Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.     

Analysis of the adenovirus type 5 terminal protein precursor and DNA polymerase by linker insertion mutagenesis.

A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late vaccinia virus promoter. The recombinant viruses were used for overexpression of the mutant genes in HeLa cells. In total, 22 different mutant pTP and 10 different Ad pol vaccinia virus recombinants were constructed, including some that expressed carboxyl-terminus-truncated forms of both proteins and one that produced the mutant H5ts149 Ad pol. To investigate the structure-function relationships of both proteins, extracts from cells infected with the recombinant viruses were tested for in vitro complementation of the initiation and elongation steps in adenovirus DNA replication. The results were in accordance with those of earlier in vivo experiments with these insertion mutants and indicate that multiple regions of both proteins are essential for adenovirus DNA replication. The carboxyl termini of both pTP and Ad pol were shown to be essential for proper functioning of these proteins during initiation of adenovirus DNA replication. Three different DNA replication-negative pTP mutants were shown to have residual activity in the initiation assay, suggesting not only that pTP is required for initiation but also that it may play a role in DNA replication after the deoxycytidylation step.     

IPTG-dependent vaccinia virus: identification of a virus protein enabling virion envelopment by Golgi membrane and egress.

A novel method has been developed to study the functional roles of individual vaccinia virus gene products that is neither limited by the possible essentiality of the target gene nor by the availability of conditional lethal mutants. The system utilises the E. coli lac repressor protein, the operator sequence to which it binds and the specific inducer IPTG. It allows the generation of recombinant viruses in which the expression of any chosen gene, and hence virus replication, can be externally controlled. In principle, this system is broadly applicable to the functional analysis of genes in any large DNA virus. This approach has demonstrated that the gene encoding the 14 kDa membrane protein of vaccinia virus is non-essential for the production of infectious intracellular virus particles, but essential for the envelopment of intracellular virions by Golgi membrane and for egress of mature extracellular viral particles. This is the first vaccinia virus protein shown to be specifically required for these processes. In vivo this system may prove useful as a means of attenuating recombinant vaccinia virus vaccines by preventing virus spread without reducing the amount of the foreign antigen expressed in each infected cell. Attenuation of other live virus vaccines may be developed in a similar way.     

The cytoplasmic and transmembrane domains of the vaccinia virus B5R protein target a chimeric human immunodeficiency virus type 1 glycoprotein to the outer envelope of nascent vaccinia virions.

The outer envelope of the extracellular form of vaccinia virus (EEV) is derived from the Golgi membrane and contains at least six viral proteins. Transfection studies indicated that the EEV protein encoded by the B5R gene associates with Golgi membranes when synthesized in the absence of other viral products. A domain swapping strategy was then used to investigate the possibility that the B5R protein contains an EEV targeting signal. We constructed chimeric genes encoding the human immunodeficiency virus (HIV) type 1 glycoprotein with the cytoplasmic and transmembrane domains replaced by the corresponding 42-amino-acid C-terminal segment of the B5R protein. Recombinant vaccinia viruses that stably express a chimeric B5R-HIV protein or a control HIV envelope protein with the original cytoplasmic and transmembrane domains were isolated. Cells infected with recombinant vaccinia viruses that expressed either the unmodified or the chimeric HIV envelope protein formed syncytia with cells expressing the CD4 receptor for HIV. However, biochemical and microscopic studies demonstrated that the HIV envelope proteins with the B5R cytoplasmic and transmembrane domains were preferentially targeted to the EEV. These data are consistent with the presence of EEV localization signals in the cytoplasmic and transmembrane domains of the B5R protein.     

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.     

Expression of structural proteins of hepatitis C virus (HCV) in mammalian cells

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Vaccinia virus nicking-joining enzyme is encoded by K4L (VACWR035)

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity.     

High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system.     

Conditional expression of foreign genes by temperature-sensitive mutants of vaccinia virus

To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.     

Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpIII, and herpes simplex virus type 1 glycoprotein H.

An open reading frame with the characteristics of a glycoprotein-coding sequence was identified by nucleotide sequencing of human cytomegalovirus (HCMV) genomic DNA. The predicted amino acid sequence was homologous with glycoprotein H of herpes simplex virus type 1 and the homologous protein of Epstein-Barr virus (BXLF2 gene product) and varicella-zoster virus (gpIII). Recombinant vaccinia viruses that expressed this gene were constructed. A glycoprotein of approximately 86 kilodaltons was immunoprecipitated from cells infected with the recombinant viruses and from HCMV-infected cells with a monoclonal antibody that efficiently neutralized HCMV infectivity. In HCMV-infected MRC5 cells, this glycoprotein was present on nuclear and cytoplasmic membranes, but in recombinant vaccinia virus-infected cells it accumulated predominantly on the nuclear membrane.     

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.     

Repression of Vaccinia Virus Holliday Junction Resolvase Inhibits Processing of Viral DNA into Unit-Length Genomes

The vaccinia virus A22R gene encodes a protein that is homologous to the bacterial enzyme RuvC and specifically cleaves and resolves four-way DNA Holliday junctions into linear duplex products. To investigate the role of the vaccinia virus Holliday junction resolvase during an infection, we constructed two recombinant viruses: vA22-HA, which has a short C-terminal epitope tag appended to the A22R open reading frame, and vA22i, in which the original A22R gene is deleted and replaced by an inducible copy. Polyacrylamide gel electrophoresis and Western blot analysis of extracts and purified virions from cells infected with vA22-HA revealed that the resolvase was expressed after the onset of DNA replication and incorporated into virion cores. vA22i exhibited a conditional replication defect. In the absence of an inducer, (i) viral protein synthesis was unaffected, (ii) late-stage viral DNA replication was reduced, (iii) most of the newly synthesized viral DNA remained in a branched or concatemeric form that caused it to be trapped at the application site during pulsed-field gel electrophoresis, (iv) cleavage of concatemer junctions was inhibited, and (v) virion morphogenesis was arrested at an immature stage. These data indicated multiple roles for the vaccinia virus Holliday junction resolvase in the replication and processing of viral DNA into unit-length genomes.