Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis
Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.
Vyšlo v časopise:
Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis. PLoS Pathog 7(10): e32767. doi:10.1371/journal.ppat.1002333
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002333
Souhrn
Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.
Zdroje
1. BlackburnEH 2005 Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett 579 859 862
2. BlackburnEH 2005 Telomerase and Cancer: Kirk A. Landon—AACR prize for basic cancer research lecture. Mol Cancer Res 3 477 482
3. GreiderCWBlackburnEH 1987 The telomere terminal transferase of tetrahymena is a ribonucleoprotein Enzyme with 2 kinds of primer specificity. Cell 51 887 898
4. ChenJLBlascoMAGreiderCW 2000 Secondary structure of vertebrate telomerase RNA. Cell 100 503 514
5. ChenJLGreiderCW 2004 Telomerase RNA structure and function: implications for dyskeratosis congenita. Trends Biochem Sci 29 183 192
6. MitchellJRChengJCollinsK 1999 A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end. Mol Cell Biol 19 567 576
7. KimNWPiatyszekMAProwseKRHarleyCBWestMD 1994 Specific association of human telomerase activity with immortal cells and cancer. Science 266 2011 2015
8. ShayJWBacchettiS 1997 A survey of telomerase activity in human cancer. Eur JCancer 33 787 791
9. BellonMNicotC 2008 Regulation of telomerase and telomeres: Human tumor viruses take control. J Natl Cancer Inst 100 98 108
10. HorikawaIBarrettJC 2003 Transcriptional regulation of the telomerase hTERT gene as a target for cellular and viral oncogenic mechanisms. Carcinogenesis 24 1167 1176
11. LiuHLuanFJuYShenHYGaoLF 2007 In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase. Biochem Biophys Res Commun 355 379 384
12. ZhuZWWilsonATGopalakrishnaKBrownKELuxonBA 2010 Hepatitis C virus core protein enhances telomerase activity in Huh7 cells. J Med Virol 82 239 248
13. ZhuZWWilsonATGopalakrishnaKNBrownKLuxonBA 2008 Hepatitis C virus core protein upregulates telomerase activity in hepatoma cells. Hepatology 48 974A 974A
14. CalnekBW 2001 Pathogenesis of Marek's disease virus infection. HiraiK Marek's disease Berlin, Germany Springer 25 55
15. OsterriederNKamilJPSchumacherDTischerBKTrappS 2006 Marek's disease virus: from miasma to model. Nat Rev Microbiol 4 283 294
16. BrownACBaigentSJSmithLPChattooJPPetherbridgeLJ 2006 Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek's disease virus. Proc Natl Acad Sci U S A 103 1687 1692
17. BrownACSmithLPKgosanaLBaigentSJNairV 2009 Homodimerization of the Meq viral oncoprotein is necessary for induction of T-cell lymphoma by Marek's disease virus. J Virol 83 11142 11151
18. JarosinskiKWTischerBKTrappSOsterriederN 2006 Marek's disease virus: lytic replication, oncogenesis and control. Expert Rev Vaccines 5 761 772
19. FragnetLBlascoMAKlapperWRasschaertD 2003 The RNA subunit of telomerase is encoded by Marek's disease virus. J Virol 77 5985 5996
20. FragnetLKutERasschaertD 2005 Comparative functional study of the viral telomerase RNA based on natural mutations. J Biol Chem 280 23502 23515
21. TrappSParcellsMSKamilJPSchumacherDTischerBK 2006 A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis. J Exp Med 203 1307 1317
22. KauferBBTrappSJarosinskiKWOsterriederN 2010 Herpesvirus telomerase RNA(vTR)-dependent lymphoma formation does not require interaction of vTR with telomerase reverse transcriptase (TERT). PloS Pathog 6 e1001073
23. KimMMRiveraMABotchkinaILShalabyRThorAD 2001 A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation. Proc Natl Acad Sci U S A 98 7982 7987
24. LinJSmithDLBlackburnEH 2004 Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response. Mol Biol Cell 15 1623 1634
