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Results for “replicative form”

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PubMed1986

Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice.

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.

Animals↗

PubMed1989

Evidence for a ligation step in the DNA replication of the autonomous parvovirus minute virus of mice.

Newly replicated DNA of the autonomous parvovirus minute virus of mice was pulse-labeled with 32PO4 during the time of maximal viral DNA replication in highly synchronized A9 cells. The subsequent processing of viral DNA-protein complexes was monitored during a chase period with no label. Several distinct classes of duplex replicative-form and progeny single-stranded DNA molecules were characterized and found to accumulate at different times during infection. Analysis of the terminal structures associated with these various forms provided new insights into the mechanism by which viral DNA replicates and, in particular, suggested that interstrand ligation occurs during this process.

Animals↗

PubMedDate not supplied

Sensitivity of Escherichia coli to viral nucleic acid, X. Ba2+-induced competence for transfecting DNA.

Effect of alkaline earth metal ions on induction of the competence for DNA transfection was investigated. Unlike spheroplasts, the bulk of the bacteria treated with these ions retains colony-forming ability. The order of effectiveness for transfection of phiA replicative-form DNA has been found to be Ba2+ greater than Ca2+ greater than Sr2+ greater than Mg2+. The competence of Ba2+-treated cells is 3 to 5 times higher that that of Ca2+-treated bacteria and about 40 times higher than that of lysozyme-EDTA spheroplasts. The Ba2+-dependent transfection is cryophilic and formation of the infective complex occurs very rapidly at 0 degrees C, But not at 37 degrees C.

Barium↗

PubMed1995

Replication, differentiation, growth and the virulence of Trypanosoma brucei infections.

This study had 2 objectives: first, to investigate how the processes of slender form replication, of differentiation from dividing slender to non-dividing stumpy forms, and of stumpy mortality, combine to determine the initial (acute-phase) growth rate of Trypanosoma brucei populations; second, to determine how acute-phase growth rates influence parasite densities during the subsequent, chronic phase of infection. During the acute phase, slender and stumpy populations both grew approximately exponentially, the latter more slowly than the former. Mathematical models showed how this difference in slender and stumpy growth rates can be explained in terms of heterogeneous replication and differentiation rates. Stumpy life-expectancy was determined for one stock and found to be age-dependent with a half-life of 48-72 h, much larger than observed population doubling times of 5-10 h. A comparison of cloned stocks showed that the highest parasite densities during the chronic phase were associated with the highest acute-phase growth rates of both the whole parasite population and of the subpopulation of slender forms. By contrast, high chronic-phase parasitaemias artificially produced following rapid syringe passage were associated with low acute-phase growth rates of slender forms. Syringe-passaging is a laboratory procedure which selects for virulent parasites, but these parasites behave differently from naturally virulent stocks.

Animals↗

PubMed2004

The DnaK-DnaJ-GrpE chaperone system activates inert wild type pi initiator protein of R6K into a form active in replication initiation.

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori gamma-specific initiation of replication by a mutant form of the initiator called pi*, the wild type (WT) pi is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT pi and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT pi. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT pi into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with pi but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that pi is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1981

Attachment of avidin-coupled spheres to linear and circular forms of mengovirus double-stranded RNA.

Picornavirus-infected cells contain a double-stranded RNA (replicative form [RF] RNA), composed of viral genomic RNA hydrogen bonded to cRNA of similar nucleotide length. Mengovirus RF RNA reacted with a succinimide ester of biotin was shown by electron microscopy to bind avidin-coupled polymethacrylate spheres. These binding sites are taken to indicate the presence of VPg protein molecular by methods previously applied to poliovirus RF RNA (Richards et al., Proc. Natl. Acad. Sci. U.S.A. 76:676-680, 1979). One sphere was bound at or very near one terminus of linear RF molecules while a second sphere was bound at a site which was 1 to 4% of the genome length from the other terminus. Assignment of VPg positions was limited by the physical dimension (ca. 60-nm diameter) of the heavy metal-contrasted sphere observed by electron microscopy. A third site of sphere binding was detected at a lower frequency of occurrence at a site which was 10 to 20% of the genome length from one or the other terminus. Circular RF RNA molecules were also detected with two spheres attached at juxtaposed sites. The termini of the linear RF RNA were located within the circular structures by sphere attachment at sites which were very close to and obscured a nucleic acid projection which we have described to occur on circular mengovirus RF RNA (D. L. Robberson, M. V. Marshall, G. B. Thornton, and R. B. Arlinghaus, manuscript submitted for publication). CsCl gradient fractions of RNA reacted with avidin-coupled spheres were highly enriched in circular structures.

Avidin↗

PubMed1998

Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

Animals↗

PubMed1975

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Intermediates of lambda DNA replication in the second half of the latent period after phage lambda infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative forms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as the first round lambda DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the lambda genome. Replicating circles with a linear DNA tail longer than one unit length of lambda genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.

Chromosome Mapping↗

PubMed1996

Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments.

Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family, Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were "circularised" within the same 105 bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts.

Base Sequence↗

PubMed1972

Replication of bacteriophage M13: specificity of the Escherichia coli dnaB function for replication of double-stranded M13 DNA.

Infection of the temperature-sensitive E. coli mutant HfrH 165/70 (dnaB) with the filamentous single-stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 41 degrees , single-stranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.

Centrifugation, Density Gradient↗

PubMed2000

Molecular analyses of a putative CTXphi precursor and evidence for independent acquisition of distinct CTX(phi)s by toxigenic Vibrio cholerae.

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi, a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi, we examined genome diversity in CTX(phi)s derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXphi replicative form, with the exception of missing ctxAB. Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5' of ctxA, as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTX(phi)s, two CTXphi-encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTX(phi)s derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTX(phi)s.

