Article Summaries - SARS-CoV-2

Sarah Jones

Bestle, D., Heindl, M. R., Limburg, H., Lam van, T., Pilgram, O., Moulton, H.,...

Bottcher-Friebertshauser. (2020). TMPRSS2 and furin are both essential for proteolytic

activation and spread of SARS-CoV-2 in human airway epithelial cells and provide

promising drug targets. doi:https://doi.org/10.1101/2020.04.15.042085

Category: virology

This article first summarizes major recent findings regarding SARS-CoV-2 and its spike

protein structure and activation. The study itself looks into the roles of host cell proteases,

specifically TMPRSS2 and furin, in SARS-CoV-2 S activation via cleavage to allow viral

entry and membrane fusion in Calu-3 human airway epithelial cells. Utilizing various

protease inhibitors like MI-432, MI-I900, MI-1851, and T-ex5 PPMO among others, it

was found that both TMPRSS2 and furin are involved in SARS-CoV-2 spike protein

activation and this viral entry, and their differing roles in cleaving different sites makes

them both essential for this entry. It is suggested that furin cleaves at the S1/S2 site on the

S protein while TMPRSS2 cleaves at the S2’ site. Based on previous research, the S1/S2

site cleaves first, creating the two S1 and S2 subunits that contain the receptor binding

domain (RBD) and membrane fusion materials, respectively, and allows for shape

changes that promote receptor binding and cleavage by TMPRSS2 at the S2’ site.

Cleavage at this site allows for membrane fusion and S protein-mediated cell entry.

Through testing protease inhibitors, it was found that inhibition of either furin or

TMPRSS2 can inhibit efficient viral entry, but inhibition of both through a combination
 of protease inhibitors can work synergistically to further decrease viral entry and

replication. The specific inhibitors with greatest success were two synthetic inhibitors of

TMPRSS2, the drug aprotinin, and MI-1851. Inhibitors for the cathepsins, like E64d, did

not seem to significantly impact viral entry or replication, so it is suggested that these

cathepsins are not integral for S protein activation.

Caly, L., Druce, J. D., Catton, M. G., Jans, D. A., & Wagstaff, K, M. (2020). The FDA-approved

drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Research, 178.

doi:https://doi.org/10.1016/j.antiviral.2020.104787

Category: virology

The researchers in this study tested the drug ivermectin against SARS-CoV-2 to see if it

had antiviral effects. Ivermectin was chosen as it is an FDA-approved antiparasitic and

has been shown to have antiviral properties against other tested viruses in vitro and is

believed to be able to inhibit IMP𝛼/𝛽1-mediated nuclear import of viral proteins in them.

Vero/hSLAM cells were infected with SARS-CoV-2 by the Australia/VIC01/2020

isolated virus and then tested against 5 uM of the drug in one trial and against 5 uM

followed by serial dilutions in another. Through looking at viral RNA loads in

supernatant and cell pellets, it was shown that the drug ivermectin correlated with over

90% decrease in viral load in both supernatant and cells after twenty-four hours of initial

exposure and even larger decrease after forty-eight hours. These values suggest that the

drug has effective anti-viral properties against SARS-CoV-2 in vitro, and due to its lack

of toxicity, could be repurposed for treatment of COVID-19 or SARS-CoV-2 infection.

Fu, Y., Cheng, Y., & Wu, Y. (2020). Understanding SARS-CoV-2-Mediated inflammatory

responses: From mechanisms to potential therapeutic tools. Virologica Sinica.

doi:https://doi.org/10.1007/s12250-020-00207-4

Category: virology & immunology

Rather than perform an experiment, this article proposes various mechanisms to explain

inflammation related to SARS-CoV-2 infection based on information known from

infected patients and SARS-CoV infection. It is assumed that the mechanisms for the

viruses are similar as both use ACE2 for viral entry, which suggests they target the same

types of cells. Patients with SARS-CoV-2 infection have been shown to have increased

levels of cytokines and chemokine secretion, and ICU patients have even higher levels,

suggesting a cytokine storm related response to infection that causes the lung injury

associated with COVID-19. One mechanistic suggestion for this inflammation is that it is

caused by mass cell apoptosis and/or pyroptosis due to rapid replication of the virus that

causes vascular leakage that causes secretion of these pro-inflammatory cytokines.

Another mechanistic suggestion is that the virus downregulates ACE2. With less ACE2,

pulmonary ACE2 function is lost to an extent, which can be associated with issues of the

renin-angiotensin system, thus causing inflammation. Lastly, inflammation could be due

to antibody response related to anti-spike protein IgGs. While there are no known

effective treatments to the inflammation that comes with infection, the authors suggest

looking into blocking FcR activation as a treatment.

