Explore the Pathogenesis and Drug Development of SARS-CoV-2 (I)

Coronaviruses are a large group of viruses that are widespread in nature. They can infect birds, humans and other mammals, and can cause respiratory, intestinal, liver, and nervous system diseases. According to the systematic classification, coronaviruses belong to the Coronaviridae family (Coronavirus, CoV) under the order Nidovirales. Coronaviruses can be divided into 4 genera according to the sequence homology of the genome, namely α (Alphacoronavirus), β (Betacoronavirus), γ (Gamacoronavirus) and δ (Deltacoronavirus). Mammalian coronaviruses are mainly alpha and beta coronaviruses, and beta coronaviruses can be further divided into four virus lineages A-D. Among the known coronaviruses before 2019, there are 6 types of coronaviruses that can be transmitted from person to person. Among them, HCoV-229E and HCoV-NL63 of the genus α, HCoV-OC43 and HCoV-HKU1 of the genus β are common influenza viruses that can cause mild upper respiratory tract diseases. Two types of β viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), are highly pathogenic. In the past 20 years, the epidemics caused by SARS-CoV and MERS-CoV— Severe Acute Respiratory Syndrome and Middle East Respiratory Syndrome, have led to more than 10,000 confirmed cases, of which the fatality rate of SARS-CoV is 10%, and the fatality rate of MERS-CoV is 35%. The newly discovered beta coronavirus SARS-CoV-2 in December 2019 is the seventh new type of coronavirus that can infect humans. The fatality rate of SARS-CoV-2 is about 3% to 4%, which is lower than that of SARS-CoV and MERS-CoV, but SARS-CoV-2 has a high transmission capacity (R0 = 1.4 ~ 3.9), a long incubation period, and has the transmission capacity in the asymptomatic period.

Coronavirus is a type of positive-strand RNA virus wrapped by a mantle, with a spirally symmetrical capsid and mantle structure. The coronavirus genome is a linear single-stranded positive-strand RNA with a methylated cap-like structure at the 5'end and a poly(A) tail at the 3'end. The length is between 27 and 32 kb. It is the longest RNA chain among the currently known RNA viruses. The total length of the RNA genome of SARS-CoV-2 is between 26,000 and 32,000 bases. According to the analysis of 6 groups of viral genomes isolated from patient samples, it is predicted that SARS-CoV-2 has at least 12 coding regions, including non-structural protein open reading frame (ORF) 1ab, 3, 7，8, 9, 10b, 13, 14 and 4 structural proteins Spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein.

The replicase complex (ORF1ab) gene of SARS-CoV-2 accounts for about 2/3 (about 7 096 amino acids) of the genome. The encoded viral non-structural protein (nsp) and the cofactor 3-chymotrypsin (nsp7), C30 enzyme-like protease (nsp8), papain-like protease, helicase, and RNA-dependent RNA polymerase (nsp12) together have high polymerase activity. The RNA synthesis complex processed by proteolysis plays a key role in the replication and transcription of viral nucleic acid. In addition, there are coding regions with unknown functions, which may be related to the immune escape mechanism of the virus. In the structural protein of SARS-CoV-2, the S protein has a total length of about 1273 to 1285 amino acids, including two subunits S1 and S2. The extracellular domain of the N-terminal S1 subunit contains a conserved receptor binding domain. (RBD), which mediates the binding of the virus to the receptor; the S2-intracellular domain at the C terminal can rivet the protein and mediate the virus into the cell. Membrane protein M determines the shape of the virus shell and is the main organizer of virus assembly. Together with nucleocapsid proteins N and E, it controls the assembly, release and infectivity of mature viruses.

1 The pathogenic mechanism of SARS-CoV-2

Studies have shown that the binding of angiotensin-converting enzyme 2 (ACE-2) receptor mediated by SARS-CoV S protein is one of the main mechanisms for the virus to invade cells. During SARS-CoV infection, the S protein on the surface of the virus binds to the ACE-2 receptor on the surface of the target cell. Type II transmembrane serine protease (TMPRSS2) binds and cleaves with ACE-2, and at the same time activates the S protein. Later ACE-2 and activated S protein help virus invade, and the expression of TMPRS2 further promotes the uptake of coronavirus. In this process, S protein is the key for SARS-CoV to invade cells. Gene sequence alignment shows that the S protein receptor binding domain (RBD) of SARS-CoV-2 has high homology (73% to 76%) with SARS-CoV, and SARS-CoV-2 retains 14 RBD keys. There are 9 fully conserved amino acids and 4 partially conserved amino acids in the region, and its receptor binding motif (RBM) has good affinity with ACE-2. The kinetic analysis of the interaction between S protein and ACE2 by further surface plasmon resonance technology showed that ACE2 binds to the extracellular domain of SARS-CoV-2 with an affinity of 15 nM, which is higher than that of ACE2. Under the high resolution of cryo-electron microscope, the complex formed by similar S protein and ACE2 can be observed. In addition, the experimental results of SARS-CoV-2 staining on HeLa cells expressing ACE2 and those that do not express ACE2 showed that SARS-CoV-2 cannot enter HeLa cells that do not express ACE2, but it can enter expressing humans, Chinese chrysanthemums, HeLa cells of civet and pig ACE2. These analysis results respectively support the conclusion that ACE2 is the cell receptor of SARS-CoV-2 at the gene, protein and cell levels, suggesting that SARS-CoV-2 may pass through the ACE-2 receptor mediated by SARS-CoV S protein. The binding mechanism invades cells and has biochemical interactions and pathogenesis similar to SARS-CoV.

Compared with SARS-CoV, the SARS-CoV-2 S protein gene contains 2 important mutations: (1) The 6 main amino acids on the RBD of the S protein, Y442, L472, N479, D480, T487 and Y4911 have occurred in 5 mutations, corresponding to L455, F486, Q493, S494, N501 and Y505, making SARS-CoV-2 have a higher affinity than SARS-CoV when RBD binds to ACE2; (2) Unlike most β-coronaviruses, there is a multi-base cleavage site at the junction of the two subunits of the SARS-CoV-2 S protein, which allows it to invade cells and increase cell-to-cell fusion. The receptor binding and membrane fusion mediated by the S protein determine the ability of different coronaviruses to target the host and the transmission ability, which may be the reason why the transmission ability of SARS-CoV-2 is higher than that of SARS-CoV.

ACE-2 is a type of transmembrane protein, which is mainly expressed in human lung type II alveolar cells (AT2). Some immune cells such as macrophages, dendritic cells, and monocytes also express ACE-2. High expression of ACE-2 is also observed in mucosal epithelial cells, ileum and colon absorptive intestinal epithelial cells. As the receptor of SARS-CoV-2, ACE-2 may mediate SARS-CoV-2 infection of the respiratory system, digestive system and immune system, leading to clinical symptoms such as upper respiratory tract and gastrointestinal diseases.

To be continued in Part II…