Coronavirus
Structural Task Force

Spike Glycoprotein: Corona’s Key for Invasion

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COVID-19 is caused by the new coronavirus SARS-CoV-2. This virus has a characteristic virus hull featuring surface proteins which are commonly called “spikes”. Protruding from the viral hull like “spikes of a crown”, they give the coronavirus its name (corona = crown).  These proteins make the first contact with human cells and are akin to keys that use a human receptor called “angiotensin-converting enzyme2” (ACE2) as a backdoor to gain access to and infect the cell.

SARS-COV2 Animated picture. Realistic surface and spike proteins with glycosylation. Image: Thomas Splettstoesser; www.scistyle.com
Fig. 1. SARS-COV2 Animated picture. Numerous spike proteins, coloured in green, protrude from the virus hull which is coloured in brown. Spikes enable the coronavirus to invade human epithelial cells. Image: Thomas Splettstoesser; www.scistyle.com

1. Fuction of ACE2

ACE2 is a membrane protein which is anchored in the human cell membrane of epithelial cells. This type of cells can be found on the surface of lung, intestine, heart and kidney tissue. As a type I membrane protein, its primary function is to take part in maturation of angiotensin, a peptide hormone which controls vasoconstriction and blood pressure. ACE2 can be compared to a lock which can be unlocked by the coronavirus spike protein. The virus can then enter the cell and hijack its functions to reproduce itself, thus causing the Covid-19 infection which poses a serious danger to humanity, especially for older people and people with pre-existing conditions. For this reason, one approach to combating SARS-CoV-2 is to target and inhibit the spike to prevent infection. In order to do so, knowledge of the structural features of the spike and its interaction processes with ACE2 are indispensable. (Further information about how macromolecular structures are visualized can be found on our homepage: https://insidecorona.net/visualizing-macromolecular-structures/)

2. Spike: Structure and Fusion Mechanism

Fig. 2. Image of a spike protein (green) protruding out of the viral envelope (brown). This image shows the structure of a spike protein divided into several subdomains. Each subdomain comprises a specific function necessary for binding and fusion. The transmembrane domain anchors the spike protein in the virus membrane.  Heptat repeat 1, 2 and the fusion peptide play key roles in mediation of the fusion process and with the RBD domain, the virus makes contact to human cells. Note that only “stumps” of carbohydrate chains are shown. Image: Thomas Splettstoesser; www.scistyle.com

The Spike protein has a trimeric shape comprising three identical monomeric structural elements. Each of these monomers can fold out akin to a modern car key with a fold-out key element with specific teeth on its surface. This fold-out key element is the so-called “receptor binding domain” (RBD). The spike can only interact with ACE2 when its RBD is in a folded-out position, exposing its teeth, or  “receptor binding motive” (RBM). As the name suggests, it comprises a motive of different amino acids which then can bind and unlock the ACE2 receptor. This key lock mechanism triggers a cascade of events initiating fusion with the host cell. First, protein scissors are recruited to the binding site. These scissors (furin & transmembrane serine protease 2) cleave the spike protein for subsequent activation. The active spike molecule then rearranges itself to form a long structural “hook” (formed of HR1/ HR2 and FP see Fig.2) that brings the epithelial cell and viral cell membrane into close proximity for fusion. Once the fusion is completed, the path for the virus is clear to transfer its genome encoded in ribonucleic acid (RNA) into the host cell. This successful transfer then enables the virus to multiply itself and finally spread from cell to cell, causeing Covid-19 in its wake.

Fig. 3. This image shows a spike protein in complex with the human ACE2 receptor. (PDB:6vsb/6lzg). Left: The structure of a spike protein coloured in orange in complex with the human ACE2 receptor coloured in light orange. The white box shows the interaction site which is shown enlarged in the image ion the right. Right: The interaction site between spike and ACE2. Spike's "receptor binding domain (RBD)" includes a "receptor binding motif (RBM)" whose amino acids interact with those of the human receptor through hydrophilic interactions. These amino acids are shown as sticks protruding from the RBM and ACE2. Image: Sabrina Stäb

3. Evading the Immune System with Carbohydrate Chains

The human immune system normally recognizes the surface proteins of foreign organisms such as viruses or bacteria and reacts with an immune response to combat them. Spike proteins are such surface proteins but because of structural peculiarities, the coronavirus evades both the innate and the adaptive human immune system. The secret of these structural peculiarities are the N-glycans. These are long carbohydrate chains which sit on spike’s surface.  Each spike comprises 66 N-glycans forming a protective shield around the protein. Hence the human immune system has problems recognizing spikes and identifying the coronavirus as an enemy.

Fig. 5. Ribbon diagrams of a spike trimer with N-glycans on its surface coloured in cyan (PDB: 6vxx). In Image a, the spike protein is shown sideways and in b, the trimer can be seen from above. Unfortunately, both X-ray crystallography and cryo-EM cannot resolve long carbohydrate chains, so the structures of the chains shown in Figure 4 contain a maximum of three sugar monomers, while in most cases, the carbohydrate chains are much longer, covering most of the contact surfaces of the upper spike protein. Image: Sabrina Stäb

The COVID 19 pandemic has a massive impact on our lives, our health and the global economy. Scientists around the world are trying to develop new drugs to combat the virus. Since the spike plays a critical role in the infection process, it is a prime target for drug development against the pandemic.  One drug approach to inhibit the interaction between spike and the ACE2 receptor is to cap the spike protein using antibodies. Antibodies are proteins, normally produced by the human immune system to fight viruses. The idea is to treat patients with antibodies that cap the RBD of spike, thus preventing interactions with ACE2. This would lead to a nonfunctional spike, blocking the coronavirus from entering the cell (The key would no longer fit the lock). Another approach includes the development of small molecules that target and inactivate the protein scissor transmembrane serine protease 2 (see chapter 2), as the spike’s functionality depends on its cleavage activity. Since the spike protein decorates the virus hull, it could even be part of a potential vaccine. For this reason,  the spike protein could also become the key in the molecular fight against COVID-19.

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Crystallography has a problem. Some amino acid side chains in our structures simply can’t be seen in our maps (Fig. 1). Crystallographic maps represent many protein molecules in a crystal lattice, thousands of copies of the same molecule averaged over measurement time and unit cells. So, what happens with inherently flexible regions of our protein? The average of many different conformations leaves us with no map to guide us in modelling our side chain. So, what is the best way to deal with this as a model builder?

Figure 1: The sequence tells us this amino acid is a lysine but there is clearly no density to support this side chain model.