Bacteriophages↗

PubMed1996

DNA double-strand break repair functions defend against parvovirus infection.

We measured parvovirus replication and sensitivity to X-ray damage in nine CHO cell lines representing a variety of DNA repair deficiencies. We found that parvovirus replication efficiency increases with radiosensitivity. Parvovirus replication is disrupted at an early stage of infection in DNA repair-proficient cells, before conversion of the single-stranded viral DNA genome into the double-stranded replicative form. Thus, status of the DNA repair machinery inversely correlates with parvovirus replication and is proportional to the host's ability to repair X-ray-induced damage.

Animals↗

PubMed1994

Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.

Bacterial Proteins↗

PubMed1987

Comparison of the DNA sequences involved in replication and packaging of the filamentous phages IKe and Ff (M13, fd, and f1).

The product of gene II of the distantly related, filamentous, single-stranded DNA phages IKe and Ff (M13, fd, and f1) is the only phage-encoded protein that is required for the replication of their double-stranded replicative form DNA. With the aid of recombinant plasmids containing the origins of viral strand replication [(+)-origins] of both IKe and Ff, we demonstrated that initiation but not termination of viral strand replication by gene II protein is restricted to its cognate (+)-origin. If the (+)-origins of IKe and Ff are present in the same orientation, fusion origins are generated upon gene II protein-instructed replication as a result of initiation at one origin and termination at the other. These fusion origins are only functional in the presence of the gene II protein encoded by the phage from which the sequence lying at the 3' side of the gene II protein cleavage site is derived. The nucleotides that determine the specificity of the replication initiation process are located between positions +17 and +49, or +17 and +40, with respect to the gene II protein cleavage site of IKe and Ff, respectively. The DNA sequence that forms the recognition signal for cleavage by gene II protein is probably located within the sequence that starts 3 nucleotides before and terminates 17 nucleotides after the cleavage site. Efficient packaging by phage IKe of plasmid DNA strands that contain the morphogenetic signal of Ff, or vice versa, indicates that, despite their only partial homology, the morphogenetic signals of IKe and Ff are interchangeable.

Bacteriophages↗

PubMed2002

Mutations in the conserved woodchuck hepatitis virus polymerase FLLA and YMDD regions conferring resistance to lamivudine.

During more than 104 weeks of treatment with lamivudine (3TC) in chronic woodchuck hepatitis virus (WHV) carrier woodchucks, viral recrudescence occurred. Analysis of WHV DNA polymerase from woodchuck serum samples by PCR followed by DNA sequencing demonstrated that all samples were wild type at the conserved YMDD motif in domain C. Four of the six 3TC-treated woodchucks showed a mixture of the wild-type Ala (GCT) and the mutant Thr (ACT) at the conserved amino acid residue 566 (FLLA) in domain B of the WHV polymerase region. The appearance of the A566T mutation was temporally associated with viral recrudescence. This change is analogous with the amino acid 181 (FLLA) in HBV where 3TC selects for a change from Ala to Thr in humans. In the woodchuck, the Ala to Thr change in the polymerase gene results in a mutation of the WHV surface protein (amino acid 377) from Trp (TGG) to an opal codon (TGA), which may prematurely terminates the polypeptide. Three WHV molecular infectious clones were constructed to study this mutation in greater detail in vitro: A566T, analogous to A181T in HBV; M589V, analogous to the M204V in HBV; and the double mutant A566T/M589V, analogous to A181T/M204V in HBV. These mutants exhibited drug-sensitivity and replication profiles that paralleled those reported for analogous HBV variants. In transfected Huh7 cells, WHV containing the M589V mutation conferred at least 100-fold increased resistance to 3TC, but replicated approximately 5-fold less efficiently than wild-type virus as judged by both extracellular virus production and intracellular DNA replicative forms. In contrast, A566T mutant was approximately 10-fold more resistant to 3TC, replicated intracellularly as well as wild type, but produced 10-fold lower levels of virions than wild type. These findings are consistent with the observation that the A566T mutation alters the overlapping WHV surface antigen reading frame. WHV carrying mutations in the conserved YMDD motif, while not directly selected during lamivudine therapy in WHV carrier woodchucks, are replication competent in cell culture indicating the potential for their emergence in treated animals. These results further illustrate the utility of the WHV/woodchuck model to studies of HBV-drug resistance.

Amino Acid Sequence↗

PubMed2000

Two forms of UvrC protein with different double-stranded DNA binding affinities.

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.

Bacterial Proteins↗

PubMed1990

The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence.

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.

Bacterial Proteins↗

PubMed1998

Effect of La Crosse virus infection on overwintering of Aedes triseriatus.

The effect of La Crosse (LAC) virus infection on Aedes triseriatus overwintering success was determined. Eggs from LAC virus transovarially infected (LAC TOT+) and uninfected (LAC TOT-) Ae. triseriatus colonies were induced into diapause, held in natural conditions, and returned to the laboratory at predetermined times for assay of diapause, mortality, and filial infection rates, and to examine viral transcription and replication during diapause. Embryos from the LAC TOT+ colony exhibited greater cumulative mortality (16.7%) than the LAC TOT- eggs (7.3%) throughout the overwintering periods. The increased mortality rate in LAC TOT+ eggs corresponded with a decrease in filial infection rates. Eggs from the LAC TOT+ colony terminated diapause more readily than the LAC TOT- colony. An RNA strand-specific reverse transcriptase-polymerase chain reaction technique was used to monitor viral transcription and replication in mosquito eggs during overwintering, and to compare viral replication in diapausing and nondiapausing embryos. Viral messenger and replicative form RNA were present in eggs in all sample periods, suggesting that some virus replication occurred during diapause.

Aedes↗