Confirmation Study Of HSF1 As A Drug Target For HIV Latent Reactivation

Highly Active Anti-retroviral Therapy(HAART)is applied in the treatment of AIDS patients successfully and controls the HIV virus in the patients’bodies effectively to lengthen patients’ life,however,the drugs have to be taken for the rest of their life due to the presence of the latent reservoir of HIV in patient’s body.Currently,the major strategy for how to remove the latent HIV virus reservoir is,Shock and Kill",that is,to activate HIV latent infection,and proceed to remove the activated latent virus reservoir by using HAART drugs or improving the immune system,so as to eventually achieve the purpose of functional cure of HIV and patients do not have to use drugs lifelong to prevent from rebounding of HIV.At present,in terms of the mechanical study of formation and maintenance of the HIV latent reservoir,latency reversing agents that reactivate HIV latent cells have been developed through different pathways.In spite of the application of a few latent infection activators in clinical tests,there still exist some defects,such as the inability to reduce the size of the latent reservoir of HIV,poor specificity and high side-effects.Therefore,the research on new HIV latent activation targets and the strategy of developing new latency reversing agents are the popular topics in the filed of HIV latent activation in the future.Heat shock factor(HSF)is a key factor in Heat shock response,which is widely existed in the transcription factor of eukaryotic cells.Under non-stress conditions,HSFI mainly exists as an inactive monomer.When they are stimulated,HSF1 will go into the nucleus in the format of active trimers and various modifications will take place insides.Our previous research has found that HSF1 may be an important transcriptional regulatory factor in the activation process of latent HIV,and drugs based on this target may become a new type of HIV latency reversing agents.However,those drugs used in previous studies are with defects of low potency owing to their certain toxic and side effects,which may be owing to the lower selectivity of the targe.This study aims to confirm that HSF1 is one kind of drug target of the new HIV latency reversing agents,which will serve as a theoretical basis for the development of a safe and efficient new latency reversing agents that specifically acts on HSF1·Firstly CRISPR/Cas9 technology was used in this topic to establish the latent HIV infection cell model to knock-out HSF1.Both classic latency reversing agents and reported drugs,which could activate HSF1 protein and promote HIV latent activation,are utilized to act on cells with HSF1 or without HSF1,in the following managed by flow cytometry technique and protein imprinting method.It is confirmed that classic latency reversing agents will not have a significantly lower level of latent HIV infection after screening the HSF1 protein,while the level of HIV latent infection in the drugs activated by HSF1 is in the trend of reducing.To investigate this phenomenon,on one hand,we constructed a HSE-driven Luciferase-HSE plasmid to integrate into HEK-293T cells through the way of lentivirus infection,and constructed a HEK-293T luciferase cell with stable Luciferase-HSE.On the other hand,CRISPR/Cas9 technology was further used to select the HSF1 protein of the cell and the HEK-293T luciferase cells were knocked out HSF1.After using the drugs above mentioned,we proceeded to compare the rate of the change of Luciferase.It was found that the ratio of Luciferase in the cells without HSF1 was decreased at the same concentration of classical LRAs compared to normal cells,whereas the Luciferase ratio was increased at the same concentration of LRAs through HSF1.It was confirmed that HSFl is one of the mechanisms by LRAs activate latent HIV reactivation,but there are still other factors in the cell that can replace HSF1 in HSE to exert latent HIV reactivation.In order to explore the replaceable protein of HSF1,we tested the relevant protein called PARP1(poly ADP ribose polymerase 1)and its ability to activate latent HIV.Using methods like flow cytometry,RT-qPCR,Western Blotting and so on,it was confirmed that the DNA damage induced by H2O2 can activate latent HIV,but the level of activation dropped after applying PARP1 inhibitors.Meanwhile,H2O2 could not down-regulate the Luciferase intensity of HEK-293T-HSELuciferase cells without HSF1.Therefore,it was proved that PARP1 could serve as one kind of replaceable protein for HSF1 in HIV latent cells.In summary,HSF1 can be used as a drug target for the study of novel HIV latency reversing agents,which also serves as a theoretical basis for the further development of HIV latency reversing agents that specifically act on HSF1 It would be expected that the discovered compounds could clear up the HIV latent infection reservoir and provide a new method of drug treatment to accomplish functional cure ultimately.