(which appeared during the revision of this manuscript) shows enhanced binding of both the mature and germline reverted version of the potent CD4bs-specific VRC01 to the 276D mutant subtype C computer virus (labeled 266c), which lacks the natural complex glycan as compared to WT N276 . of the PV constructs and phylogenetic analysis of the complete gp160 confirmed identity between the of the pseudoviruses and the original replicating viruses VI1090, VI829 and 92RW009.6 respectively. The full length sequence of the VI1090 PV construct has been deposited with GenBank (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”HQ912710″,”term_id”:”341018701″,”term_text”:”HQ912710″HQ912710). Mutagenesis Site-directed mutagenesis was carried out around the PV constructs (VI1090, VI829 and 92RW009.6) using the QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) following the guidelines of the manufacturer. Primers used to introduce the desired mutation were: VI1090_276D_Fwd (5-G -3); VI829_276D_Fwd (5–3) and VI829_276D_Rev (5-GT 3). Underlined and in strong the mutated N276 D. The presence of the mutation was confirmed by sequencing the complete gp160. Neutralization Assays of three sensitive strains from different subtypes, selected from Table 1: the original VI1090 (CRF02_AG), 92RW009.6 (subtype A) and VI829 (subtype C). Next, the impact of N276D around the sensitivity to HJ16 and other CD4bs mAbs (b12, VRC01 and VRC03) , two llama single heavy chain antibodies or VHHs (A12 and 1B5) , ; the CD4 miniprotein M48-U1  and soluble CD4 was assessed in the TZMbl neutralization assay. In addition, the TriMab combination and the CCR5 LY2795050 inhibitor Maraviroc were used. Clearly N276D mutation produced high-level resistance specifically to HJ16 in all three isolates (Table 4). There was no evidence of cross-resistance of the N276D mutants to other entry inhibitors used, as the difference in IC50 of mutant/WT was usually less than twofold. Remarkably the mutation, N276D conferred a 3 to 13 fold increase of sensitivity to both VRC01 and VRC03. The exception is usually VI829 Env made up of PV, which is not sensitive to VRC03 both in WT and mutant form. Table 4 Influence of N276D in different Envs on their sensitivity to various access inhibitors in TZMbl assay. to confirm that this mutation was responsible for the resistance to HJ16 in VI1090 and showed in addition that introducing the N276D mutation in sensitive A and C isolates also induced full resistance to HJ16. These results could be explained by the fact that this 276 position is located in the C2 part of the outer domain name of gp120, which in the three-dimensional structure is usually relatively close to, but yet just outside the CD4bs . The mutation failed to clearly alter the sensitivity towards most CD4bs targeting compounds, such as sCD4, M48-U1 (CD4 miniprotein), CD4bs mAb b12 and the VHH A12, but intriguingly the sensitivity towards novel VRC01 and VRC03 was clearly increased. As could be expected, the mutation didnt affect the activity of Maraviroc (CCR5 inhibitor), 1B5 (targeting the CD4i site C submitted for publication) or TriMab, consisting of 2G12 (realizing a specific configuration of glycans, but not including N276, on gp120) , , 2F5 (realizing the MPER in gp41) and b12 . Together with our previous findings, the present observations confirm the uniqueness of this HJ16 mAb that probably blocks the gp120-CD4 conversation by binding a glycan near the CD4bs. The importance of glycosylation in binding and LY2795050 activity of HIV-neutralizing antibodies has recently gained interest. In Lavines study, mutations of subtype B JR-FL and YU-2 viruses at position 234 and 241 (of the inner gp120 domain name) as well as 332 and 386 (outer gp120 domain name) decreased sensitivity to at least 2 out of 9 broadly neutralizing patient sera. Of these N332S and N386T also abrogated the neutralizing capacity of mAb 2G12, as could be expected. Conversely, a number of PNGS in V1 (position 160), V2 (197), V3 (301) and gp41 (616) increased sensitivity to neutralization by 5 to 9 out of the 9 patient sera. Amazingly the N276S mutation also increased sensitivity to neutralization by two patient sera . A second paper by Wang et al. analyzed the effect of glycan removal on sensitivity to numerous neutralizing mAbs and found that several mutation of PNGS in V4/V5 and C2/C3/C4 regions of gp120 from a Chinese BC LY2795050 strain alter neutralization sensitivity of these mAbs to a variable extent, but consistently reduced sensitivity to the glycan specific PG16 . Regrettably mutation of PSEN2 276 was not analyzed by Wang. A few years ago, Walker et al. explained a series of very broadly neutralizing PGT mAbs, which identify the glycans at position N332 in the C3 region of gp120 . Moore et al. showed that mutation of N332 abrogates the neutralizing.