Publicado
EXPRESIÓN DE LA PROTEÍNA CORE DEL VIRUS DE LA HEPATITIS C EN CÉLULAS HEPG2 USANDO EL VIRUS DEL BOSQUE DE SEMLIKI
Hepatitis C virus core protein expression in HepG2 cells using semliki forest virus
DOI:
https://doi.org/10.15446/abc.v26n1.79365Palabras clave:
Expresión transitoria, p53, Vector Viral, VHC (es)Transitory expression, p53, Viral Vector, HCV (en)
Descargas
El Virus de la Hepatitis C (VHC) codifica la proteína Core. Core, además de ser la subunidad de la cápside, participa en diferentes mecanismos de patogénesis de la infección por VHC. Dado que el sistema de replicación in vitrodel VHC presenta limitaciones, el uso de vectores virales podría ser una herramienta útil para estudiar las propiedades de la proteína Core. Con el fin de validar el vector con el Virus del Bosque de Semliki (SFV) para el estudio de Core en células HepG2, se evaluó la expresión de la proteína verde fluorescente (GFP) y la proteína Core utilizando este vector viral. Las expresiones de GFP y Core se detectaron en células HepG2 transducidas con rSFV de 24 a 96 horas postransducción. La expresión de la proteína Core fue inferior a la expresión de GFP en las células HepG2. Teniendo en cuenta que la proteína Core del VHC puede regular la actividad del gen p53, se evaluó el nivel transcripcional de este gen. Se observó una disminución en el nivel de mARN de p53 en las células luego de la transducción, comparado con las células control. Aunque las células transducidas con rSFV-Core presentaron el menor nivel de mARN de p53,la diferencia no fue significativa comparada con las células transducidas con rSFV-GFP. Los resultados confirman que rSFV permite la expresión transitoria de proteínas heterólogas en líneas celulares de hepatoma humano. Se necesitan estudios adicionales para determinar si la expresión disminuida de Core puede deberse a degradación de la proteína viral.
The Hepatitis C Virus (HCV) encodes the structural protein Core, which in addition to being the capsid subunit, participates in different mechanisms of HCV infection pathogenesis. Since HCV in vitroreplication system has limitations, the use of viral vectors could be a useful tool to study the Core protein properties. To validate the Semliki Forest Virus (SFV) strategy in transduced HepG2 cells to study the HCV Core protein, the expression of green fluorescent protein (GFP) and Core protein expressions were detected 24 to 96 hours post-transduction in HepG2 cells transduced with rSFV. Core protein expression was lower than GFP expression in HepG2 cells. Since HCV Core protein can regulate the activity of the p53 gene, the transcriptional level of this gene was evaluated. A decrease in the level of p53 mRNA was observed in the cells after transduction, compared to the control cells. Although the cells transduced with rSFV-Core had the lowest level of p53 mRNA, the difference was not significant compared to cells transduced with rSFV-GFP. The results confirm that rSFV allows the transient expression of heterologous proteins in human hepatoma cell lines. Additional studies are needed to determine whether the decreased expression of Core may be due to the degradation of the viral protein.
Referencias
Akkari L, Grégoire D, Floc’h N, Moreau M, Hernandez C, Simonin Y, et al. Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursors. J Hepatol. 2012;57(5):1021–1028. Doi: https://doi.org/10.1016/j.jhep.2012.06.027 DOI: https://doi.org/10.1016/j.jhep.2012.06.027
Bartenschlager R, Lohmann V. Replication of hepatitis C virus. J Gen Virol. 2000;81(7):1631–1648. Doi: https://doi.org/10.1099/0022-1317-81-7-1631 DOI: https://doi.org/10.1099/0022-1317-81-7-1631
Berglund P, Fleeton MN, Smerdou C, Liljeström P. Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice. Vaccine. 1999;17(5):497–507. Doi: https://doi.org/10.1016/S0264-410X(98)00224-2 DOI: https://doi.org/10.1016/S0264-410X(98)00224-2
Cerino A, Boender P, La Monica N, Rosa C, Habets W, Mondelli MU. A human monoclonal antibody specific for the N terminus of the hepatitis C virus nucleocapsid protein. J Immunol Baltim Md 1950. 1993;151(12):7005–7015.
