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DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER

Year 2023, Volume: 24 Issue: 1, 63 - 70, 15.04.2023

Mehmet Kara , Scott Tibbetts

https://doi.org/10.23902/trkjnat.1191873

Abstract

Gammaherpesviruses are associated with multiple types of tumor development and understanding the pathogenesis of these viruses has been the subject of various studies. Throughout the lytic and latent life cycle, these viruses utilize numerous virally encoded microRNAs (miRNAs) to regulate the key mechanisms of the infected cell in their favor. Therefore, it is important to understand the miRNA and their mRNA target interactions for developing better therapeutics. In this study, the strategy and design of a recombinant virus expressing a short hairpin RNA (shRNA) element targeting the host B-lymphocyte-induced maturation protein 1 (Blimp1) transcript was evaluated. Here we have shown that viral tRNA-driven expression of anti-Blimp1 shRNA is able to reduce the target gene expression at a statistically significant level as assessed by luciferase assay during virus infection. This proof-of-principle experiment provides a means to study important miRNA-mRNA interactions in vivo. Further, the very short promoter of the murine gammaherpesvirus 68 (MHV68) viral tRNA (vtRNA4) has the ability to generate two shRNAs from a ~180 nucleotide sequence. If there is a size limit for the shRNA construct, viral tRNA promoter provides an effective shRNA expression system.

