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PubMed1984

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin.

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.

Base Sequence↗

PubMed1970

Early intracellular events in the replication of bacteriophage T4 deoxyribonucleic acid. V. Further studies on the T4 protein-deoxyribonucleic acid complex.

Soon after infection parental deoxyribonucleic acid (DNA) enters a structure sedimenting fast to the bottom of a sucrose gradient. The addition of chloramphenicol (CM) prevents formation of this structure, whereas treatment with Pronase releases DNA which sediments thereafter with the speed characteristic of phenol-extracted replicative DNA. It is assumed therefore that the structure responsible for fast sedimentation of replicative DNA is a newly synthesized protein. Those fast-sedimenting complexes contain preferentially the replicative form of parental DNA; this was proven by density labeling experiments. Progeny DNA labeled with (3)H-thymidine added after infection can also be detected preferentially in this fast-sedimenting moiety. The association of the DNA with the complexing protein is of a colinear or quasi-colinear type. This was proven by introducing double-strand scissions into DNA embedded in the replicative complex; double-strand scissions do not liberate DNA from the fast-sedimenting complex. Despite the apparent intimate relation between protein and DNA, DNA residing in complexes is fully sensitive to the action of nucleases. Shortly prior to the appearance of the fast-sedimenting complex, parental DNA displays still another characteristic: at about 3 min after infection, it sediments faster than reference, but sizeably slower than the complex which appears at roughly 4 to 5 min after infection. The transition between these two fast-sedimenting types of moieties is not continuous. This fast-sedimenting intermediate, which appears at 3 min after infection, cannot be inhibited by the addition of CM either at the moment or prior to infection. Fast-sedimenting intermediate can be destroyed by sodium dodecyl sulfate, Pronase, or phenol extraction. The progeny DNA labeled with (3)H-thymidine between 3 and 3.5 min after infection can be recovered in fast-sedimenting intermediate. The contribution of newly synthesized progeny DNA is so small that it cannot be detected as a shift of the parental density in a density labeling experiment. Small fragments of progeny DNA recovered in fast-sedimenting intermediate are not covalentlv attached to parental molecules and represent both strands of T4 DNA.

Centrifugation, Density Gradient↗

PubMed1987

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication.

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.

Bacteriophage phi X 174↗

PubMed1977

Nucleotide sequences near the origin of replication of bacteriophage f1.

The nucleotide sequence of a region related to the initiation of the reaction in which single-stranded DNA gives rise to the replicative form (complementary strand synthesis) in bacteriophage f1 has been determined. The sequence can be drawn in an extensively base-paired structure, i.e., a single hairpin-helix 55 bases long.

Bacteriophages↗

PubMed1976

Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III.

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.

Base Sequence↗

PubMed1993

Asymmetric resolution of a parvovirus palindrome in vitro.

Cell extracts from murine A9 or human HeLa cells containing wild-type copies the NS1 polypeptide of minute virus of mice (MVM), produced from a recombinant vaccinia virus, can support the resolution of viral 3' termini from palindromic junction fragments of dimeric, replicative-form MVM DNA. Resolution resulted in the generation of two new viral termini, one associated with each arm of the junction palindrome. Telomeres were created in two configurations, "extended" forms, which were covalently associated with NS1 molecules, and smaller "turn-around" forms in which a single arm of the palindrome terminated at the axis of dyad symmetry in a covalent bond which cross-linked the two strands. The in vitro resolution reaction was asymmetric, generating predominantly extended-form termini from one arm of the palindrome but predominantly turn-around forms from the other. This asymmetry was independent of the type of cell used to prepare the in vitro extract or the orientation of the palindrome in the plasmid and was obtained for all cloned junction sequences of 156 bp or more. Two modified forms of the duplex junction fragment, which appeared to be intermediates in the resolution process since they were nicked, covalently linked to NS1, and associated with newly synthesized DNA, were identified. The structures of these intermediates suggest that resolution is initiated by preferential nicking at one of the two candidate resolution sites. The asymmetric nature of this resolution reaction is discussed in terms of current models of MVM DNA replication.

Animals↗

PubMed1999

Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement.

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.

Animals↗

PubMed2004

Specificity of the endonuclease activity of the baculovirus alkaline nuclease for single-stranded DNA.

