INTRODUCTION COVID-19

(Coronavirus Disease-2019), caused by SARS-CoV-2 (Severe Acute Respiratory SyndromeCoronavirus-2), is currently a major problem worldwide. On 11th March 2020, WHO declared the Novel Coronavirus Disease Outbreak as a Pandemic. With a high transmission rate and estimated mortality of about 3.4% (Source: worldometer), it has created much havoc across the globe.

In the first part of ‘The Warfare’ series, we have summarized some important findings from the recent (as of 10.4.2020) literature on how the virus enters into the human body and infects it.

BRIEF OVERVIEW OF THE VIRION STRUCTURE AND GENOME COMPOSITION

The SARS-CoV-2 genome encodes non-structural proteins such as 3-chymotrypsin-like protease, papain- like protease, helicase, and RNA dependent RNA polymerase [Figure 2], structural proteins [Figure 1A & 1B] such as spike glycoprotein & accessory proteins [Figure 1A].
ATTACHMENT, CELL ENTRY As shown, coronaviruses have three surface proteins, spike (S), membrane (M) and envelope (E). They use the spike glycoprotein [Figure 3] to bind to its specific receptor to enter into a cell. Spike proteins exist in trimer (homotrimer) [Figure 4] on the surface of viral particle. Each monomer consists of two subunits, S1 and S2, which mediate attachment and membrane fusion, respectively.

Depending on the virus, either N terminal or C terminal site [Figure 5] can serve as the receptor-binding domain (RBD) that binds with the specific receptor of host cell. SARS-CoV-2 uses the carboxyl domain to bind to Angiotensin converting Enzyme-2 (ACE2) receptors [Figure 6]. Upon binding through RBD of S1, the virus has two pathways to enter the host [Figure 6,7]. (i) Some specific host cell enzymes that are present on the cell surface may help in the processing of S proteins. Then, S2 will aid with the fusion of the viral envelope with the host cell membrane, eventually leading to the entry of the virus inside the host system. (ii) But, no such enzymes being present for processing, the virus will be engulfed by the host cell (Endocytosis). SARS-CoV2 mostly follows the second pathway.

In case of SARS-CoV-2, although the S protein being partially processed during its synthesis, the virus enters into the cell mainly through endocytosis. However, some shreds of evidence also point to a TMPRSS-2 mediated cell entry through virion and host cell membrane fusion. It might be depending on the cell types being studied.

Suggested mechanism for entry into host cell adopted by SARS-CoV-2: (Right)

Processing of the S protein and cleavage by protein-digesting enzymes are required for completion of infection by the viral particle. A two-step model has been proposed to describe it.

S Processing 1- Priming Cleavage: During the production of new viruses, S protein is synthesized inside the host cell . S protein is cleaved at a site between S1 & S2 subunit (S1/S2), [Figure 8] separating them by Furin enzyme. Nonetheless, the two subunits would remain joined non covalently. The presence of the furin active site might partially explain the high transmissibility of SARS-CoV-2, due to the near-ubiquitous distribution of furin-like protein-digesting enzymes & its reported effect on other virus. However, some other pieces of evidence suggest that, S1/ S2 cleavage might not be the necessary condition for S mediated viral entry.

S Processing 2- Activating Cleavage: While the virus tries to enter the host cell, activation of a site in S2 protein (S2’) [Figure 8] is performed via extensive irreversible conformational changes by the protein-digesting enzyme Cathepsin-L for mediating infection. Few studies show that Transmembrane Protease Serine Protease-2 (TMPRSS-2) enzyme might also play a role in it.

Another process associated with virus pathogenesis is Syncytium formation. While a virus is reproducing inside a host cell, its spike proteins are displayed on the host cell surface which bind to the specific receptors of neighbouring cells. Processing of S proteins (mentioned earlier), in turn, initiates a sequence of processes leading to fusion of the two cells forming a large multinucleated cell (syncytium) [Figure 9]. Thus, a viral particle goes on to infect multiple cells while residing in one through this mechanism. Few researchers claim that the S protein processing may not be necessary for syncytium mediated pathogenesis and cell-cell fusion via receptor binding might be enough for the rapid progress of the COVID-19.

