deletion of essential or non-essential genes. To analyze and control the effect of a targeted deletion, in order to exclude additional unwanted mutations in the viral genome, revertants of the generated mutants are generated. Genetic reversion is a common procedure, where the original sequence is reintroduced into the deleted region. This bears, however, the risk to ‘overlook construction flaws’ [182] that originate from overlapping gene or regulatory regions, which will be also corrected by genetic reversion but not by trans-complementation. Therefore additional information may be gained by reversion of a virus mutant phenotype through trans-
complementation. Furthermore, transient complementation of a mutant virus can help to study the function of the protein.
Especially the trans-complementation of late herpesviral proteins is a difficult task. Improper timing and expression levels on the one hand can hamper the correct localization of the proteins, while on the other hand isolate expression, i.e. without the co-expression of viral binding partners, can lead to toxicity. Particularly, construction of correctly timed expression is hindered by the very nature of herpesviral late gene expression. Only limited information about the regulation of true-late gene expression is available. As discussed earlier (see section 5.2) one remarkable feature is their dependency on DNA replication for the induction of gene expression [183]. Removal of late gene promoters from the viral genome and their insertion into the cellular genome resulted in wrong, namely early, expression [184]. Correlation of DNA replication and late gene expression was demonstrated by the fact that incoming genomes, which were not replicated yet, cannot serve as template for late gene expression [183]. Moreover, late gene expression could be restored if a late gene promoter or a minimal promoter was present together with a lytic origin in cis [185, 186]. Deviations from this principle exist in that some late gene promoters were dependent on DNA replication in trans [187]. Although trans-complementation of late herpesviral protein is so difficult to achieve with the constitutive expression cassettes, nobody has tried to construct an expression cassette mimicking herpesviral late gene expression to our knowledge.
‘Toxic’ proteins have to be expressed via conditional systems. The most common inducible expression systems are the Tet-ON/Tet-OFF system [67] or the FKBP12 [68] system, which rely on the administration of small chemical compounds. By the addition of the compound, activation takes place synchronously in all cells in the culture. This activation is
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independent from the state of virus replication in all cells. In contrast the oriLyt-based system uses viral DNA replication as signal for the induction. Moreover, the expression of the transgene follows the natural route of late kinetics in the replicon system as the expression increases in proportion with the amplification of the vector DNA. Thus each cell is activated individually upon infection with incoming virus, which leads to appropriate and correct timing of the late transgene. It has to be noted, that due to the replication dependency the system is only suitable for trans-complementation of late but not of early viral transgenes.
The TET-regulatory system has been successfully used to trans-complement the late protein M94 of MCMV [71]. In this case, the M94 gene in the viral genome was replaced by the gene encoding the tetracycline transcription activator (tTA) and a cell line encoding the M94 gene under control of the tetracycline response element (TRE) was constructed. In this setting the virus lacking M94 induces the expression of M94 upon infection in this cell line. By this elegant way the tTA protein is produced at the time point the endogenous M94 protein would be activated and binds to the TRE element, which leads to the transcription of the transgenic M94. In this case, the protein is produced at the correct time point. However, an increase of transgene expression as it is achieved with the replicon system does not take place here. While the tTA- TRE method allowed trans-complementation of the essential M94 protein, the mutant production in large scale was tedious (personal communication, C. Mohr). A further disadvantage of the system is the necessity to modify the viral genome for presence of bacterial elements in the inducing expression cassette within. Especially for vaccine production, bacterial sequences within the viral genome should be avoided. The replicon system, in contrast, is activated by wt virus and thus allows more possibilities for the design of virus mutants. Moreover the system is not marred by the presence of ‘foreign’ DNA elements.
Usage of the replicon system for protein trans-complementation was demonstrated with two viral proteins, namely gO and M50. In case of the non-essential glycoprotein O, the deletion of the gene causes a 2 to 2.5 fold order of magnitude smaller amount of virus in supernatants and a strict cell-associated spread. Growth on the complementing cell line gO-ori of the MCMV∆gO mutant restored the phenotype, meaning that the virus is no longer restricted to a focal spread pattern and releases similar amounts of virus compared to the wt situation. Thus the replicon system was suitable to trans-complement even such a difficult transgene like a glycoprotein. Due to the host-mediated silencing of the transgenes, the question whether the system is also suitable to trans-complement a toxic protein was addressed. Previous attempts to generate M50- complementing cell lines via common methods failed [150]. In contrast, the creation of M50-
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complementing cell line with the replicon system was very successful. No difference to any other non-toxic transgene was detectable regarding efficacy of cell line generation. This was due to the fast host-mediated shut-off of the M50 transgene expression. Western Blot analysis revealed the absence of the protein in uninfected cells and a high induction of the protein in infected cells. Very high titers of MCMV∆M50 virus could be grown on the M50-ori cell lines speaking for the high efficacy of trans-complementation. Furthermore, reconstitution of ∆M50 virus from transfected BACs was just as quick as the reconstitution of wt virus.
Trans-complementation bears the risk of reversion of the mutant virus to wt sequences due to recombination of homologous sequences. Viral genes are often organized in an overlapping fashion in the genome. Therefore deletion of an open reading frame is not always possible, as the neighbouring gene would be affected as well. Furthermore, the replicon vector carries the oriLyt-sequence, which of course is present in the viral genome as well. While no recombination of MCMV∆gO could be detected when propagated in the gO-ori cells, reversion of the M50 deletion was found after propagation on the M50-ori cells. In both cases there were complementary sequences in the replicon vector and the viral genome as the genes could not be completely deleted due to overlapping coding sequences. However, the selection pressure on MCMV-∆M50 is much higher, as the gene is essential for virus spread. The deletion of gO causes only a reduction in viral release and a change in the mode of virus entry. The detection of recombination is however much more sensitive in case of M50 as a few recombined genomes have a growth advantage on non-complementing cells, as these are the only virus mutants that are able to survive. The recombination rate in the cell pools was high with 1 of 104 viruses. Yet, the recombination rate in the isolated M50-ori cell clone 2.1 was very low with less than 1 of 108
viruses. Maybe, the presence of non-functional cells in the cell pool, i.e. cells only having the resistance marker integrated but not the transgene as seen in the luc-ori cl.4 line, increases the selection pressure towards recombined genomes. In case of gO, recombination via phenotypic assays is much more difficult to detect as the protein is not essential for virus amplification. Yet, even PCR analysis could not detect any recombination of MCMV∆gO with the replicon vector. Furthermore, no recombined viruses where found by immuno-histology in mice infected with
trans-complemented MCMV∆gO (personal communication B. Adler), where recombination would provide a major growth advantage and selection pressure would be also very high. It needs to be determined what favors recombination with the replicon vector and how it might be prevented.
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Single cycle viruses (see section 1.6) are of rising importance for vaccine development [71, 188]. A single cycle virus is a virus lacking an essential gene in its genome but is trans-
complemented with the respective protein in order to allow the infection of the host. Still, the virus cannot spread further to neighboring cells. The major advantage of this vaccination strategy is the presentation of almost all antigens and the high safety in comparison to attenuated vaccines. A major limitation with single cycle viruses for vaccination is the necessity to trans-
complement the missing protein. Vaccine production needs to be safe as well as efficient in order to be applicable. The trans-complementation of essential genes with the replicon vector system might help to improve the latter point, as the titers that were obtained after trans-complementing mutant viruses in replicon cell lines were comparable to wt titers. Yet, the degree of recombination of the vector might limit the usage of the system for vaccine production at this stage.