Studies Of RNA Interference On Vif Gene Of HIV

Despite years of intensive research and some therapeutic success,AIDS,caused by infection with human immunodeficiency virus 1(HIV-1) continues to be a major health and economic problem worldwide,because none of available antiretroviral drugs at present could provide a thorough control of disease.New and effective therapeutic or preventive approaches are dearly needed,and gene therapy carries considerable promises in this respect.The emergence of siRNAs as a powerful tool for posttranscriptional gene silencing has given credence to the notion of using siRNAs for certain therapies,particularly in the field of HIV treatment.This potential gene therapy renders new hope for the control of HIV,since many data has demonstrated the usefulness of siRNAs in controlling HIV by interfence on some genes of the viruses.For example,there has been some studies proved that HIV could be suppressed by RNA interfence on the target genes of gag,tat,rev or CCR5 receptor. These developments are indicative of an increasingly stronger role for siRNAs as a potential therapeutic strategy for HIV/AIDS.As one characteristics of HIV-1,various regulatory genes take an important role in the process of viruses RNA transcription,post-transcriptional processing,proteins translated processing and releaseing of virus particle from cells.HIV-1 viral infectivity factor(Vif),a highly basic 23 kDal protein composed of 192 amino acids, is one of six regulatory proteins encoded by HIV-1(the others are Tat,Rev,Nef,Vpu and Vpr) and has less mutation than other encoding genes.While essential for virus replication,its precise function is still being defined and studied.Naturally occurring HIV-1 strains with mutations in Vif are known to replicate at significantly lower levels to 1000 times compared to wild-type virus.Vif-defective virions exhibit structural abnormalities in the virus core and are incapable of completing proviral DNA synthesis in acutely infected cells.But there is no differences found in the RNA sequence structure and proteins of vif-defective virions compared to wild-type virus. Further highlighting its importance in the virus replication cycle,Vif-like proteins are encoded by all known lentiviruses except equine infectious anemia virus.Evidence exists that Vif is required during the late stage of virus formation and maturation from the producing cell.Because virions produced in the absence of Vif are incapable of completing the incorporation of proviral DNA into host chromosomes in newly infected cells,Vif activity is presumed to be required for core formation and stability, for regulating the incorporation of envelope into budding virions,and/or for modulating proviral DNA synthesis.The finding that this effect is cell-type specific led to the designation of cell lines as permissive or non-permissive for HIV replication, based upon the requirement of Vif for viral replication.Non-permissive cells require a functional Vif to complete the HIV replication cycle.Based on these observations, agents directed against Vif are likely to interfere with or inhibit viral replication. While HIV-1 Vif is predominantly localized to the cytoplasm,it also exists in a membrane-associated form.Phosphorylation is important for the interaction of Vif with cellular and viral proteins,and in targeting Vif to specific cellular compartments. Thus,in addition to HIV proteins,cellular kinases may serve as targets for developing novel anti-HIV agents.Respecting of this importance role of vif and the discovery of its interaction to APOBEC3G(apolipoprotein B mRNA editing enzyme catalytic polypeptide like 3G),more and more attentions are paid on the researches about this agent and its function in viruses reproduction of HIV-1.While the molecular mechanism of RNA silencing has been precisely defined since studied for many years,it is a newly described natural biological phenomenon mediated by small interfering siRNAs(siRNAs),which target viral mRNAs,rather than the proteins that they encode,for degradation by cellular enzymes.It has emerged as a powerful protecting tool to resist viruses or other extra-genes.The dsRNA is processed to 21-25 nt short interfering RNA(siRNA),mathed to the target mRNA,generated from doublestranded RNA by the RNAseⅢ-like protein Dicer in the initiating step of RNAi.So the process of RNAi is originally recognised as post-transcriptional gene silencing in plants(PTGS).RNAi is a new and potential gene tool,which is simple,speedy,specific,effective,low-cost and feasible,better than reverse nucleic acid technology and simpler than gene knock-out.In mammalian cells,delivery of chemically synthesized short interfering RNA specifically silences expression of the corresponding endogenous gene,thus bypassing the non-specific inhibitory mechanisms elicited by longer double-stranded RNA.Conceptually,RNAi