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) likely participates in the maturation of virus genomes and in DNA recombination. AcMNPV AN was expressed in a recombinant baculovirus as a His -tagged fusion and obtained in pure form (*AN) or as a (6)complex with the baculoviral single-stranded DNA-binding protein LEF-3 (*AN/L3). Both AN preparations possessed potent 5' --> 3'-exonuclease and weak endonuclease activities. Mutant *AN(S146A)/L3 with a change from serine to alanine at position 146 in a conservative motif was impaired in both activities. This proved that the endonuclease is an intrinsic activity of baculovirus AN. The AN endonuclease showed specificity for single-stranded DNA and converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) by a single strand break. Plasmid DNA relaxed with topoisomerase I was resistant to *AN/L3 indicating that the partially single-stranded regions in negatively supercoiled molecules served as targets for the endonuclease. Unwinding the supercoiled DNA with ethidium bromide also made DNA resistant to AN/L3. In reactions with nicked circular DNA (RFII), AN and AN/L3 hydrolyzed exonucleolytically the broken strand or cut endonucleolytically the intact strand at the position opposite the nick (gap). When LEF-3 was added to the assay, the balance between the exonucleolytic and endonucleolytic modes of hydrolysis shifted in favor of the exonuclease. The data suggest that the AN endonuclease may digest the intermediates in replication and recombination at positions of structural irregularities in DNA duplexes, whereas LEF-3 may further regulate processing of the intermediates by AN via the endonuclease and exonuclease pathways.

Alanine↗

PubMed1999

Molecular and structural basis of the evolution of parvovirus tropism.

Parvoviruses have small genomes and, consequently, are highly dependent on their host for various functions in their reproduction. Since these viruses generally use ubiquitous receptors, restrictions are usually intracellularly regulated. A lack of mitosis, and hence absence of enzymes required for DNA replication, is a powerful block of virus infection. Allotropic determinants have been identified for several parvoviruses: porcine parvovirus, canine parvovirus (CPV), feline parvovirus (feline panleukopenia virus), minute virus of mice, Aleutian disease virus, and GmDNV (an insect parvovirus). Invariably, these identifications involved the use of infectious clones of these viruses and the exchange of restriction fragments to create chimeric viruses, of which the resulting phenotype was then established by transfection in appropriate cell lines. The tropism of these viruses was found to be governed by minimal changes in the sequence of the capsid proteins and, often, only 2 or 3 critical amino acids are responsible for a given tropism. These amino acids are usually located on the outside of the capsid near or on the spike of the threefold axis for the vertebrate parvoviruses and on loops 2 or 3 for the insect parvoviruses. This tropism is not mediated via specific cellular receptors but by interactions with intracellular factors. The nature of these factors is unknown but most data point to a stage beyond the conversion of the single-stranded DNA genome by host cell DNA polymerase into monomeric duplex intermediates of the replicative form. The sudden and devastating emergence of mink enteritis virus (MEV) and CPV in the last 50 years, and the possibility of more future outbreaks, demonstrates the importance of understanding parvovirus tropism.

Animals↗

PubMed1993

Construction of an infectious DNA clone of the Y1 strain of canine parvovirus and characterization of the virus derived from the clone.

We have cloned genome fragments of canine parvovirus strain Y1 from replicative-form DNA and double-stranded DNA synthesized from virion DNA in vitro, and constructed a recombinant plasmid containing a full-length Y1 genome (pCPVY 1). When this recombinant plasmid was transfected into cell cultures, an infectious virus could be recovered. To characterize this pCPVY 1-derived virus, its biological properties were compared with those of the parental strain. No difference was observed between them in antigen expression, viral DNA replication, hemagglutination ability, and virus multiplication, indicating that the virus derived from the infectious plasmid inherited the biological properties of the authentic Y1 strain. Therefore, this recombinant plasmid appears to be useful for reverse genetics of canine parvovirus.

Animals↗

PubMed2002

Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity.

N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation.

Animals↗

PubMed2003

Evidence of parvovirus replication in cerebral neurons of cats.

The correlation between parvovirus infections and lesions in the central nervous system other than cerebellar hypoplasia was studied in 100 cats. The animals were necropsied with a history of various diseases, one third showing typical clinical and pathomorphological signs of panleukopenia. In 18 cats polyclonal antiserum against canine parvovirus consistently labeled neurons mainly in diencephalic regions, whereas the cerebellar cortex remained negative in all cases. In situ hybridization with digoxigenin-labeled minus-sense RNA probes, hybridizing with monomer-replicative form DNA or mRNA, revealed positive signals in nuclei of several neurons of the brain, again excluding the cerebellum. PCR applied to formalin-fixed and paraffin-embedded brain tissue and intestinal tissues of the diseased cats and subsequent DNA sequence analysis yielded canine parvovirus type 2 (CPV-2)-like sequences in the central nervous system. Two aspects of these findings are intriguing: (i). parvoviruses appear to be capable of replicating in neurons, cells that are considered to be terminally differentiated and (ii). CPV-like viruses of the old antigenic type CPV-2 appear to be able to infect cats.

Animals↗

PubMed1987

Deoxyhexanucleotide containing a vinyl chloride induced DNA lesion, 1,N6-ethenoadenine: synthesis, physical characterization, and incorporation into a duplex bacteriophage M13 genome as part of an amber codon.

