Molecular genetic analysis of VRK1 in mammary epithelial cells: depletion slows proliferation in vitro and tumor growth and metastasis in vivo.

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family. VRK1, a ser/thr kinase with a nuclear localization, is the most well-studied paralog and has been described as a proproliferative protein. In lower eukaryotes, a loss of VRK1 activity is associated with severe mitotic and meiotic defects. Mice that are hypomorphic for VRK1 expression are infertile, and depletion of VRK1 in tissue culture cells can impair cell proliferation and alter several signaling pathways. VRK1 has been implicated as part of a 'gene-expression signature' whose overexpression correlates with poor clinical outcome in breast cancer patients. We present here our investigation of the role of VRK1 in the growth of normal (MCF10) and malignant (MDA-MB-231) human mammary epithelial cells, and demonstrate that shRNA-mediated depletion of VRK1 slows their proliferation significantly. Conversely, stable overexpression of a FLAG-tagged VRK1 transgene imparts a survival advantage to highly malignant MDA-MB-231 cells under conditions of nutrient and growth factor deprivation. Moreover, in a murine orthotopic xenograft model of breast cancer, we demonstrate that tumors depleted of VRK1 show a 50% reduction in size from 4-13 weeks postengraftment. The incidence and burden of distal metastases in the lungs and brain was also significantly reduced in mice engrafted with VRK1-depleted cells. These studies demonstrate that VRK1 depletion or overexpression has an impact on the proliferation and survival of cell lines derived from normal or malignant mammary tissue, and moreover show that depletion of VRK1 in MDA-MB-231 cells reduces their oncogenic and metastatic properties in vivo.

The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase.

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl(2) or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of approximately 48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent approximately 45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA(-) phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308-12318, 2001).

Clustered charge-to-alanine mutagenesis of the vaccinia virus A20 gene: temperature-sensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase activity.

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298-12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA(-) phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins.

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.

Vaccinia virus blocks gamma interferon signal transduction: viral VH1 phosphatase reverses Stat1 activation.

We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-gamma) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-gamma inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-gamma. While phosphorylation of other proteins in the IFN-gamma pathway was not affected, activation of Stat1 by other ligand-receptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na(3)VO(4) but not to okadaic acid. Addition of Na(3)VO(4) but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-gamma signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-gamma signal cascade through the dephosphorylating activity of the viral phosphatase VH1.

Elucidating the essential role of the A14 phosphoprotein in vaccinia virus morphogenesis: construction and characterization of a tetracycline-inducible recombinant.

We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-beta-D-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of "empty" crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.

Clustered charge-to-alanine mutagenesis of the vaccinia virus H5 gene: isolation of a dominant, temperature-sensitive mutant with a profound defect in morphogenesis.

The vaccinia virus H5 gene encodes a 22.3-kDa phosphoprotein that is expressed during both the early and late phases of viral gene expression. It is a major component of virosomes and has been implicated in viral transcription and, as a substrate of the B1 kinase, may participate in genome replication. To enable a genetic analysis of the role of H5 during the viral life cycle, we used clustered charge-to-alanine mutagenesis in an attempt to create a temperature-sensitive (ts) virus with a lesion in the H5 gene. Five mutant viruses were isolated, with one of them, tsH5-4, having a strong ts phenotype as assayed by plaque formation and measurements of 24-h viral yield. Surprisingly, no defects in genome replication or viral gene expression were detected at the nonpermissive temperature. By electron microscopy, we observed a profound defect in the early stages of virion morphogenesis, with arrest occurring prior to the formation of crescent membranes or immature particles. Nonfunctional, "curdled" virosomes were detected in tsH5-4 infections at the nonpermissive temperature. These structures appeared to revert to functional virosomes after a temperature shift to permissive conditions. We suggest an essential role for H5 in normal virosome formation and the initiation of virion morphogenesis. By constructing recombinant genomes containing two H5 alleles, wild type and H5-4, we determined that H5-4 exerted a dominant phenotype. tsH5-4 is the first example of a dominant ts mutant isolated and characterized in vaccinia virus.

Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase.

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.

Characterization of the single-stranded DNA binding protein encoded by the vaccinia virus I3 gene.

The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in Escherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.

The vaccinia virus I1 protein is essential for the assembly of mature virions.

The product of the vaccinia virus I1 gene was characterized biochemically and genetically. This 35-kDa protein is conserved in diverse members of the poxvirus family but shows no homology to nonviral proteins. We show that recombinant I1 binds to both single-stranded and double-stranded DNA in a sequence-nonspecific manner in an electrophoretic mobility shift assay. The protein is expressed at late times during infection, and approximately 700 copies are encapsidated within the virion core. To determine the role of the I1 protein during the viral life cycle, a inducible viral recombinant in which the I1 gene was placed under the regulation of the Escherichia coli lac operator/repressor was constructed. In the absence of isopropyl-beta-D-thiogalactopyranoside, plaque formation was abolished and yields of infectious, intracellular virus were dramatically reduced. Although all phases of gene expression and DNA replication proceeded normally during nonpermissive infections, no mature virions were produced. Electron microscopic analysis confirmed the absence of mature virion assembly but revealed that apparently normal immature virions accumulated. Thus, I1 is an encapsidated DNA-binding protein required for the latest stages of vaccinia virion morphogenesis.

Characterization of a processive form of the vaccinia virus DNA polymerase.

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.

Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors.

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.

Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates.

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of > or = 200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.

The dual-specificity phosphatase encoded by vaccinia virus, VH1, is essential for viral transcription in vivo and in vitro.

The genetic complexity of vaccinia virus is such that as well as encoding its own transcription and replication machinery, it encodes two protein kinases and a protein phosphatase. The latter enzyme, designated VH1, is a prototype for the dual-specificity class of phosphatases. Here we report that the H1 phosphatase is encapsidated within vaccinia virions and describe the construction of a viral recombinant in which expression of the H1 gene is regulated by the presence or absence of isopropylthiogalactopyranoside (IPTG) in the culture medium. When expression of H1 is repressed, the number of viral particles produced is not compromised but the fraction of these particles which is infectious is significantly reduced. The lack of infectivity of the H1-deficient particles is specifically correlated with their inability to direct the transcription of early genes either in vitro or in vivo. A proximal role for the viral phosphatase in regulating the onset of viral gene expression is implied. Prominent among the encapsidated proteins found to be hyperphosphorylated in H1-deficient virions is the 11-kDa product of the F18 gene; this protein is the major DNA-binding component of the viral nucleoprotein complex. The ability of recombinant H1 phosphatase to reverse this hyperphosphorylation in permeabilized virions strengthens the conclusion that the F18 protein is a bona fide substrate for the H1 phosphatase.

Temperature-sensitive mutants with lesions in the vaccinia virus F10 kinase undergo arrest at the earliest stage of virion morphogenesis.

Vaccinia virus encodes two protein kinases; the B1 kinase is expressed early and appears to play a role during DNA replication, whereas the F10 kinase is expressed late and is encapsidated in virions. Here we report that the F10 kinase gene is the locus affected in a complementation group of temperature-sensitive mutants composed of ts15, ts28, ts54, and ts61. Although these mutants have a biochemically normal phenotype at the nonpermissive temperature, directing the full program of viral gene expression, they fail to form mature virions. Electron microscopic analysis indicates that morphogenesis undergoes arrest at a very early stage, prior to the formation of membrane crescents or immature virions. An essential role for the F10 protein kinase in orchestrating the onset of virion assembly is implied.

The vaccinia virus D5 protein, which is required for DNA replication, is a nucleic acid-independent nucleoside triphosphatase.

