Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

English version

Autoři: Lorenz Loyola ^aff001; Vasudevan Achuthan ^aff002; Kathryn Gilroy ^aff003; Gillian Borland ^aff004; Anna Kilbey ^aff004; Nancy Mackay ^aff004; Margaret Bell ^aff005; Jodie Hay ^aff004; Sriram Aiyer ^aff001; Dylan Fingerman ^aff001; Rodrigo A. Villanueva ^aff001; Ewan Cameron ^aff005; Christine A. Kozak ^aff006; Alan N. Engelman ^aff002; James Neil ^aff004; Monica J. Roth ^aff001
Působiště autorů: Rutgers-Robert Wood Johnson Medical School, Dept of Pharmacology, Piscataway, New Jersey, United States of America ^aff001; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America ^aff002; Beatson Institute for Cancer Research, Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom ^aff003; MRC Univ. of Glasgow Centre for Virus Research, College of Medicine, Veterinary Medicine and Life Sciences, University of Glasgow, Glasgow, United Kingdom ^aff004; Univ. of Glasgow School of Veterinary Medicine, Department of Veterinary Pathology Bearsden, United Kingdom ^aff005; NIH, NIAID, Bethesda, Maryland, United States of America ^aff006; Harvard Medical School, Department of Medicine, Boston, Massachusetts, United States of America ^aff007
Vyšlo v časopise: Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1008154
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008154

Souhrn

Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP^-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP^-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP^-16 tumor revealed their proximity to known MLV common insertion sites genes while maintaining the MLV IN TP^- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP^- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP^- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.

Klíčová slova:

Carcinogenesis – DNA methylation – Genomic libraries – Chromatin – Mammalian genomics – Mouse models – Polymerase chain reaction – Protein interactions

Zdroje

1. Fan H, Johnson C. Insertional oncogenesis by non-acute retroviruses: implications for gene therapy. Viruses. 2011;3(4):398–422. doi: 10.3390/v3040398 21994739

2. Guan R, Aiyer S, Cote ML, Xiao R, Jiang M, Acton TB, et al. X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition. Proteins: Structure, Function, and Bioinformatics. 2017;85(4):647–56.

3. Aiyer S, Rossi P, Malani N, Schneider WM, Chandar A, Bushman FD, et al. Structural and sequencing analysis of local target DNA recognition by MLV integrase. Nucleic Acids Res. 2015;43(11):5647–63. doi: 10.1093/nar/gkv410 25969444

4. Aiyer S, Swapna GV, Malani N, Aramini JM, Schneider WM, Plumb MR, et al. Altering murine leukemia virus integration through disruption of the integrase and BET protein family interaction. Nucleic Acids Res. 2014;42(9):5917–28. doi: 10.1093/nar/gku175 24623816

5. De Rijck J, de Kogel C, Demeulemeester J, Vets S, El Ashkar S, Malani N, et al. The BET family of proteins targets Moloney murine leukemia virus integration near transcription start sites. Cell Rep. 2013;5(4):886–94. doi: 10.1016/j.celrep.2013.09.040 24183673

6. Roth MJ, Schwartzberg PL, Goff SP. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7 2546673

7. Craigie R, Fujiwara T, Bushman F. The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell. 1990;62(4):829–37. doi: 10.1016/0092-8674(90)90126-y 2167180

8. Engelman A, Cherepanov P. Retroviral integrase structure and DNA recombination mechanism. Microbiology Spectrum. 2014;2(6):1011–33.

9. Kvaratskhelia M, Sharma A, Larue RC, Serrao E, Engelman A. Molecular mechanisms of retroviral integration site selection. Nucleic Acids Res. 2014;42(16):10209–25. doi: 10.1093/nar/gku769 25147212

10. Maetzig T, Galla M, Baum C, Schambach A. Gammaretroviral vectors: biology, technology and application. Viruses. 2011;3(6):677–713. doi: 10.3390/v3060677 21994751

11. Serrao E, Ballandras-Colas A, Cherepanov P, Maertens GN, Engelman AN. Key determinants of target DNA recognition by retroviral intasomes. Retrovirology. 2015;12:39. doi: 10.1186/s12977-015-0167-3 25924943

12. De Ravin SS, Su L, Theobald N, Choi U, Macpherson JL, Poidinger M, et al. Enhancers are major targets for murine leukemia virus vector integration. J Virol. 2014;88(8):4504–13. doi: 10.1128/JVI.00011-14 24501411