A passionate discussion within the Task Force has resulted in the following options for dealing with this situation:

  1. Set the occupancy of the unresolved atoms to 0
  2. Leave the atoms at full occupancy and allow the B-factors to inflate
  3. Trim the side chains to what can be resolved by the density
  4. Mutate the residue to a Proline, set your computer on fire, and walk away laughing maniacally.

Just to be clear, option four should only be considered in the direst of circumstances. Please consider options one to three before resorting to proline and fire, and even then, only with a computer you own. With that said, what is the best option? Sadly, none are ideal solutions to the problem so let’s discuss. 

Option 1 can be misleading as the residue appears to be present in the model (Fig. 2), despite there being no experimental evidence for it, until you check the occupancy or load the corresponding map with your model which will tell you otherwise. An occupancy of zero also adds no useful information to the model and may even exclude atoms in this position, like opening the airlock and sending it flying out into the vacuum of space.

Figure 2: Option 2, where side chain atoms with an occupancy of zero are marked in Coot by dots on the atoms

Option 2 is effectively the opposite of option 1, providing a full occupancy side chain in a sensible rotamer conformation and accept the resulting phase bias*. However, this can be equally misleading if the downstream user doesn’t check the B-factors of the sidechain, which will be very large, as they represent not only (smaller) displacement but (larger) disorder. In addition, allowing the B-factor to “explode” is not always an effective way to deal with this problem, as strong negative peaks can still be observed around the side chain in some cases. Another argument for maintaining an occupancy of 1 is that the protein sequence tells us a certain amino acid is present at a position, unless evidence of chemical clipping has been provided (mass spec, for example). Therefore, the atoms must be present in the protein so should be included in the model for the B-factors to deal with the physics of the situation. Options 1 and 2 both have the advantage of providing a complete set of atoms for downstream use in molecular modelling.

*During refinement our model will always bias the phase calculation which gives us our maps. Ideally, we would like out model to maximally affect the phases when we are confident our model is correct and minimally affect the phases when we are less confident. So, an occupancy of 1 (high confidence) where we observe no peaks in our map (low confidence) will lead to what we call phase bias. This can work both ways by underestimating the contribution of our model by setting the occupancy to 0 (option 1).

This brings us onto option 3: trimming down the side chain to what we can in the map (Fig. 3). The “make them work for it” option. If a downstream user is paying attention and realises that, for example, the side chain they are looking at is meant to be a lysine, despite the model only having atoms up to Cß, this should be the least misleading of all the options. The residue should not be mutated to, say, Alanine in this case, as that would mean you are wilfully misleading downstream users. Upon realising the atoms are missing, the downstream user can then model a (hopefully sensible) rotamer for their simulations if needed. The downside is that this approach does introduce some negative bias in favour of modelling bulk solvent into this area. Like I said, none of the options are ideal solutions.

Figure 3: Lysine following a haircut.

So, following this discussion between Nick Pearce, Dale Tronrud, Gianluca Santoni, Andrea Thorn, and I, we recommend option 3 as the best of the available solutions. We believe that the end goal of a crystallographic experiment should be to build atoms justified by the experimental data, i.e. the map, and leave the prediction of unobservable atoms to downstream users. We (crystallographers) are not here to “make it easier for users to avoid thinking about it”. However, after publishing the first iteration of this article a number of crystallographers made the case for option 2 on twitter and a poll of those involved resulted in 53.8% in favour of option 2 (Figure 4), so the matter is still far from resolved.

Figure 4: Twitter poll for options 1 to 4.

However, it’s nice to know that if we really can’t agree on the best method we can at least agree on not option 1, and there's always the fall back plan of option 4 and watch the PDB burn if we get desperate.

Figure 5: Option 4. Sorry not sorry.

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An important drug target

In the first part of this series we compared the protein nsp3 from SARS-CoV and SARS-CoV-2 by sequence. Now we delve deeper into the differences between these two proteins and follow through by analyzing the structure of one domain of nsp3 in particular: papain-like protease. This domain is a very relevant drug target because of its ability not only to cleave the polyprotein, but also remove some of the post-translational modification our cells use to fight these viruses. Without papain-like protease, the virus would be unable to spread COVID-19.

Like the entire nsp3 protein, the papain-like-protease (Pl2pro) domain is localized close to the endoplasmic reticulum’s (ER) membranes. The transmembrane domains hold it in place while the majority of the protein protrudes out of the ER membrane into the cytoplasm.[1]

SARS-CoV genome
Fig 1: Position of the nsp3 gene on the SARS-CoV-1 genome. Nsp3 is seperated into 12 domains. Picture by Thomas Splettstoesser, scistyle.com.

Ubiquitin-like-domain 2

We cannot discuss the Pl2pro domain without its little neighbor, which has been speculated to influence protease domain functionality.

In ubiquitin-specific proteases, the function of comparable Ubl2 domains is attributed to substrate recruitment or an increase in catalytic efficency. Ubiquitin-like-domain 2 (Ubl2) is the domain residing directly adjacent to the N-terminus of the Pl2pro catalytic domain. These ubiquitin-like domain seems to be more conserved compared to Ubl1 in different coronavirus species.[2]

If, in SARS-CoV and Murine coronavirus (MHV), Ubl2 is removed, Pl2pro loses its structural integrity. In addition, Pl2pro is then no longer able to act as an Interferon (IFN) antagonist (see below). However, some studies suggest that the Ubl2 domain in MERS-CoV might not be as essential as originally thought and in cell-based studies of this virus, Pl2pro could retain some of its enzymatic functions without the Ubl2 domain.[3]

To date, several inconsistent roles of Ubl2 were reported, and its exact function and inner workings remain enigmatic. This is being highlighted the fact that there are significant differences between the coronaviruses, and as a consequence, we need to exercise caution in applying our findings to SARS-CoV-2.

Combating the Host's Immune System

In the family of coronaviridae, viruses with either one or two Plpro domains can be found, with SARS-CoV and SARS-CoV-2 only having one. Confusingly, this single domain is however still called Pl2pro, even if it is the only papain-like protease domain in the viral genome.

Pl2pro cleaves the polyprotein from nsp1 (leader protein) up to nsp3. While Pl2pro cuts between nsp1-( ELNGG↓AV)-nsp2-( RLKGG↓AP)-nsp3-( SLKGG↓KI)-nsp4, the nsp5 (3c-like protease) cleaves the rest of the polyprotein. [2] The cysteine protease Plpro is similar to human ubiquitin-specific-protease (USP) in that it adopts a right-hand fold with "thumb", "palm" and "finger" subdomains.