Conti F, Buonfiglioli F, Scuteri A, Crespi C, Bolondi L, Caraceni P, et al. Early occurrence and recurrence of hepatocellular carcinoma in HCV-related cirrhosis treated with direct-acting antivirals. J Hepatol. 2016;65(4):727–733. Doi: https://doi.org/10.1016/j.jhep.2016.06.015 DOI: https://doi.org/10.1016/j.jhep.2016.06.015
DiCiommo DP, Bremner R. Rapid, high level protein production using DNA-based Semliki Forest virus vectors. J Biol Chem. 1998;273(29):18060–18066. Doi: https://doi.org/10.1074/jbc.273.29.18060 DOI: https://doi.org/10.1074/jbc.273.29.18060
Ehrengruber MU, Lundstrom K, Schweitzer C, Heuss C, Schlesinger S, Gähwiler BH. Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures. Proc Natl Acad Sci U S A. 1999;96(12):7041–7046. Doi: https://doi.org/10.1073/pnas.96.12.7041 DOI: https://doi.org/10.1073/pnas.96.12.7041
Feng S, Li M, Zhang J, Liu S, Wang Q, Quan M, et al. Regulation of HepG2 cell apoptosis by hepatitis C virus (HCV) core protein via the sirt1-p53-bax pathway. Virus Genes. 2015;51(3):338–346. Doi: https://doi.org/10.1007/s11262-015-1253-2 DOI: https://doi.org/10.1007/s11262-015-1253-2
Frelin L, Ahlén G, Alheim M, Weiland O, Barnfield C, Liljeström P, et al. Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene. Gene Ther. 2004;11(6):522–533. Doi: https://doi.org/10.1038/sj.gt.3302184 DOI: https://doi.org/10.1038/sj.gt.3302184
Gerstein AS, editor. Molecular biology problem solver: a laboratory guide. New York: Wiley; 2001. p. 575 DOI: https://doi.org/10.1002/0471223905
González-Grande R, Jiménez-Pérez M, González Arjona C, Mostazo Torres J. New approaches in the treatment of hepatitis C. World J Gastroenterol. 2016;22(4):1421–1432. Doi: https://doi.org/10.3748/wjg.v22.i4.1421 DOI: https://doi.org/10.3748/wjg.v22.i4.1421
Henao LF, Cortés F, Navas MC. Semliki Forest Virus: a viral vector with multiple applications. Colomb Médica. 2007;38(2):159–169.
Hourioux C, Ait-Goughoulte M, Patient R, Fouquenet D, Arcanger-Doudet F, Brand D, et al. Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane. Cell Microbiol. 2007;9(4):1014–1027. Doi: https://doi.org/10.1111/j.1462-5822.2006.00848.x DOI: https://doi.org/10.1111/j.1462-5822.2006.00848.x
International Agency for Research on Cancer (IARC). IARC Monographs Volume 100B Hepatitis C Virus [monografía en Internet]. 2012. Available from: https://monographs.iarc.fr/iarc-monographs-volume-100b-hepatitis-c-virus/ Cited: 12 Apr 2012.
Ip PP, Boerma A, Regts J, Meijerhof T, Wilschut J, Nijman HW, et al. Alphavirus-based Vaccines Encoding Nonstructural Proteins of Hepatitis C Virus Induce Robust and Protective T-cell Responses. Mol Ther. 2014;22(4):881–890. Doi: https://doi.org/10.1038/mt.2013.287 DOI: https://doi.org/10.1038/mt.2013.287
Jahan S, Khaliq S, Siddiqi MH, Ijaz B, Ahmad W, Ashfaq UA, et al. Anti-apoptotic effect of HCV core gene of genotype 3a in Huh-7 cell line. Virol J. 2011;8:522. Doi: https://doi.org/10.1186/1743-422X-8-522 DOI: https://doi.org/10.1186/1743-422X-8-522
Kaboev OK, Luchkina L a, Tret’iakov a N, Bahrmand a R. PCR hot start using primers with the structure of molecular beacons (hairpin-like structure). Nucleic Acids Res. 2000;28(21):E94. Doi: https://doi.org/10.1093/nar/28.21.e94 DOI: https://doi.org/10.1093/nar/28.21.e94
Kao C-F, Chen S-Y, Chen J-Y, Wu Lee Y-H. Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein. Oncogene. 2004;23(14):2472–2483. Doi: https://doi.org/10.1038/sj.onc.1207368 DOI: https://doi.org/10.1038/sj.onc.1207368
