Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion

Adequate range for sulfur-containing amino acids and biomarkers for their excess: lessons from enteral and parenteral nutrition

The adequacy range of dietary requirements of specific amino acids in disease states is difficult to determine. In health, several techniques are available allowing rather precise quantification of requirements based on growth of the organism, rises in plasma concentration, or increases in the oxidation of marker amino acids during incremental administration of the amino acid under study. Requirements may not be similar in disease with regard to protein synthesis or with regard to specific functions such as scavenging of reactive oxygen species by compounds including glutathione. Requirements for this purpose can be assessed only when such a function can be measured and related to clinical outcome. There is apparent consensus concerning normal sulfur amino acid (SAA) requirements. WHO recommendations amount to 13 mg/kg per 24 h in healthy adults. This amount is roughly doubled in artificial nutrition regimens. In disease or after trauma, requirements may be altered for methionine, cysteine, and taurine. Although in specific cases of congenital enzyme deficiency, prematurity, or diminished liver function, hypermethionemia or hyperhomocysteinemia may occur, SAA supplementation can be considered safe in amounts exceeding 2-3 times the minimal recommended daily intake. Apart from some very specific indications (e.g., acetaminophen poisoning), the usefulness of SAA supplementation is not yet established. There is a growing body of data pointing out the potential importance of oxidative stress and resulting changes in redox state in numerous diseases including sepsis, chronic inflammation, cancer, AIDS/HIV, and aging. These observations warrant continued attention for the potential role of SAA supplementation. In particular, N-acetylcysteine remains promising for these conditions

Non-reciprocal ultrafast laser writing

Photosensitivity is a material property that is relevant to many phenomena and applications, from photosynthesis and photography to optical data storage and ultrafast laser writing. It was commonly thought that, in a homogeneous medium, photosensitivity and the corresponding light-induced material modifications do not change on reversing the direction of light propagation. Here we demonstrate that when the direction of the femtosecond laser beam is reversed from the +z to -z direction, the structures written in LiNbO3 crystal when translating the beam along the +y and -y directions are mirrored. In a non-centrosymmetric medium, modification of the material can therefore differ for light propagating in opposite directions. This is the first evidence of a new optical phenomenon of non-reciprocal photosensitivity. We interpret this effect in terms of light pressure and associated heat flow, resulting in a temperature gradient in homogeneous media without inversion symmetry under uniform intense irradiation