25. DjojosubrotoMWChinACGoNSchaetzleinSMannsMP 2005 Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma. Hepatology 42 1127 1136
26. GellertGCDikmenZGWrightWEGryaznovSShayJW 2006 Effects of a novel telomerase inhibitor, GRN163L, in human breast cancer. Breast Cancer Res Treat 96 73 81
27. HimlyMFosterDNBottoliIIacovoniJSVogtPK 1998 The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology 248 295 304
28. MoscoviciCMoscoviciMGJimenezHLaiMMCHaymanMJ 1977 Continuous tissue-culture cell lines derived from chemically-induced tumors of Japanese quail. Cell 11 95 103
29. LiXHJarosinskiKWSchatKA 2006 Expression of Marek's disease virus phosphorylated polypeptide pp38 produces splice variants and enhances metabolic activity. Vet Microbiol 117 154 168
30. JarosinskiKWMargulisNGKamilJPSpatzSJNairVK 2007 Horizontal transmission of Marek's disease virus requires U S2, the UL13 protein kinase, and gC J Virol 81 10575 10587
31. TischerBKvon EinemJKauferBOsterriederN 2006 Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques 40 191 197
32. SchatKACalnekBWFabricantJ 1982 Characterization of two highly oncogenic strains of Marek's disease virus. Avian Pathol 11 593 605
33. YamaguchiTKaplanSLWakenellPSchatKA 2000 Transactivation of latent Marek's disease herpesvirus genes in QT35, a quail fibroblast cell line, by herpesvirus of turkeys. J Virol 74 10176 10186
34. WitterRLKreagerKS 2004 Serotype 1 viruses modified by backpassage or insertional mutagenesis: approaching the threshold of vaccine efficacy in Marek's disease. Avian Dis 48 768 782
35. LeeLFLupianiBSilvaRFKungHJReddySM 2008 Recombinant Marek's disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV. Vaccine 26 1887 1892
36. KatneniUSchmidtCKeelerCGolovanSReddyS Analysis of immune signaling in Marek's disease virus vaccinated chickens. SpatzSMoodyA The 5 th International Workshop on the Molecular Pathogenesis of Marek's Disease Virus and 1 st Symposium on Avian Herpesviruses 2010; Athens, GA United States Department of Agriculture, Agriculture Research Service 47 47
37. GimenoIM 2008 Marek's disease vaccines: A solution for today but a worry for tomorrow? Vaccine 26 C31 C41
38. WozniakowskiGSamorek-SalamonowiczEKozdrunW 2011 Molecular characteristics of Polish field strains of Marek's disease herpesvirus isolated from vaccinated chickens. Acta Vet Scand 53 10
39. SchatKASellersHS 2008 Cell-culture methods. Dufour-ZavalaLSwayneDEGlissonJRPearsonJEReedWM A laboratory manual for the identification and characterization of avian pathogens: American Association of Avian Pathologists, Jacksonville, FL 195 203
40. SchumacherDTischerBKFuchsWOsterriederN 2000 Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. J Virol 74 11088 11098
41. JarosinskiKWOsterriederNNairVKSchatKA 2005 Attenuation of Marek's disease virus by deletion of open reading frame RLORF4 but not RLORF5a. J Virol 79 11647 11659
42. JarosinskiKWO'ConnellPHSchatKA 2003 Impact of deletions within the Bam HI-L fragment of attenuated Marek's disease virus on vIL-8 expression and the newly identified transcript of open reading frame LORF4. Virus Genes 26 255 269
43. ChbabNEgererAVeigaIJarosinskiKWOsterriederN 2010 Viral control of vTR expression is critical for efficient formation and dissemination of lymphoma induced by Marek's disease virus (MDV). Vet Res 41
44. PiepenbrinkMSLiXHO'ConnellPHSchatKA 2009 Marek's disease virus phosphorylated polypeptide pp38 alters transcription rates of mitochondrial electron transport and oxidative phosphorylation genes. Virus Genes 39 102 112
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
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