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Background: Several approaches to gene therapy for cancer have yielded promising results in rodent models. The translation of these results to the clinical realm has been delayed by the lack of tumor models in large animals. We investigated the pattern of transgene (i. e., foreign or introduced gene) expression and virus vector elimination after systemic gene delivery using a thymidine kinase-negative vaccinia virus in a rabbit model of disseminated liver metastases., Methods: VX-2 rabbit carcinoma cells were maintained by serial transplantation in the thigh muscles of New Zealand White rabbits, and disseminated liver metastases were established by direct injection of tumor cells into the portal vein of the animals. Different doses of a recombinant thymidine kinase-negative vaccinia virus vector encoding the firefly luciferase reporter gene (i.e., transgene) were injected into tumor-bearing rabbits. Transgene activity in tumors and other organs was measured at multiple time points thereafter. The pattern of development of antibodies against the vaccinia virus vector was also examined. Two-tailed Student's paired t test was used for comparisons of transgene activity., Results: Transgene expression was increased in tumors by at least 16-fold in comparison with expression in other tissues by day 4 after vector injection (all P<. 001) and was maintained for approximately 1 week, providing evidence of tumor-specific gene delivery in this model. Rapid elimination of the circulating vector by the host immune system was observed. Anti-vector antibodies were detectable in serum as early as day 6 and were maintained for more than 3 months., Conclusions: Tumor-specific gene delivery is possible after systemic injection of a thymidine kinase-negative vaccinia virus vector in a model of rabbit liver metastases. Although the period of transgene expression appears limited because of a rapid immune response, the therapeutic window might be sufficient for an enzyme/prodrug gene therapy approach in clinical application.

HIV-1 cell tropism is determined initially at the level of fusion mediated by the viral envelope glycoprotein (Env). Cell-cell fusion assays are employed widely to study Env-mediated fusion, and generally require transfection of target cells with a reporter plasmid that is activated upon fusion with Env-expressing effector cells. Macrophages are an important target for HIV-1, but fusion studies using primary macrophages are limited by their resistance to transfection. An assay described previously used recombinant vaccinia virus to express T7 polymerase in macrophages, and effector cells transfected with a T7-driven reporter plasmid and infected with recombinant vaccinia virus expressing Env. However, this requires a recombinant vaccinia virus for each Env. We developed a method to study fusion using primary macrophages and HIV-1 env plasmid clones under control of the T7 promoter. Macrophages were infected with a recombinant vaccinia virus expressing the SP6 RNA polymerase. Effector 293T cells were infected with a recombinant vaccinia virus expressing T7 polymerase, and co-transfected with T7-driven env plasmids and an SP6-driven reporter gene plasmid. Cell-cell fusion mediated by T7-driven Env results in SP6-driven reporter gene transactivation. This approach is suitable for rapid analysis of multiple primary isolate, chimeric, or mutant env genes cloned into plasmid vectors.

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998) Genes Dev. 12, 538-546; Christen, L. M., et al. (1998) Virology 245, 360-371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure-function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparent Km for ATP is independent of DNA concentration, demonstrating that ssDNA binding alters the kcat for the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, the Kact decreases and the Vmax increases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.

Vaccinia virus encodes a eukaryotic-like RNA polymerase composed of two large and six small subunit protein species. A replication-competent virus with 10 histidine codons added to the single endogenous J4R open reading frame was constructed. The altered migration of the 22-kDa subunit of RNA polymerase on SDS-polyacrylamide gel electrophoresis confirmed that J4R encoded the RPO22 subunit and that the mutant virus was genetically stable. The histidine-tagged RNA polymerase bound quantitatively to metal-affinity resins and was eluted in an active form upon addition of imidazole. Glycerol gradient sedimentation of the eluted fraction indicated that most of the RPO22 in infected cells is associated with RNA polymerase. Using stringent washing conditions, metal-affinity chromatography resulted in a several hundred-fold increase in RNA-polymerase-specific activity, and substantially pure enzyme was obtained with an additional conventional chromatography step. When mild conditions were used for washing the metal-affinity resin, the vaccinia virus-encoded capping enzyme, early transcription factor, and nucleoside triphosphate phosphohydrolase I specifically co-eluted with the tagged RNA polymerase, consistent with their physical association. The ability to selectively bind RNA polymerase to an affinity column provided a simple and rapid method of concentrating and purifying active enzyme and protein complexes.

