A molecularly engineered antiviral banana lectin inhibits fusion and is efficacious against influenza virus infection in vivo.

There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin (H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus-endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent.

Targeted disruption of pi-pi stacking in Malaysian banana lectin reduces mitogenicity while preserving antiviral activity.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

Inhibition of Ebola Virus by a Molecularly Engineered Banana Lectin.

Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.