The results are shown as the means standard errors of three independent experiments.(9.1M, psd) 10.1186/s12977-016-0304-7 Reactivity of antibodies, VRC01, 42F9, 49G2, b12, KD247, 16G6, 0.5 and 2G12 to WT and mutant envelopes. more sensitivity Thalidomide-O-amido-C3-NH2 (TFA) to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the conformational unmasking of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples. Conclusions Mutations in gp41 that confer fusion Thalidomide-O-amido-C3-NH2 (TFA) inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users. luciferase activity was measured with a luminometer at 0, 15, 30, 45, 60, 75, 90, and 120?min time-points after co-culture. During co-culture, the expression level of envelope on the transfected cells was analyzed by staining with 2G12. The expression levels of envelope mutants were confirmed to be similar to that of WT envelope (<20?% change in MFI). The fusion percentage was calculated using the RLU value at 120?min as 100?%. Molecular dynamic (MD) simulations of the HIV-1 gp41 trimer The extracellular portion of the HIV-1JR-FL gp41 structures with and without an I37K mutation were constructed by using the homology modeling method with Molecular Operating Environment (Chemical Computing Group Inc., Montreal, QC, Canada). The crystal structure of the HIV-1 Thalidomide-O-amido-C3-NH2 (TFA) BG505 SOSIP.664 gp140 trimer at a resolution of 3.1 ? (PDB code: 4TVP) [40], which contains the extracellular portion of the gp41 trimer in association with the gp120 trimer, was used as the modeling template. MD simulations were performed as previously described to analyze changes in the structural dynamics of Rabbit Polyclonal to TFE3 protein interaction of the surface in solution [41C45]. The simulations were done by the pmemd module in the Amber 11 program package [46] with the AMBER ff99SB-ILDN force field [47] and the TIP3P water model for simulations of aqueous solutions [48]. A non-bonded cutoff of 10 ? was used. Bond lengths involving hydrogen were constrained with SHAKE, a constraint algorithm to satisfy Newtonian motion [49], and the time step for all MD simulations was set to 2?fs. After heating calculations for 20?ps until 310K using the NVT ensemble, simulations were executed using the NPT ensemble at 1?atm, at 310K, and in 150?mM NaCl for 100?ns. Root mean square fluctuation (RMSF) were calculated as previously described [41C45] to quantify the structural dynamics of the molecules in these MD simulations. RMSF of the C atoms were calculated to obtain information about the atomic fluctuations of individual amino acid residues during MD simulations [46]. The 2000 snapshots obtained from MD simulations of 80C100?ns were used to calculate RMSF. The average structures were used as reference structures for RMSF calculation. RMSF, which quantifies the differences between the average values and those obtained at given times of MD simulations, was calculated using the ptraj module in Amber, a trajectory analysis tool [46]. Results Enhanced neutralization of C34-, SC34-, and SC34EK-resistant mutants compared with WT and ENF-resistant mutants We selected HIV-1 strain JR-FL, which is a primary CCR5-tropic isolate that has been classified in the tier 2 level of neutralization sensitivity, to use as our WT for evaluating the neutralization sensitivity of drug-resistant mutants. The Env of JR-FL is relevant to subtype B.