Coronaviruses keeps on changing the conformation of its spike protein to limit recognition by the host immune response. Several studies found that the SARS-CoV-2 S trimer exists in multiple, distinct conformational states resulting from C-domain opening at the trimer apex. These structural changes are necessary for receptor engagement of the virus and lead to the initiation of fusion requiring conformational changes [Figure 10].

Previous studies have shown that S glycoprotein trimers found in highly pathogenic human coronaviruses appear to exist in partially opened states, while they remain completely closed in human coronaviruses associated with common colds. Scientists hypothesize that the most pathogenic coronaviruses would exhibit S glycoprotein trimers to be spontaneously changing between closed and open conformations, as in the case for SARSCoV-2. C-domain opening is expected to be necessary for interacting with ACE2 receptor at host cell surface, followed by pathways leading to infection, as described earlier.

Upon entering the host system, S glycoprotein would become the main target of antibodies (Abs). Therefore, S trimers are extensively decorated with small molecules of carbohydrates (N-linked glycans) that are important for proper folding and for modulating accessibility to hosts’ proteindigesting enzymes and Abs. Compared to SARS-CoV S protein, S protein of SARSCoV2 is less stable, requiring significantly lower temperature & shorter duration to be inactivated.

NOTE: Refer to COVID 19 Etiology in CONOCIMIENTO, ISSUE 1 for more details.

TROPISM In 2002 coronaviruses outbreak, SARSCoV infected mainly two kinds of lung cells: pneumocytes & macrophages [Figure 12B]. However, ACE2 expression not being limited to the lung, the spread of SARS-CoV in the specific receptor containing tissues (e.g. CNS) other than the lung was observed. The same can be expected for SARS-CoV-2 as well.

It has been suggested that the modest ACE2 expression in the upper respiratory system might limit SARS-CoV transmissibility. In light of the potentially increased transmissibility of SARSCoV-2 relative to SARS-CoV, scientists speculate that the new virus might exploit cellular attachment-promoting factors with higher efficiency than SARSCoV to ensure robust infection of ACE2 containing cells in the upper respiratory tract.

Surprisingly, ACE2 expressing levels are rather low, especially in lungs of SARS-CoV2 2019 infected patients. Hence, the SARS-COV-2 may depend on co-receptors/auxiliary proteins as ACE2 partner to facilitate the binding and entry of viruses. Apart from attacking the Respiratory System, SARS-CoV-2 was found to affect the CNS and cause neurovirulence. Expression of ACE2 receptors have been detected over glial cells and neurons in the brain, which makes them a potential target for COVID-19. The sluggish movement of the blood within the micro-circulatory system could be one of the factors to facilitate the interaction of the spike protein with the ACE2, expressed in the capillary endothelium. Subsequent budding of the viral particles from the capillary endothelium leads to the damage of the endothelial lining & might favor the entry of viral progeny in the brain. Once within the milieu of the neuronal tissues, viral interaction with the ACE2 receptors, expressed in neurons, can initiate a cycle of viral budding, accompanied by neuronal damage without substantial inflammation. In fact, before the anticipated neuronal damages occur, the endothelial ruptures in cerebral capillaries, that can have fatal consequences in patients with COVID19 infections.

Although the cerebral damage may complicate the infection, the widespread dysregulation of homeostasis, caused by pulmonary, cardiac, renal & circulatory damage proves to be fatal for the patient. However, a dominant cerebral involvement alone with the potential of cerebral edema can lead to death long before homeostasis dysregulation sets in.

CONCLUSION

These were some aspects on the mechanisms through which SARS-CoV-2 enters into the host cell. Also there was a short discussion on the cellular tropism of the virus. In the next part of this series, we will be discussing some aspects of the SARS-CoV-2 pathogenesis and the host’s immune response against it.

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WEBSITES REFERRED

1. https://www.sinobiological. com/research/virus/ hcov-spike-protein-overview
2. https://www.pinterest.com/ pin/728527677209302088/
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