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.

Dual-acting virucidal entry inhibitors (DAVEIs) have previously been shown to cause irreversible inactivation of HIV-1 Env-presenting pseudovirus by lytic membrane transformation. This study examined whether this transformation could be generalized to include membranes of Envpresenting cells. Flow cytometry was used to analyze HEK293T cells transiently transfected with increasing amounts of DNA encoding JRFL Env, loaded with calcein dye, and treated with serial dilutions of microvirin (Q831K/M83R)-DAVEI. Comparing calcein retention against intact Env expression (via Ab 35O22) on individual cells revealed effects proportional to Env expression. "Low-Env" cells experienced transient poration and calcein leakage, while "high-Env" cells were killed. The cell-killing effect was confirmed with an independent mitochondrial activity-based cell viability assay, showing dose-dependent cytotoxicity in response to DAVEI treatment. Transfection with increasing quantities of Env DNA showed further shifts toward "High-Env" expression and cytotoxicity, further reinforcing the Env dependence of the observed effect. Controls with unlinked DAVEI components showed no effect on calcein leakage or cell viability, confirming a requirement for covalently linked DAVEI compounds to achieve Env transformation. These data demonstrate that the metastability of Env is an intrinsic property of the transmembrane protein complex and can be perturbed to cause membrane disruption in both virus and cell contexts.