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Latent Macrophage and Immature B Cell Lines Generated with Hygromycin-Resistant Murine Gammaherpesvirus 68 Genome Expresses Modest Levels of Viral miRNAs

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Abstract

Murine gammaherpesvirus 68 (MHV68) establishes latency mainly in B cells and causes lymphomas reminiscent of human gammaherpesvirus diseases in laboratory mice. To study the molecular mechanism of virus infection and how the viral determinants control cell and eventually cause tumorigenesis, readily available latently infected cell lines are essential. For in vitro MHV68 latency studies, only two cell culture systems have been available. Gammaherpesviruses are known to infect developing B cells and macrophages, therefore we aimed to expand the MHV68 latently infected cell line repertoire. Here, several latently infected immature B cell and macrophage-like cell line clones were generated. Hygromycin-resistant recombinant MHV68 was isolated from a laboratory-made latent cell line, HE2.1, and propagated to develop stable cell lines that carry the viral genome under hygromycin selection. Subclones of these cells lines were analyzed for viral miRNA expression by TaqMan qPCR and assessed for expression of a lytic viral transcript M3. The cell lines maintain the viral genome as an episome shown by the digestion-circularization PCR assay. Latently infected cell lines generated here do not express viral miRNAs higher than the parental cell line. However, these cell lines may provide an alternative tool to study latency mechanisms and miRNA target identification studies.

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REFERENCES

  1. Thorley-Lawson D.A., Gross A. 2004. Persistence of the Epstein–Barr virus and the origins of associated lymphomas. N. Engl. J. Med. 350, 1328–1337.

    CAS  PubMed  Google Scholar 

  2. Miller G. 1982. Immortalization of human lymphocytes by Epstein−Barr virus. Yale J. Biol. Med. 55, 305–310.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bechtel J.T., Liang Y., Hvidding J., Ganem D. 2003. Host range of Kaposi’s sarcoma-associated herpesvirus in cultured cells. J. Virol. 77, 6474–6481.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Coleman C.B., Nealy M.S., Tibbetts S.A. 2010. Immature and transitional B cells are latency reservoirs for a gammaherpesvirus. J. Virol. 84, 13045–13052.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Hwang S., Wu T.-T., Tong L.M., Kim K.S., Martinez-Guzman D., Colantonio A.D., Uittenbogaart C.H., Sun R. 2008. Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo. J. Virol. 82, 12498–12509.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Usherwood E.J., Stewart J.P., Nash A.A. 1996. Characterization of tumor cell lines derived from murine gammaherpesvirus-68-infected mice. J. Virol. 70, 6516–6518.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Forrest J.C., Speck S.H. 2008. Establishment of B-cell lines latently infected with reactivation-competent murine gammaherpesvirus 68 provides evidence for viral alteration of a DNA damage-signaling cascade. J. Virol. 82, 7688–7699.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Barton E., Mandal P., Speck S.H. 2011. Pathogenesis and host control of gammaherpesviruses: Lessons from the mouse. Annu. Rev. Immunol. 29, 351–397.

    CAS  PubMed  Google Scholar 

  9. Bartel D.P. 2009. MicroRNAs: Target recognition and regulatory functions. Cell. 136, 215–233.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kincaid R.P., Sullivan C.S. 2012. Virus-encoded microRNAs: An overview and a look to the future. PLoS Pathog. 8, e1003018.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Cai X., Schäfer A., Lu S., Bilello J.P., Desrosiers R.C., Edwards R., Raab-Traub N., Cullen B.R. 2006. Epstein–Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog. 2, e23.

    PubMed  PubMed Central  Google Scholar 

  12. Haecker I., Gay L.A., Yang Y., Hu J., Morse A.M., McIntyre L.M., Renne R. 2012. Ago HITS-CLIP expands understanding of Kaposi’s sarcoma-associated herpesvirus miRNA function in primary effusion lymphomas. PLoS Pathog. 8, e1002884.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Ramalingam D., Kieffer-Kwon P., Ziegelbauer J.M. 2012. Emerging themes from EBV and KSHV microRNA targets. Viruses. 4, 1687–1710.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Coleman C.B., McGraw J.E., Feldman E.R., Roth A.N., Keyes L.R., Grau K.R., Cochran S.L., Waldschmidt T.J., Liang C., Forrest J.C., Tibbetts S.A. 2014. A gammaherpesvirus Bcl-2 ortholog blocks B cell receptor-mediated apoptosis and promotes the survival of developing B cells in vivo. PLoS Pathog. 10, e1003916.