Hoffman, M., Kleine-Weber, H., & Pohlmann, S. (2020). A multibasic cleavage site in the spike

protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular Cell,

78(4). 779-784. doi:https://doi.org/10.1016/j.molcel.2020.04.022

Category: virology

The authors of this paper investigated the role of host cell proteases in coronavirus spike

protein activation and how that impacts virus entry into target cells. They altered the S

protein at the Sl/S1 cleavage site in order to create various mutants to see the mutation

effects on viral entry. Looking at entry efficiency and syncytium formation, they found

that in order for efficient proteolytic processing of SARS-2-S in human lung cells,

arginine residues at the S1/S2 site are necessary. Also, cleavage at this S1/S2 site is done

by the protease furin and essential for cell-cell fusion and viral entry. Both SARS-CoV-2

and MERS-CoV utilize furin for this essential cleavage as well as TMPRSS2 for

following cleavage. Even though this cleavage at the S1/S2 site drives viral entry, the site

must be multibasic for SARS-2-S, unlike SARS-S, which has been shown to efficiently

fuse with only a monobasic site.

Hoffman, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S.,... Pohlmann,

S. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a

clinically proven protease inhibitor. Cell, 181(2). 271-280. doi:https://doi.org/10.1016/j.

Cell.2020.02.052

Category: virology
 The virus SARS-CoV-2 utilizes the human ACE2 cell receptor for entry via its S protein,

which is primed by the host cell serine protease TMPRSS2. SARS-CoV primarily infects

pneumocytes and lung macrophages, which have high ACE2 expression, so similar target

cells are assumed for SARS-CoV-2. Based on the importance of TMPRSS2 in viral entry,

the researchers investigated a TMPRSS2 inhibitor as a treatment option, looking into its

effectiveness with regard to SARS-CoV-2 infection. Using immunoblot analysis of 293T

cells, they found that there is proteolytic processing of the S protein of SARS-CoV-2 in

these cells, and that the cleavage at the S2’ site is similar to the S protein priming of

SARS-CoV. Having found that both TMPRSS2 and endosomal cysteine proteases

cathepsin B and L are involved in S protein priming, they used inhibitors for both to see

whether they are essential and the efficacy of the inhibitors. Ammonium chloride and

E-64d, which are known to block cathepsin B and L activity, was shown to significantly

block SARS-2-S-mediated entry. Camostat mesylate, a protease inhibitor against

TMPRSS2, also partially blocked SARS-2-S driven entry. The greatest inhibition of the

virus was achieved when using camostat mesylate and E-64d together.

Jiang, S., Hillyer, C., & Du, L. (2020). Neutralizing antibodies against SARS-CoV-2 and other

human coronaviruses. Trends in Immunology 41(5). 355-359. doi:https://doi.org/10.1016/

j.it.2020.03.007

Category: virology, immunology, epidemiology

This article summarizes major findings on SARS-CoV-2 and the virus’s similarity to

other coronaviruses like SARS-CoV and MERS-CoV. It explains neutralizing antibodies

against the latter two viruses and the possibility of using them for treatment and further

research of SARS-CoV-2. These coronaviruses are highly pathogenic, zoonotic viruses

that have bats as their natural reservoirs and utilize intermediate hosts for transmission to

humans; however, the intermediate host for SARS-CoV-2 is unknown. SARS-CoV-2 is

primarily transmitted from human to human via respiratory droplets and contact with

infected surfaces, and infection can result in pneumonia. Most symptoms of infection

revolve around the respiratory system, with the primary symptoms being cough and fever

and others including shortness of breath and sore throat. All three coronaviruses listed

encode a variety of proteins, with the spike protein being essential for viral entry, as the

S1 subunit is involved in viral binding to the target receptor, which is ACE2 for

SARS-CoV and SARS-CoV-2 and DPP4 for MERS-CoV, and the S2 subunit is involved

in cell membrane and virus fusion. In terms of neutralizing antibodies, all developed

neutralizing antibodies against SARS-CoV target the S protein, with most of these

targeting the RBD specifically. Most neutralizing antibodies against MERS-CoV also

target its S protein RBD. There are no neutralizing antibodies currently developed for

SARS-CoV-2, but due to the similarity in sequence identity to SARS-CoV and its S

protein in particular, it is suggested that SARS-CoV neutralizing antibodies might have

cross-reactivity against SARS-CoV-2 infection and should be looked into for research

and treatment. There has even been evidence for the SARS-CoV neutralizing antibody

CR3022 exhibiting interaction with the RBD of SARS-CoV-2.

Lukassen, S., Chua, R. L., Trefzer, T., Khan, N. C., Schneider, M. A., Muley, T.,... Elis, R.

