 mug of RNase A per ml, both RFs and RIs yielded the same distribution of the three cores, RFI, RFII, and RFIII. The minus-strand RNA of both RFI and RFIII contained a poly(U) sequence. That from RFII did not. It is known that RFI is the double-stranded form of the 42S plus-strand RNA and that RFIII is the experimetnally derived double-stranded form of 26S mRNA. The poly(A) sequences in each are most likely transcribed directly from the poly(U) at the 5'-end of the 42S minus-strand RNA. The 26S mRNA thus represents the nucleotide sequence in that one-third of the 42S plus-strand RNA that includes its 3'-terminus.

Base Sequence↗

PubMed1971

Transfection of Escherichia coli spheroplasts. I. General facilitation of double-stranded deoxyribonucleic acid infectivity by protamine sulfate.

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.

Centrifugation, Density Gradient↗

PubMed1989

State of hepatitis B virus DNA in hepatocytes of patients with noncarcinomatous liver disease. Its special relationship with necroinflammatory activity and the stage of disease.

By Southern blot hybridization, the state of hepatitis B virus (HBV) in the liver was investigated by utilizing needle biopsy specimens of 59 patients with hepatitis B surface antigen-positive noncarcinomatous liver disease. The tissue HBV DNA revealed a replicative form in 23 patients with chronic active hepatitis (48%) and an integrated form in 23 (48%) of these 48 patients. A liver with a replicative form was more frequently associated with hepatitis Be antigen (HBeAg), serum HBV DNA, and expression of hepatitis B core antigen than one without it. Integration of HBV DNA was more common in the anti-hepatitis Be phase or in the absence of necroinflammatory activity, and its frequency of occurrence roughly paralleled progression of liver disease: early, 21%; advanced, 56%; and cirrhotic stage, 61%.

Adult↗

PubMed1983

Studies on the replication of Escherichia coli phage lambda DNA. I. The kinetics of DNA replication and requirements for the generation of rolling circles.

Escherichia coli phage lambda DNA has been isolated from infected bacteria using a new technique by which virtually all phage DNA is recovered. Isolated DNA is examined by electron microscopy. Addition of phi X174 RF1 molecules as a counting standard enables us to determine the average number of lambda DNA molecules present in an infected cell. In this study, we have followed the kinetics of lambda DNA replication and examined rolling circle replication. The most important findings are the following: (1) Rolling circle replication is initiated at roughly the same time as is theta replication, indicating that the rolling circle is not solely a late-replicating form. (2) theta replication stops at about 16 min after infection. (3) Early in infection the number of DNA molecules per cell doubles every 2-3 min until theta replication stops, at which point most DNA synthesis consists of growth of the tails of about three rolling circles per cell. (4) Neither the timing of rolling circle replication nor the number of molecules is affected by the activity of the lambda red genes. (5). The red genes are responsible for the production of oligomeric circles late in infection.

Bacteriophage lambda↗

PubMed1990

Modified subcellular localization of interferon-induced p68 kinase during encephalomyocarditis virus infection.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68,000 Mr protein (p68 kinase) induced by interferon. When autophosphorylated, p68 kinase catalyzes the phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. The level of p68 kinase is dramatically reduced in nonionic detergent NP-40 extracts, obtained from interferon-treated cells during infection with encephalomyocarditis virus (EMCV) (A. G. Hovanessian, J. Galabru, E. Meurs, C. Buffet-Janvresse, J. Svab and N. Robert, Virology 159, 126-136, 1987). Here we show that such reduction of p68 kinase is in fact due to its reduced NP-40 solubility occurring during EMCV infection. However, p68 kinase can be recovered by extraction with an ionic detergent. Reduced NP-40 extractibility of p68 kinase is dependent on the multiplicity of virus infection and seems to be specific, since other cellular proteins as well as the 100-kDa 2',5'-oligoadenylate synthetase also induced by interferon are not modified. Immunofluorescence studies using specific antibodies demonstrated that p68 kinase which is distributed evenly in the cytoplasm of HeLa cells becomes concentrated around the nuclei after EMCV infection. As a consequence of aggregating around the nuclei, p68 kinase might then resist extraction by NP-40. The aggregated kinase is found to be already activated probably due to binding to the replicative form and/or to replicative intermediates of EMCV RNA. Through this process, the functioning of p68 kinase might be guaranteed by a localized activation in the replication complexes of EMCV.

2',5'-Oligoadenylate Synthetase↗

PubMed1978

Insertion of the Tn3 transposon into the genome of the single-stranded DNA phage M13.

The transposable genetic element Tn3, which carries an ampicillin (Ap) resistance determinant, has been translocated from a ColE1-Apr plasmid, RSF2124, to the genome of the filamentous single-stranded DNA phage M13. The site orientation of the inserted element has been determined for one such phage, M13::Tn3-15. The insertion is within the intergenic space separating genes 2 and 4 and containing both the viral strand and complementary strand origins. The lengths of both the filamentous phage and the duplex replicative form (RF) DNA are 1.7--1.8 times those of M13 phage and replicative form DNA. Both plaque formation and transduction of sensitive cells to ampicillin resistance by M13::Tn3-15 are sensitive to purified antibodies to the M13 major coat protein.

Ampicillin↗