Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism.  These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

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TNF␣ is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-B, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNF␣-induced activation of NF-B, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-B activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-B activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.apoptosis ͉ necrosis ͉ herpesvirus ͉ ribonucleotide reductase A ntiviral innate immune responses are triggered by receptors and sensors that recognize pathogen-, damage-, or stressassociated molecular patterns (1). These receptors can be located at endosomal or cell surface membranes, as is the case for the Toll-like receptors (TLRs). Other receptors, such as the double-stranded RNA (dsRNA)-activated helicases RIG-I and Mda5, sense the presence of potentially dangerous molecular patterns inside of the cell. The receptors initiate specific signaling cascades that lead to the activation of transcription factors, (such as NF-B and IFN regulatory factors) and MAPKs, or the initiation of programmed cell death (PCD). Very similar responses are triggered by proinflammatory cytokines such as TNF␣, which also play important roles in controlling viral infections.The receptor-interacting protein RIP1 (also called RIP) is located at the intersection of several signaling pathways [supporting information (SI) Fig. 7]. It integrates signals from membrane-bound receptors and intracellular stress sensors (reviewed in refs. 2 and 3). RIP1 has been investigated extensively because of its crucial role in the TNF receptor (TNFR)1 signaling pathway (4). Stimulation with TNF␣ initially induces the recruitment of RIP1, the TNFR-associated factor (TRAF)2, and the TNFR-associated death domain to the plasma membrane (5, 6). The subsequent ubiquitination of RIP1 by TRAF2 (7, 8) is required for activation of IB kinase and NF-B (9). RIP1 also activates the MAPKs p38 and ERK and participates in the activation of JNK (10, 11). In addition, RIP1 mediates NF-B activation upon stimulation of TLR3 and TLR4 via the TIR domain-containing adaptor-inducing INF-␤ (TRIF) (12, 13). RIP1 also transmits signals derived from DNA-damage sensors (14) and from intracellular sensors of dsRNA (15, 16).TNFR1 and other death receptors can trigger apoptosis by inducing the formation of a complex containing the Fasassociated death domain and procaspase-8, in which the latter is activated autocatalytically (5)....

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Background:The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results: Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion: Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

Nanomedicine opens new therapeutic avenues for attacking viral diseases and for improving treatment success rates. Nanoparticulate-based systems might change the release kinetics of antivirals, increase their bioavailability, improve their efficacy, restrict adverse drug side effects and reduce treatment costs. Moreover, they could permit the delivery of antiviral drugs to specific target sites and viral reservoirs in the body. These features are particularly relevant in viral diseases where high drug doses are needed, drugs are expensive and the success of a therapy is associated with a patient's adherence to the administration protocol. This review presents the current status in the emerging area of nanoparticulate delivery systems in antiviral therapy, providing their definition and description, and highlighting some peculiar features. The paper closes with a discussion on the future challenges that must be addressed before the potential of nanotechnology can be translated into safe and effective antiviral formulations for clinical use.

Recent studies reported a broad but selective antiviral activity of 25-hydroxycholesterol (25HC) against enveloped viruses, being apparently inactive against non-enveloped viruses. Here we show that 25HC is endowed with a marked antiviral activity against three pathogenic non-enveloped viruses, i.e. human papillomavirus-16 (HPV-16), human rotavirus (HRoV), and human rhinovirus (HRhV), thus significantly expanding its broad antiviral spectrum, so far recognized to be limited to viruses with envelope. Moreover, here we disclose the remarkable antiviral activity of another oxysterol of physiological origin, i.e. 27-hydroxycholesterol (27HC), against HPV-16, HRoV and HRhV. We have also identified a much weaker antiviral activity of other oxysterols of pathophysiological relevance, i.e 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol. These findings suggest that appropriate modulation of endogenous production of oxysterols might be a primary host strategy to counteract a broad panel of viral infections. Moreover, 25HC and 27HC could be considered for new therapeutic strategies against HPV-16, HRoV and HRhV.