A Mouse Model to Study the Pathogenesis of γ-herpesviral Infections in Germinal Center B Cells.

CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein-Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies.

Different roles of the human Orc6 protein in the replication initiation process.

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency.

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.