A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

Zoonotic coronavirus (CoV) infections, like those responsible for the current SARS-CoV-2 epidemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). While double-membrane vesicles (DMVs) appear to be a pan-coronavirus RO element, studies to date describe an assortment of additional coronavirus-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in coronavirus infection.

The incorporation of 3H-palmitic acid into Ornithogalum umbellatum lipotubuloids, which are a cytoplasmic domain rich in lipid bodies and microtubules. Light and EM autoradiography

<p>In the ovary epidermis of <em>Ornithogalum umbellatum </em>L. lipotubuloids were found, i.e. distinguished cytoplasmic domain with an agglomeration of half unit membrane-surrounded lipid bodies, entwined and held together by a system of microtubules (Protoplasma 75: 345-357; 77: 473-476).</p><p>Using light and EM-autoradiography with ³H-palmitic acid (25 μCi/ml) it was found that lipotubuloids were the site of intense incorporation of this isotope. After extraction of lipids with lipid solvent the lipotubuloids were not labeled. Localization of autoradiographic grains after 15-h postincubation with isotope-free medium indicated a migration of the labeled substances from the lipotubuloids to the whole cells. Ultrastructural studies demonstrated that most autoradiographic grains after 2-h incubation were localized over the site of the microtubules adjoining closely the half unit membranes of lipid bodies. These observations suggest, that the surface of lipid bodies may be the active site in lipid synthesis and involvement of the microtubules in these processes is possible.</p>

Biogenesis of the Semliki Forest Virus RNA Replication Complex

ABSTRACT The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326–338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Development of a cryosection EM autoradiography technique and its application for the subcellular localization of receptors

Recent progress in immunocytochemistry and cryo-techniques has made it possible to study receptor localization at the subcellular level. For many receptor-ligand systems suitable antibodies are not available and it would be more appropriate to use radioligands to study these receptors. Although fresh frozen sections have been widely used in light microscopy (LM) autoradiography studies, to our knowledge, no one has established a technique using electron microscope (EM) autoradiography with ultrathin frozen sections.Unlike conventional EM approaches which can extract many biological molecules during dehydration and plastic embedding steps, we have adopted the method of Tokuyasu combined with LM autoradiography protocol for frozen sections to develop a new EM autoradiography technique using ultrathin frozen sections. Heart blocks were fixed in 2% periodate-lysine-paraformaldehyde (PLP) and 0.1% glutaraldehyde (GA), sucrose infused, and frozen in liquid nitrogen. They were sectioned in a Reichert Ultracut S cryo-microtome equipped with a Reichert FCS cryo-unit.

Cell fractionation and electron microscope studies of kidney folate-binding protein

The subcellular distribution of folate-binding protein (FBP) and [3H]folate in the proximal tubule was examined using cell fractionation and different electron microscope (EM) techniques. Cell fractionation of rabbit proximal tubules revealed that FBP distributed into two modes: 50% of FBP distributed with alanylaminopeptidase activity (brush border), and the remaining FBP distributed with organelles of lower density that did not show a large digitonin-induced shift to greater density. Infusion of [3H]folate into the kidney followed by isolation and fractionation of the proximal tubules revealed a time-dependent shift of [3H]folate from the heavy (brush border) mode to the lighter organelle mode. By EM immunocytochemistry, rat kidney FBP locates in the brush border, endocytic invaginations, endocytic vacuoles, and dense apical tubules of proximal tubule cells. EM autoradiography of rat kidney 10 min after intravenous infusion of [3H]folate revealed that the label was significantly concentrated only in the brush border, endocytic vesicles, and lysosomes. These data support a mechanism of receptor-mediated endocytosis for the process of FBP-mediated folate transport in the kidney.