Tern blot analysis of VLP Env. Below the gel, the densityTern blot analysis of VLP

Tern blot analysis of VLP Env. Below the gel, the density
Tern blot analysis of VLP Env. Below the gel, the density of trimeric gp41 stumps (indicated by an arrow on the gel) is shown, as determined using ImageJ densitometry software (NIH, Bethesda, USA, http://imagej.nih.gov/ij/). Second, we coated the same trimer VLPs of JR-FL B) or the subtype B transmitted/founder viruses REJO and WITO (C) on an ELISA plate at 20x the concentration present in transfection supernatants, treated then with graded doses of M48U1 or sCD4, as indicated and then measured ELISA binding by mAb 7B2 that reacts with a cryptic epitope of gp41 that only becomes exposed upon the loss of gp120 from native trimers (i.e. gp120 shedding). D) Finally, virus particles were isolated with magnetic CD44 microbeads from PBMC culture supernatant at 0 h, 24 h and 72 h after M48U1 exposure. Gp120 was then quantified in virion and virion-free HIV-1 integrase inhibitor 2MedChemExpress HIV-1 integrase inhibitor 2 fractions using a D7324-based gp120-ELISA. Shedding was expressed as a ratio of free gp120 in the supernatant and intact Env in the virion fraction and compared to the untreated control cultures (i.e., medium). Values are the means +/- SEM of two independent measurements. E) Env incorporation in the virion fraction was determined by quantifying both the gp120 and p24 content in ELISA and plotting the gp120/p24 ratio.sustained after multiple rounds of viral replication, supernatant containing de novo virus was collected from the same cell cultures at 48 h and 72 h and again titrated on TZM-bl cells and PBMCs (Figure 1A and C). For all viruses that showed a reduced titer at 24 h, a clear increase in infectivity was observed over time. Whereas in PI-treated cultures, virion infectivity was rapidly restored to normal levels; the virions produced by griffithsin- and M48U1-treated cultures remained respectively two and four times less infectious than virions from the control cultures, as late as 72 hours post-exposure (Figure 1C). To exclude a strain-specific effect of the lab-adapted Bal virus, the experiment was repeated for M48U1 using the more relevant transmitted/founder (T/F) viruses REJO and THRO (subtype B). Similar results were obtained with both viruses, showing a severely reduced relative titer at 24 h (3.7 and 3.1 , respectively) that restores to normal infectivity levels over time (Figure 1D).The memory effect of M48U1 depends on direct interaction between M48U1 and gpfunctional envelope proteins (Env) on infected cell surfaces.M48U1 induces gp120 sheddingTo assess the role of M48U1:gp120 interaction in relation to the sustained reduction in infectivity, we used a miniCD4 resistant, but PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 CD4 receptor binding competent mutant virus bearing the S375R mutation (BalS375R). This mutation is located next to the CD4 binding loop and disrupts the molecular interaction of M48U1 with gp120 [18]. Similar to the previous experiments, PBMCs were infected with wild type (WT), and mutant HIV-1 Bal, subsequently treated with a variety of ARVs and washed 24 h later. As before, cultures were then left to produce new virions in the absence of additional ARV pressure. For most ARVs no significant differences in WT or mutant virus production were found after ARV treatment. However, contrasting the low infectious titer (<1 ) of WT virus after M48U1 treatment, a normal titer (i.e., 100 ) was found for the S375R mutant virus (Figure 1E). This result clearly indicates that the gp120: M48U1 interaction is a prerequisite for the memory effect observed with M48U1. As CFV is removed after treatment, the most logical.