13. LaFave MC, Varshney GK, Gildea DE, Wolfsberg TG, Baxevanis AD, Burgess SM. MLV integration site selection is driven by strong enhancers and active promoters. Nucleic Acids Res. 2014;42(7):4257–69. doi: 10.1093/nar/gkt1399 24464997

14. Hsu SC, Blobel GA. The role of Bromodomain and Extraterminal Motif (BET) proteins in chromatin structure. Cold Spring Harb Symp Quant Biol. 2017;82:37–43. doi: 10.1101/sqb.2017.82.033829 29196562

15. El Ashkar S, De Rijck J, Demeulemeester J, Vets S, Madlala P, Cermakova K, et al. BET-independent MLV-based vectors target away from promoters and regulatory elements. Mol Ther Nucleic Acids. 2014;3:e179. doi: 10.1038/mtna.2014.33 25072693

16. Steffen D. Proviruses are adjacent to c-myc in some murine leukemia virus-induced lymphomas. Proc Natl Acad Sci U S A. 1984;81(7):2097–101. doi: 10.1073/pnas.81.7.2097 6326104

17. Hay J, Gilroy K, Huser C, Kilbey A, Mcdonald A, MacCallum A, et al. Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity. J Cell Biochem 2019, 120(10):18332–18345. doi: 10.1002/jcb.29143 31257681

18. Neil JC, Gilroy K, Borland G, Hay J, Terry A, Kilbey A. The RUNX Genes as conditional oncogenes: Insights from retroviral targeting and mouse models. In: Groner Y, Ito Y, Liu P, Neil JC, Speck NA, van Wijnen A, editors. RUNX Proteins in Development and Cancer. Singapore: Springer Singapore; 2017. p. 247–64.

19. Blyth K, Vaillant F, Hanlon L, Mackay N, Bell M, Jenkins A, et al. Runx2 and MYC collaborate in lymphoma development by suppressing apoptotic and growth arrest pathways in vivo. Cancer Res. 2006;66(4):2195–201. doi: 10.1158/0008-5472.CAN-05-3558 16489021

20. Huser CA, Gilroy KL, de Ridder J, Kilbey A, Borland G, Mackay N, et al. Insertional mutagenesis and deep profiling reveals gene hierarchies and a Myc/p53-dependent bottleneck in lymphomagenesis. PLoS Genet. 2014;10(2):e1004167. doi: 10.1371/journal.pgen.1004167 24586197

21. Stewart M, Mackay N, Hanlon L, Blyth K, Scobie L, Cameron E, et al. Insertional mutagenesis reveals progression genes and checkpoints in MYC/Runx2 lymphomas. Cancer Res. 2007;67(11):5126–33. doi: 10.1158/0008-5472.CAN-07-0433 17545590

22. Kozak CA. Origins of the endogenous and infectious laboratory mouse gammaretroviruses. Viruses. 2014;7(1):1–26. doi: 10.3390/v7010001 25549291

23. Greenwood AD, Ishida Y, O'Brien SP, Roca AL, Eiden MV. Transmission, evolution, and endogenization: Lessons learned from recent retroviral invasions. Microbiol Mol Biol Rev. 2018;82(1):e00044–17. doi: 10.1128/MMBR.00044-17 29237726

24. Stoye JP, Coffin JM. The four classes of endogenous murine leukemia virus: structural relationships and potential for recombination. J Virol. 1987;61(9):2659–69. 3039159

25. Giovannini D, Touhami J, Charnet P, Sitbon M, Battini JL. Inorganic phosphate export by the retrovirus receptor XPR1 in metazoans. Cell Rep. 2013;3(6):1866–73. doi: 10.1016/j.celrep.2013.05.035 23791524

26. Fan H. Leukemogenesis by Moloney murine leukemia virus: a multistep process. Trends Microbiol. 1997;5(2):74–82. doi: 10.1016/S0966-842X(96)10076-7 9108934

27. Stoye JP, Moroni C, Coffin JM. Virological events leading to spontaneous AKR thymomas. J Virol. 1991;65(3):1273–85. 1847454

28. Ott D, Friedrich R, Rein A. Sequence analysis of Amphotropic and lOAl murine leukemia viruses: Close relationship to Mink Cell Focus-Inducing Viruses. J Virol. 1990;64(2):757–66. 2153240