Different regions of Plpro
Fig. 2: Plpro of nsp3 SARS-CoV (PDB-ID: 5E6J) with the catalytic triad marked in red. The Finger domain (blue), palm domain (light green) and thumb domain (forest green). Picture by Kristopher Nolte

Despite the variations of Pl2pro in different coronaviridae, the same catalytic motif of three amino acid residues is essential for the stability and proteolytic activity of the domain: Cys112 is located in the thumb, His273 and Asp287 are located in the palm subdomain. (The numbers identifying these residues can vary between species.)

nsp3Plpro catalytic Mechanism
Fig 3: Catalytic cycle and proposed chemical mechanism of SARS-CoV PL2pro proteolysis. Active site residues of the catalytic triad (Cys112, His273, Asp287) and oxyanion hole residue Trp107 are shown in black. The peptide substrate is shown in green and a catalytic water molecule is shown in blue. [1] Source: The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds, Beaz-Santos et al.

In addition, Pl2pro has deubiquitinating and deISGylating (removal of ISG15 from target proteins) abilities.[4] Both ubiquitin and ISG15 regulate facets of the immune response and through their removal Pl2pro poses as an antagonist to the human immune response. They can stimulate the production of cytokines, chemokines and other IFN-stimulated gene products which have antiviral properties. [6] ISG 15 is an ubiquitin-like modifier composed of two ubiquitin-like folds that has an essential role in marking newly synthesized proteins during the antiviral response.[3] Post-translational modification by ubiquitin and interferon-stimulating gene 15 (ISG15) is reversed by isopeptide bond hydrolysis. Figure 3 shows a proposed mechanism for the cleaving of isopeptide bonds by SARS-CoV.

Ubiquitin bound to Plpro
Fig. 4: Ubiquitin (light blue) bound to Plpro (green) with the catalytic triad marked red. (PDB-ID: 5E6J) Picture by Kristopher Nolte

An example

Toll-like receptors (TLRs) are an important part of the machinery of the human immune response, which recognizes the pathogen-associated molecular patterns. The ability of the host cell to transduce the so-called Toll-like receptor 7 (TLR7) mediated immune response is diminished (Fig. 5) by Pl2pro as it removes Lys63-linked-ubiquitin from the TNF receptor associated factors TRAF3 and TRAF6. [5]

In addition, SARS-CoV can hamper the antiviral activities of interferon. The Pl2pro domain inhibits in combination with a transmembrane (TM) domain the STING mediated activation of interferon expression. PL2pro-TM interacts with TRAF3, TBK1, IKKε, STING and IRF3, the key components assembling a regulatory complex for activation of IFN expression.[5]

Fig. 5: Different ways in which Pl2pro of various coronaviruses interact with the human immune response. A pointed circle symbol means the binding of one protein to another. If the binding has positive effect on the protein it is marked with a plus. The triangle marks the cleavage of ubiquitin from the target protein. Also,nsp3 cleaves ISG15 off target shown on the right. Picture by Kristopher Nolte.

Another tool to fight the coronavirus in human cells is the "guardian of the genome", p53. The tumor supressor protein p53 impedes the replication of SARS-CoV, though the virus fights back with Pl2pro, which binds a p53 degradation stimulator named "RING finger and CHY zinc finger domain-containing protein 1" (or short: RCHY1). Enhanced by the Macro somains in NSP3, this binding enhances the stability of RCHY1 and hence promotes the degradation of p53. In addition, Pl2pro blocks another crucial cellular defense mechanism: The NF-κB pathway, which regulates immune responses to infections. SARS-CoV Pl2pro can stabilize IκBα, an inhibitor of NF-κB.[3]

Although all Pl2pro in different coronaviridae suppress the immune response, the targets differ between various species. For example, SARS-CoV Pl2pro preferentially processes Lys48 linked poly-ubiquitin chains, which are markers for proteasome degradation. MERS, on the other hand, shows no differences in effectivity between Lys48 and Lys63 linked di-Ubq chains. Lys63-linked chains are related to signal transduction cascades of the host immune system. Studies have shown that specificity among Pl2pro for Ubiquitin and ISG15 substrates can be altered with as little as a single amino acid change.[6] However, even though there are differences, for SARS-CoV-2, it is likely that at least some of the functions are similar.

Structural comparison

In order to predict Pl2Pro function for the novel Coronavirus SARS-CoV-2, we start by aligning their sequence like we did in the first part of this series to comapare the sequence with the one from SARS-CoV-2. Both domains share a similarity of 82.8% over the length of 313 amino acids. However, this time, we go for a more detailed analysis of the 54 individual differences, which are:

T3R N14I V20V N48N H49S V56Y D60N E66V D75T S77P P95Y G99N S114A V115T L116A L119T E123I K125L P129P A134D A143E N155C H170S L171Y Q173F S179D K181C C191T T195Q T196Q G200K N214E L215Q K216F G218K I221Q C225T D228K A229Q Y232K F240P Y250Q L252E Q254K G255H C259T E262S H274K K278S I284C L289L S293S T300I S308N
(The first letter refers to SARS-CoV, and the second to the amino acid residue in SARS-CoV-2.)

Figure 6: SARS-CoV (PDB-ID: 5Y3Q) and SARS-CoV-2 (PDB-ID: 6WZU) Pl2pro overlaid over each other. RMSD = 0.758. Differences in SARS-CoV and SARS-CoV-2 marked in red. Picture by Kristopher Nolte

The mutations are evenly spread over the protein. None of the catalytic triad (Cys 112, His 273, Asp287) are changed as is to be expected given their conservation in all other coronaviruses. On further investigation, however, in the motif which interacts with ubiquitin six sites are different: S170T, Y171H, F216L, Q195K, T225V, and K232Q. Earlier studies concluded that the mutation of position 232 from Glutamine to Lysine increases the affinity for ubiquitin at the expense of the de-ubiquitination effectiveness.[6] The kinetics of SARS-CoV-2 nsp3 Pl2pro were studied to test if the protease domain of nsp3 has a reduced effectiveness in binding ubiquitin compared to nsp3 from SARS-CoV, MERS-CoV.
All three Pl2Pro variants cleave more ISG15 than ubiquitin. SARS-CoV has the fastest kinetics of the three viruses. And, the slower kinetics of SARS-CoV-2 resemble those of MERS-CoV rather more than SARS-CoV, having a 10 times higher turnover rate (kcat) as a deISGylase than as a deubiquitase.[6]

