Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drugresistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase II␤. One can prevent the interaction by introducing a C 11 -to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase II␣/␤ in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase II␣ and II␤ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases II␣/␤ in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.Topoisomerases play a critical role in modulating DNA superhelical density and in unknotting and decatenating the structures formed during replication, recombination, and repair (reviewed in references 2 and 5). Mammalian cells carry several different kinds of topoisomerases, classified as being either type I or type II enzymes. Both classes of enzyme catalyze DNA strand breakage and rejoining, but type I enzymes catalyze reactions involving single-strand breaks, while the type II topoisomerases catalyze double-strand cleavage reactions. The mammalian type IB enzyme (Topo I) is well adapted for removing the superhelical stress created by the replication and transcription machinery. The two closely related type IIA enzymes (Topo II␣ and Topo II␤) may also catalyze these same reactions but probably play a more critical role in processes like chromatin reorganization and chromosome segregation. A third class of type IA enzymes (Topo III␣ and Topo III␤)

Vaccinia virus DNA polymerase (VVpol) encodes a 3'-to-5' proofreading exonuclease that can degrade the ends of duplex DNA and expose single-stranded DNA tails. The reaction plays a critical role in promoting virus recombination in vivo because single-strand annealing reactions can then fuse molecules sharing complementary tails into recombinant precursors called joint molecules. We have shown that this reaction can also occur in vitro, providing a simple method for the directional cloning of PCR products into any vector of interest. A commercial form of this recombineering technology called In-Fusion(®) that facilitates high-throughput directional cloning of PCR products has been commercialized by Clontech. To effect the in vitro cloning reaction, PCR products are prepared using primers that add 16-18 bp of sequence to each end of the PCR amplicon that are homologous to the two ends of a linearized vector. The linearized vector and PCR products are coincubated with VVpol, which exposes the complementary ends and promotes joint molecule formation. Vaccinia virus single-stranded DNA binding protein can be added to enhance this reaction, although it is not an essential component. The resulting joint molecules are used to transform E. coli, which convert these noncovalently joined molecules into stable recombinants. We illustrate how this technology works by using, as an example, the cloning of the vaccinia N2L gene into the vector pETBlue-2.

Bladder cancer has a recurrence rate of up to 80% and many patients require multiple treatments that often fail, eventually leading to disease progression. In particular, standard of care for high‐grade disease, Bacillus Calmette–Guérin (BCG), fails in 30% of patients. We have generated a novel oncolytic vaccinia virus (VACV) by mutating the F4L gene that encodes the virus homolog of the cell‐cycle‐regulated small subunit of ribonucleotide reductase (RRM2). The F4L‐deleted VACVs are highly attenuated in normal tissues, and since cancer cells commonly express elevated RRM2 levels, have tumor‐selective replication and cell killing. These F4L‐deleted VACVs replicated selectively in immune‐competent rat AY‐27 and xenografted human RT112‐luc orthotopic bladder cancer models, causing significant tumor regression or complete ablation with no toxicity. It was also observed that rats cured of AY‐27 tumors by VACV treatment developed anti‐tumor immunity as evidenced by tumor rejection upon challenge and by ex vivo cytotoxic T‐lymphocyte assays. Finally, F4L‐deleted VACVs replicated in primary human bladder cancer explants. Our findings demonstrate the enhanced safety and selectivity of F4L‐deleted VACVs, with application as a promising therapy for patients with BCG‐refractory cancers and immune dysregulation.

Vaccinia virus (VACV) produces large plaques consisting of a rapidly expanding ring of infected cells surrounding a lytic core, whereas myxoma virus (MYXV) produces small plaques that resemble a focus of transformed cells. This is odd, because bioinformatics suggests that MYXV carries homologs of nearly all of the genes regulating Orthopoxvirus attachment, entry, and exit. So why does MYXV produce foci? One notable difference is that MYXV-infected cells produce few of the actin microfilaments that promote VACV exit and spread. This suggested that although MYXV carries homologs of the required genes (A33R, A34R, A36R, and B5R), they are dysfunctional. To test this, we produced MYXV recombinants expressing these genes, but we could not enhance actin projectile formation even in cells expressing all four VACV proteins. Another notable difference between these viruses is that MYXV lacks a homolog of the F11L gene. F11 inhibits the RhoA-mDia signaling that maintains the integrity of the cortical actin layer. We constructed an MYXV strain encoding F11L and observed that, unlike wild-type MYXV, the recombinant virus disrupted actin stress fibers and produced plaques up to 4-fold larger than those of controls, and these plaques expanded ϳ6-fold faster. These viruses also grew to higher titers in multistep growth conditions, produced higher levels of actin projectiles, and promoted infected cell movement, although neither process was to the extent of that observed in VACV-infected cells. Thus, one reason for why MYXV produces small plaques is that it cannot spread via actin filaments, although the reason for this deficiency remains obscure. A second reason is that leporipoxviruses lack vaccinia's capacity to disrupt cortical actin. P oxviruses produce two kinds of plaques in culture. The first kind is formed rapidly by viruses like vaccinia virus (VACV), and typically they comprise a ring of infected cells surrounding a large central clearing or lytic zone. The second type of poxvirus plaque is smaller, grows slower, and consists of a clump of virusinfected cells. These plaques look more like a cluster of transformed cells and are sometimes called foci. Such plaques are produced by tumorigenic poxviruses like the leporipoxviruses myxoma virus (MYXV) and Shope fibroma virus. Although it is well established that the appearance of poxvirus plaques is determined by the genetics of both the host and the virus, why MYXV plaques look so different from VACV plaques, even when plated on the same cell type, is not well understood.The process of plaque formation depends (in part) upon how well viruses can engage the host machinery to spread efficiently from cell to cell, processes that are best understood for VACV (reviewed in references 44, 51, and 58). VACV characteristically produces several different forms of infectious virus. Mature viruses (MV) are bounded by just a single lipid bilayer. MV comprise the most abundant infectious form and are probably released by cell lysis. However, some MV migrate away from viral factories, w...

The rapid growth of tumors depends upon elevated levels of dNTPs, and while dNTP concentrations are tightly regulated in normal cells, this control is often lost in transformed cells. This feature of cancer cells has been used to advantage to develop oncolytic DNA viruses. DNA viruses employ many different mechanisms to increase dNTP levels in infected cells, because the low concentration of dNTPs found in non-cycling cells can inhibit virus replication. By disrupting the virus-encoded gene(s) that normally promote dNTP biosynthesis, one can assemble oncolytic versions of these agents that replicate selectively in cancer cells. This review covers the pathways involved in dNTP production, how they are dysregulated in cancer cells, and the various approaches that have been used to exploit this biology to improve the tumor specificity of oncolytic viruses. In particular, we compare and contrast the ways that the different types of oncolytic virus candidates can directly modulate these processes. We limit our review to the large DNA viruses that naturally encode homologs of the cellular enzymes that catalyze dNTP biogenesis. Lastly, we consider how this knowledge might guide future development of oncolytic viruses.