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

Abstract

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...

Inhibition of the Ubiquitin-Proteasome System Prevents Vaccinia Virus DNA Replication and Expression of Intermediate and Late Genes

“…However, this could not explain the effects of the proteasome inhibitors since VACV-induced plasmid replication was also prevented by the addition of MG132. Previous studies had demonstrated the ability of transfected plasmids to replicate in the cytoplasm of poxvirus-infected cells (6,32). Importantly, plasmid replication requires each factor known to be required for VACV genome replication (10).…”

“…To test this model, we took advantage of previous observations that any circular DNA can be replicated in cells infected with poxviruses (6,32). Plasmid replication occurs in cytoplasmic viral factories and requires all viral proteins known to be necessary for VACV genome replication, making it a surrogate for the latter (10).…”

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

“…In Saccharomyces cerevisiae and some viruses, origins were identified as autonomous replication sequences that confer replication ability to transfected episomes (31). However, the autonomous replication strategy to define origins was unsuccessful when applied to poxviruses: plasmids regardless of sequence efficiently replicate in poxvirus-infected cells, raising questions of whether specific origins are needed for the viral replication machinery (14)(15)(16). To investigate the mechanism of VACV replication, we exploited approaches recently proven effective for mapping eukaryotic replication origins by deep sequencing (26,32).…”

“…Collectively, these data are compatible with a discontinuous or semidiscontinuous mode of genome replication, with origins located near the termini. Subsequently, however, the existence of specific VACV origins came into question by the demonstration that any circular DNA replicates in VACV or Shope fibroma virusinfected cell cytoplasm, and that replication is not enhanced by inclusion of any VACV DNA sequence (14,15). Moreover, all VACV proteins known to be required for genome replication are also required for origin-independent plasmid replication (16).…”

The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination

“…Second, one knows precisely where the CDV is located and need not make any assumptions regarding differential rates of CDV uptake and incorporation into DNA. Finally, poxviruses cannot replicate linearized plasmid DNAs that are transfected into infected cells unless they are first recombined into circles (10,12,64). Thus, the system we are studying becomes recombination dependent, since circularization, through recombination, must precede the replication of these DNAs.…”