Besides the kinetics, the Pl2pro’s affinity for different poly-ubiquitin linkage sites was measured. The result shows that while SARS-CoV-2 can cut K48-Ub linked polyproteins, it seems to lack an ability to cut other polyubiquitin chains. Those K48-Ub linked polyproteins are cleaved at a slower rate than by SARS-CoV. In this regard, SARS-CoV-2 distinguishes itself from MERS-CoV which has the ability to cleave K63-linkages. It is suggested that the decrease in deubiquitinase effectiveness may not be irrelevant, but could lead to the often-mild symptoms that are a factor in why SARS-CoV-2 has been able to evade our efforts in quarantine. But this is mere speculation and a lot more research is needed to resolve the matter.[6]

PL2pro as a drug target

Pl2pro was a potential drug target early on in SARS-CoV-2 research. Hilgenfeld et al. name two major challenges we have to overcome to find a drug targeting Pl2pro. One is that the binding sites are tailor-made to bind glycine residues. Also, this very specific binding motif is rather ubiquitious in our cells. These two problems make it difficult to find an inhibitor which fits and is specific to Pl2pro. However, scientists found a weak spot: a loop called Blocking Loop 2 (BL2) regulates substrate binding and may be a promising target to inhibit PL2pro.[2] Naphthalene based inhibitors, which were earlier proposed to inhibit the BL2 of SARS-CoV, were shown to also inhibit SARS-CoV-2 Pl2pro, in particular an inhibitor called GRL-0617.[6]

For in-silico drug development, it might be prudent to choose high-resolution structures which already have a ligand or inhibitor bound, such as 6yva, 6wuu, 6wx4 or 6yaa. Technically speaking, 6wrh, albeit being a mutant, is one of the highest-quality structures available for SARS-CoV-2 Pl2pro.

In fact, a lot of research is still required to consolidate our understanding of this protein and its domains. In spite of that, we are making progress in our endeavor to fight this virus - and every step we take is one more to win this fight.

Sources

[1] Báez-Santos YM, St John SE, Mesecar AD. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antiviral Res. 2015;115:21-38. doi:10.1016/j.antiviral.2014.12.015, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896749/

[2] Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018;149:58-74. doi:10.1016/j.antiviral.2017.11.001, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113668/

[3] Clasman JR, Báez-Santos YM, Mettelman RC, O'Brien A, Baker SC, Mesecar AD. X-ray Structure and Enzymatic Activity Profile of a Core Papain-like Protease of MERS Coronavirus with utility for structure-based drug design. Sci Rep. 2017;7:40292. Published 2017 Jan 12. doi:10.1038/srep40292, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5228125/

[4] Lei J, Hilgenfeld R. RNA-virus proteases counteracting host innate immunity. FEBS Lett. 2017;591(20):3190-3210. doi:10.1002/1873-3468.12827, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163997/

[5] Chen X, Yang X, Zheng Y, Yang Y, Xing Y, Chen Z. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex. Protein Cell. 2014;5(5):369-381. doi:10.1007/s13238-014-0026-3, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996160/

[6] Freitas BT, Durie IA, Murray J, et al. Characterization and Noncovalent Inhibition of the Deubiquitinase and deISGylase Activity of SARS-CoV-2 Papain-Like Protease [published online ahead of print, 2020 Jun 4]. ACS Infect Dis. 2020;acsinfecdis.0c00168. doi:10.1021/acsinfecdis.0c00168, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274171/

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The world holds its breath as the novel Coronavirus continues to spread across the world, bringing our lives to a halt. We have gathered a lot of knowledge about the virus but there are still many gaps to fill. The non-structural-protein 3 (nsp3) represents one of these gaps in our knowledge. As the largest protein encoded by the coronaviruses genome, untangling its structure and function poses a huge task.

However, we can glean some knowledge around the specific function of SARS-CoV-2 nsp3 by looking at the virus‘s subfamily,  Orthocoronaviridae. As related viruses do share some common traits, academics were not completely unprepared when SARS-CoV-2 came. In the background, while only very few people were worried about a new corona virus, scientists around the world had been investigating the invisible enemy for decades. Building on this past work we look at the functions of proteins from other coronaviruse, like Murine Hepatitis Virus (MHV) and SARS-CoV, to learn more about how best to fight against SARS-CoV-2.

Fig. 1: The crystal structure of papain-like protease of SARS CoV-2 nsp3 (PDB-ID: 6w9c). Picture by Kristopher Nolte.

The gene which produces nsp3 lies on the open reading frame 1a (ORF1a) which encodes polyprotein 1a. The sequence for nsp3 of SARS-CoV is 1922 amino acids long and sandwiched between nsp2 and nsp4. It not only cleaves itself from the polyprotein by its papain-like protease domain but also nsp1 and nsp2. In coronaviruses, 18 different domains have been found in nsp3. Each virus type has 10 to 16 of these, out of which eight domains and two transmembrane regions form the conserved part of nsp3, which can be found in every coronavirus known to date [1]:

  1. Ubiquitin-like-domian (Ubl1)
  2. Ubiquitin-like-domain (Ubl2)
  3. Papain-like protease (PlPro)
  4. Macro domain / X domain (Mac)
  5. Hypervariable region / Glu-rich acidic domain (HVR)
  6. Transmembrane regions (TM1)
  7. Transmembrane regions (TM2)
  8. Ectodomain / Zinc finger domain (3ecto)
  9. Nidovirus-conserved domain of unknown function (Y1)
  10. Coronvirus specific carboxyl-terminal domain (CoV-Y)

To start our investigation on SARS-CoV-2 related structural data, we will look into the protein sequences of SARS-CoV and SARS-CoV-2 to learn where they are similar and where they differ.

Genetic Comparsion of SARS-CoV and SARS-CoV-2

SARS-CoV has 16 domains which span 1922 amino acids. The nsp3 protein of SARS-CoV-2 is a bit longer at 1945 amino acids. When compared to each other, there is an overall similarity of 75,97%.[2] In Addition to the ten conserved domains the nsp3 gene of SARS-CoV-2 codes for four domains:

Fig 1: Position of the nsp3 gene on the SARS-CoV-1 genome. Nsp3 is seperated into 12 domains. Picture by Thomas Splettstoesser, scistyle.com.
  1. Nucleic-acidic-binding domain (NAB)
  2. Betacoronavirus specific marker domain (βSM)
  3. Domain preceding Ubl2 and PL2pro (DPUP)
  4. Amphipathic helix 1 (AH1)

The two domains at the N-terminal end, Ubl1 and HVR, have an alignment of 79% and 64%, respectively. There seems to be a trend in coronaviridae for these domains to be poorly conserved, but Ubl1 still adopts the expected conserved fold.[4] If this proves true, could be analysed by comparing the sequence alignment and the structural similarity. It is unsurprising that the "high variable region" lives up to its name and shows the worst alignment of all. In the related MHV nsp3, this domain is dispensable for replication.[5]
It has been speculated that the Mac1 domain functions as an ADP ribose 1"-phosphatase, however, the effects of mutation in this region differ from virus to virus.[4] As a result, it is difficult to judge what significance the bad alignment of this domain will have on our understanding of SARS-CoV-2 without further research.