A vaccinia virus-bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus. The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase. The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures. The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3. However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression. Infections using vtsT7 at a low m.o.i. at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter. The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates.

Vaccinia virus infection results in the synthesis of a protein that promotes joint molecule formation and strand-transfer reactions in vitro. We show here that this activity is also expressed by vaccinia DNA polymerase (gpE9L). Recombinant vaccinia polymerase was produced using a hybrid vaccinia/T7 expression system and purified to homogeneity. This protein catalyzed joint molecule formation and strand transfer in vitro in reactions containing single-stranded circular and linear duplex DNAs. The reaction required homologous substrates and magnesium ions and was stimulated by DNA aggregating agents such as spermidine HCl and Escherichia coli single-strand DNA binding protein. There was no requirement for a nucleoside triphosphate cofactor. The reaction ceased when approximately 20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantities of DNA polymerase (0.5-1 molecules of polymerase per double-stranded DNA end). Electron microscopy showed that the joint molecules formed during these reactions contained displaced strands and thus represented the products of a strand-exchange reaction. We also reexamined the link between replication and recombination using a luciferase-based transfection assay and cells infected with DNA polymerase Cts42 mutant viruses. These data substantiate the claim that there exists an inextricable link between replication and recombination in poxvirus-infected cells. Together, these biochemical and genetic data suggest a way of linking poxviral DNA replication with genetic recombination., (Copyright 1999 Academic Press.)

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.

Topoisomerases relax the DNA superhelical tension that arises in cells as a result of several nuclear processes, including transcription, replication and recombination. Recently determined crystal structures of human topoisomerase I in complex with DNA and of the 27 kDa catalytic domain of the vaccinia virus topoisomerase have advanced our understanding of the eukaryotic type IB topoisomerases. These recent structural results provide insights into functional aspects of these topoisomerases, including their DNA binding, strand cleavage and religation activities, as well as the mechanism that these enzymes use to relax DNA superhelical tension. In addition, two proposed models of the anticancer drug camptothecin bound to a covalent complex of human topoisomerase I and DNA suggest a structural basis for the mode of action of the drug.

Defective vaccinia viruses were constructed that express functional Moloney murine leukemia virus-based vector genomes, giving rise to substantial titers of transduction-competent retrovirus particles after infection of a retroviral packaging cell line. For this purpose, the proviral retrovirus genome, engineered into the vaccinia virus mutant, was subjected to several modifications, including the replacement of retroviral promoter sequences by vaccinia virus sequences and the precise fusion of the transcription stop signal downstream of and the removal of such signals within the transcription unit, allowing cytoplasmic transcription of distinct full-length retroviral transcripts. Vaccinia-mediated expression of retroviral vector particles could be observed as early as 3 h postinfection and resulted in stable transduction of NIH/3T3 target cells at higher titers than the control performed by conventional plasmid transfections. Thus at least part of the vaccinia life cycle and retroviral assembly can occur concomitantly. Due to the favorable properties of vaccinia vectors, including high coding capacity, stability, and wide host range, defective vaccinia viral/retroviral chimeric vectors are promising tools for gene therapy applications., (Copyright 1999 Academic Press.)

Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, the hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium-dependent and has a turnover number of 1 s-1. Here we show that purified recombinant Cet1p possesses a robust ATPase activity (Km = 2.8 microM; Vmax = 25 s-1) in the presence of manganese. Cobalt is also an effective cofactor, but magnesium, calcium, copper, and zinc are not. Cet1p displays broad specificity in converting ribonucleoside triphosphates and deoxynucleoside triphosphates to their respective diphosphates. The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs. Mutational analysis establishes that conserved glutamate and arginine side chains within these motifs are essential for the RNA triphosphatase and ATPase activities of Cet1p in vitro and for Cet1p function in vivo. These findings are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel family of phosphohydrolase enzymes with a common active site.

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.

Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea.

As determined by simultaneous monitoring of its four activities, vaccinia virus-coded ribonucleoside diphosphate (rNDP) reductase shows responses to individual nucleoside triphosphate effectors-ATP, dATP, dGTP, and dTTP-similar to those previously reported for rNDP reductase of mouse, which the viral enzyme closely resembles. This investigation uses the vaccinia enzyme as a readily available and convenient model for understanding the cellular enzyme. As previously reported for T4 phage aerobic rNDP reductase, we found the relative activities of ADP, CDP, GDP, and UDP reduction to be reasonably close to the proportions of the four deoxyribonucleotides in the vaccinia virus genome, but only when the four substrates and the four allosteric effectors were all provided at their approximate intracellular concentrations. GDP reductase levels were somewhat higher, proportionately, than the representation of dGMP in vaccinia virus DNA. To understand this behavior and also to evaluate possible relationships between ribonucleotide reductase control and the very low dGTP pools seen in eukaryotic cells, we carried out substrate saturation experiments with a "bioproportional" mixture containing the four rNDP substrates at their relative in vivo concentrations as determined from rNDP pool measurements. Reduction of the two purine substrates was inhibited at high concentrations of this mixture, and data suggest that ADP acts as a specific inhibitor of its own reduction and that of GDP. Use of the four-substrate assay revealed also that a mixture of vaccinia virus R1 protein and mouse R2 protein is catalytically active, making this the first reported chimeric rNDP reductase to show biological activity.

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.

Over the last several years topoisomerases have finally begun to yield to high-resolution structural studies. These models have greatly aided our understanding of the mechanisms of topoisomerase catalysis and drug interactions. This review will cover advances in the structural biology of topoisomerases and discuss their implications for topoisomerase function.

Vaccinia topoisomerase has proven to be an instructive model system for mechanistic studies of the type IB family of DNA topoisomerases. The catalytically relevant functional groups at the active site and the circumferential topoisomerase-DNA interface were correctly surmised by mutational and footprint analysis of vaccinia topoisomerase in advance of structure determinations by X-ray crystallography. It is now evident from multiple crystal structures that the catalytic domains of type IB topoisomerases and site specific recombinases derive from a common ancestral strand transferase capable of forming a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids catalyzes attack of the tyrosine nucleophile on the scissile phosphate. Domain dynamics and DNA-induced conformational changes within the catalytic domain are likely to play a role in triggering strand scission and coordinating the strand exchange or strand passage steps.

Poxvirus-encoded DNA ligases are assumed to play a role in viral DNA replication; however mutational inactivation of vaccinia ligase has not been reported to affect viral growth rates in culture. This communication re-examines this surprising aspect of poxviral biology using both Shope fibroma virus (SFV) and vaccinia virus. SFV and vaccinia ligase deficiencies create essentially identical phenotypes. In particular, ligase-deficient SFV strains are mildly UV sensitive and etoposide resistant, phenotypes previously shown to characterize ligase-deficient vaccinia strains. Moreover, we find that ligase mutations can inhibit the growth of both SFV and vaccinia virus in vitro. The poor growth observed in the absence of a viral ligase is correlated with a two- to tenfold reduction in viral and extragenomic DNA synthesis. This phenotype is host dependent. No differences in viral growth or DNA yield were seen when vaccinia strains were cultured on rabbit (SIRC) cells, but ligase deficiencies reduced growth and DNA yields when vaccinia was plated on BSC-40 cells or SFV on SIRC cells. Despite these replicative defects, mutational inactivation of SFV ligase produced no detectable increase in the number of viral DNA breaks and had no effect on virus-catalyzed extragenomic DNA recombination or UV repair. We conclude that poxviral ligases do play a role in viral DNA replication, but the replicative defect is obscured in some cell lines.

Vaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than approximately 35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates.

We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2'-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2'-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA-protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), as a dramatic elevation of KM at pH values above 7.5. KM values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt from the 5' terminus to a previously unknown site on the VP39 surface.

The recently determined crystal structures of fragments of the human and vaccinia virus type IB topoisomerases reveal unexpected similarity with the lambda family of site-specific recombinases. The conservation of structure suggests a common mechanism, indicating that topoisomerase activity may be the consequence of uncoupling DNA strand cleavage/religation from synapsis.

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.

Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphotyrosine from specific signal-transducing proteins. Although regulatory mechanisms for protein kinases have been described, no general mechanism for controlling PTPs has been demonstrated. Numerous reports have shown that cellular redox status plays an important role in tyrosine phosphorylation-dependent signal transduction pathways. This study explores the proposal that PTPs may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2). Recent reports indicated that H2O2 is transiently generated during growth factor stimulation and that H2O2 production is concomitant with relevant tyrosine phosphorylation. By use of recombinant enzymes, the effects of H2O2 on three PTPs [PTP1, LAR (leukocyte antigen-related), and VHR (vaccinia H1-related)] and three distinct serine/threonine protein phosphatases (PPs: PP2Calpha, calcineurin, and lambda phosphatase) were determined. Hydrogen peroxide had no apparent effect on PP activity. In contrast, PTPs were rapidly inactivated (kinact = 10-20 M-1 s-1) with low micromolar concentrations of H2O2 but not with large alkyl hydroperoxides. PTP inactivation was fully reversible with glutathione and other thiols. Because of the slower rate of reduction, modification occurred even in the presence of physiological thiol concentrations. By utilization of a variety of biochemical techniques including chemical modification, pH kinetic studies, and mutagenesis, the catalytic cysteine thiolate of PTPs was determined to be the selective target of oxidation by H2O2. By use of the electrophilic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl), it was shown that a cysteine sulfenic acid intermediate (Cys-SOH) is formed after attack of the catalytic thiolate on H2O2. A chemical mechanism for reversible inactivation involving a cysteine sulfenic acid intermediate is proposed.

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).

We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers. The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase. The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s). This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25. The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei. Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver. These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily., (Copyright 1998 Academic Press.)

Vaccinia virus RNA polymerase terminates transcription in response to a specific signal UUUUUNU in the nascent RNA. Transduction of this signal to the elongating polymerase requires a trans-acting viral termination factor (VTF/capping enzyme), and is coupled to the hydrolysis of ATP. Recent studies suggest that ATP hydrolysis is catalyzed by a novel termination protein (factor X), which is tightly associated with the elongation complex. Here, we identify factor X as NPH-I (nucleoside triphosphate phosphohydrolase-I), a virus-encoded DNA-dependent ATPase of the DExH-box family. We report that NPH-I serves two roles in transcription (1) it acts in concert with VTF/CE to catalyze release of UUUUUNU-containing nascent RNA from the elongation complex, and (2) it acts by itself as a polymerase elongation factor to facilitate readthrough of intrinsic pause sites. A mutation (K61A) in the GxGKT motif of NPH-I abolishes ATP hydrolysis and eliminates the termination and elongation factor activities. Related DExH proteins may have similar roles at postinitiation steps during cellular mRNA synthesis.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.