Kwak J, Tiwari I, Jang KL. Hepatitis C virus core activates proteasomal activator 28γ expression via upregulation of p53 levels to control virus propagation. J Gen Virol. 2017;98(1):56–67. Doi: https://doi.org/10.1099/jgv.0.000655 DOI: https://doi.org/10.1099/jgv.0.000655
Kwun HJ, Jang KL. Dual effects of hepatitis C virus Core protein on the transcription of cyclin-dependent kinase inhibitor p21 gene. J Viral Hepat. 2003;10(4):249–255. Doi: https://doi.org/10.1046/j.1365-2893.2003.00434.x DOI: https://doi.org/10.1046/j.1365-2893.2003.00434.x
Lee MN, Jung EY, Kwun HJ, Jun HK, Yu D-Y, Choi YH, et al. Hepatitis C virus core protein represses the p21 promoter through inhibition of a TGF-beta pathway. J Gen Virol. 2002;83(Pt 9):2145–2151. Doi: https://doi.org/10.1099/0022-1317-83-9-2145 DOI: https://doi.org/10.1099/0022-1317-83-9-2145
Lemmonier F, Chen M, Liljeström P, Brinster C, Paranhos-Baccala G, Inchauspé G, et al. Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles. J Gen Virol. 2002;83(2):369–381. Doi: https://doi.org/10.1099/0022-1317-83-2-369 DOI: https://doi.org/10.1099/0022-1317-83-2-369
Liljeström P, Garoff H. SFV expression systems. Technical manual. Huddinge, Sweden Novum Research Center, Huddinge; 1993. p. 31.
Lin C. HCV NS3-4A Serine Protease. In: Tan S-L, editor. Hepat C Viruses Genomes Mol Biol [Internet]. Norfolk (UK): Horizon Bioscience; 2006. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1623/. Cited 2017 Mar 25
Lu W, Lo SY, Chen M, Wu K j, Fung YK, Ou JH. Activation of p53 tumor suppressor by hepatitis C virus core protein. Virology. 1999;264(1):134–141. Doi: https://doi.org/10.1006/viro.1999.9979 DOI: https://doi.org/10.1006/viro.1999.9979
McGivern DR, Lemon SM. Virus-specific mechanisms of carcinogenesis in hepatitis C virus associated liver cancer. Oncogene. 2011;30(17):1969–1983. Doi: https://doi.org/10.1038/onc.2010.594 DOI: https://doi.org/10.1038/onc.2010.594
Navas M-C, Stoll-Keller F, Pavlovic J, Navas M-C, Stoll-Keller F, Pavlovic J. Lack Of Expression Of Hepatitis C Virus Core Protein In Human Monocyte-Derived Dendritic Cells Using Recombinant Semliki Forest Virus. Acta biol. Colomb. 2019;24(3):493–502. Doi: https://doi.org/10.15446/abc.v24n3.79368 DOI: https://doi.org/10.15446/abc.v24n3.79368
Poole MI, Sorribes I, Jain HV. Modeling hepatitis C virus protein and p53 interactions in hepatocytes: Implications for carcinogenesis. Math Biosci. 2018;306:186–196. Doi: https://doi.org/10.1016/j.mbs.2018.10.003 DOI: https://doi.org/10.1016/j.mbs.2018.10.003
Ríos-Ocampo WA, Daemen T, Buist-Homan M, Faber KN, Navas M-C, Moshage H. Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress. Redox Rep. 2019;24(1):17–26. Doi: https://doi.org/10.1080/13510002.2019.1596431 DOI: https://doi.org/10.1080/13510002.2019.1596431
Rosenthal ES, Graham CS. Price and affordability of direct-acting antiviral regimens for hepatitis C virus in the United States. Infect Agent Cancer. 2016;11:24. Doi: https://doi.org/10.1186/s13027-016-0071-z DOI: https://doi.org/10.1186/s13027-016-0071-z
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning a laboratory manual [Internet]. 2nd ed. Cold Spring Harbor, N.Y Cold Spring Harbor Laboratory; 1989. Available from: https://trove.nla.gov.au/version/264232692. Cited 2020 Jan 13
Sukowati CH. Significance of hepatitis virus infection in the oncogenic initiation of hepatocellular carcinoma. World J Gastroenterol. 2016;22(4):1497. Doi: https://doi.org/10.3748/wjg.v22.i4.1497 DOI: https://doi.org/10.3748/wjg.v22.i4.1497
Tornesello ML, Buonaguro L, Tatangelo F, Botti G, Izzo F, Buonaguro FM. Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections. Genomics. 2013;102(2):74–83. Doi: https://doi.org/10.1016/j.ygeno.2013.04.001 DOI: https://doi.org/10.1016/j.ygeno.2013.04.001
