The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants.

Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines. Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication. When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Construction of recombinant vaccinia viruses using PUV-inactivated virus as a helper

Recombinant vaccinia viruses (VVs) are widely used as expression vectors in molecular biology and immunology and are now under evaluation for gene therapy. The current techniques for inserting foreign DNA into the large VV genome are based on either homologous recombination between transfer plasmids and VVgenomes or direct DNA ligation and packaging using replication-deficient poxviruses. Here, we describe efficient new versions of both methods that produce 90%-100% of the recombinant viruses. In the new homologous recombination method, VV DNA "arms" obtained by NotI digestion and intact transfer plasmids were used for co-transfection. In the direct DNA ligation method, foreign DNA was inserted into a unique NotI restriction site of the VVgenome. In both methods, the generation of recombinant viruses was carried out in cells infected with a non-replicating, psoralen-UV (PUV)-inactivated helper VV. The convenience of these new techniques is demonstrated by the construction of recombinant VVs that produce E. coli beta-galactosidase. An important feature of these strategies is that any VV strain can be used as a helper virus after PUV inactivation.     

Protection of Mice against Lethal Coxsackievirus B3 Infection by Using DNA Immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.     

5-Variable genome sequence of the LIVP vaccinia virus. A possible role for short direct repeats in the formation of deletions]

HindIII-O/N DNA fragments of vaccinia virus (VV) of the LIVP strain were mapped using thirteen restriction endonucleases. Nucleotide sequences of the HindIII-O fragment (1530 bp) as well as of a site of the HindIII-N genome fragment 353 bp in size were determined. Comparison of restriction maps and nucleotide sequences of VV strains (WR and LIVP) demonstrated that DNA of VV LIVP contained % deletions and 2 insertions. "Reliable" short direct repeats were localized and their possible role in formation of DNA deletions was shown. It was suggested that VV endonuclease and DNA-ligase participate in replication and repair processes. Mechanism of formation of variable sequences of viral genomes is discussed.     

Physical mapping of DNA-temperature-sensitive mutations of vaccinia virus

Four DNA-temperature-sensitive (ts) mutations were mapped in the genome of vaccinia virus (VV). Physical mapping of these mutations was performed by restriction analysis of the genomes of recombinants between VV DNA- ts mutants and ectromelia virus as well as by the marker rescue with cloned restriction fragments of VV DNA. One of the mutations was mapped on the HindIII-E-fragment. Biochemical studies of this mutant indicate that the mutation is not in the DNA polymerase gene which is located on the same fragment. The other three mutations were mapped in a 10 kilobase region in the middle of the HindIII-D-fragment. As shown previously, these mutations inactivate different genes, and the products of these genes participate directly in the DNA synthesis. Thus, at least three proteins involved in the VV DNA synthesis are encoded by neighboring genes in the central part of the viral genome.     

Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus

Modified vaccinia virus Ankara (MVA) is an attenuated strain of vaccinia virus (VV) that has attracted significant attention as a candidate viral vector vaccine for immunization against infectious diseases and treatment of malignancies. Although MVA is unable to replicate in most nonavian cells, vaccination with MVA elicits immune responses that approximate those seen after the administration of replication-competent strains of VV. However, the mechanisms by which these viruses elicit immune responses and the determinants of their relative immunogenicity are incompletely understood. Studying the interactions of VV and MVA with cells of the human immune system may elucidate these mechanisms, as well as provide a rational basis for the further enhancement of the immunogenicity of recombinant MVA vectors. Toward this end, we investigated the consequences of MVA or VV infection of human dendritic cells (DCs), key professional antigen-presenting cells essential for the generation of immune responses. We determined that a block to the formation of intracellular viral replication centers results in abortive infection of DCs with both VV and MVA. MVA inhibited cellular protein synthesis more rapidly than VV and displayed a distinct pattern of viral protein expression in infected DCs. MVA also induced apoptosis in DCs more rapidly than VV, and DC apoptosis after MVA infection was associated with an accelerated decline in the levels of intracellular Bcl-2 and Bcl-X(L). These findings suggest that antigen presentation pathways may contribute differentially to the immunogenicity of VV and MVA and that targeted modifications of virus-induced DC apoptosis may further increase the immunogenicity of MVA-vectored vaccines.     

Vaccinia and ectromelia recombinant viruses, causing an infection, characteristic for ectromelia, in mice

Ten recombinants between the viruses of vaccinia and ectromelia were isolated that cause the ectromelia virus specific lesions in mice. The structure of recombinant viral genomes, the efficiency of viral propagation in mice, the nature of lesions induced by viruses have been studied. Eight of obtained recombinants have a DNA insertion originating from the right end of ectromelia viral genome, nine recombinants have an insertion originating from the left end, seven recombinants possess both insertions. The latter recombinants have more pronounced pathogenicity for mice. Both revealed regions are supposed to define the specific pathogenicity of ectromelia virus for mice.     

Identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.     

The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses

Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related.     

The adenovirus E3 14.7-kilodalton protein which inhibits cytolysis by tumor necrosis factor increases the virulence of vaccinia virus in a murine pneumonia model.

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.     

Puromycin resistance (pac) gene as a selectable marker in vaccinia virus

The antibiotic puromycin, an inhibitor of protein synthesis, was shown to inhibit vaccinia virus (VV) replication. We evaluated the use of puromycin-resistance (pac) gene as a selectable marker in VV. A recombinant vaccinia virus expressing pac (VV-pac) under the control of a viral early/late promoter was constructed and characterized. VV-pac grew in the presence of puromycin at concentrations that were inhibitory for the parental VV and toxic for the cells. Isolation of recombinant VV usually relies on plaque purification under selective conditions. Because virus plaquing was not feasible under inhibitory puromycin concentration, a protocol based on serial passage of virus was devised. The usefulness of this procedure in selecting pac expressing viruses was tested by isolating a recombinant VV.     

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses.

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.