Table. 1: The domain amino acid range for SARS-CoV-1 was taken from Hilgenfeld et al.,2018 [2]. The range for SARS-CoV-2 was determined by taking the amino acid ranges of CoV-1 and using BLAST [2] to search for the best alignment of the domain sequences. Picture by Kristopher Nolte

The Mac1 domain, also known as the X-domain, is followed by two macrodomains which were originally called "SARS-CoV Unique domains" (SUD-N and SUD-M), but were renamed when they were found to not be unique to SARS-CoV. It has since been observed that only Mac3 plays an essential role in viral RNA replication[6], which could explain why Mac3 is one the most conserved domains in the alignment of SARS-CoV and SARS-CoV-2.

Pl2Pro and its neighbouring domain Ubl2 show some of the highest sequence alignments of all domain comaprisons. This could be explained by their essential function to cleave nsp3 from the polyprotein.
Little is known about the domains following Pl2Pro and our current structural knowledge is limited to a nuclear magnetic resonance (NMR) structure of NAB. While the structure and function of Y1 and CoV-Y from SARS-CoV-2 are currently unknown, their sequence, which compromises a fifth of the genome, is highly conserved in all coronaviruses.

Fig. 2: The location of the aligned domains of SARS-CoV (abbreviated CoV-1) and SARS-CoV-2 (abbreviated CoV-2) is shown over the length of nsp3 (TM1 = 1, TM2 = 2, AH1 =A). Picture by Tim Scharf.

In the second part of the series of Untangling Nsp3 of SARS-CoV-2 we will delve deeper into some structures of nsp3 of SARS-CoV-1 and SARS-CoV-2 and will try to find out how the differences in the sequence may have influenced some structures of the protein. For a further in-depth reading on the topics discussed here I highly recommend the sources below.  

Table. 2: For each domain and their respective counterpart in SARS-CoV-2 a BLAST search was contucted to search for fitting PDB-IDs. Last Update: 18.05.2020. The scripts and the PDB-data can be found in our Git repository [3]
Picture by Kristopher Nolte

Sources

  • [1] Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018 Jan;149:58-74. doi: 10.1016/j.antiviral.2017.11.001. Epub 2017 Nov 8. PMID: 29128390; PMCID: PMC7113668.
  • [2] Madden T. The BLAST Sequence Analysis Tool. 2002 Oct 9 [Updated 2003 Aug 13]. In: McEntyre J, Ostell J, editors. The NCBI Handbook [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2002-. Chapter 16. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21097/
  • [3] https://github.com/thorn-lab/coronavirus_structural_task_force
  • [4] Benjamin W. Neuman, Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles, Antiviral Research, Volume 135, 2016, Pages 97-107, ISSN 0166-3542, https://doi.org/10.1016/j.antiviral.2016.10.005.
  • [5] K.R. Hurst, C.A. Koetzner, P.S. Masters, Characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein 3 of the viral replicase-transcriptase complex J. Virol., 87 (2013), pp. 9159-9172
  • [6] Kusov Y, Tan J, Alvarez E, Enjuanes L, Hilgenfeld R. A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex. Virology. 2015 Oct;484:313-22. doi: 10.1016/j.virol.2015.06.016. Epub 2015 Jul 3. PMID: 26149721; PMCID: PMC4567502.

Für diesen Beitrag steht leider keine deutsche Übersetzung zur Verfügung.

Introduction

Before I started writing this article, the first thing I did was to google the name of my protein “NendoU” and was greeted by Figure 1. Needless to say, this is not what I was expecting. So, if you’re an anime fan looking for Riki Nendou, a dutiful yet dull-witted boy who likes helping people, particularly prioritising the weak, from The Disastrous Life of Saiki K: I’m afraid you have come to the wrong place. However, now that you’re here, maybe you’d like to learn about an interesting protein involved in SARS-CoV-2 viral replication? It can bind to and process six RNA molecules at a time! Six!

Figure 1: Not the NendoU you were looking for

After that interlude, I should get this blog post back on track! So… viruses and proteins. SARS-CoV-2 is an enveloped coronavirus with a non-segmented positive-sense RNA genome, in English this means the RNA genome in SARS-COV-2 can be used “as is” to make viral proteins without prior modification. SARS-CoV-2 has one of the largest RNA genomes among RNA viruses, made up of a replicase gene encoding non-structural proteins (nsps), as well as various structural and accessory genes. During viral replication, depending on the starting point (a.k.a. a ribosomal frame shift), the replicase gene can produce one of two poly-protein chains, which are then cleaved to produce 15-16 individual viral nsps (non-structural Proteins). These nsps then form a large membrane-bound replicase complex with multiple enzymatic activities, like a tiny viral Voltron.

What’s in a Name?

This blog post will focus on SARS-CoV-2 Nsp15, a nidoviral RNA uridylate‐specific endoribonuclease (NendoU). That is a very long and complicated name which conveys a lot of information, so let’s break it down into its individual parts, like when Voltron separates to become several small robots. It’s possible I’ve watched too many cartoons during lockdown:

  • Nidoviral – An order of RNA viruses which infect vertebrates and invertebrates.
  • RNA – Genetic material used to produce proteins
  • Uridylate-specific – Cuts Uridine (U) in RNA, not Cytosine (C), Adenine (A) or Guanine (G)
  • Endo – A Greek word meaning inside or within
  • Ribonuclease – An enzyme that cuts RNA into smaller pieces.

So, what’s in a name? Well, Nsp15 is a viral enzyme that likes to cut at uridine (a building block of RNA) in the middle of an RNA sequence. Quite a lot really. The final bit of the name “NendoU” goes into even more specifics on our protein, as it defines a common family of proteins which share certain traits. The first is that when Nsp15 cuts RNA, it gives a 2′‐3′ cyclic phosphodiester and 5′‐hydroxyl terminus. If we look at Figure 2, you’ll see a purple RNA chain made of two bases linked by an orange phosphate in the middle. When RNA is cleaved by Nsp15, a 2′‐3′ cyclic phosphodiester is made: in the two resulting molecules, the phosphate ion has been incorporated into a 5-membered ring (orange), and the other half of the RNA has a 5′‐hydroxyl, or and OH- group on another 5-membered ring (green). The second thing being a member of the NendoU family tells us is that the catalytic domain of the protein (the business end) is found on the C-terminal end of the protein (the latter half) as this is a shared trait within the NendoU family.

