Viral vectors: a meeting review

The use of viruses as vectors in plants and animals received comprehensive review in a recent Banbury Conference held at Cold Spring Harbor Laboratory. In addition to the expected concentrated attention on retroviruses, a number of other viruses were discussed as expression or vaccine vectors—nondefective and defective viral vectors, as well as vectors made from various promoters and other regulatory elements derived from DNA and RNA virus sources. Virus-based vectors capable of expressing high levels of recombinant proteins also were presented. Large DNA viruses (poxviruses, herpesviruses, adenoviruses, and baculoviruses) have become widely used as expression and vaccine vectors. Bernard Moss (National Institutes of Health) described recent work on vaccinia transcription, including a factor capable of binding specifically to and activating transcription via the TATA box equivalent of vaccinia early genes (Yuen et al. 1987) and a transcription termination factor that is the same as the previously identified vaccinia capping enzyme (Shuman et al. 1987). The capping/termination protein is associated with the viral RNA polymerase and functions cotranscriptionally. As a result, there is very little capping and polyadenylation in vaccinia virus-bacteriophage T7 hybrid vectors that will otherwise generate extremely high levels of transcripts (Fuerst et al. 1987). Recombinant herpesvirus vectors have not been exploited to the same degree as vaccinia. Bernard Roizman (University of Chicago) discussed the engineering of the herpes simplex virus (HSV-1) genome as a live vaccine and vector for foreign antigens. He described an attenuated recombinant virus lacking a large segment of the viral genome, including both repeated and nonrepeated regions. Even though the recombinant HSV was incapable of killing highly susceptible owl monkeys, it was fully capable of inducing protective immunity to wildtype challenge. Leonard Post (Upjohn Company) addressed similar issues of animal vaccination with herpesviruses and the use of pseudorabies virus (PRV) of pigs as a vaccine vector (Thomsen et al. 1987). Recombinant PRV vaccines have been constructed by deletion mutagenesis. Edward Mocarski (Stanford University School of Medicine) described methods for engineering cytomegalovirus (CMV) (Spaete and Mocarski 1987), work that makes use of a strong p (delayed-early) viral promoter that has been harnessed for expression of p-galactosidase to levels approaching 10^ molecules per infected cell (1% of cell protein). In uninfected cells, another CMV promoter, the immediate early promoter-enhancer, is expressed at relatively high levels and has been used for manipulations in a wide range of cell types. Expression from plasmid constructs using this promoter/enhancer can be further increased 10to 20fold by tJflizs-activation with the viral major immediate early gene itself. A herpesvirus with the capacity to maintain episomal genomes in human cells, Epstein-Barr virus (EBV), was shown to have a lytic DNA replication origin (oriLyr) mapping separately from the previously characterized latent origin (oriP) in work described by Wolfgang Hammerschmidt (McArdle Laboratory, University of Wisconsin). The lytic cycle of EBV, analogous to that already defined in HSV, utilizes viral rather than host cell replication machinery. This region maps between bases 53,000 and 55,000 on the EBV genome and shares no sequence homology with the lytic origins of HSV, the only other herpesvirus whose origins have been characterized. Finally, Robert Margolskee (Roche Institute) described the utility of full-length cDNA cloning vectors that incorporate EBV episome replication signals. As described in a recently published paper (Margolskee et al. 1988), this modified pcD vector system (EBV-pcD-X) is remarkably efficient for expressing full-length cDNA libraries in human cells for cloning receptor genes. Both fluorescent antibody and fluorescent ligand reagents have been used in conjunction with cell sorting to identify and separate mammalian cells carrying and expressing cloned cDNAs. Stable transformants as well as transient expression systems can be used, depending on the particular situation and mRNA abundance. Adenoviruses (Ad), for which two vaccines (Ad4 and Ad7) are licensed, have been subjected to insertion and deletion mutagenesis for years, but have not been very widely used as recombinant virus vectors. Bruce Mason (Wyeth Ayerst Laboratories) described further investigations on the introduction of the hepatitis B surface antigen gene into dispensable regions of the Ad genome. Current efforts are directed at the E3 region or between the E4 region and the genomic terminus. High-level expression was obtained by placing this foreign gene under the Ad major late promoter and tripartite leader. Thus, adenoviruses already in use as vaccines may be engineered readily to express other antigens without disrupting essential viral genes. Vectors based on regulatory signals derived from adenovirus have been used widely in transient expression systems and in stably transformed cell lines. Randy Kaufman (Genetics Institute), who has generated expression vectors for transient transfection and transformation of cells (Kaufman 1985), described further work on the mechanisms of adenovirus VA RNA function in transiently transfected cells, an effect that seems reminiscent of the translational role of VA RNA during viral replication. Finally, Kathleen Berkner (ZymoGenetics) expanded upon her recent observations (Boel et al. 1987) suggesting that, in contrast to accepted dogma, certain mammalian mRNAs may be functionally bicistronic and may express two (or more) large proteins. The particular intercistronic sequences appear to be important. Her work suggests that a region with potential hairpin