Isolation and structure elucidation of Dm-CVNH, a new cyanovirin-N homolog with activity against SARS-CoV-2 and HIV-1.

Abstract

An anti-HIV screen of natural product extracts resulted in the discovery of a new antiviral protein through bioassay-guided fractionation of an aqueous extract of the ascidian Didemnum molle. The protein was sequenced through a combination of tandem mass spectroscopy and N-terminal Edman degradation of peptide fragments after a series of endoproteinase digestions. The primary amino acid sequence and disulfide bonding pattern of the 102-amino acid protein were closely related to the antiviral protein cyanovirin-N (CV-N). This new CV-N homolog was named Dm-CVNH. Alphafold2 prediction resulted in a tertiary structure, highly similar to CV-N, comprised of two symmetrically related domains that contained five β-strands and two α-helical turns each. Dm-CVNH showed specificity for high mannose and oligomannose structures, bound to HIV-1 gp-120 and potently inactivated HIV in neutralization assays (EC₅₀ of 0.95 nM). Dm-CVNH inhibited infection in a SARS-CoV-2 live virus assays and was shown to bind to the S1 domain of SARS-CoV-2 Spike glycoprotein. Dm-CVNH behaved in a manner similar to CV-N, binding with a 2:1 stoichiometry to Spike (both to WH-1 and Omicron variants) and preferring the Omicron variant (Kd 42 nM) to original WH-1 (Kd = 89 nM) Spike. This sensitivity to emergent strains was mirrored in viral neutralization assays where Dm-CVNH potently inhibited the infection of Omicron strains XBB.1.16 and JN.1 (IC₅₀ = 11-18 nM).