Organic synthesis and recombinant DNA techniques have been used to situate a single 1,N6-ethenoadenine (epsilon Ade) DNA adduct at an amber codon in the genome of an M13mp19 phage derivative. The deoxyhexanucleotide d[GCT(epsilon A)GC] was chemically synthesized by the phosphotriester method. Mild nonaqueous conditions were employed for deprotection because of the unstable nature of the epsilon Ade adduct in aqueous basic milieu. Physical studies involving fluorescence, circular dichroism, and 1H NMR indicated epsilon Ade to be very efficiently stacked in the hexamer, especially with the 5'-thymine. Melting profile and circular dichroism studies provided evidence of the loss of base-pairing capabilities attendant with formation of the etheno ring. The modified hexanucleotide was incorporated into a six-base gap formed in the genome of an M13mp19 insertion mutant; the latter was constructed by blunt-end ligation of d(GCTAGC) in the center of the unique SmaI site of M13mp19. Phage of the insertion mutant, M13mp19-NheI, produced light blue plaques on SupE strains because of the introduced amber codon. Formation of a hybrid between the single-strand DNA (plus strand) of M13mp19-NheI with SmaI-linearized M13mp19 replicative form produced a heteroduplex with a six-base gap in the minus strand. The modified hexamer [5'-32P]d-[GCT(epsilon A)GC], after 5'-phosphorylation, was ligated into this gap by using bacteriophage T4 DNA ligase to generate a singly adducted genome with epsilon Ade at minus strand position 6274. Introduction of the radiolabel provided a useful marker for characterization of the singly adducted genome, and indeed the label appeared in the anticipated fragments when digested by several restriction endonucleases. Evidence that ligation occurred on both 5' and 3' sides of the oligonucleotide also was obtained. The adduct was introduced into a unique NheI site, and it was observed that this restriction endonuclease was able to cleave the adducted genome, albeit at a lower rate compared to unmodified DNA. The M13mp19-NheI genome containing epsilon Ade will be used as a probe for studying mutagenesis and repair of this DNA adduct in Escherichia coli.

Adenosine↗

PubMed1985

Transfection of REP- mycoplasmas with viral single-stranded DNA.

Double-stranded DNA from mycoplasma virus L2 can transfect Acholeplasma laidlawii cells in the presence of polyethylene glycol (T. L. Sladek and J. Maniloff, J. Bacteriol. 155:734-741, 1983). We report here that both single-stranded DNA and double-stranded replicative form DNA, from the single-stranded DNA mycoplasma virus L51, are also infectious in this system. For both DNAs transfection frequencies were in the range of 10(-8) transfectants per DNA molecule and 10(-3) transfectants per CFU. An unexpected finding was that both DNAs could transfect A. laidlawii strain REP-, a variant which is a nonpermissive host for single-stranded DNA mycoplasma viruses due to a block in viral DNA replication (Nowak et al., J. Bacteriol. 127:832-836, 1976). The number of viruses produced by transfected REP- cells was comparable to the number produced by both transfected and infected wild-type cells. Therefore, transfected L51 DNAs are able to bypass the replication block in REP- cells that occurs when these cells are infected by L51 virions.

Acholeplasma laidlawii↗

PubMed1987

Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth.

Integration of retroviral DNA is a site-specific reaction involving an endonuclease encoded by the viral pol gene (pol-endo). In vitro the pol-endo from avian sarcoma and leukosis viruses (ASLVs) cleaves both DNA strands near the U5-U3 junction of tandem long terminal repeats (LTR-LTR junction) in single-stranded and replicative form (RF)-I substrates. We have reported previously that the sequences that are required for cleavage of single-stranded substrates by the alpha beta form of the pol-endo differ for the plus and minus strands (G. Duyk, M. Longiaru, D. Cobrinik, R. Kowal, P. deHaseth, A. M. Skalka, and J. Leis, J. Virol. 56:589-599, 1985). This is not the case with RF-I substrates, in which a maximum of 22 base pairs of U5 and 8 base pairs of U3 were required for alpha beta pol-endo cleavage in each strand. Insertion of a palindromic octanucleotide (CATCGATG) at the LTR-LTR junction abolished cleavage in RF-I but not in single-stranded DNA substrates. Deletion of the four nucleotides (TTAA) at the junction prevented cleavage in the plus strand of RF-I DNA, but did not affect cleavage of single-stranded DNA. Furthermore, the alpha beta form of ASLV pol-endo did not recognize heterologous LTR-LTR junction sequences from the reticuloendotheliosis virus or Moloney murine leukemia virus in either substrate form, despite their sequence and structural similarities to the ASLV junction. These results support a role for a sequence-specific interaction between the ASLV pol-endo and the LTR-LTR junction domains that are required for cleavage. By using the infectious Rous sarcoma virus clone pATV8-K, we introduced a set of deletions into the U5 region that would be incorporated into the LTR-LTR junction on viral replication. In the unintegrated provirus, the deletions started 43 base pairs from the LTR-LTR junction and extended various lengths toward the junction. Results of transfection studies with these clones indicated that the U5 sequences that are required for virus production in vivo correspond to those that are required for cleavage of RF-I DNA in vitro.

