Study On The Mechanism Of HIV-1 Membrane Fusion Inhibitor SC29EK And SFT Resistance

In the process of HIV-1 virus entering the host' s cell,the envelope protein(Env)plays a critical role,it mediates the combining of virus and receptor of host' s cell and co-receptors and the membrane fusion of viral envelope and cell membrane of host cell.In the whole process,the forming of 6-HB structure is a vital step,so it became the key target for drug design of fusion inhibitor.SC29EK and SFT belong to the third generation fusion inhibitor which both can inhibit the forming of viral 6-HB structure and have the design of salt bridge in helix.Compared to the last generation inhibitors,they have better structural stability and antiviral activity.We have found amino acid mutations in the variants selected by SC29EK and SFT,which are Q39R,N43K,E49A,V38A,A47I,Q52R which are located in Gln39?Asn43?Glu49?Val38?Ala47?Gln52 of gp41 respectively.Using site mutation technology we got 15 kinds of plasmids came from the template plasmid which could express the Env protein including the single mutations or mutation combinations to be researched.Mutations had no effect on expression and cleavage of Env protein was confirmed by performing capture ELISA.We identified the effects made by single mutation or combined mutations on ability of viral cell entry and cell fusion and the resistance to SC29EK and SFT.By synthesizing the natural NHR peptide N36 or N36 peptides including the mutations,the changes of the structural stability of 6-HB and binding affinity of N peptide and fusion inhibitors caused by mutations was researched.By conforming ELISA,the influence made by mutations on the conformational change of 6-HB was explored.Based on analyzing the experimental data,we concluded that:(1)N43K decides the viral high resistance to SC29EK,when combined with Q39R or E49A,the resistance would be increased further.Q52R resulted in the viral high resistance to SFT,when combined with V38A and A47I the resistance to SFT could be increased much more;(2)The variants had cross-resistance to different inhibitors including T20,C34 and 2P23;(3)some mutations could decrease the binding affinity of N peptides and inhibitors and some mutations could increase the binding affinity of N peptides and endogenous C peptide,they could act synergistically to improve the resistance;(4)the acquirement of resistance was often accompanied with the loss of Env' s function;(5)mutations caused the conformational changes of 6-HB.The study provides new insights into the molecular mechanisms of HIV-1 resistance to different inhibitors and will help us understand the structure-function relationship of gp41.