29. Kozak C, Rowe WP. Genetic mapping of xenotropic leukemia virus-inducing loci in two mouse strains. Science. 1978;199(4336):1448. doi: 10.1126/science.204014 204014

30. Kohn DB. Historical perspective on the current renaissance for hematopoietic stem cell gene therapy. Hematol Oncol Clin North Am. 2017;31(5):721–35. doi: 10.1016/j.hoc.2017.06.006 28895843

31. Milone MC, Bhoj VG. The pharmacology of T cell therapies. Mol Ther—Methods Clin Dev. 2018;8:210–21. doi: 10.1016/j.omtm.2018.01.010 29552577

32. Hacein-Bey-Abina S Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302((5644)):415–9. doi: 10.1126/science.1088547 14564000

33. Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008;118(9):3132–42. doi: 10.1172/JCI35700 18688285

34. Hacein-Bey-Abina S, Hauer J, Lim A, Picard C, Wang GP, Berry CC, et al. Efficacy of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2010;363(4):355–64. doi: 10.1056/NEJMoa1000164 20660403

35. Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B, Kinner A, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med. 2010;16(2):198–204. doi: 10.1038/nm.2088 20098431

36. Braun CJ, Boztug K, Paruzynski A, Witzel M, Schwarzer A, Rothe M, et al. Gene therapy for Wiskott-Aldrich syndrome—long-term efficacy and genotoxicity. Sci Transl Med. 2014;6(227):227ra33.

37. Wu C, Dunbar CE. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity. Front Med. 2011;5(4):356–71. doi: 10.1007/s11684-011-0159-1 22198747

38. Hacein-Bey-Abina S, Pai SY, Gaspar HB, Armant M, Berry CC, Blanche S, et al. A modified gamma-retrovirus vector for X-linked severe combined immunodeficiency. N Engl J Med. 2014;371(15):1407–17. doi: 10.1056/NEJMoa1404588 25295500

39. El Ashkar S, Van Looveren D, Schenk F, Vranckx LS, Demeulemeester J, De Rijck J, et al. Engineering next-generation BET-independent MLV vectors for safer gene therapy. Mol Ther Nucleic Acids. 2017;7:231–45. doi: 10.1016/j.omtn.2017.04.002 28624199

40. Roth MJ. Mutational analysis of the carboxyl terminus of the Moloney murine leukemia virus integration protein. J Virol. 1991;65:2141–5. 2002557

41. Schneider WM, Wu D-t, Amin V, Aiyer S, Roth MJ. MuLV IN mutants responsive to HDAC inhibitors enhance transcription from unintegrated retroviral DNA. Virology. 2012;426(2):188–96. doi: 10.1016/j.virol.2012.01.034 22365328

42. Gupta SS, Maetzig T, Maertens GN, Sharif A, Rothe M, Weidner-Glunde M, et al. Bromo- and extraterminal domain chromatin regulators serve as cofactors for murine leukemia virus integration. J Virol. 2013;87(23):12721–36. doi: 10.1128/JVI.01942-13 24049186

43. O'Reilly L, Roth MJ. Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses. J Virol. 2000;74((2)):899–913. doi: 10.1128/jvi.74.2.899-913.2000 10623753

44. Moiani A, Miccio A, Rizzi E, Severgnini M, Pellin D, Suerth JD, et al. Deletion of the LTR enhancer/promoter has no impact on the integration profile of MLV vectors in human hematopoietic progenitors. PLoS One. 2013;8(1):e55721. doi: 10.1371/journal.pone.0055721 23383272

45. Peredo C, O'Reilly L, Gray K, Roth MJ. Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins. J Virol. 1996;70(5):3142–52. 8627794

46. Bamunusinghe D, Liu Q, Plishka R, Dolan MA, Skorski M, Oler AJ, et al. Recombinant origins of pathogenic and nonpathogenic mouse gammaretroviruses with polytropic host range. J Virol. 2017;91(21):e00855–17. doi: 10.1128/JVI.00855-17 28794032

47. Jern P, Stoye JP, Coffin JM. Role of APOBEC3 in genetic diversity among endogenous murine leukemia viruses. PLoS Genet. 2007;3(10):2014–22. doi: 10.1371/journal.pgen.0030183 17967065

48. Santoni FA, Hartley O, Luban J. Deciphering the code for retroviral integration target site selection. PLoS Comput Biol. 2010;6(11):e1001008. doi: 10.1371/journal.pcbi.1001008 21124862