    PubMed  PubMed Central  Google Scholar 

  15. Paden C.R., Forrest J.C., Moorman N.J., Speck S.H. 2010. Murine gammaherpesvirus 68 LANA is essential for virus reactivation from splenocytes but not long-term carriage of viral genome. J. Virol. 84, 7214–7224.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Feldman E.R., Kara M., Coleman C.B., Grau K.R., Oko L.M., Krueger B.J., Renne R., van Dyk L.F., Tibbetts S.A. 2014. Virus-encoded microRNAs facilitate gammaherpesvirus latency and pathogenesis in vivo. mBio. 5, e00981-14.

    PubMed  PubMed Central  Google Scholar 

  17. Kara M., Tibbetts S.A. 2021. Empirical validation of overlapping virus lncRNAs and coding transcripts by northern blot. In: Long Non-Coding RNAs in Cancer. Methods in Molecular Biology. Navarro A., Ed. New York: Springer, pp. 243–253.

    Google Scholar 

  18. Hasbold J., Klaus G.G. 1990. Anti-immunoglobulin antibodies induce apoptosis in immature B cell lymphomas. Eur. J. Immunol. 20, 1685–1690.

    CAS  PubMed  Google Scholar 

  19. Flaño E., Husain S.M., Sample J.T., Woodland D.L., Blackman M.A. 2000. Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages. J. Immunol. 165, 1074–1081.

    PubMed  Google Scholar 

  20. Weck K.E., Kim S.S., Virgin HW I.V., Speck S.H. 1999. Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells. J. Virol. 73, 3273–3283.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Kim K.J., Kanellopoulos-Langevin C., Merwin R.M., Sachs D.H., Asofsky R. 1979. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J. Immunol. 122, 549–554.

    CAS  PubMed  Google Scholar 

  22. Humme S., Reisbach G., Feederle R., Delecluse H.-J., Bousset K., Hammerschmidt W., Schepers A. 2003. The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold. Proc. Natl. Acad. Sci. U. S. A. 100, 10989–10994.

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hu J., Garber A.C., Renne R. 2002. The latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus supports latent DNA replication in dividing cells. J. Virol. 76, 11677–11687.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Forrest J.C., Paden C.R., Allen R.D., Collins J., Speck S.H. 2007. ORF73-null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication. J. Virol. 81, 11957–11971.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Salinas E., Gupta A., Sifford J.M., Oldenburg D.G., White D.W., Forrest J.C. 2018. Conditional mutagenesis in vivo reveals cell type- and infection stage-specific requirements for LANA in chronic MHV68 infection. PLoS Pathog. 14, e1006865.

    PubMed  PubMed Central  Google Scholar 

  26. Henderson A., Ripley S., Heller M., Kieff E. 1983. Chromosome site for Epstein−Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro. Proc. Natl. Acad. Sci. U. S. A. 80, 1987–1991.

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bullard W.L., Kara M., Gay L.A., Sethuraman S., Wang Y., Nirmalan S., Esemenli A., Feswick A., Hoffman B.A., Renne R., Tibbetts S.A. 2019. Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery. PLoS Pathog. 15, e1007843.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Gay L.A., Sethuraman S., Thomas M., Turner P.C., Renne R. 2018. Modified cross-linking, ligation, and sequencing of hybrids (qCLASH) identifies Kaposi’s sarcoma-associated herpesvirus microRNA targets in endothelial cells. J. Virol. 92, e02138-17.

    PubMed  PubMed Central  Google Scholar 

  29. Ungerleider N., Bullard W., Kara M., Wang X., Roberts C., Renne R., Tibbetts S., Flemington E.K. 2021. EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape. PLoS Pathog. 17 (5), e1009217.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Skalsky R.L., Corcoran D.L., Gottwein E., Frank C.L., Kang D., Hafner M., Nusbaum J.D., Feederle R., Delecluse H.J., Luftig M.A., Tuschl T., Ohler U., Cullen B.R. 2012. The viral and cellular microRNA targetome in lymphoblastoid cell lines. PLoS Pathog. 8, e1002484.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Feldman E.R., Tibbetts S.A. 2015. Emerging roles of herpesvirus microRNAs during in vivo infection and pathogenesis. Curr. Pathobiol. Rep. 3, 209–217.

    PubMed  PubMed Central  Google Scholar 

  32. Simas J.P., Swann D., Bowden R., Efstathiou S. 1999. Analysis of murine gammaherpesvirus-68 transcription during lytic and latent infection. J. Gen. Virol. 80, 75–82.

