encoded FL expression
5.4 Usage of the replicon vector to study MCMV oriLyt function
DNA replication of the β- herpesvirus subfamily is not completely understood yet. While some work has been published on human CMV (HCMV), almost no data is available for any of the
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other cytomegaloviruses used as models for HCMV disease, like rat CMV, guinea pig CMV or MCMV. While eleven proteins are necessary to replicate an oriLyt-containing vector in HCMV, no information is available, which proteins are necessary for the DNA replication of MCMV, although the core set of proteins is conserved [30]. The study of DNA replication on MCMV might be of special interest as HCMV, like MCMV, does not harbor a special latent origin of replication. Moreover, only one replication origin has been identified for cytomegaloviruses in contrast to other herpesviruses, which harbor up to three origins of replication. Still, HCMV resides latently in actively replicating cells as for example myeloid stem cells [173]. To prevent the loss of the viral genome during division of the host cell an active DNA replication process appears to be necessary. Therefore, the oriLyt of HCMV, like that of MCMV must provide the factors for latent DNA replication. Although there is no sequence homology between the oriLyt of MCMV and HCMV, the structural composition and functional elements, such as direct and indirect repeats, A/T-rich regions, Y-block and transcription factor-rich binding sites are shared [45, 46]. Studying HCMV latency in humans is extremely difficult: information on time-point and course of infection in patients without pathological findings and availability of tissue-samples is restricted. Therefore a detailed knowledge of MCMV DNA replication and in vivo studies might be helpful to elucidate the general mechanism of replication in latency.
Identification of the proteins necessary for DNA replication of HSV-1 has been performed by a method proposed by Challberg [156]. Viral genome fragments were ligated, cells transfected with the plasmids, and super-infected with the respective herpesvirus. Amplification of the plasmid with the potential origin of replication was analyzed by Southern blot hybridization and several oriLyt sequences were identified by this way [45, 46, 63, 174-177]. Later on, plasmids containing oriLyt sequences were co-transfected with several plasmids containing herpesviral genes, to clarify which individual proteins were necessary for the amplification [156]. Although often successful, this strategy is very tedious and time consuming. In this work, a strict correlation of DNA replication and induction of gene expression of the replicon vector was found. Applying the replicon vector principle would simplify the identification all replication proteins. Superinfection of the luc-ori cells and subsequent bioluminescence assays would simplify the procedure. While the Challberg-method relies on Southern Blot analysis as read-out of DNA amplification, the DNA replication of the replicon vector is simply monitored by FL expression. Southern blot experiments extend over a period of four to five days, a bioluminescence assay of the replicon vector system can be performed in less than one hour. Optionally, quantitative PCR, which is also the more precise and direct proof for DNA amplification compared to the bioluminescence experiments can be added to verify findings from
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the bioluminescence assay. Yet, the bioluminescence assay is cheaper and less prone to errors compared to qPCR, which makes it very interesting for a fast high-throughput screening.
Notably, even the minimal oriLyt sequence of MCMV has been mapped only roughly and no key elements have been assigned yet. The HCMV oriLyt possesses two important regions, whereby one region can be replaced by an SV40 promoter [52]. The replicon vector contains also a SV40 promoter close to the oriLyt sequence. This could contribute to the connection of replication and gene induction, if the SV40 promoter directing FL expression would mimic part of the oriLyt sequence. Several viral as well as cellular transcription factor binding sites have been mapped to the HCMV oriLyt sequence, but none of these have been analyzed in detail. Mutagenesis of the oriLyt sequence in the replicon vector instead of mutagenesis of the viral genome should give clearer results, as there is no risk of analyzing side effects due to overlapping ORFs or promoters. A RNA/DNA hybrid has been found in the HCMV oriLyt [178], thus it would be interesting to know if MCMV possess also RNA/DNA hybrid regions and how these elements regulate DNA replication. Furthermore, miRNAs have been identified close to the oriLyt [179]. There is no information about their role, yet. Again, these questions can be addressed in the replicon system using the FL expression of the pEpibo-luc-ori vector as a simple read-out.
The induction of DNA replication varies between the herpesvirus subfamilies. While the mode of action is very well analyzed in α-herpesviruses only little knowledge is available for the other subfamilies. The origin binding protein of the α-subfamily and also that of the roseoloviruses initiates DNA replication by forming a cruciform/hairpin structure by complementary intrastrand base pairing, which leads to strand separation and recruitment of the core replication proteins [180]. In γ-herpesviruses a hairpin structure is also a key element to the initiation of DNA replication. There, an imperfect preformed hairpin within a repetitive element is recognized by a viral transcription factor and is an important feature of DNA replication initiation [181]. A common herpesviral mechanism of DNA initiation based on the formation and stabilization of such secondary elements has been proposed [36]. In cytomegaloviruses no hairpin structure has been identified yet, although there are several inverted repeats that would allow such a conformation. DNA conformation analysis of the oriLyt might be more easily accessible in the replicon vector than in the large MCMV genome, with a size of about 230 kb.
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