Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells

Abstract

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

Keywords: COVID-19; electron microscopy; lipids; pathology.

FIG 1

Subcellular localization of viral proteins in infected Vero cells. Vero cells were infected with SARS-CoV-2 for 24 h and immuno-EM labeled with antibodies against SARS-CoV-1 proteins, followed by secondary antibodies conjugated to 10-nm gold particles. (A) Clusters of N-protein labeling in cytosol (open arrows) and (enlarged in A’) on double-membrane spherules (right-most black arrow), or virus particles enclosed in a single membrane (two left-most black arrows). From the e-lucent compartment (red *), a “virus-like” particle (as it is without N-protein labeling) is budding (white arrow). (A’’) Enlarged and not enlarged area with MViB containing labeled and unlabeled virus-like particles. (B) M-protein immunogold labeling on e-lucent compartments (gold is circled in red); in enlarged box, immunogold labeling on convoluted membrane structure (CM). Note virus-like particles are not labeled. (C) Immunogold labeling of nsp4 on e-lucent compartments (circled in red) and various virus-like particles enclosed in a membrane without nsp4 labeling, also enlarged in panel C’. (D) Immunolabeling of nsp13 on e-lucent compartments containing lipid-like structures (red arrows). (D’) Higher magnification of panel D. Immunogold decoration on e-lucent compartments is indicated by red circles, mitochondria by m, multiple virus body by MViB, convoluted membrane structure by CM, lipid-like structures by red arrows, N-protein in cytosol by open arrows, N-protein labeled virus by black arrows, and enlarged area by black boxes.

FIG 2

Lipid accumulates in e-lucent compartments more densely in infected Vero cells. Fluorescence microscopy of DNA and lipid staining with Nile red in (A) the uninfected control (Con) Vero cells and (B) cells infected with SARS-CoV-2 for 24 h. (C) Electron microscopy of infected cells. (D) Fluorescence microscopy of the same cells. (E) Correlative light-electron microscopy (CLEM) showing lipid staining at e-lucent compartments in the electron microscope. Immuno-EM labeling for lipid droplet marker perilipin-2 in (F) uninfected Vero cells and (G) cells infected with SARS-CoV-2 for 24 h. Blue color in panels A, B, D, and E shows the nuclei stained with Hoechst, and red shows the lipids stained with Nile red. In electron micrographs, lipid-like structure is denoted by red arrows, virus particles by black arrows, immunogold labeling of perilipin-2 by red circles, mitochondria by m, and lipid droplets by LD.

FIG 3

Release of virus particles from Vero cells infected with SARS-CoV-2 for 24 h. EM micrographs demonstrate (A) lack of immunogold labeling on extracellular virus particle using anti-nsp4, a nonstructural protein of SARS-CoV-2 (black arrow), (B) extracellular virus particles labeled with anti-N-protein, and (C) anti-M-protein also labeled on extracellular virus particles. Here, m represents mitochondrion, MViB multivirus body.

FIG 4

N-protein in e-lucent compartments in lung COVID-19 patient. Lung from control and infected patients was sectioned semithin for FM (A to C) or CLEM (D) and stained with Hoechst (blue) to identify nuclei, Nile red (red) to denote lipid, or anti-N-protein (green) to show N-protein, or it was ultrathin-sectioned for EM (E and F) and immunogold labeled using anti-N-protein followed by secondary antibody tagged with 10-nm gold particles. (A) COVID-19-infected lung showing accumulations of N-protein and Nile red stained lipids. (B) Overview of an uninfected control lung with no N-protein or lipid accumulation. (C) Overview of infected lung with lipid accumulation. (D) Identical section analysed by CLEM of infected lung demonstrate by EM e-lucent compartments (D) by FM Nile red and N-protein labelling (D’) and by the overlay of the FM on the EM micrograph the presence of Nile Red and N-protein on e-lucent compartments (D’’). Immunogold labeling of infected lung with antibody against N-protein at low magnification (E) and magnified region from boxed area where lipid-like structures (open red arrows) are visible (E’) and a single virus particle with N labeling (E’’). (F) Low magnification of N-protein labeling on membrane structures near the e-lucent compartments. (F’) high magnification of clusters of N-protein labeling. Erythrocytes represented by e, nucleus by n, open red arrow lipid-like structures, and boxed areas enlarged region.

FIG 5

Nonstructural protein 4 in e-lucent compartments of infected lung. Lung tissue of COVID-19 patient 58 was sectioned semithin for FM with (A) nuclei, stained with Hoechst (blue), nsp4 stained with Alexa 488 (green) in nsp4 positive cells indicated by white arrows and in black and white, and erythrocytes represented by e. Separate channels of nsp4 (B) and DNA (C). Ultrathin sections of infected lung immunogold labeled against nsp4 and 10-nm gold particles in overview (D) and at higher magnification (E). E-lucent compartments with nsp4 labeling on membrane and lipid-like structures (open red arrows); erythrocytes represented by e. (F) Schematic representation of uninfected Vero cells, SARS-CoV-2-infected Vero cells, and lung tissue of COVID-19 patient summarizing presence cellular organelles and subcellular localization viral proteins. In black, host compartments; in green, viral compartments; in pink, lipid-like structures; CM, convoluted membrane; G, Golgi; LD, lipid droplet; MLB, multilamellar bodies; MViB, multivirus body; LFC, lipid-filled compartment and immunolabeling viral proteins. Dark green triangle, nsp13; light green triangle, nsp4; blue circle, N-protein; yellow square, M-protein.