Question

8) The Replication of Coronavirus is depicted in Figure 1 (See below). As you can see in Comment 2, it is not clear whether the virus gets into the host cell by FUSION or RECEPTOR-MEDIATED ENDOCYTOSIS. Please define & describe the entry of a virus by FUSION and by RECEPTOR-MEDIATED ENDOCYTOSIS. How could a Coronavirus enter a cell? Which mechanism do YOU THINK is most likely correct? Why? (20 points)

Answer 1

Entry via endocytosis

Viral entry via endocytosis.

Viruses with no viral envelope enter the cell through endocytosis; they are ingested by the host cell through the cell membrane. In essence, the virus tricks the cell into thinking that the virus knocking at the door is nothing more than nutrition or harmless goods. A cell, which naturally takes in resources from the environment by attaching goods onto surface receptors and bringing them into the cell, will engulf the virus. Once inside the cell, the virus must now break out of the vesicle by which it was taken up in order to gain access to the cytoplasm. Examples include the poliovirus, Hepatitis C virus, Foot-and-mouth disease virus., and SARS-CoV-2.

Many enveloped viruses also enter the cell through endocytosis. Entry via the endosome guarantees low pH and exposure to proteases which are needed to open the viral capsid and release the genetic material inside. Further, endosomes transport the virus through the cell and ensure that no trace of the virus is left on the surface, which could be a substrate for immune recognition

SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway

The entry of enveloped viruses into cells is known to occur via two primary pathways: some viruses deliver their genomes to the cytosol after their envelopes fuse with the plasma membrane at the cell surface, whereas others take advantage of the cell's endocytic machinery. In the latter mechanism, the endocytosed virions are subjected to an activation step in the endosome, which is typically mediated by the acidic endosomal pH, resulting in fusion of the viral and endosomal membranes and release of the viral genome into the cytosol. Therefore, the endocytic mechanism is thought to be a pH-sensitive process, while direct membrane fusion is pH-independent. The endocytic pathways exploited by animal viruses to gain entry into host cells include macropinocytosis, clathrin-dependent endocytosis, and caveolae-dependent endocytosis, as well as poorly characterized routes such as clathrin- and caveolae-independent endocytosis . Although most viruses use only one of these pathways to enter cells, recent studies have shown that some viruses use multiple mechanisms to gain entry into host cells.

The entry of SARS-CoV into cells was first identified to occur by direct fusion at the plasma membrane . Some later studies have shown that entry of SARS-CoV may be pH-dependent , and that the endosomal protease cathepsin L might be involved, suggesting that this virus may employ endocytosis. Because conflicting results have been obtained by various research groups using similar experimental methods, such as fluorospectrometric monitoring of GFP-labeled pseudovirus entry into host cells, we decided to use a different approach. We labeled the SARS-CoV functional receptor angiotensin converting enzyme 2 (ACE2) with GFP by stable transfection of human embryonic kidney 293E (HEK293E) cells. Receptor recycling was then tracked after the cells were treated with pseudoviruses or the spike protein, a membrane component of SARS-CoV that mediates membrane fusion and is required for viral entry . This new approach greatly improves experimental stability and reliability. The results showed that SARS-CoV entered cells via receptor-dependent, pH-sensitive endocytosis. We also showed that the specific endocytic pathway used by SARS-CoV to enter cells is clathrin- and caveolae-independent. Moreover, lipid rafts were found to play an important role in this process

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