3-beta-Hydroxysteroid dehydrogenase (3-beta-HSD) activity coded for by the A44L gene of vaccinia virus (VV) was demonstrated in CV-1 cultures infected by all five VV strains tested, viz. WR, Praha virus, DRYVAX Wyeth-derived virus (DD), LIVP and MVA. Deletion of the A44L gene in two Praha virus-derived clones (the moderately virulent P13 and the highly attenuated P20), the WR and DD viruses resulted in absence of 3-beta-HSD activity from infected cultures. The virulence for mice of P13 was not affected, and that of WR was only slightly decreased, by the A44L gene deletion. Recombinant VVs expressing either varicella-zoster virus glycoprotein E (VZV-gE) or hepatitis B virus preS2-S protein (HBV-preS2-S) and their respective A44L deleted mutants were used in immunogenicity tests in mice. In terms of antibody response to VV and the recombinant proteins, the deletion resulted in a lowering the immunogenicity in the moderately virulent clone P13 virus and its progenies. In the highly attenuated P20 and DD viruses and their progenies no effects were apparent.

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.

p37, the major protein of the extracellular enveloped form of vaccinia virus, is involved in the biogenesis of the viral double membrane and in egress of virus from the cell. p37 was expressed as a glutathione S-transferase fusion protein and was purified to homogeneity by silver staining using glutathione-agarose, Sephacryl S-200, and DEAE-cellulose chromatography. Incubation of p37 with phosphatidylcholine labeled in the fatty acyl side chains resulted in the production of multiple lipid products that were identified by thin layer chromatography and mass spectrometry as diacylglycerol, free fatty acid, monoacylglycerol, and lysophosphatidylcholine. Lipid-metabolizing activities colocalized with p37-containing fractions throughout the chromatographic steps. p37 also metabolized phosphatidylethanolamine efficiently, but it had less activity toward phosphatidylinositol and little or no activity toward phosphatidylserine. The purified enzyme also metabolized triacylglycerol to diacylglycerol but was inactive toward sn-1, 2-diacylglycerol. p37 was also expressed in insect cells as a poly-His fusion protein; cell lysates and partially purified proteins also generated products expected from phospholipase C and A activities. Thus, p37 is a broad specificity lipase with phospholipase C, phospholipase A, and triacylglycerol lipase activities.

We have analyzed the elongation properties of vaccinia virus RNA polymerase during a single round of transcription in vitro. RNA-labeled ternary complexes were halted at a unique template position located upstream of a T-run (TTTTTTTTT) in the nontemplate strand; this element encodes an RNA signal for factor-dependent transcription termination at distal sites on the template. The halted ternary complexes were purified and allowed to resume elongation under a variety of conditions. We found that the T-run constituted a strong elongation block, even at high nucleotide concentrations. The principal sites of pausing were at a C position situated two nucleotides upstream of the first T in the T-run and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. Intrinsic pausing was exacerbated at suboptimal nucleotide concentrations. Ternary complexes arrested by the T-run at 10 microM NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 microM) resulted in polymerase stuttering at the 3' margin of the T-run, immediately prior to a templated G position; this generated a ladder of slippage synthesis products. We found that vaccinia ternary complexes remained intact after elongating to the very end of a linear DNA template and that such complexes catalyzed the addition of extra nucleotides to the 3' end of the RNA chain. The 3' end addition required much higher concentrations of NTPs than did templated chain elongation. Finally, we report that factor-dependent transcription termination by vaccinia RNA polymerase downstream of the T-run was affected by nucleotide concentration. Limiting UTP caused the polymerase to terminate at sites closer to the UUUUUNU termination signal. This is consistent with the kinetic coupling model for factor-dependent termination.

VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5'-rU2-N15-rU-3' motif (where N = any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29-30 nucleotides (nt), or greater, in length. Nine residues separate the 3'-most ribouridylate of the optimally positioned motif from the primer 3'-OH. A ribose sugar at the extreme 3'-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position -3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU2-N15-rU motif was identified within the uridylate-rich 3' end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the approximately 30-35-nt burst observed with the naturally occurring 3' end.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.

Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein-DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3'-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2' OH of the ribose sugar to yield a free 2', 3' cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.

We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554-4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis. In vitro assays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

A cDNA library enriched for human fetal-specific liver genes was constructed by suppressive subtractive hybridization. EST fls223 generated from this library was found to represent a novel putative serine/threonine (Ser/Thr) kinase. A full-length clone isolated for this gene encodes a protein of 396 amino acids. The amino acid sequence has 40% identity over 305 amino acids with the B1R Ser/Thr protein kinase of vaccinia virus. This gene has therefore been named VRK1 (vaccinia virus B1R kinase related kinase). VRK1 was also found to have sequence identity (62.0% over 481 nucleotides) to a database EST. A full-length clone for this EST was isolated and sequenced. Conceptual translation predicts a protein of 508 amino acids that, like VRK1, has similarity to B1R kinase (38.7% identity over 300 amino acids). This gene has been named VRK2. Comparison of VRK1 with VRK2 indicates that they encode structurally related putative Ser/Thr protein kinases. Northern analysis shows that expression of both genes is widespread and elevated in highly proliferative cells, such as testis, thymus, and fetal liver. B1R kinase is reported to be essential for DNA replication of vaccinia virus. The similarity of VRK1 and VRK2 to B1R indicates that these genes may have similar functions., (Copyright 1997 Academic Press.)

The interferon (IFN)-induced enzyme RNase L produced by a recombinant vaccinia virus (VV) causes death of mammalian cells with morphological and biochemical characteristics of apoptosis. Coexpression of 2-5A-synthetase enhances apoptosis induced by RNase L Activation of endogenous RNase L by infection with a VV ts mutant (ts22) or with wild-type virus in the presence of the antipoxvirus drug isatin-beta-thiosemicarbazone, a treatment known to significantly increase the amount of double-stranded RNA late during infection, also causes pronounced apoptosis of infected cells. The effects observed with recombinant virus-derived RNase L or with the endogenous enzyme are specific, since apoptosis also occurs in cells derived from mice lacking the IFN-induced protein kinase (PKR). The apoptosis antagonist Bcl-2 prevents induction of cell death by RNase L activation. Apoptosis of mammalian cells by RNase L activation could be a mechanism mediating anticellular actions of IFN.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.

VP39 is a single-domain, bifunctional viral protein, which acts at both ends of nascent mRNA. At the 5' end, it acts as a cap-specific 2'-O-methyltransferase. At the 3' end, it acts as a poly(A) polymerase processivity factor, requiring its direct association with poly(A) polymerase. Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase function to be juxtaposed around a superficial cleft on the protein surface, surface regions required for VP39's mRNA 3' end modifying functions are not known. Here, we identify a surface region that interfaces directly with poly(A) polymerase, taking three independent approaches: (i) development of a direct in vitro dimerization assay, which is applied to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization and further mutagenesis based upon these data; (iii) site-specific photo-cross-linking of VP39 to VP55. We find that the dimerization interface lies on a surface region remote from the methyltransferase cleft and contains a 3-5-residue "hot-spot," which is very sensitive to amino acid substitutions. Various other sites within VP39 consistently became hypersensitive to protease cleavage upon interaction with VP55, indicating the occurrence of extensive conformational changes.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Vaccinia virus DNA ligase repairs nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand. This study addresses the ability of vaccinia DNA ligase to seal nicked substrates containing one or more RNA strands. We found that the viral enzyme rapidly and efficiently joined a 3'-OH RNA to 5'-phosphate DNA when the reacting polynucleotides were annealed to a bridging DNA strand. In contrast, ligation of 3'-OH DNA to 5'-phosphate RNA was slow (0.2% of the rate of RNA-to-DNA ligation) and entailed the accumulation of high levels of RNA-adenylate intermediate. A native gel mobility shift assay showed that vaccinia DNA ligase discriminates at the substrate binding step between ligands containing 5'-phosphate DNA versus 5'-phosphate RNA at the nick. The enzyme displayed weak activity in RNA-to-RNA ligation on a bridging DNA template (0.01% of RNA-to-DNA activity). Vaccinia DNA ligase was incapable of joining two DNAs annealed on an RNA template. These results can be explained by a requirement for B-form helical conformation on the 5'-phosphate side of the nick. The robust RNA-to-DNA strand joining activity underscores the potential for vaccinia DNA ligase to catalyze RNA-based integration of host cell genetic information into the genome of cytoplasmic poxviruses.

