Summary

طريقتين لتشكيل متغايرة النوى إلى اكتشاف العوامل تقييد HCV

Published: July 16, 2012
doi:

Summary

ونحن تصف طريقتين لشرطي<em> العابرة</em>-تكامل من التهاب الكبد الوبائي جمعية (سي) فيروس والانتهاء من دورة الحياة الفيروسية كامل، والتي تعتمد على تشكيل متغايرة النوى. هذه التقنيات هي مناسبة للكشف عن خطوط الخلايا التي تعبر عن عوامل تقييد المهيمنة، والتي تحول دون إنتاج ذرية HCV المعدية.

Abstract

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.

We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.

Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected naïve cells in a receptor-dependent fashion.

To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

Protocol

فيوجن الخلية بواسطة PEG 1. خلية ثقافة ثقافة هوه-7.5، الحله وHep56.1D خطوط الخلايا ساذجا أو التعبئة والتغليف 12 في 15 سم أطباق ثقافة خلية كاملة في DMEM (DMEM cplt) المتوسطة، على أن تستكمل مع DMEM 2…

Discussion

نقدم طريقتين للحث على تشكيل متغايرة النوى في الخلايا المزروعة لتحليل القيود السلبية السائدة التي تحول دون تكرار سي. باستخدام هذه الإجراءات استبعدنا وجود عامل مهيمن أعرب constitutively أو فيروس من صنع الإنسان في مختلف الكبد غير والفئران في خطوط خلايا الكبد. الفحص 1 يحلل في ا…

Disclosures

The authors have nothing to disclose.

Acknowledgements

ونحن ممتنون لWakita Takaji وينس لBukh JFH1 والعزلات J6CF، على التوالي. وعلاوة على ذلك نشكر تشارلز رايس للخلايا 7.5 هوه والأجسام المضادة 9E10، ستيفن Foung للأجسام المضادة E2 محددة CBH-23، وجميع أعضاء قسم علم الفيروسات التجريبي، Twincore للحصول على اقتراحات مفيدة والمناقشات.

Materials

Name of the reagent Company Catalogue number
DMEM Invitrogen, Karlsruhe, Germany 41965-039
L-glutamine Invitrogen, Karlsruhe, Germany 25030-024
Non-essential amino acids Invitrogen, Karlsruhe, Germany 11140-035
Penicillin/ streptomycin Invitrogen, Karlsruhe, Germany 15140-122
Fetal calf serum PAA, Cölbe, Germany A15151
α-E2 (CBH23) kindly provided by Steven Foung 10  
ATP Sigma, Steinheim, Germany A2833-106
Glutathione Sigma, Steinheim, Germany G4251-1G
Blasticidin Invivo Gen, San Diego, USA Ant-bl-1
G418 (geneticin) Invitrogen, Karlsruhe, Germany 11811-064
Polyethylene-glycol-1500 Roche, Mannheim, Germany 10783641001
Paraformaldehyde Roth, Karlsruhe, Germany 0335.3
Triton X-100 Roth, Karlsruhe, Germany 3051.2
Goat serum Sigma, Steinheim, Germany G9023-5mL
α-NS5A (9E10) Kindly provided by Charles Rice 7  
DAPI (4′,6′- diamidino-2-phenylindole dihydrochloride) Invitrogen D1306
Alexa-Fluor 546 – goat anti-human IgG Invitrogen, Karlsruhe, Germany A21089
Alexa-Fluor 488 – goat anti-mouse IgG Invitrogen, Karlsruhe, Germany A10680
Lipofectamine 2000 Invitrogen, Karlsruhe, Germany 11668-019
CellTracker CMTMR Invitrogen, Karlsruhe, Germany C2927
CellTracker CMFDA Invitrogen, Karlsruhe, Germany C2925
Fluoromount Sigma, Steinheim, Germany F4680-25ML
All other chemicals Roth, Karlsruhe, Germany  
Cell culture materials Sarstedt, Nümbrecht, Germany  

References

  1. Zhu, Q., Guo, J. T., Seeger, C. Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J. Virol. 77, 9204-9210 (2003).
  2. Kato, T. Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon. J. Virol. 79, 592-596 (2005).
  3. Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line. J. Virol. 78, 491-501 (2004).
  4. Date, T. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells. J. Biol. Chem. 279, 22371-22376 (2004).
  5. Chang, K. S. Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities. J. Virol. 80, 7364-7374 (2006).
  6. Zhong, J. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U.S.A. 102, 9294-9299 (2005).
  7. Lindenbach, B. D. Complete replication of hepatitis C virus in cell culture. Science. 309, 623-626 (2005).
  8. Wakita, T. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11, 791-796 (2005).
  9. Witteveldt, J. CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells. J. Gen. Virol. 90, 48-58 (2009).
  10. Bitzegeio, J. Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors. PLoS Pathog. 6, e1000978 (2010).
  11. Lindemann, D., Goepfert, P. A. The foamy virus envelope glycoproteins. Curr Top Microbiol Immunol. 277, 111-129 (2003).
  12. Brohm, C. Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation. J. Virol. 83, 11682-11693 (2009).
  13. Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R., Pietschmann, T. Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J. Virol. 82, 7034-7046 (2008).
  14. Koutsoudakis, G. Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses. J. Virol. 80, 5308-5320 (2006).
  15. Frentzen, A. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines. PLoS Pathog. 7, e1002029 (2011).
  16. Lindemann, D. A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity. J. Virol. 75, 5762-5771 (2001).
  17. Blight, K. J., McKeating, J. A., Rice, C. M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76, 13001-13014 (2002).

Play Video

Cite This Article
Frentzen, A., Hueging, K., Bitzegeio, J., Pietschmann, T., Steinmann, E. Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors. J. Vis. Exp. (65), e4029, doi:10.3791/4029 (2012).

View Video