(2020). SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial

transient secretory cells. The EMBO journal, 39(10). e105114. doi:10.15252/embj.

20105114

Category: epidemiology

Using single nuclei RNA sequencing on surgical samples of healthy lung tissue and

air-liquid interface cultures from primary HBECs, ACE2, TMPRSS2, and FURIN

expression was investigated. Within the lung tissue, ACE2 expression was highest in AT2

cells, and TMPRSS2 expression had a specificity for AT2 cells. Within the subsegmental

bronchial branches, ACE2 expression was highest in a subset of secretory cells deemed

“secretory3” cells; considering that these cells exist at a point shortly before terminal

differentiation is completed, they are also referred to as “transient secretory cells.” These

transient secretory cells are thought to be more susceptible to SARS-CoV-2 infection due

to the increased RHO GTPases found as they are related to the viral replication cycle.

ACE2 expression is similar or slightly stronger in these cells as compared to those in lung

tissue. On the other hand, TMPRSS2 expression is lesser in these HBECs than the lung

tissue cells, but it is still higher in expression than ACE2. Next, coexpression of these

proteins and with furin was investigated. In HBECs, there was enrichment of the number

of double positive ACE2+/TMPRSS2+ cells. Triple-positive expression with furin was

not significant in these HBECs. However, in lung tissue, there was high overall furin

expression and enrichment of the number of double-positive and triple-positive cells with

ACE2+/TMPRSS2+/FURIN+ expression. Lastly, any correlation between ACE2

 expression and sex, age, or history of smoking was investigated based on the sample

donors, and no correlation was found with any individual factor.

Sungnak, W., Huang, N., Becavin, C., Berg, M., Queen, R.,... HCA Lung Biological Network.

(2020). SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together

with innate immune genes. Nature Medicine, 26. 681-687. doi:https://doi.org/10.1038/

s41591-020-0868-6

Category: epidemiology

Based on the knowledge that nasal swabs of both symptomatic and asymptomatic patients

with COVID-19 yielded higher viral loads than throat swabs, the researchers used

single-cell RNA-sequencing to analyze expression of ACE2 and TMPRSS2 on various

datasets from different tissues of healthy donors to see how this expression could relate to

initial infection location and transmission. They found that TMPRSS2 has a broader

expression than ACE2, indicating that ACE2, the receptor for SARS-CoV-2, could be a

limiting factor for initial infection via viral entry. When analyzing ACE2 expression in

the lung and airway epithelium, it was observed that ACE2 expression was relatively low

but the highest expression was in nasal secretory cells, which consisted of two clusters of

goblet cells, and ciliated cells. Based on these expression patterns, these nasal secretory

cells act as the portal for initial infection and could be the main source of viral

transmission through secreted droplets carrying the virus. There was also coexpression of

ACE2 and TMPRSS2 in other barrier surface tissues so other transmission pathways

could be observed, but the primary transmission route is most likely through these
 droplets. Considering this reservoir for the virus in the nasal cells, it is suggested that

perhaps drugs be administered intranasally.

Walls, A. C., Park, Y., Tortorici, M. A., Wall, A., McGuire, A. T., & Veesler, D. (2020).

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2),

281-292. doi: https://doi.org/10.1016/j.cell.2020.02.058

Category: virology

SARS-CoV-2 is suggested to use hACE2 as a functional receptor for transmembrane

spike (S) glycoprotein mediated entry. This S glycoprotein is shown to be highly similar

to SARS-CoV S, sharing 75% amino acid sequence identity, but it differs with its

insertion of four amino acid residues at the S1/S2 subunit boundary. A furin cleavage in

the SARS-CoV-2 S is most likely entirely primed and processed by furin proteases in the

golgi body at this S1/S2 site during biosynthesis (shown in HEK293T cells), and it could

be changing the virus pathogenicity in comparison to SARS-CoV by increasing cell and

tissue tropism. Even with these spike glycoprotein differences, in the context of the

experiment, the cleavage was not necessary for S-mediated virus entry into target cells.

Also, the spike protein is the primary target of neutralizing antibodies at the start of

infection, specifically at the S2 subunit. By using polyclonal mouse sera, SARS-CoV S

was shown to elicit polyclonal antibody responses, thus neutralizing SARS-CoV-2

S-mediated entry into cells to an extent. Some of the methods implemented in the study

include using a murine leukemia virus (MLV) pseudotyping system that compared

transduction of SARS-CoV-2 S-MLV and SARS-CoV S-MLV into VeroE6 cells,

 sequence analysis, western blot analysis, designing of an S mutant lacking the four amino

acid residue insertion and the furin cleavage site, and cryo-EM.