Torresi J, Meanger J, Lambert P, Li F, Locarnini SA, Anderson DA. High level expression of the capsid protein of hepatitis E virus in diverse eukaryotic cells using the Semliki Forest virus replicon. J Virol Methods. 1997;69(1–2):81–91. Doi: https://doi.org/10.1016/S0166-0934(97)00142-0 DOI: https://doi.org/10.1016/S0166-0934(97)00142-0
Vescovo T, Refolo G, Vitagliano G, Fimia GM, Piacentini M. Molecular mechanisms of hepatitis C virus-induced hepatocellular carcinoma. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2016;22(10):853–861. Doi: https://doi.org/10.1016/j.cmi.2016.07.019 DOI: https://doi.org/10.1016/j.cmi.2016.07.019
Vidalin O, Fournillier A, Renard N, Chen M, Depla E, Boucreux D, et al. Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens. Virology. 2000;276(2):259–270. Doi: https://doi.org/10.1006/viro.2000.0566 DOI: https://doi.org/10.1006/viro.2000.0566
Wahlfors JJ, Zullo SA, Loimas S, Nelson DM, Morgan RA. Evaluation of recombinant alphaviruses as vectors in gene therapy. Gene Ther. 2000;7(6):472–480. Doi: https://doi.org/10.1038/sj.gt.3301122 DOI: https://doi.org/10.1038/sj.gt.3301122
Welbourn S, Pause A. HCV NS2/3 Protease. In: Tan S-L, editor. Hepat C Viruses Genomes Mol Biol [Internet]. Norfolk (UK): Horizon Bioscience; 2006. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1622/. Cited 2017 Mar 25
World Health Organization, International Agency for Research on Cancer. Globocan. International Agency for Research on Cancer (IARC); 2018. Report No.: 263.
World Health Organization (WHO). Hepatitis C [Internet]. 2018. Available from: https://www.who.int/news-room/fact-sheets/detail/hepatitis-c. Cited 2019 Apr 12
World Health Organization, World Health Organization, Global Hepatitis Programme. Global hepatitis report, 2017 [Internet]. 2017. Available from: http://apps.who.int/iris/bitstream/10665/255016/1/9789241565455-eng.pdf?ua=1. Cited 2019 Apr 12
Cómo citar
APA
ACM
ACS
ABNT
Chicago
Harvard
IEEE
MLA
Turabian
Vancouver
Descargar cita
Licencia
Derechos de autor 2020 Acta Biológica Colombiana
Esta obra está bajo una licencia internacional Creative Commons Atribución-NoComercial-CompartirIgual 4.0.
1. La aceptación de manuscritos por parte de la revista implicará, además de su edición electrónica de acceso abierto bajo licencia Attribution-NonCommercial-ShareAlike 4.0 (CC BY NC SA), la inclusión y difusión del texto completo a través del repositorio institucional de la Universidad Nacional de Colombia y en todas aquellas bases de datos especializadas que el editor considere adecuadas para su indización con miras a incrementar la visibilidad de la revista.
2. Acta Biológica Colombiana permite a los autores archivar, descargar y compartir, la versión final publicada, así como las versiones pre-print y post-print incluyendo un encabezado con la referencia bibliográfica del articulo publicado.
3. Los autores/as podrán adoptar otros acuerdos de licencia no exclusiva de distribución de la versión de la obra publicada (p. ej.: depositarla en un archivo telemático institucional o publicarla en un volumen monográfico) siempre que se indique la publicación inicial en esta revista.
4. Se permite y recomienda a los autores/as difundir su obra a través de Internet (p. ej.: en archivos institucionales, en su página web o en redes sociales cientificas como Academia, Researchgate; Mendelay) lo cual puede producir intercambios interesantes y aumentar las citas de la obra publicada. (Véase El efecto del acceso abierto).