Catching Common Cold Virus with a Net: Pyridostatin Forms Filaments in Tris Buffer That Trap Viruses-A Novel Antiviral Strategy?

The neutrophil extracellular trap (ET) is a eukaryotic host defense machinery that operates by capturing and concentrating pathogens in a filamentous network manufactured by neutrophils and made of DNA, histones, and many other components. Respiratory virus-induced ETs are involved in tissue damage and impairment of the alveolar-capillary barrier, but they also aid in fending off infection. We found that the small organic compound pyridostatin (PDS) forms somewhat similar fibrillary structures in Tris buffer in a concentration-dependent manner. Common cold viruses promote this process and become entrapped in the network, decreasing their infectivity by about 70% in tissue culture. We propose studying this novel mechanism of virus inhibition for its utility in preventing viral infection.

Rhinovirus and Innate Immune Function of Airway Epithelium.

Airway epithelial cells, which lines the respiratory mucosa is in direct contact with the environment. Airway epithelial cells are the primary target for rhinovirus and other inhaled pathogens. In response to rhinovirus infection, airway epithelial cells mount both pro-inflammatory responses and antiviral innate immune responses to clear the virus efficiently. Some of the antiviral responses include the expression of IFNs, endoplasmic reticulum stress induced unfolded protein response and autophagy. Airway epithelial cells also recruits other innate immune cells to establish antiviral state and resolve the inflammation in the lungs. In patients with chronic lung disease, these responses may be either defective or induced in excess leading to deficient clearing of virus and sustained inflammation. In this review, we will discuss the mechanisms underlying antiviral innate immunity and the dysregulation of some of these mechanisms in patients with chronic lung diseases.

ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating.

Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.

ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments.

UNLABELLED: Human rhinovirus A89 (HRV-A89) and HRV-B14 bind to and are internalized by intercellular adhesion molecule 1 (ICAM-1); as demonstrated earlier, the RNA genome of HRV-B14 penetrates into the cytoplasm from endosomal compartments of the lysosomal pathway. Here, we show by immunofluorescence microscopy that HRV-A89 but not HRV-B14 colocalizes with transferrin in the endocytic recycling compartment (ERC). Applying drugs differentially interfering with endosomal recycling and with the pathway to lysosomes, we demonstrate that these two major-group HRVs productively uncoat in distinct endosomal compartments. Overexpression of constitutively active (Rab11-GTP) and dominant negative (Rab11-GDP) mutants revealed that uncoating of HRV-A89 depends on functional Rab11. Thus, two ICAM-1 binding HRVs are routed into distinct endosomal compartments for productive uncoating. IMPORTANCE: Based on similarity of their RNA genomic sequences, the more than 150 currently known common cold virus serotypes were classified as species A, B, and C. The majority of HRV-A viruses and all HRV-B viruses use ICAM-1 for cell attachment and entry. Our results highlight important differences of two ICAM-1 binding HRVs with respect to their intracellular trafficking and productive uncoating; they demonstrate that serotypes belonging to species A and B, but entering the cell via the same receptors, direct the endocytosis machinery to ferry them along distinct pathways toward different endocytic compartments for uncoating.

Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles-based nonviral vectors.

BACKGROUND: As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. METHODS: We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. RESULTS: Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. CONCLUSIONS: DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.