Keywords

Blimp1 protein Gammaherpesvirus MHV68 Red mediated mutagenesis shRNA

References

  • 1. Arvin, A., Campadelli-Fiume, G., Mocarski, E., Moore, P.S., Roizman, B., Whitley, R. & Yamanishi, K. (2007). Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge University Press, 1408 pp. http://www.ncbi.nlm.nih.gov/books/NBK47376/
  • 2. Bartel, D.P. (2009). MicroRNAs: Target recognition and regulatory functions. Cell, 136(2): 215-233. https://doi.org/10.1016/j.cell.2009.01.002
  • 3. Barton, E., Mandal, P. & Speck, S.H. (2011). Pathogenesis and host control of gammaherpesviruses: Lessons from the mouse. Annual Review of Immunology, 29: 351-397. https://doi.org/10.1146/annurev-immunol-072710-081639
  • 4. Bogerd, H.P., Karnowski, H.W., Cai, X., Shin, J., Pohlers, M. & Cullen, B.R. (2010). A mammalian herpesvirus uses non-canonical expression and processing mechanisms to generate viral microRNAs. Molecular Cell: 37(1): 135. https://doi.org/10.1016/j.molcel.2009.12.016
  • 5. 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. Journal of Virology, 85(19): 9877-9886. https://doi.org/10.1128/JVI.05558-11
  • 6. 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 Pathogens, 15(8), e1007843. https://doi.org/10.1371/journal.ppat.1007843
  • 7. Calame, K. (2006). Transcription factors that regulate memory in humoral responses. Immunological Reviews, 211(1), 269-279. https://doi.org/10.1111/j.0105-2896.2006.00377.x
  • 8. Carnero, E., Sutherland, J.D. & Fortes, P. (2011). Adenovirus and miRNAs. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1809(11-12), 660-667. https://doi.org/10.1016/j.bbagrm.2011.05.004
  • 9. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. (2009). Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature, 460(7254): 479-486. https://doi.org/10.1038/nature08170
  • 10. Cullen, B.R. (2011). Viruses and microRNAs: RISCy interactions with serious consequences. Genes & Development, 25(18): 1881-1894. https://doi.org/10.1101/gad.17352611
  • 11. 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(3), e00981-00914. https://doi.org/10.1128/mBio.00981-14
  • 12. Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. (2013). Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell, 153(3), 654-665. https://doi.org/10.1016/j.cell.2013.03.043
  • 13. Jurak, I., Griffiths, A. & Coen, D.M. (2011). Mammalian Alphaherpesvirus miRNAs. Biochimica et Biophysica Acta, 1809(11-12): 641-653. https://doi.org/10.1016/j.bbagrm.2011.06.010
  • 14. Kallies, A. & Nutt, S.L. (2007). Terminal differentiation of lymphocytes depends on Blimp-1. Current Opinion in Immunology, 19(2): 156-162. https://doi.org/10.1016/j.coi.2007.01.003
  • 15. Moore, C.B., Guthrie, E.H., Huang, M.T.-H. & Taxman, D.J. (2010). Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown. Methods in Molecular Biology (Clifton, N.J.), 629: 141-158. https://doi.org/10.1007/978-1-60761-657-3_10
  • 16. Nealy, M.S., Coleman, C.B., Li, H. & Tibbetts, S.A. (2010). Use of a virus-encoded enzymatic marker reveals that a stable fraction of memory B cells expresses latency-associated nuclear antigen throughout chronic gammaherpesvirus infection. Journal of Virology, 84(15): 7523-7534. https://doi.org/10.1128/JVI.02572-09
  • 17. Parnas, O., Corcoran, D.L. & Cullen, B.R. (2014). Analysis of the mRNA Targetome of MicroRNAs Expressed by Marek’s Disease Virus. MBio, 5(1): e01060-13. https://doi.org/10.1128/mBio.01060-13
  • 18. Pfeffer, S., Zavolan, M., Grässer, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C. & Tuschl, T. (2004). Identification of Virus-Encoded MicroRNAs. Science, 304(5671): 734-736. https://doi.org/10.1126/science.1096781
  • 19. Reese, T.A., Xia, J., Johnson, L.S., Zhou, X., Zhang, W. & Virgin, H.W. (2010). Identification of Novel MicroRNA-Like Molecules Generated from Herpesvirus and Host tRNA Transcripts. Journal of Virology, 84(19): 10344-10353. https://doi.org/10.1128/JVI.00707-10
  • 20. RNA Folding Form for 88.230.102.221. (n.d.). Retrieved October 8, 2022, from http://www.unafold.org/RNA_form.php
  • 21. Samols, M.A., Hu, J., Skalsky, R.L. & Renne, R. (2005). Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi’s sarcoma-associated herpesvirus. Journal of Virology, 79(14): 9301-9305. https://doi.org/10.1128/JVI.79.14.9301-9305.2005
  • 22. Scherer, L.J., Frank, R. & Rossi, J.J. (2007). Optimization and characterization of tRNA-shRNA expression constructs. Nucleic Acids Research, 35(8): 2620-2628. https://doi.org/10.1093/nar/gkm103
  • 23. Simas, J.P. & Efstathiou, S. (1998). Murine gammaherpesvirus 68: A model for the study of gammaherpesvirus pathogenesis. Trends in Microbiology, 6(7): 276-282. https://doi.org/10.1016/S0966-842X(98)01306-7
  • 24. SiRNA Wizard—Design hairpin insert—InvivoGen. (n.d.). Retrieved October 8, 2022, from https://www.invivogen.com/sirnawizard/construct.php
  • 25. Speck, S.H. & Ganem, D. (2010). Viral latency and its regulation: Lessons from the gamma-herpesviruses. Cell Host & Microbe, 8(1): 100-115. https://doi.org/10.1016/j.chom.2010.06.014
  • 26. Tischer, B.K., von Einem, J., Kaufer, B. & Osterrieder, N. (2006). Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques, 40(2): 191-197.
  • 27. Yang, Y., Boss, I.W., McIntyre, L.M. & Renne, R. (2014). A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells. BMC Genomics, 15(1): 668. https://doi.org/10.1186/1471-2164-15-668
  • 28. Zhou, Z., Li, A., Wang, Z., Pei, F., Xia, Q., Liu, G., Ren, Y. & Hu, Z. (2013). Blimp-1 siRNA inhibits B cell differentiation and prevents the development of lupus in mice. Human Immunology, 74(3): 297-301. https://doi.org/10.1016/j.humimm.2012.11.019
  • 29. Zhu, J.Y., Strehle, M., Frohn, A., Kremmer, E., Höfig, K.P., Meister, G. & Adler, H. (2010). Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68. Journal of Virology, 84(19): 10266-10275. https://doi.org/10.1128/JVI.01119-10

Year 2023, Volume: 24 Issue: 1, 63 - 70, 15.04.2023

Mehmet Kara , Scott Tibbetts

https://doi.org/10.23902/trkjnat.1191873

Abstract

Gammaherpesvirüsler çeşitli tiplerde kanserlerin gelişimi ile ilişkilidir ve bu virüslerin patogenezi birçok çalışmaya konu olmuştur. Bu virüsler litik ve latent adı verilen iki farklı yaşam döngüleriyle enfekte hücrenin anahtar mekanizmalarını kendi lehlerine düzenlemek için çok sayıda viral kökenli mikroRNA (miRNA) kodlarlar. Bu nedenle, daha iyi terapötik maddeler geliştirmek için miRNA ve bunların mRNA hedef etkileşimlerinin anlaşılması önemlidir. Burada, viral tRNA tarafından üretilen ve Blimp1 transkriptini hedefleyen, bir shRNA’nın viral enfeksiyon aşamasında hedef genin ifadesini istatistiksel açıdan anlamlı oranda azaltabildiğini lusiferaz deneyi ile gösterdik. Bu deney dizaynı önemli olan miRNA-mRNA etkileşimlerini test etmek açısından bir kavram ispatı sunmayı hedeflemektedir. Ayrıca oldukça kısa bir promotör boyutuna sahip olan Murine Gammaherpesvirus 68 viral tRNA4, yaklaşık 180 nukleotid uzunluğunda bir diziden iki adet shRNA üretebilmektedir. İstenilen shRNA’nın anlatımı için yalnızca sınırlı bir alan mevcutsa, viral tRNA promotrü etkin bir shRNA anlatımı sistemi sunmaktadır.

