| Neutralizing antibodies specific for HIV can bind the virus and inhibit HIV entry into host cells. They play an important role in the control of HIV infection. The targets of HIV-specific neutralizing antibodies are the sites associated with receptor binding or membrane fusion on HIV's envelop protein. The membrane protein gp120 plays an important role in HIV-1 entry into host cells. It is also the main target of HIV-1 neutralizing antibodies for suppressing viral infection. V3-region of gp120 is related to the binding of host-receptors and many neutralizing antibody epitopes are located in V3 loop. However, roles of V2-region of gp120 are not well understood. The previous study found a naturally occurred V2-region mutant virus, L175P env, in the large time culture of HIV-1. The V2 mutation dramatically changed the neutralizing ability of KD-247. But weather the V2 mutation can generally change the binding of V3 antibodies is still unknown.Part 1.293T cells were transfected by pJR-FL WT env, pJR-FL L175P env, pSG3 and pRSV-Rev to get the pseudovirus. Pseudovirus suspension, MAbs at various concentrations and TZM-bl cells were mixed and cultured. The luciferase activity was measured by microplate luminometer. The reduction in infectivity was determined by comparing the relative light units in the presence and absence of MAbs and was expressed as the percentage of neutralization. Comparing with JF-FL WT, the JF-FL L175P became neutralization-sensitive to anti-V3 neutralizing antibodies and could be neutralized in very low concentration of V3 Mabs. For 0.5y, KD-247,717G2 and 5G2, their IC50 of JR-FL WT were 4.0252μg/ml,12.2132μg/ml, 10.8875μg/ml and 7.6555μg/ml, respectively. On the contrary, their IC50 of JR-FL L175P were<0.0016μg/ml,0.0024μg/ml,0.0061μg/ml and<0.0016μg/ml. The IC50 of JR-FL L175P were 1000 times lower than the IC50 of JR-FL WT. These results illustrated that L175P mutation dramatically changed the neutralizing ability of V3-specific antibodies.Part 2.293T cells were transfected by pJR-FL WT env, pJR-FL L175P env, pSG3 and pRSV-Rev and lysed by 0.5% nonionic Nonidet P-40 to get gp120 monomer. In the ELISA test, monomeric gp120 were captured by Anti-C5 antibody and bound with MAb, phosphatase-conjugated goat anti-human IgG, and phosphatase substrate. A405 measurements were taken using a microplate reader. We found that the ELISA figures of JR-FL WT monomeric gp120 and JR-FL L175P monomeric gp120 were almost overlapped, which means that L175P mutation showed no effects on the binding affinity of these antibodies to gp120 monomer.Part 3. In the present study, we used a series of eukaryotic cell expression plasmids to concatenate HIV-1 env gene and GFP marker gene in those plasmids. The expression plasmids were transfected into 293T cells to express HIV-1 gp120 protein onto the surface of the cells. The transfected cells were incubated with anti-V3 antibodies and PE-CyTM5 Mouse Anti-Human IgG, separately. Those successfully transfected cells were detected by GFP florescence marker. Immunochemistry staining and flow cytometry were used to test the binding affinity of several common V3-region specific neutralizing antibodies with wild type gp120 or mutant gp120 proteins. We found that the V2-region L175P mutation yielded higher binding affinity of V3-region specific antibodies, indicating the increasing sensitivity of the V2-region mutant virus to V3-region specific neutralizing antibodies. Above all, our results revealed the L175P mutation at V2 loop changed the natural trimmer structure of gp120 and enhanced the neutralizing ability of V3-specific antibodies. V2 loop of gp120 have a significant role in the structure of gp120 natural trimmer. These data showed the significance of V2-region in the maintenance of normal structure and biological function of whole gpl20. The results also revealed the influence of V2-region for the recognition and neutralization of V3-specific antibodies to gpl20 protein of HIV-1. |
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