The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2.

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 µM) with little concomitant cytotoxicity (CC50 ~ 2 µM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 µM) but also the viral inhibition (IC50 ~ 1 µM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

Endothelial Nitric Oxide Synthase Knockdown in Human Stem Cells Impacts Mitochondrial Biogenesis and Adipogenesis: Live-Cell Real-Time Fluorescence Imaging.

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

Robotic therapy for chronic stroke: general recovery of impairment or improved task-specific skill?

There is a great need to develop new approaches for rehabilitation of the upper limb after stroke. Robotic therapy is a promising form of neurorehabilitation that can be delivered in higher doses than conventional therapy. Here we sought to determine whether the reported effects of robotic therapy, which have been based on clinical measures of impairment and function, are accompanied by improved motor control. Patients with chronic hemiparesis were trained for 3 wk, 3 days a week, with titrated assistive robotic therapy in two and three dimensions. Motor control improvements (i.e., skill) in both arms were assessed with a separate untrained visually guided reaching task. We devised a novel PCA-based analysis of arm trajectories that is sensitive to changes in the quality of entire movement trajectories without needing to prespecify particular kinematic features. Robotic therapy led to skill improvements in the contralesional arm. These changes were not accompanied by changes in clinical measures of impairment or function. There are two possible interpretations of these results. One is that robotic therapy only leads to small task-specific improvements in motor control via normal skill-learning mechanisms. The other is that kinematic assays are more sensitive than clinical measures to a small general improvement in motor control.

Live-cell real-time imaging reveals role of mitochondria in cell-to-cell transmission of HIV-1.

We used live-cell, real-time fluorescence imaging of co-cultures of HIV-1 infected T cells and uninfected target cells to examine the action of mitochondria during cell-to-cell transmission of the virus. We find that mitochondria of HIV infected cells enter uninfected target cells and advance viral spread. We show that human mitochondria serve as viral reservoirs and carriers and that they can move between cells. This was confirmed by our results that purified mitochondria from HIV infected cells are infectious, and that mitochondrial inhibitors block HIV transmission. Viral infection and replication in the target cells were verified by syncytial formation and HIV-1 core protein p24 production. Our results offer new insights into the cellular mechanisms of viral transmission and identify mitochondria as new host targets for viral infection.

Computational study of bindings of HL9, a nonapeptide fragment of human lysozyme, to HIV-1 fusion protein gp41.

HL9 is a nonapeptide fragment of human lysozyme which has been shown to have anti-HIV-1 activity in nanomolar concentration. This study aims to explain this inhibitory activity by using molecular dynamics (MD) simulation, focusing on the ectodomain of gp41, the envelope glycoprotein of HIV-1 crucial to membrane fusion. It was found that in HL9, two Trp residues separated by two others occupy the conserved hydrophobic pocket on gp41 and thus inhibit fusion in dominant-negative manner. Detailed HL9-gp41 binding interactions and free energies of binding were obtained through MD simulation and solvated interaction energies (SIE) calculation, giving a binding free energy of -8.25 kcal/mol which is in close agreement with the experimental value of -9.96 kcal/mol. Since C-helical region (C34) of gp41 also has two Trp residues separated by two others, this arrangement may be generalised and used to scan peptide library and to find those having similar manner of inhibition.

A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase--structural and modeling insight into its functions.

We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. fusion [corrected] inhibition.

We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC(50)s of 66-58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article.

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: part II. integrase inhibition.

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.

Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity.

We previously reported that lysozyme accounts for anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin [Lee-Huang, S., Huang, P. L., Sun, Y., Kung, H. F., Blithe, D. L. & Chen, H. C. (1999) Proc Natl Acad Sci U S A 96, 2678-81]. To define the structural and sequence requirements for anti-HIV activity, we carried out peptide fragmentation and activity mapping of human lysozyme. We identified two peptides that consist of 18 and 9 amino acids of human lysozyme (HL18 and HL9), corresponding to residues 98-115 and 107-115. HL18 and HL9 are potent inhibitors of HIV-1 infection and replication with EC(50)s of 50 to 55 nM, comparable to intact lysozyme. Scrambling the sequence or substitution of key arginine or tryptophan residues results in loss of antiviral activity. HL9, with the sequence RAWVAWRNR, is the smallest peptide we identified with full anti-HIV activity. It forms a pocket with its basic residues on the surface of the molecule. HL9 exists as an alpha-helix in native human lysozyme, in a region of the protein distinct from the muramidase catalytic site. Monte Carlo peptide folding energy minimizing simulation modeling and CD studies indicate that helical propensity does not correlate with antiviral activity. HL9 blocks HIV-1 viral entrance and replication, and modulates gene expression of HIV-infected cells, affecting pathways involved in survival, stress, TGFbeta, p53, NFkappaB, protein kinase C and hedgehog signaling.

Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment.

We investigated the antiviral activity of olive leaf extract (OLE) preparations standardized by liquid chromatography-coupled mass spectrometry (LC-MS) against HIV-1 infection and replication. We find that OLE inhibits acute infection and cell-to-cell transmission of HIV-1 as assayed by syncytia formation using uninfected MT2 cells co-cultured with HIV-1-infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose dependent, with EC(50)s of around 0.2 microg/ml. In the effective dose range, no cytotoxicity on uninfected target cells was detected. The therapeutic index of OLE is above 5000. To identify viral and host targets for OLE, we characterized gene expression profiles associated with HIV-1 infection and OLE treatment using cDNA microarrays. HIV-1 infection modulates the expression patterns of cellular genes involved in apoptosis, stress, cytokine, protein kinase C, and hedgehog signaling. HIV-1 infection up-regulates the expression of the heat-shock proteins hsp27 and hsp90, the DNA damage inducible transcript 1 gadd45, the p53-binding protein mdm2, and the hedgehog signal protein patched 1, while it down-regulates the expression of the anti-apoptotic BCL2-associated X protein Bax. Treatment with OLE reverses many of these HIV-1 infection-associated changes. Treatment of HIV-1-infected cells with OLE also up-regulates the expression of the apoptosis inhibitor proteins IAP1 and 2, as well as the calcium and protein kinase C pathway signaling molecules IL-2, IL-2Ralpha, and ornithine decarboxylase ODC1.

Production of antiviral and antitumor proteins MAP30 and GAP31 in cucurbits using the plant virus vector ZYMV-AGII.

ZYMV-AGII (zucchini yellow mosaic virus-AGII) is a recombinant nonpathogenic potyvirus-based vector system for the expression of foreign genes in cucurbit plants and their edible fruits, including squash, cucumber, melon, watermelon, and pumpkin. MAP30 (Momordica anti-HIV protein, 30 kDa) and GAP31 (Gelonium anti-HIV protein 31 kDa) are multifunctional plant proteins with activity against HIV-1 virus. These proteins are also effective against other viruses, tumor cells, and microbes. We report here the production and characterization of biologically active MAP30 and GAP31 in squash plant by expression of their genes using the ZYMV-AGII vector. Recombinant expressed MAP30 and GAP31 exhibit comparable antiviral, antitumor, and antimicrobial activities as their counterparts from their original plant sources, with EC(50)s in the ranges of 0.2-0.3 nM for HIV-1. These results demonstrate for the first time the amplification and production of therapeutic proteins, MAP30 and GAP31, in common vegetables. This provides valuable alternative food sources of these antiviral, antitumor, and antimicrobial agents for therapeutic applications.