Improved Human Immunodeficiency Virus(HIV) Outgrowth Assay And Murine Models By A Dcas9 Activation System

Background Acquired Immunodeficiency Syndrome(AIDS)can be caused by human immunodeficiency virus(HIV)infection in humans.Combination Antiretroviral Therapy(cART)can be reduced the replication of virus lower than the level of HIV-1 detection in HIV-infected individuals,but can't completely eliminate it.One of the main challenges in the fight against HIV infection is to cure that are able to eliminate the persistent viral reservoir that harbours integrated,replication-competent provirus within host cellular DNA.At present,there are many shortcomings in the approaches of detecting viral reservoir.How to find viral reservoir correctly and rapidly?Therefore,in this paper,we posed questions and improved the viral outgrowth assay(VOA),which is currently regarded as the 'gold standard,to detect viral reservoir.As a result of the complexity of HIV-1,there is no effective vaccine.Animal models are needed to explore the complications of HIV infected individuals,especially after anti-retroviral therapy becomes a chronic disease.Based on dCas9 activation system,we improved HIV-1 outgrowth assay and HIV murine model by means of combining dCas9 activation system with Eco-HIV.Objective In order to improve the HIV-1 outgrowth assay and murine cell models by a dCas9 activation system.Methods 20 gRNAs sequences were constructed targeting the LTR sequence of HIV-DNA.Screening of gRNAs was performed based on luciferase intensity,acquiring the optimal gRNA design.The optimal gRNA-L along with the dCas9 activation system were transfected into Jurkat cells,establish working cell lines.HIV-luciferase infected Jurkat cells were cultured for the replication of HIV latency.Luciferase intensity was observed and measured to characterize the detection capability of the amplification model on day7 and dayl4.A chemical based activation reagent was adopted to further evaluate the performance of the dCas9 activation system.In the direction of testing whether murine cell lines infected by Eco-HIV can complete virus growth cycle when the dCas9 activation system was used.Results Acquired optimal gRNA designs named gRNA-L and gRNA-O.Compared with the Luciferase activity intensity of blank control(9876 ± 437 pg/ml)on day6,significant amplification of HIV-Luc was observed(85530 ± 979.2)and(96925 ± 2759)(p<0.01).gRNA-L was eventually selected for superior performance.The namely Jurkat 6465L cell line was constructed by transfecting gRNA-L and the dCas9 activation protein complex as the working cells for HIV latency analysis.On day7 and 14,The frequency of activating HIV latent model cells was 1/864 and 1/755 which was closer to the pre-set frequency(1:500).The dCas9 activation model was further evaluated in comparison with a chemical-based HIV induction reagent SAHA.The dCas9 model yielded results with frequency readings(1:615)closer to the pre-set frequency(1:500)than the regent-based control(1:749).dCas9 activation system can activate C6 cells of rats and BsB8 cells of mice infected by Eco-HIV.In uninfected group,the fluorescence intensity(RFU)of C6-6465-L(74.67±5.812)was not higher than C6-6465-O(84±8)compared with C6-6465-control(86.67±3.528);Compared with the fluorescence intensity(RFU)of blank control(105.3 ±3.528)in infected group,significant amplification of HIV-Luc was observed(C6-6465-L(2839±25.75),C6-6465-0(3173±61.2))(p<0.01).The dCas9 activation system can activate the BsB8 cell line of mice infected by Eco-HIV.The fluorescence intensity(RFU)of BsB8-6465-L(929.7 ± 11.55)was higher than BsB8-6465-control(179±12.49)comparedwith C6-6465-control(169.3±8.353)(p<0.01).The dCas9 activation system can be determined the transcription and replication of C6 cells in rats infected by EcoHIV.the fluorescence intensity(RFU)of C6-LTR-L(788±41.63),C6-LTR-O(836 ±28.1),C6-LTR-empty(85.33±7.055),Comparedwith C6 without infection(83.67+8.11)respectively,p<0.01,p<0.01,N.Conclusion Compared with the traditional methods,dCas9 activation model improves the HIV outgrowth assay by promoting HIV transcription to detectable levels with proper accuracy and efficiency.The capability to amplify and detect latent virus in such a manner serves great significance in the field.Solved the difficulty that the EcoHIV murine model could not be transcribed and duplicated.