We have previously shown that the purified, 116-kDa DNA polymerase encoded by vaccinia virus is inherently distributive, synthesizing only a few nucleotides per template binding event under moderate reaction conditions (W. F. McDonald and P. Traktman, J. Biol. Chem. 269, 31190-31197). These properties would be incompatible with efficient DNA replication in vivo and suggest that the polymerase most probably interacts with accessory proteins that stabilize the template/polymerase interaction. Here we show that a highly processive form of the enzyme is indeed present with cytoplasmic lysates prepared from infected cells, and demonstrate that this form of the enzyme is likely to comprise the DNA polymerase in association with an early viral protein with a native molecular weight of approximately 48K.

Vaccinia DNA topoisomerase, a 314 amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. To identify amino acids that participate in the DNA binding and transesterification steps, we introduced alanine substitutions at 18 positions within a centrally located 27 amino acid segment (181-RLYKPLLKLTDDSSPEEFLFNKLSERK-207) and at 8 positions near the N-terminus (1-MRALFYKDGK-10). All mutant proteins except two displayed wild-type activity in relaxing supercoiled DNA. F200A and S204A exhibited reduced rates of relaxation and were subjected to a kinetic analysis of the strand cleavage reaction under single-turnover and equilibrium conditions. The F200A and S204A mutations reduced the rate of single-turnover DNA cleavage by factors of 5 and 70, respectively. Both mutations shifted the cleavage-religation equilibrium in favor of the noncovalently bound state. The S204A mutation reduced the affinity of topoisomerase for CCCTT-containing DNA, but did not alter the site-specificity of DNA cleavage. Vaccinia residue Ser-204, which is conserved in all poxvirus topoisomerases, but not in the cellular homologues, may contribute to the unique cleavage site specificity of the poxvirus enzymes. Phe-200 is conserved in all members of the type IB topoisomerase family.

Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3'-phosphotyrosyl) protein adduct at the sequence 5'-CCCTT downward arrow. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5' and RNA 3' of the scissile bond. However, a 36-mer composed of RNA 5' and DNA 3' of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5'-OH-terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5' end of RNA in vitro. Religation of the covalently bound CCCTT-containing DNA strand to a 5'-OH-terminated DNA acceptor is efficient and rapid (krel > 0.5 s-1), provided that the acceptor DNA is capable of base pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5' nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5' end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is as follows: +1 insertion > -1 deletion > +2 insertion >> -2 deletion. These findings underscore the importance of a properly positioned 5'-OH terminus in transesterification reaction chemistry, but they also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.

Vaccinia topoisomerase relaxes DNA through the formation of a covalent DNA-(3'-phosphotyrosyl)protein intermediate at sites containing the sequence 5'-(T/C)CCTT/. The active site, Tyr-274, is situated near the carboxyl terminus of the 314 amino acid enzyme. Here, we report the effects of serial C-terminal deletions. Removal of five amino acids had no effect on topoisomerase activity. However, deletion of 10, 15, or 20 amino acids rendered the enzyme distributive in DNA relaxation, incrementally slowed the rate of single-turnover DNA cleavage, and progressively diminished DNA binding affinity, without altering the sequence specificity of DNA cleavage. These effects lead us to speculate that the region downstream of the active site, which is not well-conserved among the poxvirus-encoded topoisomerases, is a component of the proposed circumferential interface between the enzyme and duplex DNA.