Most 20-fold increase in virus assembly and 3-fold increase in virus entry compared to the WT (Table 3). Deletion of CT in the WT Env significantly enhanced pseudovirus assembly and subsequent entry into the cells, which isconsistent with the observation previously reported by others. But deletion of CT did not rescue DV1V2 and DlpD viruses to enter the cells although DlpDDCT had 5-fold increase in pseudovirus assembly compared to DlpD alone (Table 3). Interestingly, we found that DCT significantly enhanced assembly of DV2, DV3, DV2C, and DV3C pseudoviruses (3-6-fold increases), and enhanced the entry of DV2C and DV3C pseudoviruses into the cells (Table 3). Combination of DCD4bl with DCT (DCD4blDCT) also significantly enhanced pseudovirus assembly compared to the WT (50-fold increase) although the assembled pseudoviruses could not enter the cells, as expected (Table 3).Deletion of V4 or V5 did not Cause gp120 SheddingTo 16574785 investigate the AN 3199 possibility that DV4 and DV5 may enhance gp120 shedding, resulting in lack of Env cell surface display, we did a capture ELISA to detect soluble gp120 that may be present in the BI-78D3 culture supernatant of 293T cells co-transfected with recombinant pSVIII plasmid encoding JRFL gp160 WT, or DV4, or DV5, and pcTAT plasmid. IgG1 2G12 was used as a primary antibody in the capture ELISA. The result showed that gp120 was absent in the culture supernatant of 293T cells co-transfected with the pSVIII gp160 DV4 or DV5 mutant plasmid, and pcTAT, while gp120 was present in the culture supernatant of 293T cells co-transfected with Env WT plasmid and pcTAT (data not shown). This result indicates that undetectable DV4 and DV5 Envs on cell surface may be attributed to the lack of Env cell surface display, not to gp120 shedding.Importance of HIV-1 Env Variable LoopsFigure 2. Effects of various loop deletions on total Env expression in 293T cells, Env binding to mAb 2G12, and pseudovirus assembly. A: Immunostaining of 293T cells co-transfected with DV4 or DV5 Env plasmid and pcTAT with or without permeabilization prior to staining with mAb 2G12; B: Binding of Env loop deletion mutant proteins in the whole cell lysates to 2G12 by capture ELISA; C: Titration of pseudovirus in the culture supernatants by capture ELISA. The volume of each supernatant that contains the same amount of pseudovirus is indicated with a flat line when OD405nm = 0.95. doi:10.1371/journal.pone.0069789.gImportance of HIV-1 Env Variable LoopsEffects of V4 and V5 Loop Deletions on Env Structural IntegritySince DV4 and DV5 Env proteins can be expressed in cells, but cannot be displayed on cell surface, we examined the possible conformational changes occurred to the Env proteins by measuring their bindings to various mAbs by ELISA. We found that DV4 and DV5 Env proteins lost the binding to CD4bs mAbs b12 and VRC01, and CD4i mAbs X5 and 17b, but had increased binding to gp41-specific mAbs m47 (N-trimer-specific)(unpublished), 2F5 and 4E10 (MPER-specific) (Fig. 3A-G). The result indicates that deletion of V4 or V5 may destroy Env structural integrity, resulting in loss of the receptor and coreceptor binding sites, and enhanced exposure of the N-trimer structure and the MPER. Binding of glycan-specific mAb 2G12 to DV4 and DV5 Env proteins decreased, to some extent, which may be attributed to the decreased number of PNGS in the Envs due to the deletion of V4 and V5 (Fig. 3H).DiscussionEngineering Env is one of approaches for HIV-1 vaccine development. Understandin.Most 20-fold increase in virus assembly and 3-fold increase in virus entry compared to the WT (Table 3). Deletion of CT in the WT Env significantly enhanced pseudovirus assembly and subsequent entry into the cells, which isconsistent with the observation previously reported by others. But deletion of CT did not rescue DV1V2 and DlpD viruses to enter the cells although DlpDDCT had 5-fold increase in pseudovirus assembly compared to DlpD alone (Table 3). Interestingly, we found that DCT significantly enhanced assembly of DV2, DV3, DV2C, and DV3C pseudoviruses (3-6-fold increases), and enhanced the entry of DV2C and DV3C pseudoviruses into the cells (Table 3). Combination of DCD4bl with DCT (DCD4blDCT) also significantly enhanced pseudovirus assembly compared to the WT (50-fold increase) although the assembled pseudoviruses could not enter the cells, as expected (Table 3).Deletion of V4 or V5 did not Cause gp120 SheddingTo 16574785 investigate the possibility that DV4 and DV5 may enhance gp120 shedding, resulting in lack of Env cell surface display, we did a capture ELISA to detect soluble gp120 that may be present in the culture supernatant of 293T cells co-transfected with recombinant pSVIII plasmid encoding JRFL gp160 WT, or DV4, or DV5, and pcTAT plasmid. IgG1 2G12 was used as a primary antibody in the capture ELISA. The result showed that gp120 was absent in the culture supernatant of 293T cells co-transfected with the pSVIII gp160 DV4 or DV5 mutant plasmid, and pcTAT, while gp120 was present in the culture supernatant of 293T cells co-transfected with Env WT plasmid and pcTAT (data not shown). This result indicates that undetectable DV4 and DV5 Envs on cell surface may be attributed to the lack of Env cell surface display, not to gp120 shedding.Importance of HIV-1 Env Variable LoopsFigure 2. Effects of various loop deletions on total Env expression in 293T cells, Env binding to mAb 2G12, and pseudovirus assembly. A: Immunostaining of 293T cells co-transfected with DV4 or DV5 Env plasmid and pcTAT with or without permeabilization prior to staining with mAb 2G12; B: Binding of Env loop deletion mutant proteins in the whole cell lysates to 2G12 by capture ELISA; C: Titration of pseudovirus in the culture supernatants by capture ELISA. The volume of each supernatant that contains the same amount of pseudovirus is indicated with a flat line when OD405nm = 0.95. doi:10.1371/journal.pone.0069789.gImportance of HIV-1 Env Variable LoopsEffects of V4 and V5 Loop Deletions on Env Structural IntegritySince DV4 and DV5 Env proteins can be expressed in cells, but cannot be displayed on cell surface, we examined the possible conformational changes occurred to the Env proteins by measuring their bindings to various mAbs by ELISA. We found that DV4 and DV5 Env proteins lost the binding to CD4bs mAbs b12 and VRC01, and CD4i mAbs X5 and 17b, but had increased binding to gp41-specific mAbs m47 (N-trimer-specific)(unpublished), 2F5 and 4E10 (MPER-specific) (Fig. 3A-G). The result indicates that deletion of V4 or V5 may destroy Env structural integrity, resulting in loss of the receptor and coreceptor binding sites, and enhanced exposure of the N-trimer structure and the MPER. Binding of glycan-specific mAb 2G12 to DV4 and DV5 Env proteins decreased, to some extent, which may be attributed to the decreased number of PNGS in the Envs due to the deletion of V4 and V5 (Fig. 3H).DiscussionEngineering Env is one of approaches for HIV-1 vaccine development. Understandin.