49. LeRoy G, Chepelev I, DiMaggio PA, Blanco MA, Zee BM, Zhao K, et al. Proteogenomic characterization and mapping of nucleosomes decoded by Brd and HP1 proteins. Genome Biol. 2012;13(8):R68. doi: 10.1186/gb-2012-13-8-r68 22897906

50. Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J, et al. Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell. 2013;24(6):777–90. doi: 10.1016/j.ccr.2013.11.003 24332044

51. Gilroy KL, Terry A, Naseer A, de Ridder J, Allahyar A, Wang W, et al. Gamma-retrovirus integration marks cell type-specific cancer genes: A novel profiling tool in cancer genomics. PLoS One. 2016;11(4):e0154070. doi: 10.1371/journal.pone.0154070 27097319

52. Ranzani M, Annunziato S, Adams DJ, Montini E. Cancer gene discovery: exploiting insertional mutagenesis. Mol Cancer Res. 2013;11(10):1141–58. doi: 10.1158/1541-7786.MCR-13-0244 23928056

53. Touw IP, Erkeland SJ. Retroviral insertion mutagenesis in mice as a comparative oncogenomics tool to identify disease genes in human leukemia. Mol Ther. 2007;15(1):13–9. doi: 10.1038/sj.mt.6300040 17164770

54. Uren AG, Kool J, Berns A, van Lohuizen M. Retroviral insertional mutagenesis: past, present and future. Oncogene. 2005;24(52):7656–72. doi: 10.1038/sj.onc.1209043 16299527

55. Ernst J, Kheradpour P, Mikkelsen TS, Shoresh N, Ward LD, Epstein CB, et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011;473(7345):43–9. doi: 10.1038/nature09906 21441907

56. Ey P, Freeman N, Bela B, Li P, McInnes J. Nucleotide sequence of the murine leukemia virus amphotropic strain 4070A integrase (IN) coding region and comparative structural analysis of the inferred polypeptide. Arch Virol. 1997;142:1757–70. doi: 10.1007/s007050050195 9672635

57. Puglia J, Wang T, Smith-Snyder C, Cote M, Scher M, Pelletier J, et al. Revealing domain structure through linker-scanning analysis of the murine leukemia virus (MuLV) RNase H and MuLV and human immunodeficiency virus type 1 integrase proteins. J Virol. 2006;80(19):9497–510. doi: 10.1128/JVI.00856-06 16973554

58. Pattison JM, Wright JB, Cole MD. Retroviruses hijack chromatin loops to drive oncogene expression and highlight the chromatin architecture around proto-oncogenic loci. PLoS One. 2015;10(3):e0120256. doi: 10.1371/journal.pone.0120256 25799187

59. Arita K, Maeda-Kasugai Y, Ohshima K, Tsuzuki S, Suguro-Katayama M, Karube K, et al. Generation of mouse models of lymphoid neoplasm using retroviral gene transduction of in vitro-induced germinal center B and T cells. Exp Hematol. 2013;41(8):731–41 e9. doi: 10.1016/j.exphem.2013.04.001 23583576

60. Junk DJ, Cipriano R, Stampfer M, Jackson MW. Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells. PLoS One. 2013;8(2):e53776. doi: 10.1371/journal.pone.0053776 23390492

61. Nakagawa M, Tsuzuki S, Honma K, Taguchi O, Seto M. Synergistic effect of Bcl2, Myc and Ccnd1 transforms mouse primary B cells into malignant cells. Haematologica. 2011;96(9):1318–26. doi: 10.3324/haematol.2011.041053 21606168

62. Melamed A, Yaguchi H, Miura M, Witkover A, Fitzgerald TW, Birney E, et al. The human leukemia virus HTLV-1 alters the structure and transcription of host chromatin in cis. Elife. 2018;7: e36245. doi: 10.7554/eLife.36245 29941091

63. Satou Y, Miyazato P, Ishihara K, Yaguchi H, Melamed A, Miura M, et al. The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome. Proc Natl Acad Sci U S A. 2016;113(11):3054–9. doi: 10.1073/pnas.1423199113 26929370

64. Ong CT, Corces VG. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat Rev Genet. 2011;12(4):283–93. doi: 10.1038/nrg2957 21358745

65. Ong CT, Corces VG. CTCF: an architectural protein bridging genome topology and function. Nat Rev Genet. 2014;15(4):234–46. doi: 10.1038/nrg3663 24614316

66. Tang Z, Luo OJ, Li X, Zheng M, Zhu JJ, Szalaj P, et al. CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell. 2015;163(7):1611–27. doi: 10.1016/j.cell.2015.11.024 26686651

67. Ozaki T, Wu D, Sugimoto H, Nagase H, Nakagawara A. Runt-related transcription factor 2 (RUNX2) inhibits p53-dependent apoptosis through the collaboration with HDAC6 in response to DNA damage. Cell Death Dis. 2013;4(4):e610.

