An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy

Abstract

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

SARS-CoV-2: A Master of Immune Evasion

“…After newly generated viruses are discharged from the cell, the ACE2 attachment process is used to disseminate the infection, and emerging viruses merge their membrane with the cell membrane of the objective cell and then deliver their RNA inside the cytoplasm [ 121 ]. The new viral reproduction “sabotages” several intracellular vesicle machinery components.…”

“…Immunization reduces the seriousness of the illness, although SARS-CoV-2 can re-infect vaccinated individuals, resulting in dangerous sickness and even death [ 126 ]. Furthermore, the virus entrance pathways into permissive cells are unclear, and the way the pathogen spreads and multiplies in a subject with a powerful immunological response is still unknown [ 121 ].…”

“…The latest research has demonstrated that this virus produces infection to other cells using tunneling nanotubes (TNTs), which are nanometer- to micrometer-diameter cylindrical formations that connect adjacent or remote cells and promote cytoplasm communication between connected cells ( Figure 2 ), enabling biomolecules to be shared and/or transported intra-cytoplasmically [ 121 , 127 ]. TNTs are defined as transitory formations that emerge and dissolve in a few minutes [ 128 ].…”

“…In a related way to transport by the axons, TNT thickness increases when a large object is transported inside it. The cylindrical structure of some of these components has been successfully evidenced by scanning helium-ion microscopy (HeIM), demonstrating that TNTs can transport vesicles or viruses [ 121 ]. The role of TNT in a SARS-CoV-2 infection was explored in two recent papers [ 129 , 143 ].…”

“…TNTs can also reduce virus-cell connections, which might activate host defense mechanisms and antiviral responses. TNTs are used by several viruses, including influenza, HIV, and herpes simplex virus (HSV), to transfer their genetic material to new cells, permitting them to bypass host immune surveillance and pharmaceutical targeting [ 121 ].…”