Alpharetrovirus↗

PubMed1981

In vitro conversion of S13 viral DNA in phage particles to the double-stranded DNA.

The conversion of single-stranded DNA in S13 intact phage particles to the double-stranded replicative form DNA was observed in cell extracts prepared from Escherichia coli H560 (S13s, polA, endA) cells lysed with lysozyme and the non-ionic detergent, Brij 58. The DNA product, which associated with a rapidly sedimenting component, was identified as RFII-DNA with a gap by sedimentation analysis. The conversion was inhibited by N-ethylmaleimide, but not by rifampicin, nicotinamide mononucleotide or polymyxin B. The dnaB gene product was involved in the replicative system. Similar extracts prepared from a S13-resistant E. coli strain K12W6 also catalyzed this synthesis.

Bacterial Proteins↗

PubMed2000

A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains.

A specific serotype, O3:K6, of Vibrio parahaemolyticus has recently been causing epidemics of gastroenteritis in Southeast Asia, Japan, and North America. To examine whether the new O3:K6 strains possess characteristics that may exacerbate outbreaks, we compared V. parahaemolyticus O3:K6 strains with non-O3:K6 strains using strains isolated from individuals with traveler's diarrhea at Kansai Airport Quarantine Station, Osaka, Japan. All 24 O3:K6 strains possessed a common plasmid, pO3K6 (DNA size, 8,782 bp, with 10 open reading frames [ORFs]). The gene organization of pO3K6 was similar to that of Vf33, a filamentous phage previously described in V. parahaemolyticus. We isolated a phage (phage f237) from the culture supernatant of V. parahaemolyticus O3:K6 strain KXV237, which formed a turbid plaque on an indicator strain. The genome of f237 was single-stranded DNA, and the double-stranded DNA obtained by treatment of the genome with DNA polymerase was identical to that of pO3K6 when analyzed by agarose gel electrophoresis after HindIII digestion. Furthermore, the N-terminal amino acid sequence of the f237 major coat protein was found in ORF4 of pO3K6. Our results showed that pO3K6 is a replicative form of f237. Among the ORFs found in the f237 genome, the sequence of ORF8 had no significant homology to those of any proteins in databases. ORF8 was located on a region corresponding to the distinctive region of Vf33, and its G+C content was apparently lower than that of the remaining DNA sequence of f237. By colony hybridization, ORF8 was detected only in O3:K6 strains isolated since 1996 and was not found in O3:K6 strains isolated before 1996 and clinical V. parahaemolyticus strains other than those of serotype O3:K6. Thus, this study shows that f237 is exclusively associated with recent V. parahaemolyticus O3:K6 strains. The ORF8 gene can be a useful genetic marker for the identification of the recently widespread O3:K6 strains of V. parahaemolyticus.

Diarrhea↗

PubMed2002

Potency of nonnucleoside reverse transcriptase inhibitors (NNRTIs) used in combination with other human immunodeficiency virus NNRTIs, NRTIs, or protease inhibitors.

Efavirenz and a series of related quinazolinone nonnucleoside inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were evaluated in a series of two-drug combinations with several nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and protease inhibitors (PIs). These combinations were tested in an established HIV-1 RT enzyme assay and a cell-based yield reduction assay with HIV-1 (replicative form [RF])-infected MT-2 cells. Synergy, additivity, and antagonism were determined in the two different assay systems by the method of Chou and Talalay (T.-C. Chou and P. Talalay, Adv. Enzyme Reg. 22:27-55, 1984). Efavirenz, DPC082, DPC083, DPC961, and DPC963 used in combination with the NRTIs zidovudine and lamivudine acted synergistically to inhibit RT activity in the HIV-1 RT enzyme assay and additively to slightly synergistically to inhibit HIV-1 (RF) replication in the yield reduction assay. The five NNRTIs in combination with the PI nelfinavir acted additively in the yield reduction assay to inhibit HIV-1 replication. Interestingly, efavirenz in combination with a second NNRTI acted additively to inhibit HIV-1 RT function in the enzyme assay, while it acted antagonistically to inhibit HIV-1 (RF) replication in the yield reduction assay. These data suggest that antiretroviral combination regimens containing multiple NNTRIs should be given thorough consideration before being used.

Alkynes↗