Marco Cantisani

Silver Nanoparticles as Potential Antiviral Agents

Virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. This makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. In the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. Silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis B virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. The use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. Since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. The present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses.

Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

ABSTRACT Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

Microbe-Host Interactions: Structure and Role of Gram-Negative Bacterial Porins

Gram negative bacteria have evolved many mechanisms of attaching to and invading host epithelial and immune cells. In particular, many outer membrane proteins (OMPs) are involved in this initial interaction between the pathogen and their host. The outer membrane (OM) of Gram-negative bacteria performs the crucial role of providing an extra layer of protection to the organism without compromising the exchange of material required for sustaining life. The OM, therefore, represents a sophisticated macromolecular assembly, whose complexity has yet to be fully elucidated. This review will summarize the structural information available for porins, a class of OMP, and highlight their role in bacterial pathogenesis and their potential as therapeutic targets. The functional role of porins in microbe-host interactions during various bacterial infections has emerged only during the last few decades, and their interaction with a variety of host tissues for adhesion to and invasion of the cell and for evasion of host-defense mechanisms have placed bacterial porins at the forefront of research in bacterial pathogenesis. This review will discuss the role that porins play in activating immunological responses, in inducing signaling pathways and their influence on antibiotic resistance mechanisms that involve modifications of the properties of the OM lipid barrier.

A peptide derived from herpes simplex virus type 1 glycoprotein H: membrane translocation and applications to the delivery of quantum dots

UNLABELLED Cell membranes are impermeable to most molecules that are not actively imported by living cells, including all macromolecules and even small molecules whose physiochemical properties prevent passive membrane diffusion. However, recently, we have seen the development of increasingly sophisticated methodology for intracellular drug delivery. Cell-penetrating peptides (CPPs), short peptides believed to enter cells by penetrating cell membranes, have attracted great interest in the hope of enhancing gene therapy, vaccine development and drug delivery. Nevertheless, to achieve an efficient intracellular delivery, further strategies to bypass the endocytotic pathway must be investigated. We report on a novel peptide molecule derived from glycoprotein gH of herpes simplex type I virus that is able to traverse the membrane bilayer and to transport a cargo into the cytoplasm with novel properties in comparison with existing CPPs. We use as cargo molecule quantum dots that do not significantly traverse the membrane bilayer on their own. FROM THE CLINICAL EDITOR Cell-penetrating peptides have recently attracted great interest in optimizing gene therapy, vaccine development and drug delivery. In this study, a peptide derived from glycoprotein gH of herpes simplex I is investigated from this standpoint.

The Identification and Characterization of Fusogenic Domains in Herpes Virus Glycoprotein B Molecules

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell–cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV‐1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post‐translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV‐1) and herpes simplex virus type 1 (HSV‐1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

Peptide-Lipid Interactions: Experiments and Applications

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.

Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity

Abstract Herpes simplex virus (HSV) membrane fusion represents an attractive target for anti-HSV therapy. To investigate the structural basis of HSV membrane fusion and identify new targets for inhibition, we have investigated the different membranotropic domains of HSV-1 gH envelope glycoprotein. We observed that fusion peptides when added exogenously are able to inhibit viral fusion likely by intercalating with viral fusion peptides upon adopting functional structure in membranes. Interestingly, peptides analogous to the predicted HSV-1 gH loop region inhibited viral plaque formation more significantly. Their inhibitory effect appears to be a consequence of their ability to partition into membranes and aggregate within them. Circular dichroism spectra showed that peptides self-associate in aqueous and lipidic solutions, therefore the inhibition of viral entry may occur via peptides association with their counterpart on wild-type gH. The antiviral activity of HSV-1 peptides tested provides an attractive basis for the development of new fusion peptide inhibitors corresponding to regions outside the fusion protein heptad repeat regions.

Intracellular Delivery: Exploiting Viral Membranotropic Peptides

Recent advances in the understanding of cellular and molecular mechanisms of the pathogenesis of several diseases offer the possibility to address novel molecular targets for an improved diagnosis and therapy. In fact, in order to fulfill their function, macromolecular drugs, reporter molecules, and imaging agents often require to be delivered into specific intracellular compartments, usually the cytoplasm or the nucleus. From a medical perspective, biological membranes represent a critical hindrance due to their barrier-like behaviour not easily circumvented by many pharmacologically-active molecules. Therefore, identifying strategies for membrane translocation is essential. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). These peptides, which are able to efficiently translocate macromolecules through the plasma membrane, have attracted a lot of attention, and new translocating peptides are continuously described. In this review, we will focus on the viral derived peptides, and in particular those derived by viral entry proteins that may be useful as delivery vehicles due to their intrinsic properties of inducing membrane perturbation.

Membrane Fusion and Fission: Enveloped Viruses

Membrane fusion and fission are two key processes that occur during the replication of enveloped viruses, namely access to the interior of the host-cell (entry, which requires fusion of the viral envelope with the target cell envelope) and dissemination of viral progeny after replication (egress, which involves budding and fission). These dynamic processes are mediated by specialized proteins that modify and bend the lipid bilayer transiently and locally. This review focuses on fusion and fission reactions and on the hypothetical shared mechanism that generates their driving force.

Structure and orientation of the gH625-644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system.

Glycoprotein H (gH) of the herpes simplex virus type 1 is involved in the complex mechanism of membrane fusion of the viral envelope with host cells. The virus requires four glycoproteins (gB, gD, gH, gL) to execute fusion and the role played by gH remains mysterious. Mutational studies have revealed several regions of gH ectodomain required for fusion and identified the segment from amino acid 625 to 644 as the most fusogenic region. Here, we studied the behavior in a membrane-mimicking DPC micellar environment of a peptide encompassing this region (gH625-644) and determined its NMR solution structure and its orientation within the micelles.