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.

[1]  Daniel G. Sausen,et al.  EBV and Lymphomagenesis , 2023, Cancers.

[2]  L. Leoncini,et al.  Epstein–Barr Virus and the Pathogenesis of Diffuse Large B-Cell Lymphoma , 2023, Life.

[3]  C. Münz,et al.  Co-Infection of the Epstein–Barr Virus and the Kaposi Sarcoma-Associated Herpesvirus , 2022, Viruses.

[4]  S. Kenney,et al.  Epstein-Barr virus: Biology and clinical disease , 2022, Cell.

[5]  L. T. Krug,et al.  Conquering the Host: Determinants of Pathogenesis Learned from Murine Gammaherpesvirus 68 , 2021, Annual review of virology.

[6]  E. Cesarman,et al.  KSHV/HHV8-mediated hematologic diseases. , 2021, Blood.

[7]  R. Küppers,et al.  Molecular biology of Hodgkin lymphoma , 2021, Leukemia.

[8]  S. Barta,et al.  2021 Update on Diffuse large B cell lymphoma: A review of current data and potential applications on risk stratification and management , 2021, American journal of hematology.

[9]  T. Molina,et al.  Rapid identification and characterization of infected cells in blood during chronic active Epstein-Barr virus infection , 2020, The Journal of experimental medicine.

[10]  M. Zelazowska,et al.  Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire , 2020, Viruses.

[11]  B. Falini,et al.  Frequent traces of EBV infection in Hodgkin and non-Hodgkin lymphomas classified as EBV-negative by routine methods: expanding the landscape of EBV-related lymphomas , 2020, Modern Pathology.

[12]  M. Zelazowska,et al.  Gammaherpesvirus-infected germinal center cells express a distinct immunoglobulin repertoire , 2020, Life Science Alliance.

[13]  A. Feuchtinger,et al.  Chronic CD30 signaling in B cells results in lymphomagenesis by driving the expansion of plasmablasts and B1 cells. , 2019, Blood.

[14]  R. Küppers,et al.  Human CD30+ B cells represent a unique subset related to Hodgkin lymphoma cells , 2018, The Journal of clinical investigation.

[15]  J. Whisstock,et al.  Understanding CD30 biology and therapeutic targeting: a historical perspective providing insight into future directions , 2017, Blood Cancer Journal.

[16]  Sizun Jiang,et al.  Epstein-Barr Virus LMP1-Mediated Oncogenicity , 2017, Journal of Virology.

[17]  T. Kurosaki,et al.  Regulated selection of germinal-center cells into the memory B cell compartment , 2016, Nature Immunology.

[18]  B. McFarlin,et al.  Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry , 2016, Current protocols in cytometry.

[19]  I. de Vries,et al.  A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors , 2015, European journal of immunology.

[20]  G. Belz,et al.  A murid gamma-herpesviruses exploits normal splenic immune communication routes for systemic spread. , 2014, Cell host & microbe.

[21]  U. Koszinowski,et al.  Systemic and Local Infection Routes Govern Different Cellular Dissemination Pathways during Gammaherpesvirus Infection In Vivo , 2013, Journal of Virology.

[22]  S. Speck,et al.  Tracking Murine Gammaherpesvirus 68 Infection of Germinal Center B Cells In Vivo , 2012, PloS one.

[23]  V. Connor,et al.  Vaccination against a hit-and-run viral cancer , 2010, The Journal of general virology.

[24]  F. Delbos,et al.  Multiple layers of B cell memory with different effector functions , 2009, Nature Immunology.

[25]  S. Speck,et al.  Gammaherpesvirus-Driven Plasma Cell Differentiation Regulates Virus Reactivation from Latently Infected B Lymphocytes , 2009, PLoS pathogens.

[26]  B. Steer,et al.  The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection , 2009, Journal of Virology.

[27]  M. Alenquer,et al.  In vivo imaging of murid herpesvirus-4 infection , 2009, The Journal of general virology.

[28]  C. Staib,et al.  In vivo attenuation of recombinant murine gammaherpesvirus 68 (MHV-68) is due to the expression and immunogenicity but not to the insertion of foreign sequences. , 2008, Virology.

[29]  K. Rajewsky,et al.  LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1. , 2008, Blood.

[30]  W. Hammerschmidt,et al.  Epstein-Barr virus latent membrane protein 2A is a B-cell receptor mimic and essential for B-cell survival. , 2007, Blood.

[31]  K. Rajewsky,et al.  Tracking germinal center B cells expressing germ-line immunoglobulin gamma1 transcripts by conditional gene targeting. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[32]  B. Jungnickel,et al.  Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus. , 2005, Blood.

[33]  Ralf Küppers,et al.  Mechanisms of B-cell lymphoma pathogenesis , 2005, Nature Reviews Cancer.

[34]  J. Kutok,et al.  B cell receptor signal strength determines B cell fate , 2004, Nature Immunology.

[35]  Patricia A. Dyck,et al.  Epstein-Barr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma. , 2003, Blood.

[36]  S. Speck,et al.  Long-Term Latent Murine Gammaherpesvirus 68 Infection Is Preferentially Found within the Surface Immunoglobulin D-Negative Subset of Splenic B Cells In Vivo , 2003, Journal of Virology.

[37]  K. Rajewsky,et al.  Epstein–Barr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Stewart,et al.  Natural history of murine gamma-herpesvirus infection. , 2001, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[39]  D. Thorley-Lawson,et al.  The expression pattern of Epstein-Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell. , 2000, Immunity.

[40]  K. Rajewsky,et al.  EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency. , 2000, Immunity.

[41]  U. Koszinowski,et al.  Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome , 2000, Journal of Virology.

[42]  S. Anderson,et al.  Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. , 1998, Immunity.

[43]  K. Toellner,et al.  T Helper 1 (Th1) and Th2 Characteristics Start to Develop During T Cell Priming and Are Associated with an Immediate Ability to Induce Immunoglobulin Class Switching , 1998, The Journal of experimental medicine.

[44]  M. Ueffing,et al.  Latent membrane protein 1 of Epstein–Barr virus mimics a constitutively active receptor molecule , 1997, The EMBO journal.

[45]  R. Bowden,et al.  Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency. , 1997, The Journal of general virology.

[46]  P. Doherty,et al.  Cytokine production in the immune response to murine gammaherpesvirus 68 , 1996, Journal of virology.

[47]  E. Kieff,et al.  Epstein-Barr virus latent membrane protein 1 is essential for B-lymphocyte growth transformation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[48]  J. Steitz,et al.  Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[49]  K. Rajewsky,et al.  Idiotypic analysis of the response of C57BL/6 mice to the (4‐hydroxy‐3‐nitrophenyl)acetyl group , 1977, European journal of immunology.

[50]  C. Münz.  Epstein Barr Virus Volume 2 , 2015, Current Topics in Microbiology and Immunology.

[51]  D. Thorley-Lawson.  EBV Persistence--Introducing the Virus. , 2015, Current topics in microbiology and immunology.

[52]  R. Longnecker,et al.  Latent Membrane Protein 2 (LMP2). , 2015, Current topics in microbiology and immunology.

[53]  A. Kieser,et al.  The Latent Membrane Protein 1 (LMP1). , 2015, Current topics in microbiology and immunology.

[54]  J. Yates,et al.  Stable replication of plasmids derived from Epstein–Barr virus in various mammalian cells , 1985, Nature.