Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.     

Mutations in the herpes simplex virus DNA polymerase gene can confer resistance to 9-beta-D-arabinofuranosyladenine.

Mutants of herpes simplex virus type 1 resistant to the antiviral drug 9-beta-D-arabinofuranosyladenine (araA) have been isolated and characterized. AraA-resistant mutants can be isolated readily and appear at an appreciable frequency in low-passage stocks of wild-type virus. Of 13 newly isolated mutants, at least 11 were also resistant to phosphonoacetic acid (PAA). Of four previously described PAA-resistant mutants, two exhibited substantial araA resistance. The araA resistance phenotype of one of these mutants, PAAr5, has been mapped to the HpaI-B fragment of herpes simplex virus DNA by marker transfer, and araA resistance behaved in marker transfer experiments as if it were closely linked to PAA resistance, a recognized marker for the viral DNA polymerase locus. PAAr5 induced viral DNA polymerase activity which was much less susceptible to inhibition by the triphosphate derivative of araA than was wild-type DNA polymerase. These genetic and biochemical data indicate that the herpes simplex virus DNA polymerase gene is a locus which, when mutated, can confer resistance to araA and thus that the herpes simplex virus DNA polymerase is a target for this antiviral drug.     

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.     

Isolation of vaccinia virus mutants capable of replicating independently of the host cell nucleus

alpha-Amanitin-resistant vaccinia virus mutants were isolated after serial viral passages in BSC-40 cells that were carried out in the presence of inhibitory levels (6 micrograms/ml) of alpha-amanitin. One such mutant, alpha-27, was highly refractory (greater than 95%) to alpha-amanitin-mediated inhibition and was selected for further study. In the absence of drug, the phenotypes of alpha-27 and wild-type vaccinia virus were indistinguishable with respect to growth kinetics. DNA synthesis, protein synthesis, and morphogenesis. Infections in the presence of alpha-amanitin revealed two striking differences, however. First, wild-type virus was unable to catalyze proteolytic processing of the two major capsid proteins VP62 and VP60, whereas alpha-27 was most efficient at this process. Second, wild-type viral morphogenesis within the infected cells was arrested by alpha-amanitin at an apparently analogous step to that previously described for enucleated cells. This observation was supported by the ability of alpha-27 virus to replicate in enucleated BSC-40 cells. Restriction enzyme analyses of alpha-27 versus wild-type genomes revealed that a XhoI cleavage site was altered in the alpha-27 DNA molecule, suggesting a possible location for the alpha-amanitin resistance locus.     

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.     

Preferential Inhibition of Herpes-Group Viruses by Phosphonoacetic Acid: Effect on Virus DNA Synthesis and Virus-Induced DNA Polymerase Activity

In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.     

The human cytomegalovirus UL97 protein kinase,an antiviral drug target,is required at the stage of nuclear egress

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.     

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.     

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

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.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

Vaccinia DNA ligase complements Saccharomyces cerevisiae cdc9, localizes in cytoplasmic factories and affects virulence and virus sensitivity to DNA damaging agents.

The functional compatibility of vaccinia virus DNA ligase with eukaryotic counterparts was demonstrated by its ability to complement Saccharomyces cerevisiae cdc9. The vaccinia DNA ligase is a 63 kDa protein expressed early during infection that is non-essential for virus DNA replication and recombination in cultured cells. This implies complementation by a mammalian DNA ligase, yet no obvious recruitment of host DNA ligase I from the nucleus to the cytoplasm was observed during infection. An antiserum raised against a peptide conserved in eukaryotic DNA ligases identified the virus enzyme in discrete cytoplasmic 'factories', the sites of virus DNA synthesis, demonstrating immunological cross-reactivity between host DNA ligase I and the vaccinia enzyme. DNA ligase was not detected in the factories of a mutant virus lacking the ligase gene. Despite this, no difference in growth between wild-type (WT) and mutant virus was detectable even in Bloom's syndrome cells which have reduced DNA ligase I activity. However, DNA ligase negative virus showed an increased sensitivity to UV or bleomycin in cultured cells, and the importance of DNA ligase for virus virulence in vivo was demonstrated by the attenuated phenotype of the deletion mutant in intranasally infected mice.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.