68. Westendorf JJ, Zaidi SK, Cascino JE, Kahler R, van Wijnen AJ, Lian JB, et al. Runx2 (Cbfa1, AML-3) interacts with histone deacetylase 6 and represses the p21(CIP1/WAF1) promoter. Mol Cell Biol. 2002;22(22):7982–92. doi: 10.1128/MCB.22.22.7982-7992.2002 12391164

69. Valenzuela-Fernandez A, Cabrero JR, Serrador JM, Sanchez-Madrid F. HDAC6: a key regulator of cytoskeleton, cell migration and cell-cell interactions. Trends Cell Biol. 2008;18(6):291–7. doi: 10.1016/j.tcb.2008.04.003 18472263

70. Ernst J, Kellis M. Large-scale imputation of epigenomic datasets for systematic annotation of diverse human tissues. Nat Biotechnol. 2015;33(4):364–76. doi: 10.1038/nbt.3157 25690853

71. Hoffman MM, Ernst J, Wilder SP, Kundaje A, Harris RS, Libbrecht M, et al. Integrative annotation of chromatin elements from ENCODE data. Nucleic Acids Res. 2013;41(2):827–41. doi: 10.1093/nar/gks1284 23221638

72. Poletti V, Mavilio F. Interactions between retroviruses and the host cell genome. Mol Ther Methods Clin Dev. 2018;8:31–41. doi: 10.1016/j.omtm.2017.10.001 29159201

73. Maskell DP, Renault L, Serrao E, Lesbats P, Matadeen R, Hare S, et al. Structural basis for retroviral integration into nucleosomes. Nature. 2015;523(7560):366–9. doi: 10.1038/nature14495 26061770

74. McGinty RK, Tan S. Recognition of the nucleosome by chromatin factors and enzymes. Current Opinion in Structural Biology. 2016;37:54–61. doi: 10.1016/j.sbi.2015.11.014 26764865

75. Wilson MD, Renault L, Maskell DP, Ghoneim M, Pye VE, Nans A, et al. Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat Commun. 2019;10(1):4138. doi: 10.1038/s41467-019-11557-3

76. Lesbats P, Serrao E, Maskell DP, Pye VE, O'Reilly N, Lindemann D, et al. Structural basis for spumavirus GAG tethering to chromatin. Proc Natl Acad Sci U S A. 2017;114(21):5509–14. doi: 10.1073/pnas.1621159114 28490494

77. Shaytan AK, Landsman D, Panchenko AR. Nucleosome adaptability conferred by sequence and structural variations in histone H2A-H2B dimers. Curr Opin Struct Biol. 2015;32:48–57. doi: 10.1016/j.sbi.2015.02.004 25731851

78. King SR, Berson BJ, Risser R. Mechanism of interaction between endogenous ecotropic murine leukemia viruses in (Balb/C X C57bl/6) hybrid-cells. Virology. 1988;162(1):1–11. doi: 10.1016/0042-6822(88)90388-1 2447699

79. Biasco L, Rothe M, Buning H, Schambach A. Analyzing the genotoxicity of retroviral vectors in hematopoietic cell gene therapy. Mol Ther Methods Clin Dev. 2018;8:21–30. doi: 10.1016/j.omtm.2017.10.002 29159200

80. Rothe M, Schambach A, Biasco L. Safety of gene therapy: new insights to a puzzling case. Curr Gene Ther. 2014;14(6):429–36. doi: 10.2174/1566523214666140918110905 25245088

81. Cavazza A, Cocchiarella F, Bartholomae C, Schmidt M, Pincelli C, Larcher F, et al. Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes. Gene Ther. 2013;20(9):949–57. doi: 10.1038/gt.2013.18 23615186