Figure 2: RNA Cleavage to give a 2′‐3′ cyclic phosphodiester and 5′‐hydroxyl terminus. Image generated in PyMOL using molecules made with Coot’s Ligand builder by Sam Horrell.

Domains

One Nsp15 monomer is made up of three distinct domains, the aforementioned N-terminal oligomerisation domain (green), a middle domain in… well, the middle (orange), and the catalytic NendoU domain at the C-terminal (purple, Figure 3b). Overall SARS-CoV-2 Nsp15 shows high sequence identity with SARS-CoV Nsp15 (88%) and, somewhat lower identity with MERS-CoV (51%) (Youngchang 2020), but the overall structural similarity is very high between the three viruses. For a more detailed breakdown of the secondary structure that makes up individual Nsp15 domains, check out our proteopedia entry!

Figure 3: Nsp15 monomer coloured by domain. Image generated in PyMOL using PDB 6X4I by Sam Horrell. 

Tertiary Structure

Nsp15 forms a double-ring hexamer made up of a dimer of trimers stabilised by an N-terminal oligomerisation domain. So, three monomers form a trimer which then binds another trimer of monomers. However, If you open a crystal structures this can be confusing as you might not be presented with the whole complex. A crystal is composed of an infinite array of identical (or near enough) molecules related to each other by symmetry. To eliminate the need to store an infinite number of atoms on your computer the PDB file gives you just enough of the crystal to define the unique part. You are then expected to remember that the rest are generated by symmetry. This subset is called the asymmetric unit. Should you want to try and generate the whole crystal you can try, but your computer will likely grind to a halt on its way to infinity (and beyond).

For most structures the asymmetric unit is the interesting part. Often, when the biologically relevant complex has symmetry itself, like Nsp15 does, only part of the complex will be present in the file from the PDB. In the case of the PDB model 6X4I the molecules of each trimer obey the crystal’s three-fold symmetry. The file you download contains two molecules, one monomer from each trimer, and you must generate the symmetry related molecules (shown in green and orange in figure 3) to build the entire complex. These six monomers all come together to form the active enzyme, a 100 Å long and 10-15 Å wide channel, open to solvent from the top, bottom, and three separate side openings in the middle of the hexamer (Figure 4). Formation of the hexamer has been shown to be essential for enzymatic activity, making the oligomerisation interfaces a potential target for structure-based drug design. I’m not sure if I should be proud or disappointed that I didn’t mention Voltron once back there.

Figure 4: The Structure of the Nsp15 hexamer showing a side on view generated by crystallographic symmetry (a) and a top down view (b) looking down the 10-15 Å wide channel. Image generated in PyMOL using PDB 6X4I by Sam Horrell. 

The Active Site

SARS-CoV-2 Nsp15 is a Mn2+ dependent endoribonuclease, meaning it relies on the coordination of manganese to perform the transesterification reaction (cutting RNA). Unfortunately, the structure of SARS-CoV-2 Nsp15 has not been solved with manganese present, but we do have a structure with 3’ uridine monophosphate in the active site (PDBID: 6X4I). It has been proposed that the presence of manganese help stabilise the active site and substrate, but it is yet to been seen. Based on sequence alignment against related enzymes from other viruses we know the active site is made up of six conserved residues that sit in a shallow groove between two β-sheets (His235, His250, Lys290, Thr341, Tyr343, and Ser294), as shown in Figure 5. His235, His250, and Lys290 are predicted to act as a catalytic triad, His235 as a general acid, and His250 as a base with Lys290 governing U specificity.

Figure 5: SARS-CoV-2 Nsp15 active site conserved residues without (top) and with (bottom) 3’ uridine monophosphate. β-sheets are coloured purple, α-helices in orange, loops and ligands in green and waters in red. Image by Sam Horrell generated in PyMOL using PDB 6X4I.

But What Does it do?

After all that we have a pretty clear picture of what Nsp15 NendoU looks like, but what does it actually do? The fact that it cuts RNA would immediately suggest a role in viral replication, but Nsp15 deficient coronaviruses are still able to replicate. So maybe not, at least it's not essential for replication. Another suggestion is that Nsp15 is involved in interfering with the hosts innate immune response, but other studies suggest this is independent of Nsp15 activity. Finally, it has been suggested that Nsp15 degrades viral RNA as a means of hiding viral infection from the host immune system. So why does coronavirus bother with Nsp15? I’m afraid we don’t exactly know yet, but we’re working on it.

With that I’m going to leave you with one final Voltron reference for making it to the end. Good job, you earned this.

Figure 6: A perfectly good use of my time. Nsp15 coloured as Voltron featuring the arm monomers (forest and firebrick), leg monomers (skyblue and yelloworange), chest/back monomers (aquamarine and grey70), all loops (black), waters (white), and bound ligands (cyan). Image by Sam Horrell generated in PyMOL using PDB 6X4I.

Für diesen Beitrag steht leider keine deutsche Übersetzung zur Verfügung.

Introduction

Have you heard that the coronavirus “mutates”? Or that there are “several strains” of it around the world? Sounds scary, right? However, the reality is that everything “mutates”. All organisms, over time, acquire differences in their genes, from bacteria to humans. You might be aware that this can happen when your DNA (Deoxyribonucleic Acid) is exposed to UV light (like from the sun!), but this can also happen during DNA replication. This is when a cell uses the template of one of the two DNA strands to make a new complimentary copy of the other strand. Mutation is common to all living organisms (and viruses) and a driver of evolution. This is the first post in a series that will explore coronavirus replication with a focus on the proteins involved. 

How does the coronavirus make more of itself?

SARS-CoV-2 uses single-strand Ribonucleic acid (RNA) to encode its genome, not DNA, and hence belongs to a class of “single-strand RNA viruses”. For this reason, the virus needs a different way to copy its genome than “normal” cells have. The viral protein that copies the RNA is called an “RNA-dependent RNA polymerase” (RdRp). This protein uses the viral RNA as a template to make a new copy of viral RNA, by stringing single ribonucleotides together like beads on a string. This process is called polymerization.

A study by the Morse lab at Texas A&M University showed that SARS-CoV-2 RNA polymerase has a remarkable similarity to the RNA polymerase of SARS-CoV (>95%) as well as MERS-CoV [1], the virus which causes Middle-Eastern Respiratory Syndrome. This means that research performed in response to the SARS and MERS epidemics can inform our response to SARS-CoV-2. Unfortunately, a lack of consistent pandemic-preparedness funding means that we didn’t learn as much about RdRp in time as we could have. Still, RNA polymerase might be a viable drug target for halting the spread and reducing the fatality rate of COVID-19.

