Effect Of CCR5 Site-directed Mutagenesis On The Anti HIIV-1 Activity Of Thioraviroc In Vitro

Objective:CCR5 has a wide range of genetic polymorphisms,and there are obvious ethnic differences.Single nucleotide polymorphisms in the coding region of CCR5 have been found in the Chinese Han population.These mutation sites may lead to conformational changes in the protein,which may alter the structure and function of the receptor.Thioraviroc is the first CCR5 antagonist approved for clinical trial in China.The study on the effect of site-directed CCR5 mutants on the anti-HIV-1activity of Thioraviroc can provide a basis for the efficacy of personalized medicine.Methods:Firstly,the lentiviral expression vector of CCR5 and its mutants were constructed,the lentiviral particles of CCR5 and its mutants were packaged in the three-plasmid system and then infected HOS-CD4 cells;Cells with stable expression of CCR5 and its mutant were screened by antibiotic G418,after further screening by limiting dilution method,the cell membrane proteins were extracted for western blot identification,and monoclonal cells HOS-CD4-CCR5 and its mutant strains were obtained.Western blot and flow cytometry were used to detect the expression of CCR5 protein on the surface of HOS-CD4-CCR5 and its mutant cells;quantitative detection of the infectivity of HOS-CD4-CCR5 and its mutant cells against R5tropic virus HIV-1_SF162 by ELISA.The inhibitory effect of Thioraviroc（DC521022）on the entry of HIV-1_SF162 into HOS-CD4-CCR5 and its mutant cells was detected by real-time fluorescent quantitative PCR;HIV-1 p24 antigen was captured by ELISA method to detect the effect of Thioraviroc（DC521022）on inhibiting HIV-1_SF162 entry into HOS-CD4-CCR5 and its mutant cells.All experiments used Maraviroc（MVC）as a positive control drug.Results:（1）The lentivirus expression vector Plvx-IRES-NEO-CCR5 and its mutant were successfully constructed,and it was proved that both of them could successfully express CCR5 protein.（2）After continuous screening of antibiotics and western blot identification,HOS-CD4-Plvx 7 strains,HOS-CD4-CCR5 5 strains,HOS-CD4-CCR5/503G/T2 strains,HOS-CD4-CCR5/668G/A 1 strain,HOS-CD4-CCR5/Δ894C 7 strains,HOS-CD4-CCR5/999G/T 4 strains monoclonal cells were obtained respectively.（3）Compared with wild-type CCR5 cells,the surface expression of CCR5 protein in site-directed mutant cell lines was reduced.（4）Compared with wild-type CCR5 cells,the susceptibility of site-directed mutant cell lines to HIV-1_SF162 was reduced.（5）At the level of HIV-1 virus RNA,DC521022 could effectively inhibit the entry of HIV-1_SF162 virus into HOS-CD4-CCR5 and its mutant cells at the concentration of 100 n M;Among them,in HOS-CD4-CCR5 and HOS-CD4-CCR5/668G/A cells,DC521022 had a better inhibitiory effect on HIV-1_SF162 virus than the positive control drug MVC at the same concentration,while the inhibitiory effects of two drugs in remaining CCR5 mutant cells are equivalent.（6）At the level of HIV-1 virus p24 protein,DC521022 could effectively inhibit the entry of HIV-1_SF162 into HOS-CD4-CCR5 and its mutant cells,and the inhibitiory effect was equivalent to that of the positive control drug MVC.The EC50 values of DC521022 and MVC in HOS-CD4-CCR5 cells were 1.58±1.10 n M and 1.85±0.54 n M,respectively.In HOS-CD4-CCR5/503G/T cells were 3.23±0.88 n M and 2.66±0.03 n M,respectively.And 2.74±0.02 n M and 2.34±0.40 n M in HOS-CD4-CCR5/668G/A cells,respectively.In HOS-CD4-CCR5/Δ894C cells were 0.40±0.04 n M and 0.46±0.03 n M;In HOS-CD4-CCR5/999G/T cells were 1.43±0.27 n M and 1.33±0.53 n M,respectively.Conclusion:Through the construction of CCR5 and four kinds of site-directed mutation plasmids,the monoclonal cell lines of CCR5 and its mutants were screened.It was detected that these CCR5 mutants could reduce the expression of CCR5protein on the cell surface and reduce the infectivity of the cells to R5 tropic virus HIV-1_SF162.Under the effect of CCR5 antagonist,DC521022 can effectively inhibit HIV-1_SF162 from entering HOS-CD4-CCR5 and its mutant cells,and the inhibitiory effect is better than or equivalent to MVC.The site-directed mutations of CCR5 did not produce significant resistance to CCR5 antagonists DC521022 and MVC,theΔ894C mutant can significantly improve the anti-HIV-1 effects of DC521022 and MVC.