These linked dimers show enhanced HIV neutralization compared to WT CV-N against 33 strains from 3 clades

These linked dimers show enhanced HIV neutralization compared to WT CV-N against 33 strains from 3 clades. is usually uniquely positioned to become a therapeutic and prophylactic SPL-B for diseases caused by enveloped viruses. CV-N is usually a small, two-domain protein that neutralizes HIV by specifically binding to high mannose glycans around the envelope glycoprotein gp120, thereby preventing conversation of the virus with a host cell (1, 2). In addition to its potent activity against HIV, CV-N is also active against a number of other enveloped viruses including influenza (3, 4), Ebola (5, 6), hepatitis C (7), and herpesvirus 6 (2). The two domains of CV-N are homologous in both their sequence [32% sequence identity and 58% sequence similarity (8)] and their three-dimensional structure (9, 10). Wild-type (WT) CV-N exists mainly as a monomer in solution and a domain-swapped dimer in crystals (Fig.?S1). NMR structures of the monomer show that the protein is an ellipsoid with ten -strands and four 310-helical turns, approximately 55?? in length and 25?? wide (Fig.?S1and and of about 1.5?M (9). Numerous studies have shown, however, that both sites are necessary for viral neutralization and that destruction of either site renders the CV-N variant inactive (16, 17). However, a recent study showed that in the context of a CV-N dimer that was covalently crosslinked using disulfide bonds, two out of the four possible binding sites are sufficient to maintain neutralization activity, indicating that it is the number and not the identity of sites that is important for neutralization (18, 19). These results point toward a key role for avidity in the viral neutralization activity of CV-N. A number of groups have attempted to study the oligomerization of CV-N to determine whether the domain name swapping is usually a crystallization artifact or a biologically relevant state. However, because the domain-swapped dimer of WT CV-N is not stable at physiological temperatures, a significant amount of purified dimer may revert to MGF monomer during the course of a viral neutralization assay (14). Therefore, mutations have been used to stabilize either the monomer (14) or the domain-swapped dimer (14, 20, 21). The effect of dimerization remains unclear, as some groups have concluded SPL-B that the dimeric state is usually more active than monomeric WT CV-N (21), whereas others find that monomeric and dimeric SPL-B variants have comparable antiviral activities (20). In this study, we show that by linking two CV-N molecules together in a head-to-tail fashion, we can stabilize the domain-swapped dimeric form of the protein in solution. These linked dimers show enhanced HIV neutralization compared to WT CV-N against 33 strains from 3 clades. In addition, we show that although two carbohydrate binding sites are sufficient for activity as previously reported (18, 19), variants with more binding sites (three or four) have increased neutralization activity. Results Design and Construction of CV-N Oligomers. To directly assay the effects of multimerization on the activity of CV-N, we generated CV-N dimers (CVN2s) made up of tandem repeats of CV-N in which the C terminus of one copy of CV-N was linked to the N terminus of the next copy through a flexible polypeptide linker. Because WT CV-N has the ability to domain name swap, we hypothesized that this oligomeric molecules would adopt either a monomeric-like linked structure in which the two CV-N repeats are folded as monomers and connected through the linker (Fig.?1 and and and for Man1-2Man than binding site A (9) and may indicate that the overall activity of CV-N could be improved by improving the affinity of site A. An alternate mechanism for increased neutralization could result from the SPL-B fact that this binding sites in CVN2s can potentially sample distances farther apart than the binding sites in monomeric WT CV-N. The wider spacing could allow CVN2s to crosslink glycosylation sites within a single gp120, across multiple gp120 subunits on an envelope spike or, less likely, across multiple spikes. This crosslinking would prevent a larger number of gp120 subunits from binding to CD4, the primary receptor for HIV, than would be blocked by WT CV-N, thus decreasing the IC50. An interesting note is usually that in one conformation of the domain-swapped structure of WT CV-N (Fig.?S1delivery (36, 37). In addition to the increase in potency of CVN2L0, the lack of a proteolytically sensitive linker between the CV-N repeats suggests that this variant will probably have similar stability in vivo as WT CV-N. CVN2L0 shows.