Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina

Abstract

Background: Plants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity.

Methods: We employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina.

Results: Here, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels.

Conclusions: CBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Keywords: Anti-viral; CBA; Glycan; HIV-1; Lectin; MoMo30-plant; Momordica; gp120.

Fig. 1

Plant extracts primarily contain a 30 kDa protein as visualized by Coomassie stain on a 4–20% SDS-PAGE gel. A A Coomassie blue stained gel of the extract and extract passed through a 30kD cutoff filter shows a predominantly single band. The band is reactive with an N-terminal antibody to the MoMo30-plant (Ab 1) and a second antibody made from a sequence from the gene of the predicted MoMo30-plant (Ab 2) B. The IC₅₀ of the protein was determined by exposing HIV-1_NL43 (equivalent to 1 ng p24) to concentrations of MoMo30-plant from 1 to 100 nM and determining the percent infectivity by MAGI assay. The IC₅₀ value was determined by curve-fitting using Dr. Fit software. The blue curve is MoMo30-plant, and the red curve is MoMo30-HEK. The green curve is the commercially available fusion inhibitor Enfuvirtide. C MoMo30-plant is adsorbed into the serum of Rhesus macaques. Two macaques were given herbal therapy in the same regimen as in the field for humans (adjusted for weight). Three microliters of serum were tested by MAGI assay (in triplicate) for antiviral effects from 0 to 183 days. The inset shows a western blot using N-terminal ab and 15 µL of the sample in crosshatched bars. D MoMo30-plant shows no cellular toxicity at therapeutic levels. MoMo30-plant was tested at concentrations from 1 to 1000 nm in an MTT assay. Control is untreated cells

Fig. 2

MoMo30-plant is heat stable and stays bound to the virus for long periods. A MoMo30-plant was mixed with HIV-1_NL43 (equivalent to 1 ng p24) at concentrations of 0.1 (red) and 1 (blue) nM sufficient to cause 50% (blue) or 70% inhibition (red). The mixture was then heated at temperatures from 25° to 120° for 30 min, allowed to cool to 25 °C. An aliquot of 100 µL was then tested in the MAGI infectivity assay, B MoMo30-plant (3 nM) was mixed with HIV-1_NL43 (equivalent to 1 ng p24) and allowed to interact for 5 min before centrifuging the complex through a 40% sucrose cushion to remove from free MoMo30-plant. The virus-MoMo30-plant complex (in the pellet) was removed from 5 min to 72 h at 4 °C before testing by MAGI cell assay. All measurements were done in triplicate

Fig. 3

MoMo30-plant is homologous to the Hevamine A-like protein. A The N-terminal sequence from MoMo30-plant was used to search protein BLAST. The top ten hits are shown. The yellow box highlights the signal sequence, and the blue highlights the homologous portion of the N-terminal sequence. B The N-terminal sequence from MoMo30-plant was compared to the Hevamine A-like protein from M. chantaria and the ribosome-inactivating protein (RIP) from M. chantaria both alpha and beta forms. Conserved amino acids are highlighted in blue

Fig. 4

Clustal omega alignment of DNA sequences from MoMo30-plant, Hevamine A-like protein from M. charantia. Nucleotides that differ from the MoMo30-plant sequence are highlighted in yellow. MoMo30-plant is 92% identical to the M. charantia Hevamine A-like gene

Fig. 5

Translation of the DNA sequence of MoMo30-plant, including structural prediction of the resulting protein. Amino acids highlighted in red are differences between the two sequences. Arrows denote the predicted beta-sheet structure, and hatched boxes denote predicted alpha-helical structure. The two yellow shaded boxes denote domains of conservation in this class of proteins. The asterisks denote the highly conserved catalytic residues

Fig. 6

In vitro translation of the MoMo30-plant gene produces a 30 kD protein with antiviral activity. A An in vitro synthesized gene was inserted into a pGenLenti vector and used as a template for coupled transcription/translation. The reaction was run on a 20% SDS-PAGE gel, and a western blot was probed with an N-terminal ab to MoMo30-plant. B One hundred µL of the reaction mix was tested in a MAGI assay. C The MoMo30-plant pGenLenti plasmid was used to transfect HEK 293 cells. Supernatant and cell lysates were run on a 20% SDS-PAGE gel and probed with the N-terminal ab. A sample of purified MoMo30-plant is used as a marker. D One hundred µL of the cell-free conditioned medium as tested by MAGI assay

Fig. 7

MoMo30-plant can bind to purified FITC labeled gp120 and blocks its interaction with Jurkat cells. A Labeled gp120 was added to Jurkat cells and allowed to bind to the surface, making it visible. B The same cells were stained with Hoechst 33342 nuclear stain. C Phase contrast image. D Pre-incubation of fluorescent gp120 was done with a stock of 200 µg of MoMo30-plant, which blocks its interaction with the cell. E The same cells were stained with Hoechst 33342 nuclear stain. F Phase contrast image

Fig. 8

MoMo30-plant binds to purified gp120. A Gp120 was bound to a Biacore chip surface, and MoMo30-plant was allowed to flow across the chip at concentrations from 1 to 100 nM. Changes in surface plasmon resonance monitored binding. B Gp120 pre-treated with PNGase F (an N-linked glycosylase) dramatically reduces binding. The three lines represent triplicate measurements. C Mannose can block the activity of MoMo30-plant. HIV-1_NL4-3, 2 nM of MoMo30-plant, and different concentrations of D-mannose were added simultaneously and incubated for 5 min at 37 °C before testing by MAGI cell assay for inhibition of infection. Inhibition as a percentage of the untreated control (Infection in the presence of 2 nM Momo30-plant) is plotted