Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus

Abstract

We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide.

Roles of variable linker length in dual acting virucidal entry inhibitors on HIV‐1 potency via on‐the‐fly free energy molecular simulations

“…This finding provides a plausible structural rationale for the loss in virolytic activity of the lectin-based DAVEI molecules when moving from the L2 to the L0 linkers. 14 We did not perform free-energy calculations for the L4 linkers because of high computational expense. We speculate that an L4-based DAVEI might be able to use more glycans than just the one at N448, which might explain why L4 has a lower EC 50 for virolysis.…”

“…3,4,11 The original DAVEI molecules used cyanovirin-N (CVN) as a glycan binder, while second-generation DAVEI molecules use microvirin (MVN), which has more specific binding to mannose-9 glycan structures like those on Env. 3,4,[12][13][14] The MPER-like region of DAVEI is believed to bind to Env MPER determined by competition assays with MPER specific antibodies (4E10 and 10E8) and alanine mutation studies. 4,11 The linker is used to provide space between the two functional ends of DAVEI to allow them access to their binding partners.…”

“…4,11 The linker is used to provide space between the two functional ends of DAVEI to allow them access to their binding partners. 3,4,11,14 As such, there is a minimal linker length that must be achieved to allow for the two active ends to function coordinately. Previous studies have shown that a repeat unit of zero (L0) is not functional while a repeat unit of two (L2) is functional with IC50's similar to repeat units of four and eight (L4 and L8); thus, L2 is the minimal effective linker length for DAVEI.…”

Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate

“…Furthermore, to overcome the relatively complex glycan binding pattern of CVN [22][23][24][25][26][27] , we identified a second generation protein fusion construct-MVN-DLI 28 . MVN-DLI is also a chimera made up of MVN fused to Trp3 sequence (DKWALSWNW) by a glycine 4 serine linker 28 . The second generation reengineered molecule MVN-DLI, denoted as MVN*-DLI, has optimal Env gp120 binding affinity and strong antiviral and virolytic potency as well 28 .…”

“…MVN-DLI is also a chimera made up of MVN fused to Trp3 sequence (DKWALSWNW) by a glycine 4 serine linker 28 . The second generation reengineered molecule MVN-DLI, denoted as MVN*-DLI, has optimal Env gp120 binding affinity and strong antiviral and virolytic potency as well 28 .…”