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(22). EEVs are believed to be responsible for cell-to-cell spread and long-range transmission of vaccinia virus in vivo (26). Six proteins (encoded by A33R, A34R, A36R, A56R, B5R, and F13L) are EEV specific (11). However, A56R has no effect on infectivity or spread if mutated (27).

It may be possible to circumvent normal receptor requirements by engineering vaccinia virus to bind to alternative cell surface molecules. Consistent with this, expression of an ScFv to erbB2 on the surface of the EEV (created as a fusion with A56R) was shown to bind erbB2 by enzyme-linked immuno-sorbent assay (27). Creation of a fusion protein between an ScFv and A56R is technically feasible and may direct binding of the EEV to a specific antigen or cell type. Fusions of other surface proteins, including B5R have been reported (28).

The life cycle of vaccinia is illustrated in Fig. 1. Vaccinia virus (as with all poxviruses) spends its entire life cycle in the cytoplasm of the host cell and has never been shown to integrate into the host genome. Vaccinia has very few interactions with host cellular proteins, allowing for rapid, efficient replication without negative effects from host cell defenses. The virus induces a profound cytopathic effect very soon after viral entry, as early viral enzymes completely shut down host cell functions. By 4 to 6 h after infection, there is almost complete inhibition of host protein synthesis. This allows for very efficient expression of viral genes and viral replication. In fact, approximately 10,000 copies of the viral genome are made within 12 h of infection (29). Half of these are incorporated into mature virions and released.

After vaccinia enters the cell, it undergoes a process of DNA uncoating and transcription begins. Three stages of transcription—early, intermediate, and late—have been described, each with its own specific promoters and transcription factors (30). The enzymes required for initiation of transcription are encapsulated in viral particles and released on viral entry. Proteins needed for viral replication are synthesized at the early (prereplicative) stage of infection. A DNA-depen-dent RNA polymerase is injected with the virus into the cytoplasm leading to the synthesis of early mRNA. Translation of this RNA forms early proteins, which are involved in un-coating of the viral DNA, DNA replication, and intermediate transactivation for transcription of intermediate mRNA. Intermediate mRNA is then expressed, which encodes for late transactivators leading to late mRNA synthesis. Late proteins include structural proteins for membrane formation and early transcription factors to be incorporated into the new virus particle. Only a relatively small number of proteins are required for DNA synthesis, making the system simple and largely autonomous.

Unlike many viruses, which rely on cellular machinery to replicate, vaccinia replicates virtually as an independent unit. Artificial transcriptional control for gene therapy applications is therefore challenging. It is clear, however, that some cellular protein interaction with viral transcription may occur. One group has identified a cellular protein transcription factor YY1, with vaccinia late promoter-binding activity (31). YY1 has been shown to bind the vaccinia ILl late promoter (32).

Figure 1 Schematic of vaccinia life cycle. (From Ref. 144.)

The vaccinia double-stranded DNA genome replicates in the cytoplasm, forming multiple concatamers of the genome. These concatamers are then resolved into individual genomes, which are encapsulated along with the early transcription factors into golgi-derived membranes. The first stage in the formation of infectious particles is the development of viral crescents composed of lipid and viral protein. To date, the origin of these crescents is disputed. Currently, the crescent is believed to be composed of a single lipid bilayer without continuity to cellular membranes (33). These crescents then coalesce into immature virus that lack infectivity. Immature virus then matures into IMV by condensation of the core and processing of core proteins (Fig. 2). IMV is transported to sites at which it becomes wrapped with 2 additional membranes. These membranes are derived from trans-golgi network membranes that have been modified by the inclusion of virus-encoded proteins and ultimately become part of the EEV outer envelope. These wrapped intracellular enveloped virus move to the cell surface where the outer membrane fuses with the plasma membrane, exposing the virus on the cell surface. If the virus is retained or reattaches it is called the cell-associated enveloped virus (CEV), but if released, becomes EEV. The A34R gene product plays a role in holding the virus to the cell surface. Mutations in A34R lead to increased EEV released and decreased CEV (34).

This entire life cycle occurs very rapidly. Initial RNA transcripts are detectible within 20 min of infection and DNA replication begins 1 to 2 h after infection. Within 12 h after infection, the majority of mRNA within the cytoplasm is from vaccinia-encoded genes. The entire replication cycle occurs in approximately 12 h (6).