Xia, S., Liu, M., Wang, C., Xu, W., Lan, Q., Feng, S.,... Lu, L. (2020). Inhibition of

SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent-pan coronavirus

fusion inhibitor targeting its spike protein that harbors a high capacity to mediate

membrane fusion. Cell Research, 30. 343-255. doi:https://doi.org/10.1038/s41422-

020-0305-x

Category: virology

In this paper, the researchers sought to better understand the structures that make up the S

protein of SARS-CoV-2 and similar coronaviruses in order to understand viral entry

mechanisms and possible treatment options through inhibitors. Using the full

SARS-CoV-2 sequence and x-ray crystallographic analysis, they found that this virus has

a S protein with a S1 and S2 subunit with a cleavage site between them at aR685/S686.

The S1 subunit is located towards the N-terminal of the protein, containing an n-terminal

domain, receptor binding domain, and receptor binding motif. The S2 subunit contains

fusion peptide, heptad repeat 1, heptad repeat 2, transmembrane domain, and cytoplasmic

domain. These HR1 and HR2 domains make up the 6-HB fusion core in the S2 subunit

and interact in a way that promotes viral entry into target cells via membrane fusion. By

cloning the S gene and infecting cells with live virus, this mechanistic pathway was

supported as syncytium formed in the SARS-CoV-2 infected cells but not the SARS-CoV,

suggesting that cell-cell fusion occurs. For treatment option purposes, the researchers
 utilized SAR analysis of lipopeptides and found that the peptide EK1 has inhibitory

activity against 6-HB formation, thus inhibiting SARS-CoV-2 infection partially. To

increase the inhibitor efficacy, they attached cholesterol to EK1, and it proved to work;

the new lipopeptide completely inhibited the virus up to a certain concentration. Even

further, the synthesized lipopeptide EK1C4 had the greeted inhibitory effects. Overall,

HR1 and HR2 are essential for viral membrane fusion and entry for infection and thus are

good targets for drug treatment through inhibition means.

Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., & Zhou, Q. (2020). Structural basis for the

recognition of SARS-CoV-2 by full length human ACE2. Science, 367(6485).

1444-1448. doi: 10.1126/science.abb2762

Category: virology

This study focused on the structures of ACE2 and its complexes with SARS-CoV and

SARS-Cov-2 receptor binding domains on the surface spike glycoprotein that form

during cell entry by the virus. Using cryo-electron microscopy, the structures and

conformations of these complexes could be seen and analyzed. The images suggest that

ACE2 is the receptor for both SARS-CoV and SARS-CoV-2 entry. The S1 subunit of

SARS-CoV contains the receptor binding domain (RBD), and allows for protease

cleavage after conformational shape changes take place during binding. More

specifically, the interaction between the viruses and the host cell takes place with the

N-terminal peptidase domain of ACE2 and the RBD of the viruses, with each PD

interacting with only one RBD. The S protein of SARS-CoV-2 is however trimeric in
 structure with the possibility of both up and down conformations of RBD, and in order to

bind with ACE2 it must contain an up RBD conformation. The ACE2 mediated entry for

SARS-CoV and SARS-CoV-2 are similar in process and complexes formed. The

differences in amino acid sequence of the S protein in SARS-CoV-2 is suggested to

change the affinity for ACE2 in comparison to SARS-CoV, both strengthening and

weakening it, but the overall change is not clear or suggested to be significant.

Yuan, M., Wu, N. C., Zhu, X., Lee, C. D., So, R. T. Y., Lv, H.,... Wilson, I. A. (2020). A highly

conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and

SARS-CoV. Science, 368(6491). 630-633. doi:10.1126/science.abb7269

Category: virology & immunology

CR3022 is a neutralizing antibody that targets a highly conserved epitope distal from the

receptor binding site of SARS-CoV S protein. This paper looked into the structure of the

complex made when CR3022 interacts with the RBD of SARS-CoV-2 and how it

compares to the complex with SARS-CoV. Interactions were found to be fairly similar

considering 24 out of 28 residues in the epitope of SARS-CoV-2 RBD is conserved from

SARS-CoV, with the biggest difference being an addition of an n-glycosylation site at

N370 on SARS-CoV. To bind with the RBD of the S proteins, CR3022 utilizes six

complementarity determining region loops. For proper binding that is most effective, the

S protein needs to have two adjacent trimers in an up conformation and slight overall

rotation as this avoids steric clash. These interactions allow for neutralization

mechanisms to take place without blocking or interacting with the ACE2 binding site on
the protein. In the experiment, however, neutralization by CR3022 was only effective

against SARS-CoV, not SARS-CoV-2, even though the antibody could interact with

SARS-CoV-2.