Replication of vaccinia DNA in mouse L cells I. In vivo DNA synthesis

Esteban, Mariano; Holowczak, John A.

doi:10.1016/0042-6822(77)90078-2

Cited by 55 publications

(25 citation statements)

References 57 publications

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“…The observation that these viruses are capable of replication in enucleated cells suggests that poxvirus DNA synthesis is a relatively autonomous process and may not require host functions (4)(5)(6). Electron microscopic observation of the replicating vaccinia genome (7)(8)(9) and in vivo labeling experiments (10)(11)(12) suggest that DNA replication starts near the genomic termini, but definitive evidence that a specific poxviral origin sequence exists is lacking. To further localize and examine the role of viral sequences during replication, we have utilized the transfection of exogenous plasmids into virus-infected cells, an approach that has previously been used with success to identify and characterize various functional viral DNA domains, including replication origins (13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

DeLange¹,

McFadden²

1986

Proc. Natl. Acad. Sci. U.S.A.

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A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (it) amplification of intracellular plasmid DNA; and (Wi) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2, 4X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.Poxviruses replicate their double-stranded linear genomes in factories or "micro-nuclei" within the cytoplasm of infected cells (1-3). The observation that these viruses are capable of replication in enucleated cells suggests that poxvirus DNA synthesis is a relatively autonomous process and may not require host functions (4-6). Electron microscopic observation of the replicating vaccinia genome (7-9) and in vivo labeling experiments (10-12) suggest that DNA replication starts near the genomic termini, but definitive evidence that a specific poxviral origin sequence exists is lacking. To further localize and examine the role of viral sequences during replication, we have utilized the transfection of exogenous plasmids into virus-infected cells, an approach that has previously been used with success to identify and characterize various functional viral DNA domains, including replication origins (13)(14)(15)(16)(17)(18).In this communication, we report on the fate of transfected plasmids with and without viral DNA inserts after their introduction into cells infected with either vaccinia virus or the tumorigenic poxvirus, Shope fibroma virus (SFV). Al-though preliminary experiments using standard calcium phosphate (CaPO4) precipitation protocols had indic...

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mentioning

confidence: 99%

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

DeLange¹,

McFadden²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The evidence of nick formation near the ends of the genome (21,22) and the resolution of concatemeric forms of DNA by a virus-encoded Holliday junction endonuclease (23,24) are compatible with this rolling hairpin mechanism. However, there are reports of short nascent VACV DNA segments resembling Okasaki fragments that could be chased into larger molecules in vivo (25). The recent identification of a DNA primase encoded by VACV (26) suggests the use of RNA primers for the initiation of lagging-strand synthesis (27).…”

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confidence: 99%

Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Sele

Gabel

Gutsche

et al. 2013

J Virol

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c Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.

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“…This model (Fig. 3) departs from the current rolling hairpin model, yet is strongly supported by an earlier electron microscopic study, which showed a variable length double-stranded DNA loop at one end of replicating VACV DNA molecules (13) and by biochemical studies (10)(11)(12). Moreover, the sequences determined to be optimal for replication of a linear DNA template with hairpin ends (17) contained the origin sequences described here.…”

Section: Consensus Nucleotides At the Start And End Of Vacv Dna Fragmmentioning

confidence: 66%

“…Although not followed up on for nearly 4 decades, early studies on poxvirus DNA replication described putative Okazaki fragments of about 1,000 nt in length and RNA primers on the 5′-ends of newly made chains of VACV DNA (10,11). Additionally, the specific activity of [ 3 H]thymidine incorporated during a short pulse under conditions of synchronous VACV DNA synthesis was highest in fragments from the ends of the genome, suggesting that replication originated close by (12).…”

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confidence: 99%

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

Senkevich

Bruno

Martens

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNAprimed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.vaccinia virus | DNA replication | DNA replication fork | DNA replication origin | Okazaki fragments P oxviruses comprise a large family of complex enveloped DNA viruses that infect vertebrates and insects and includes the agent responsible for human smallpox (1). In contrast to the nuclear location exploited for genome replication by many other DNA viruses, the 130-to 230-kbp linear double-stranded DNA genomes of poxviruses are synthesized within discrete, specialized regions of the cytoplasm known as virus factories or virosomes. Furthermore, most, if not all, proteins required for DNA replication are virus-encoded (2). Poxvirus genomes, as shown for the prototype species vaccinia virus (VACV), have covalently closed hairpin termini, so that the DNA forms a continuous polynucleotide chain (3). The hairpin is a 104-nucleotide (nt) A+T-rich incompletely base-paired structure that exists in two inverted and complementary forms. As expected for a genome with such covalently closed ends, VACV replicative intermediates are headto-head or tail-to-tail concatemers (4, 5). The concatemers exist only transiently because they are cleaved by a virus-encoded Holliday junction resolvase into unit length genomes with hairpin ends before incorporation into virus particles (6, 7). Because VACV genomes and concatemers resemble the replicative intermediates of the much smaller parvoviruses, the rolling hairpin model of replication originally proposed for the latter family was e...

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Replication of vaccinia DNA in mouse L cells I. In vivo DNA synthesis

Cited by 55 publications

References 57 publications

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing

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