References

  • 1. Arvin, A., Campadelli-Fiume, G., Mocarski, E., Moore, P.S., Roizman, B., Whitley, R. & Yamanishi, K. (2007). Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge University Press, 1408 pp. http://www.ncbi.nlm.nih.gov/books/NBK47376/
  • 2. Bartel, D.P. (2009). MicroRNAs: Target recognition and regulatory functions. Cell, 136(2): 215-233. https://doi.org/10.1016/j.cell.2009.01.002
  • 3. Barton, E., Mandal, P. & Speck, S.H. (2011). Pathogenesis and host control of gammaherpesviruses: Lessons from the mouse. Annual Review of Immunology, 29: 351-397. https://doi.org/10.1146/annurev-immunol-072710-081639
  • 4. Bogerd, H.P., Karnowski, H.W., Cai, X., Shin, J., Pohlers, M. & Cullen, B.R. (2010). A mammalian herpesvirus uses non-canonical expression and processing mechanisms to generate viral microRNAs. Molecular Cell: 37(1): 135. https://doi.org/10.1016/j.molcel.2009.12.016
  • 5. 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. Journal of Virology, 85(19): 9877-9886. https://doi.org/10.1128/JVI.05558-11
  • 6. 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 Pathogens, 15(8), e1007843. https://doi.org/10.1371/journal.ppat.1007843
  • 7. Calame, K. (2006). Transcription factors that regulate memory in humoral responses. Immunological Reviews, 211(1), 269-279. https://doi.org/10.1111/j.0105-2896.2006.00377.x
  • 8. Carnero, E., Sutherland, J.D. & Fortes, P. (2011). Adenovirus and miRNAs. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1809(11-12), 660-667. https://doi.org/10.1016/j.bbagrm.2011.05.004
  • 9. Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. (2009). Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature, 460(7254): 479-486. https://doi.org/10.1038/nature08170
  • 10. Cullen, B.R. (2011). Viruses and microRNAs: RISCy interactions with serious consequences. Genes & Development, 25(18): 1881-1894. https://doi.org/10.1101/gad.17352611
  • 11. 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(3), e00981-00914. https://doi.org/10.1128/mBio.00981-14
  • 12. Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. (2013). Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell, 153(3), 654-665. https://doi.org/10.1016/j.cell.2013.03.043
  • 13. Jurak, I., Griffiths, A. & Coen, D.M. (2011). Mammalian Alphaherpesvirus miRNAs. Biochimica et Biophysica Acta, 1809(11-12): 641-653. https://doi.org/10.1016/j.bbagrm.2011.06.010
  • 14. Kallies, A. & Nutt, S.L. (2007). Terminal differentiation of lymphocytes depends on Blimp-1. Current Opinion in Immunology, 19(2): 156-162. https://doi.org/10.1016/j.coi.2007.01.003
  • 15. Moore, C.B., Guthrie, E.H., Huang, M.T.-H. & Taxman, D.J. (2010). Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown. Methods in Molecular Biology (Clifton, N.J.), 629: 141-158. https://doi.org/10.1007/978-1-60761-657-3_10
  • 16. Nealy, M.S., Coleman, C.B., Li, H. & Tibbetts, S.A. (2010). Use of a virus-encoded enzymatic marker reveals that a stable fraction of memory B cells expresses latency-associated nuclear antigen throughout chronic gammaherpesvirus infection. Journal of Virology, 84(15): 7523-7534. https://doi.org/10.1128/JVI.02572-09
  • 17. Parnas, O., Corcoran, D.L. & Cullen, B.R. (2014). Analysis of the mRNA Targetome of MicroRNAs Expressed by Marek’s Disease Virus. MBio, 5(1): e01060-13. https://doi.org/10.1128/mBio.01060-13
  • 18. Pfeffer, S., Zavolan, M., Grässer, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D., Sander, C. & Tuschl, T. (2004). Identification of Virus-Encoded MicroRNAs. Science, 304(5671): 734-736. https://doi.org/10.1126/science.1096781
  • 19. Reese, T.A., Xia, J., Johnson, L.S., Zhou, X., Zhang, W. & Virgin, H.W. (2010). Identification of Novel MicroRNA-Like Molecules Generated from Herpesvirus and Host tRNA Transcripts. Journal of Virology, 84(19): 10344-10353. https://doi.org/10.1128/JVI.00707-10
  • 20. RNA Folding Form for 88.230.102.221. (n.d.). Retrieved October 8, 2022, from http://www.unafold.org/RNA_form.php
  • 21. Samols, M.A., Hu, J., Skalsky, R.L. & Renne, R. (2005). Cloning and identification of a microRNA cluster within the latency-associated region of Kaposi’s sarcoma-associated herpesvirus. Journal of Virology, 79(14): 9301-9305. https://doi.org/10.1128/JVI.79.14.9301-9305.2005
  • 22. Scherer, L.J., Frank, R. & Rossi, J.J. (2007). Optimization and characterization of tRNA-shRNA expression constructs. Nucleic Acids Research, 35(8): 2620-2628. https://doi.org/10.1093/nar/gkm103
  • 23. Simas, J.P. & Efstathiou, S. (1998). Murine gammaherpesvirus 68: A model for the study of gammaherpesvirus pathogenesis. Trends in Microbiology, 6(7): 276-282. https://doi.org/10.1016/S0966-842X(98)01306-7
  • 24. SiRNA Wizard—Design hairpin insert—InvivoGen. (n.d.). Retrieved October 8, 2022, from https://www.invivogen.com/sirnawizard/construct.php
  • 25. Speck, S.H. & Ganem, D. (2010). Viral latency and its regulation: Lessons from the gamma-herpesviruses. Cell Host & Microbe, 8(1): 100-115. https://doi.org/10.1016/j.chom.2010.06.014
  • 26. Tischer, B.K., von Einem, J., Kaufer, B. & Osterrieder, N. (2006). Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques, 40(2): 191-197.
  • 27. Yang, Y., Boss, I.W., McIntyre, L.M. & Renne, R. (2014). A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells. BMC Genomics, 15(1): 668. https://doi.org/10.1186/1471-2164-15-668
  • 28. Zhou, Z., Li, A., Wang, Z., Pei, F., Xia, Q., Liu, G., Ren, Y. & Hu, Z. (2013). Blimp-1 siRNA inhibits B cell differentiation and prevents the development of lupus in mice. Human Immunology, 74(3): 297-301. https://doi.org/10.1016/j.humimm.2012.11.019
  • 29. Zhu, J.Y., Strehle, M., Frohn, A., Kremmer, E., Höfig, K.P., Meister, G. & Adler, H. (2010). Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68. Journal of Virology, 84(19): 10266-10275. https://doi.org/10.1128/JVI.01119-10
There are 29 citations in total.