technology can be used alone,or in combination with other existing therapeutic tools, to curb diseases that are caused by dominantly acting mutant alleles,such as cancer and neurodegenerative diseases,as well as to combat viral infection.Additionally, RNAi can be used to analyze gene function,investigate molecule conducting process, explore new drugs and gene therapy,and so on.As one gene therapy tool,SiRNA has been proved to be used in HIV treatment,and no interaction could occur when combining with available antiretroviral agents.This potential antiviral technology is believed to be other effective and powerful therapy tool for HIV in the future.So in present study,the aim is to achieve sustained and synergistic siRNA-mediated inhibition of HIV-1 replication by targeting at vif gene.There were 3 SiRNAs specific to HIV-1 vif designed in the study,all of which showed suppression effects on replication of HIV-1 by cotransfected with pEGFP-N1/vif plasmid.At last,this potential suppression was validated at mRNA and protein level demonstrating siRNA-mediated inhibition of HIV-1 replication by targeting at vif gene.To study the suppression of specific SiRNA on HIV-1 by directed to vif gene and explore the potential effects of gene therapy for HIV-1,SiRNA and the plasmid expressing vif gene and EGFP were cotransfected into HEK 293T cell,to express the vif protein and investigate the suppression effect of SiRNA on HIV-1 vif.At first, pEGFP-N1-vif plasmid was constructed and then transfected into HEK 293T cell alone.The cells then were harvested and observed transfected yield under fluorescence microscope and western blot was used to assess for vif protein production.Notably,HIV-1 vif protein was exhibited to highly express in HEK 293T cells.Six sequences of SiRNA(21nt) synthesized through transcription in vitro from DNA templates,four sequences specific to vif and one positive control,one negative control,were contransfected with pEGFP-N1-vif plasmid into HEK 293T cella. Under fluorescence microscope,effective suppression on expression of vif protein was investigated for weakened green light.Then cells were harvested and extracted total RNA,to quantitate by Real-time PCR and assess protein production by western blot.Consequently,there were 3 sequences SiRNA targeted to HIV-1 vif exhibiting moderate inhibited ability on vif mRMA,S-SiRNA1,S-SiRNA2 and S-SiRNA3 with 53%,60%and 63%of supression effect rate,respectively.Meanwhile,Vif protein production was observed to decrease obviously.These results suggested the SiRNA targeted to vif could suppress HIV-1 vif replication specifically.The pEGFP-N1-HIV-1/vif,an expression vector for HIV-1 vif gene,was construceted and expressed in mammalian cell successfully in this study.Vif protein was exhibited highly production in this mammalian cell expression system.The use of siRNAs,a new gene tool,to target HIV-1 vif was proved useful in inhibition of vif expression.While it has been proposed that highly conserved regions of HIV-1 might be better targets despite limitation in sites suitable for siRNA targeting.Regions in HIV-1 vif may be a better targets,which was suggested in this study.It indicated that RNAi,as a gene therapeutic tool,would be potential and useful for anti-HIV treatment in the future.The characteristics of RNAi,speedy,specific,effective, low-cost and feasible,could also be suggested in present study.However,the interference rates were keeping around 50%.This meaned HIV-1 vif of this study was not knockouted completely,not as some researches demonstrating knockout rate 80%～95%.It was explained to be likely related to my conditions and operations,or the low RNAi-effect of vif.No matter what the reason was,RNAi was deserved for further study,as a potential therapeutic tool for HIV-1.These data suggest that siRNAs,a breakthrough in lifescience field,may be widely used as alternate therapeutic agents,particularly for anti-HIV treatment. Although RNAi does work effectively as an antiviral agent in cell cultures and many data were also proved successfully,there are many problems existing in the road to the day of RNAi application in clinic.So many works and preparasion should be made at theory and practice levels for RNAi application as a therapeutic method. HIV-1 infection is a worldwide disease that requires alternate therapeutic strategies. Despite significant advances,current treatments are limited by toxicity,complexity, cost,and resistance and they do not afford a complete cure.So intracellular immunization by gene therapy strategies offers a promising alternative approach for controlling and managing HIV/AIDS.With the discovery that the RNAi phenomenon operates in mammalian cells and is highly effective in selective gene silencing,new potent siRNA molecules,which may be combined with other antiretroviral agents, have become available to add to the anti-HIV arsenal.