Structure of the RNA-Dependent RNA Polymerase

By determining the structure of RdRp, and deeply understanding how it works, we can optimize a drug to specifically target it and hinder its function. To this end, in the last few months, several structures of SARS-CoV-2 RNA polymerase have been published. 

One interesting structure shows RNA polymerase in action, in the process of elongating an RNA strand (see Figure 1).[2] This structure clearly show the polymerase in complex with smaller proteins, non-structural protein 7 and 8 (nsp7 and nsp8). These proteins improve how well the RNA polymerase binds the template RNA and also how long it stays bound before dissociating – a feature called “processivity”.[3]

Figure 1. Front and back views of the structure of elongating RdRp with RNA and two cofactors, nsp7 and nsp8 (PDB ID: 6yyt). Two copies of nsp8 (grey) form sliding poles that help stabilize the RNA (orange ball-and-stick model). One copy of nsp8 binds to the polymerase (blue) directly, but the other copy uses nsp7 (pink) to anchor to a second position on the polymerase.

In the center of the protein is the area where the main action happens, called the “active site”. The amino acids of the polymerase that form the active site have a particular shape and chemical properties, which enable the polymerization reaction to occur very rapidly. In fact, the polymerase can string together as many as 100 nucleotides per second! [3] New RNA molecules can enter the active site through a little window to be added to the growing RNA chain. It is here that the antiviral drugs make their move!

Figure 2. The third view shows the window into the active site through which new nucleotides must enter!

How do antiviral drugs attack RNA-dependent RNA polymerase?

First, let’s talk about Gilead’s FDA-approved drug, Remdesivir, which has taken the spotlight in the search for COVID-19 cures. Remdesivir (which has a fancy chemistry ID, GS-5734, and is sold under the brand name Veklury), is a “nucleotide analog”, which means that it mimics the shape and chemistry of the nucleotides that make up RNA and DNA (see figure). 

Remdesivir was developed originally as a general antiviral drug and was later shown to protect cells (in a test tube) and monkeys (not in a test tube) from the Ebola Virus [4]. However, this was recent enough, and science is slow enough that, until the COVID-19 pandemic, large-scale clinical trials of Remdesivir hadn’t been done yet. So scientists and doctors have been rushing to test the drug in COVID-19 patients. In fact, the US and Japan both approved the drug for “Emergency Use Authorization'' for severe COVID-19 patients as early as May [5], [6]. And, in July, the European Medicines Agency gave Remdesivir a “conditional marketing authorization” (used for drugs that meet an unmet medical need but have insufficient data for normal approval). This allows the use of Remdesivir in severe COVID-19 patients through the next year [7]. So, how the heck does a drug for Ebola, Influenza, or some other viruses also work against COVID-19? I was concerned by this when the news about all the drug trials were coming out – and I’m sure I wasn’t the only one...

The simple answer to that is all these viruses need to do the same thing - copy their RNA genome from an RNA template. And in order to do that, they all end up using basically the same tool, an RNA-Dependent RNA polymerase. And all drugs that are nucleotide analogs use the very same trick: they dress up like ribonucleotides (the "beads on a string" from before) and fool the RNA polymerase into letting them into the active site. Once inside, they get “stuck” in the active site, jamming the polymerase machine. Since this trick should work for any viral RNA polymerase, we can use these drugs for any RNA virus, and call them ‘general antivirals’. Of course, in practice, this doesn't always work, because there are differences between the different RNA polymerases. However, it is a great place to start! In the future, if we have general antivirals for SARS-CoV-2 all ready-to-go, we may be better equipped to deal with another coronavirus outbreak!

Figure 3. We all see what we want to see, I guess.

The Chemistry of Remdesivir

Remdesivir resembles the nucleotide adenine in structure, although it has some fancy chemical add-ons which help make it a better drug (thank you, medicinal chemistry!). When Remdesivir is injected into a vein, it travels through the bloodstream and enters into our cells, which recognize it as a foreign substance and try to digest it. However, what ends up happening is that the cells remove just the fancy chemical add-ons, and then confuse it for a normal adenine nucleotide. In infected cells, the viral RNA-dependent RNA polymerase then starts grabbing these molecules and inserting them into the new viral RNA strand in place of adenine molecules. Remdesivir, now attached to the RNA, jams the polymerase, rendering the virus unable to make more copies of its genome. Ultimately, this halts viral replication and helps the patient fight off the virus.

Figure 4. (A) The red part of Remdesivir makes it a better drug by helping it get from the blood stream into human cells, but it isn’t necessary for jamming the polymerase. It was designed on purpose so that when it gets inside human cells, the cells try to digest it. When they do, they cleave off the red bits, causing it to get confused for an adenine nucleotide.  (B) This causes the cell to add two more phosphates to the molecule, making it the ‘tri’-phosphate form. This is the active form of the molecule, which mimics ATP (C), and is incorporated into the growing RNA chain in the place of ATP. The extra bit sticking off the side (in blue) is called a 1’-cyano group, and makes the RNA get stuck inside the polymerase, jamming it.
Figure 5. Structure of Remdesivir (cyan) in the active site of RNA-dependent RNA polymerase. The window through which new nucleotides enter is to the bottom left of the image. The RNA (orange ball-and-stick model) template strand enters from the bottom right. Remdesivir makes base-pair hydrogen bonds with the opposite uracil base.

Another drug that inhibits the RNA polymerase activity is Favipiravir, sold under brand names Avigan, Abigan, and FabiFlu. Favipiravir has been discovered by Toyama Chemical Co., Ltd. in Japan and it has a similar mechanism to Remdesivir, except that it mimics a guanosine nucleoside instead of an adenine nucleotide [8]. This drug was approved in Japan back in 2014 for use in resistant cases of Influenza A and B, but still remains unapproved in the US (still in Phase II and Phase III clinical trials) and the UK [9]. This drug is also being tested for use against Ebola virus, Lassa virus, and currently SARS-CoV-2 in 43 countries. The approval of Favipiravir for  COVID-19 has been much faster in China (Mar 15, 2020), Russia (Jun 3, 2020), and India (Jun 20, 2020)[10], [11]. Nonetheless, other countries, including Japan, are in various stages of clinical trials, and the results are anticipated to be out by the end of July [10].

So...do we have a cure for SARS-CoV-2?

