| HIV Envelope mediates receptor-binding and membrane fusion, which plays an essential role in the entry step. HIV envelope fusing with the target cells’ membrane is a key stage in the release of viral capsid into target cells. It is shown that formation of 6-HB is crucial to membrane fusion, and peptides derived from NHR or CHR can interrupt that structure, making it an ideal target for drug. T20 (Enfuvirtide, FUZEON) is a fusion inhibitor that is the first-and-only-available drug of this kind approved by FDA in curing AIDS. However, T20, with low resistant gene barrier, induces resistant mutants easily both in vivo and in vitro. Based on the newly found structure, the M-T hook, a short peptide called MTSC22 was designed with SC22 as a template, which owns not only high potency but also high genetic barrier to resistance. Two sites of amino acid resistant mutations have been selected by SC22EK and MTSC22, and they are confirmed as E49K (Glu49) and L57R (Leu57) respectively. The amino acid Glu49 locates on the N-terminal helix adhering to the pocket domain, while Leu57 actually forms the pocket domain. On the other hand, these two mutants are proved to have huge effects on both Env’s functions and virus’s sensitivity to short-peptide inhibitors.Based on our previous works, we studied the functions of naturally-occurring mutations of E49 and L57 residues. On the E49 site, seven substitutions were identified, including E49A, E49D, E49G, E49K, E49N, E49Q, and E49S; On the L57 site, there were four substitutions:L57F, L57P, L57R, L57V. Additionally, L57A was included for comparison. Totally,12 substitutions were introduced into the Env of HIV-1NL4-3 template. Firstly, Western Blot and capture ELIS A were performed to determine the effects on Env expression and protein cleavage and no obvious effects were found. Then, the effects of each mutations on the ability of HIV-1 entry and its sensitivity on fusion inhibtitors were determined. We found that several mutations could significantly affect the virus entry and mediated high-resistantance to fusion inhibitors.. Besides, the NHR peptide N36 and mutant N36 peptides earring 12 corresponding substitutions (N36F49A/D/F/K/N/Q/S and N36L57A/F/P/R/V) were synthesized and their inhibitory potency and thermol stability were tested. The results demonstrated the changes in the NHR stability and the affinity between trimeric NHR coiled-coil and CHR. Briefly, we discussed the mechanisms of HIV-1 resistance to fusion inhibitors from four aspects as following:the effects of mutations on the Env function, the helical stability of NHR helix,6-HB stability and the binding affinity of inhibitors.Based on the comprehensive analysis, we conlude that (a) Natural substitutions on E49 and L57 caused high resistance level to short-peptide inhibitors, while only mild resistance to the first- and next-generation inhibitors;(b) Drug resistance of mutants was a balanced result between the interaction of two specific amino acids and the impact on 6-HB; (c) Our results partly matched the theory that the poor Env function may cause drug resistance; (d) For the substitutions E49S/N/G, the stability of NHR coiled-coil was mainly responsible fordrug resistance; (e) For the substitutions E49A/Q/D, the 6-HB stability caused by amino acids’ charges accounts for the effects (f) As the amino acid Proline can break the a-helix, the mutant L57P lost the infectivity completely (g) As the amino acids A, F, V are hydrophobicsimilar to the residue L, the mutants L57A/F/V performed mild resistance to short-peptide inhibitors (h) The mutant L57F resisted to C34 and SFT slightly, which may be caused by the steric obstruction of the Benzene ring on Phenylalanine. Taken together, our data provide important information for understanding the structure and fusntion of HIV-1 gp41, the mechanism of gp41-mediated cell fusion, and the mechanism HIV-1 resistance to the fusion inhbitors, and will afficiate the development of novel HIV-1 fusion inhibitors. |
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