    CAS  PubMed  Google Scholar 

  33. Kara M., O’Grady T., Feldman E.R., Feswick A., Wang Y., Flemington E.K., Tibbetts S.A. 2019. Gammaherpesvirus readthrough transcription generates a long non-coding RNA that is regulated by antisense miRNAs and correlates with enhanced lytic replication in vivo. Noncoding RNA. 5, 6.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Speck S.H., Ganem D. 2010. Viral latency and its regulation: Lessons from the gamma-herpesviruses. Cell Host Microbe. 8, 100–115.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Feederle R., Haar J., Bernhardt K., Linnstaedt S.D., Bannert H., Lips H., Cullen B.R., Delecluse H.-J. 2011. The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocyte. J. Virol. 85, 9801–9810.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Feederle R., Linnstaedt S.D., Bannert H., Lips H., Bencun M., Cullen B.R., Delecluse H.-J. 2011. A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog. 7, e1001294.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Gottwein E., Corcoran D.L., Mukherjee N., Skalsky R.L., Hafner M., Nusbaum J.D., Shamulailatpam P., Love C.L., Dave S.S., Tuschl T., Ohler U., Cullen B.R. 2011. Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines. Cell Host Microbe. 10, 515–526.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Boss I.W., Nadeau P.E., Abbott J.R., Yang Y., Mergia A., Renne R. 2011. A Kaposi’s sarcoma-associated herpesvirus-encoded ortholog of microRNA miR-155 induces human splenic B-cell expansion in NOD/LtSz-scid IL2Rγnull mice. J. Virol. 85, 9877–9886.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Moody R., Zhu Y., Huang Y., Cui X., Jones T., Bedol-la R., Lei X., Bai Z., Gao S.-J. 2013. KSHV microRNAs mediate cellular transformation and tumorigenesis by redundantly targeting cell growth and survival pathways. PLoS Pathog. 9, e1003857.

    PubMed  PubMed Central  Google Scholar 

  40. Preiss N.K., Kang T., Usherwood Y.-K., Huang Y.H., Branchini B.R., Usherwood E.J. 2020. Control of B cell lymphoma by gammaherpesvirus-induced memory CD8 T cells. J. Immunol. 205, 3372–3382.

    CAS  PubMed  Google Scholar 

  41. Mrázová V., Betáková T., Kúdelová M., Šupolíková M., Lachová V., Lapuníková B., Golais F. 2015. Murine gammaherpesvirus (MHV-68) transforms cultured cells in vitro. Intervirology. 58, 69–72.

    PubMed  Google Scholar 

  42. Mrázová V., Kúdelová M., Smolinská M., Nováková E., Šupolíková M., Vrbová M., Golais F. 2017. Transformation of cells by photoinactivated murine gamma-herpesvirus 68 during nonproductive and quiescent infection. Intervirology. 60, 61–68.

    PubMed  Google Scholar 

  43. Sunil-Chandra N.P., Efstathiou S., Nash A.A. 1993. Interactions of murine gammaherpesvirus 68 with B and T cell lines. Virology. 193, 825–833.

    CAS  PubMed  Google Scholar 

  44. Wu T.T., Usherwood E.J., Stewart J.P., Nash A.A., Sun R. 2000. Rta of murine gammaherpesvirus 68 reactivates the complete lytic cycle from latency. J. Virol. 74, 3659–3667.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Pavlova I.V., Virgin IV H.W., Speck S.H. 2003. Disruption of gammaherpesvirus 68 gene 50 demonstrates that Rta is essential for virus replication. J. Virol. 77, 5731–5739.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Adler H., Messerle M., Wagner M., Koszinowski U.H. 2000. Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome. J. Virol. 74, 6964–6974.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Jain V., Plaisance-Bonstaff K., Sangani R., Lanier C., Dolce A., Hu J., Brulois K., Haecker I., Turner P., Renne R., Krueger B. 2016. A toolbox for herpesvirus miRNA research: Construction of a complete set of KSHV miRNA deletion mutants. Viruses. 8, 54.

    PubMed  PubMed Central  Google Scholar 

  48. Wu T.-T., Liao H.-I., Tong L., Leang R.S., Smith G., Sun R. 2010. Construction and characterization of an infectious murine gammaherpesivrus-68 bacterial artificial chromosome. BioMed. Res. Int. 2011, e926258.

    Google Scholar 

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ACKNOWLEDGMENTS

I would like to thank my PhD advisor Prof. Dr. Scott Tibbetts and Tibbetts Lab members. The laboratory work was performed at the University of Florida during my research at Tibbetts Lab.

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The author declared that this study received no specific financial support.

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  1. Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bursa Uludag University, 16059, Bursa, Turkey

    M. Kara

  2. Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, FL 3261, Gainesville, USA

    M. Kara

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Kara, M. Latent Macrophage and Immature B Cell Lines Generated with Hygromycin-Resistant Murine Gammaherpesvirus 68 Genome Expresses Modest Levels of Viral miRNAs. Mol Biol 58, 123–132 (2024). https://doi.org/10.1134/S0026893324010138

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