The vaccinia virus D5 gene encodes a 90-kDa protein that is transiently expressed at early times after infection. Temperature-sensitive mutants with lesions in the D5 gene exhibit a fast-stop DNA- phenotype and are also impaired in homologous recombination. Here we report the overexpression of the D5 protein within the context of a vaccinia virus infection and its purification to apparent homogeneity. The purified protein has an intrinsic nucleoside triphosphatase activity which is independent of, and not stimulated by, any common nucleic acid cofactors. All eight common ribo- and deoxyribonucleoside triphosphates are hydrolyzed to the diphosphate form in the presence of a divalent cation. Implications for the role of D5 in viral DNA replication are addressed.

Identification and characterization of the orf virus type I topoisomerase.

Vaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively, encode type I DNA topoisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT.

Vaccinia virus DNA polymerase. In vitro analysis of parameters affecting processivity.

The polymerization and proofreading activities of the vaccinia virus DNA polymerase reside within a 116-kDa catalytic polypeptide. We report here an investigation of the intrinsic processivity of this enzyme on both natural and homopolymeric DNA templates. Inclusion of the Escherichia coli helix destabilizing protein allowed the viral enzyme, which lacks strand displacement activity, to utilize a singly primed M13 DNA template. In the presence of either 10 mM MgCl2 or 1 mM MgCl2 + 40 mM NaCl, synthesis was achieved in a highly distributive manner. RFII formation required a significant excess of enzyme, and < or = 10 nucleotides (nt) were added per primer-template binding event. The apparent rate of primer elongation varied with the enzyme/template ratio and reached a maximum of 8 nt/s. A similar lack of processivity was observed on a poly(dA390)-oligo(dT12-18) template. In contrast, highly processive synthesis was achieved on both templates in the presence of 1 mM MgCl2 and the absence of NaCl. A primer extension rate of 30 nt/s was observed, and > or = 2000 nt were added per binding event. These studies suggest that the catalytic polypeptide of the vaccinia virus DNA polymerase will require accessory protein(s) to form a stable enzyme-template interaction and direct processive DNA synthesis under isotonic conditions in vivo.

Overexpression and purification of the vaccinia virus DNA polymerase.

We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.

Biochemical analysis of mutant alleles of the vaccinia virus topoisomerase I carrying targeted substitutions in a highly conserved domain.

The 32-kDa topoisomerase I encoded by vaccinia virus relaxes supercoiled DNA in a manner which is mechanistically equivalent to that utilized by eucaryotic enzymes. Its amino acid sequence contains significant homology to the enzymes encoded by Saccharomyces cerevisiae, Saccharomyces pombe, human cells, and other poxviruses. The small size of the viral enzyme, and its essentiality in the viral life cycle, make it ideally suited for structural and functional analysis. In this report we present the construction and analysis of 15 mutant alleles of the topoisomerase containing amino acid substitutions in a highly conserved region. The enzymes encoded by these alleles were expressed in Escherichia coli and various parameters of their activity were examined. All of the alleles which show diminished (seven alleles) or abrogated (three alleles) DNA relaxation activity are deficient in DNA cleavage and the concomitant formation of the covalent enzyme/DNA intermediate. None are deficient in the prior step of noncovalent interaction with substrate DNA. Five of the mutant enzymes show significant temperature sensitivity in vitro. The extent of in vitro activity of the enzymes shows a good but incomplete correlation with the enzymes' abilities to lethally induce the resident lambda prophage within E. coli BL21(DE3) (via illegitimate recombination). Mutations in 1 amino acid, in particular, impair prophage induction in vivo more significantly than DNA relaxation in vitro. In sum, these studies suggest that this region of the topoisomerase (amino acids 216-225) plays a proximal role in mediating DNA cleavage and the covalent interaction between the 3'-phosphoryl of the nicked DNA and tyrosine 274 of the vaccinia topoisomerase I. The studies also provide useful reagents for the molecular genetic analysis of the role of the topoisomerase within the context of vaccinia virus infection.