طريقتين لتشكيل متغايرة النوى إلى اكتشاف العوامل تقييد HCV
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
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 |
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