Figure 2 Electron micrograph of intracytoplasmic IMV form of vaccinia. The virions have a characteristic brick shape with a biconcave central core. (Courtesy of Maria Tsokos, MD, and Mones Abu-Asab, PhD, Laboratory of Pathology, NCI.)

III. CONSTRUCTION OF RECOMBINANT VECTORS

Homologous recombination occurs naturally during the replication of vaccinia virus, thus lending itself toward efficient insertion of foreign DNA. The creation of recombinant vaccinia vectors is relatively simple. The issues to be considered when creating a recombinant vaccinia vector include choosing a site for proposed recombination, a selection method(s), and a promoter for the foreign gene.

A shuttle plasmid is first created where a foreign gene expressed off a vaccinia promoter is flanked by vaccinia DNA sequences. Care must be taken that the foreign gene does not contain vaccinia transcription termination signals for early promoters (TTTTTNT) (35). The most common site of recombination has been the vaccinia thymidine kinase (TK) gene. Insertion of genes into the TK locus eliminates functional viral TK, leading to attenuation of the virus in vivo (36). Recombinations into numerous other loci have been performed, including intergenic segments such that no functional deletion occurs (37,38). The functional analysis of many vaccinia genes has been defined through insertional deletion.

A wide range of vaccinia promoters are available for expression of transgenes. It is necessary to use vaccinia promoters for creation of the recombinant vectors because these are specific for vaccinia polymerase. Eukaryotic promoters will not function in vaccinia infection because the host cell polymerase is not present in the cytoplasm where vaccinia transcription occurs. Several natural and synthetic early and late promoters have been described with various levels of activity (39-41). The native vaccinia promoters are generally very strong and compare favorably to other viral promoters used in other viral vectors. The synthetic early/late promoter described by Chakrabarti et al. (41) has led to consistent, reliable high levels of gene expression in numerous systems tested.

Several methods for selection of recombinant viruses are available. Growth in the presence of the thymidine analog BdUr can be used to select for a TK-negative phenotype in select cells after recombination into the TK locus (42). Others have commonly used the selection gene xanthine-guanine phosphoribosyltransferase, which allows for selective growth in media containing mycophenolic acid (43). Positive selection through replacement of an essential gene previously deleted from a backbone virus grown on permissive cell lines is also available (44). p-galactosidase and green fluorescent protein can aid in selection of recombinants. Once the shuttle plasmid is constructed, it can be transfected into a cell that has been infected with vaccinia. Homologous recombination leads to the insertion of the foreign gene into 0.1 % of progeny virus genomes (6). The use of at least 3 rounds of selection ensures that there is no contaminating parental virus.

IV. IN VITRO GENE TRANSFER VECTOR

It has long been recognized that vaccinia is a valuable tool for expression of foreign genes in vitro (45,46). It is relatively easy to make recombinant viruses, and to grow and purify the virus. Large inserts can be accepted in the genome, and strong synthetic promoters lead to high levels of protein expression from infected cells. The virus has a broad tropism, and will infect and replicate in most mammalian cells. The high efficiency of expression with vaccinia obviated the need for other vectors, which permanently integrated into the genome and the cloning of high expressing cells. Infection with recombinant vaccinia leads to high levels of expression of foreign genes that are processed in the appropriate way such that their function can be studied. Vaccinia infection of cells at an multiplicity of infection (MOI) of 1.0 leads to >99% of cells expressing the gene of interest in most cell lines. Rather than make recombinants, simple plasmid transfection in a virally infected cell leads to efficient expression of foreign genes. The gene must be placed under control of a vaccinia promoter. The backdrop of vaccinia infection allows for cytoplasmic transcription, avoiding the inefficient process of trafficking into the nucleus. This leads to >90% of cells expressing foreign genes after simple plasmid transfection (47). This efficient transient expression system has been used for the functional analysis of innumerable foreign proteins over the years. Inactivation of the virus with psoralen and ultraviolet (UV) light can result in an efficient expression vector that does not cause a cyto-pathic effect (48). This may be important for the functional analysis of some proteins.

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