Details

Primary Language English
Subjects Virology
Journal Section Research Article/Araştırma Makalesi
Authors

Mehmet Kara 0000-0002-9646-4584

Scott Tibbetts This is me 0000-0001-8889-1642

Publication Date April 15, 2023
Submission Date December 30, 2022
Acceptance Date April 5, 2023
Published in Issue Year 2023 Volume: 24 Issue: 1

Cite

APA Kara, M., & Tibbetts, S. (2023). DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER. Trakya University Journal of Natural Sciences, 24(1), 63-70. https://doi.org/10.23902/trkjnat.1191873
AMA Kara M, Tibbetts S. DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER. Trakya Univ J Nat Sci. April 2023;24(1):63-70. doi:10.23902/trkjnat.1191873
Chicago Kara, Mehmet, and Scott Tibbetts. “DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING ShRNA FROM A NATIVE VIRAL TRNA PROMOTER”. Trakya University Journal of Natural Sciences 24, no. 1 (April 2023): 63-70. https://doi.org/10.23902/trkjnat.1191873.
EndNote Kara M, Tibbetts S (April 1, 2023) DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER. Trakya University Journal of Natural Sciences 24 1 63–70.
IEEE M. Kara and S. Tibbetts, “DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER”, Trakya Univ J Nat Sci, vol. 24, no. 1, pp. 63–70, 2023, doi: 10.23902/trkjnat.1191873.
ISNAD Kara, Mehmet - Tibbetts, Scott. “DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING ShRNA FROM A NATIVE VIRAL TRNA PROMOTER”. Trakya University Journal of Natural Sciences 24/1 (April 2023), 63-70. https://doi.org/10.23902/trkjnat.1191873.
JAMA Kara M, Tibbetts S. DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER. Trakya Univ J Nat Sci. 2023;24:63–70.
MLA Kara, Mehmet and Scott Tibbetts. “DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING ShRNA FROM A NATIVE VIRAL TRNA PROMOTER”. Trakya University Journal of Natural Sciences, vol. 24, no. 1, 2023, pp. 63-70, doi:10.23902/trkjnat.1191873.
Vancouver Kara M, Tibbetts S. DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER. Trakya Univ J Nat Sci. 2023;24(1):63-70.

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