82. Nam JS, Lee JE, Lee KH, Yang Y, Kim SH, Bae GU, et al. Shifting retroviral Vector integrations away from transcriptional start sites via DNA-binding protein domain insertion into Integrase. Mol Ther Methods Clin Dev. 2019;12:58–70. doi: 10.1016/j.omtm.2018.11.001 30534579

83. Schneider WM, Brzezinski JD, Aiyer S, Malani N, Gyuricza M, Bushman FD, et al. Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag. Proc Natl Acad Sci U S A. 2013;110(23):9487–92. doi: 10.1073/pnas.1221736110 23661057

84. Felkner RH, Roth MJ. Mutational analysis of the N-linked glycosylation sites of the SU envelope protein of Moloney murine leukemia virus. J Virol. 1992;66(7):4258–64. 1318404

85. McCutchan JH, Pagano JS. Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968;41:351–7. 4299537

86. Wu DT, Aiyer S, Villanueva RA, Roth MJ. Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein. J Virol Methods. 2013;193(2):332–6. doi: 10.1016/j.jviromet.2013.06.020 23810854

87. Ting YT, Wilson CA, Farrell KB, Chaudry GJ, Eiden MV. Simian sarcoma-associated virus fails to infect Chinese hamster cells despite the presence of functional gibbon ape leukemia virus receptors. J Virol. 1998;72(12):9453–8. 9811678

88. Valdivieso-Torres L, Sarangi A, Whidby J, Marcotrigiano J, Roth MJ. Role of cysteines in stabilizing the randomized receptor binding domains within feline leukemia virus Envelope proteins. J Virol. 2015;90(6):2971–80. doi: 10.1128/JVI.02544-15 26719270

89. Tanese N, Roth MJ, Goff SP. Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli. J Virol. 1986;59:328–40. 2426463

90. Uphoff CC, Lange S, Denkmann SA, Garritsen HS, Drexler HG. Prevalence and characterization of murine leukemia virus contamination in human cell lines. PLoS One. 2015;10(4):e0125622. doi: 10.1371/journal.pone.0125622 25927683

91. Zheng H, Jia H, Shankar A, Heneine W, Switzer WM. Detection of murine leukemia virus or mouse DNA in commercial RT-PCR reagents and human DNAs. PLoS One. 2011;6(12):e29050. doi: 10.1371/journal.pone.0029050 22205995

92. Stoye JP, Coffin SM. Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes. J Virol. 1988;62:168–75. 2824845

93. Serrao E, Cherepanov P, Engelman AN. Amplification, next-generation sequencing, and genomic DNA mapping of retroviral integration sites. J Vis Exp. 2016(109) doi: 10.3791/53840 27023428

94. Wang GG, Calvo KR, Pasillas MP, Sykes DB, Hacker H, Kamps MP. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nature Methods. 2006;3(4):287–93. doi: 10.1038/nmeth865 16554834

95. Achuthan V, Perreira JM, Sowd GA, Puray-Chavez M, McDougall WM, Paulucci-Holthauzen A, et al. Capsid-CPSF6 interaction licenses nuclear HIV-1 trafficking to sites of viral DNA integration. Cell Host Microbe. 2018;24(3):392–404 e8. doi: 10.1016/j.chom.2018.08.002 30173955

96. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60. doi: 10.1038/nmeth.3317 25751142

97. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. doi: 10.1093/bioinformatics/btp352 19505943

98. Maldarelli F, Wu X, Su L, Simonetti FR, Shao W, Hill S, et al. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science. 2014;345(6193):179–83. doi: 10.1126/science.1254194 24968937

99. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841–2. doi: 10.1093/bioinformatics/btq033 20110278

100. Wickham H. ggplot2. New York: Springer-Verlag; 2016.

101. Achuthan V, DeStefano JJ. Alternative divalent cations (Zn(2)(+), Co(2)(+), and Mn(2)(+)) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity. BMC Biochem. 2015;16:12. doi: 10.1186/s12858-015-0041-x 25934642

102. Jia X, Lin X, Chen J. Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites. AMB Express. 2017;7(1):195. doi: 10.1186/s13568-017-0495-x 29098449

103. Gouet P, Robert X. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42(W1):W320–W4.

104. Pei J, Kim B-H, Grishin NV. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 2008;36(7):2295–300. doi: 10.1093/nar/gkn072 18287115

105. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. doi: 10.1002/jcc.20084 15264254