Sadly, not yet. While the speed at which Remdesivir has gone through clinical trials is unprecedented, more work needs to be done to make sure it is safe and effective. Since (in the big scope of things) not a lot have people have taken Remdesivir, we aren’t really sure what all the side effects are, although there is emerging evidence for liver and kidney damage [12, 13]. The most common side effects are nausea (10% and 9% of patients), indigestion (7%) and increase of transaminases (6% and 8%). In one study, 3.6% of patients in a 10-day trial needed to stop taking therapy due to the latter. However, serious viral infections can also cause liver damage, so separating the two causes is a challenge! Remdesivir is not a cure-all, either. In one study it improved the recovery time from 15 days to 11 days, but it showed no effect for patients with mild to moderate disease, and no difference in median recovery time for patients who were already on a ventilator [14]. Since the drug has to be given by infusion over several days, there is a pretty small window in which Remdesivir can actually help. 

Likewise, Favipiravir has its own side effects such as liver damage, elevated uric acid levels, kidney damage, skin allergies, etc. [15]. These effects restrict it for use by severe diabetes and heart patients. On top of that, it is not suitable for pregnant women because it can cause potential fetal deaths and deformities. It has been shown that Favipiravir works only during the earlier stages of SARS-CoV-2 infection when the body’s immune system isn’t totally drained, whereas it can result in a cytokine storm (when your immune system really freaks out) in severely ill patients. But, unfortunately, the virus doesn’t differentiate between humans while attacking, so a universal drug for COVID-19 has to be safe for use by all people. 

However, these drugs are better than nothing, and by understanding the mechanisms involved, scientists can continue to improve upon the existing drugs for the benefit of all. While most of the ‘general antivirals’ that target RNA Polymerase have failed with SARS-CoV-2, Remdesivir has been relatively successful. Scientists think that this is actually because of a proofreading protein in SARS-CoV-2 called exonuclease. Immediately after the RNA-polymerase makes new RNA, exnuclease checks to make sure the new RNA is correct. In one study, another drug that mimics RNA called Ribivarin was shown to be removed from newly synthesized RNA by exonuclease [16]. Thankfully, Remdesivir is not excised , which is likely why it has been more successful than the other options [17], [18]. To read more about how nsp14 maintains the integrity and virulence of SARS-CoV-2, tune in to a future blog entry!

Figure 6. Hey, we've all been there.

Recommended Structures

For those interested in reviewing the structures further, they are available in our GitHub repo, along with information about validation and, where relevant, improved structures. For a high-resolution comparison of the active site with and without Remdesivir, 7BV2 and 7BV1 (respectively) were published together at 2.5 and 2.8 Å. The elongating structure of the complex shown above (6YYT) has the polymerase as well as the cofactors and RNA very well resolved, with little "missing" density and a resolution of 2.9 Å. It is likely preferable to 6M71 and 7BTF, which were published with a similar resolution but with less of the complex resolved, and no RNA. For those interested, 7C2K and 7BZF (at 2.93 Å and 3.26 Å) show the complex bound to RNA in a pre- and post-translocation state.

Sources

[1] J. S. Morse, T. Lalonde, S. Xu, and W. R. Liu, “Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV,” ChemBioChem, vol. 21, no. 5, pp. 730–738, Mar. 2020, doi: 10.1002/cbic.202000047.

[2] H. S. Hillen, G. Kokic, L. Farnung, C. Dienemann, D. Tegunov, and P. Cramer, “Structure of replicating SARS-CoV-2 polymerase,” Nature, May 2020, doi: 10.1038/s41586-020-2368-8.

[3] W. Yin et al., “Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir,” Science, p. eabc1560, May 2020, doi: 10.1126/science.abc1560.

[4] R. T. Eastman et al., “Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19,” ACS Cent. Sci., May 2020, doi: 10.1021/acscentsci.0c00489.

[5] O. of the Commissioner, “Coronavirus (COVID-19) Update: FDA Issues Emergency Use Authorization for Potential COVID-19 Treatment,” FDA, May 04, 2020. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-issues-emergency-use-authorization-potential-covid-19-treatment (accessed Jul. 08, 2020).

[6] A. Sternlicht, “Japan Approves Remdesivir For Use On Severe COVID-19 Patients,” Forbes. https://www.forbes.com/sites/alexandrasternlicht/2020/05/07/japan-approves-remdesivir-for-use-on-severe-covid-19-patients/ (accessed Jul. 08, 2020).

[7] D. CZARSKA-THORLEY, “First COVID-19 treatment recommended for EU authorisation,” European Medicines Agency, Jun. 25, 2020. https://www.ema.europa.eu/en/news/first-covid-19-treatment-recommended-eu-authorisation (accessed Jul. 10, 2020).

[8] E. De Clercq, “New Nucleoside Analogues for the Treatment of Hemorrhagic Fever Virus Infections,” Chem. Asian J., vol. 14, no. 22, pp. 3962–3968, Nov. 2019, doi: 10.1002/asia.201900841.

[9] K. Shiraki and T. Daikoku, “Favipiravir, an anti-influenza drug against life-threatening RNA virus infections,” Pharmacol. Ther., vol. 209, p. 107512, May 2020, doi: 10.1016/j.pharmthera.2020.107512.

[10] T. Hornyak, “Japan sending Fujifilm’s flu drug favipiravir to over 40 countries for Covid-19 trials,” CNBC, May 04, 2020. https://www.cnbc.com/2020/05/04/fujifilms-flu-drug-favipiravir-sent-to-43-nations-for-covid-19-trials.html (accessed Jul. 14, 2020).

[11] G. P. Ltd, “Glenmark Becomes the First Pharmaceutical Company in India to Receive Regulatory Approval for Oral Antiviral Favipiravir, for the Treatment of Mild to Moderate COVID-19.” https://www.prnewswire.com/in/news-releases/glenmark-becomes-the-first-pharmaceutical-company-in-india-to-receive-regulatory-approval-for-oral-antiviral-favipiravir-for-the-treatment-of-mild-to-moderate-covid-19-855346546.html (accessed Jul. 14, 2020).

[12] Goldman, J. D. et al. Remdesivir for 5 or 10 Days in Patients with Severe Covid-19. N. Engl. J. Med. (2020) doi:10.1056/NEJMoa2015301

[13] Remdesivir Safety Forecast: Watch the Liver, Kidneys | MedPage Today. https://www.medpagetoday.com/infectiousdisease/covid19/86582

[14] J. H. Beigel et al., “Remdesivir for the Treatment of Covid-19 — Preliminary Report,” N. Engl. J. Med., vol. 0, no. 0, p. null, May 2020, doi: 10.1056/NEJMoa2007764.

[15] Sandhya Ramesh, “Favipiravir, Japanese drug that’s the new Covid treatment hope your chemist will soon stock,” ThePrint, Jun. 25, 2020. https://theprint.in/health/favipiravir-japanese-drug-thats-the-new-covid-treatment-hope-your-chemist-will-soon-stock/447987/ (accessed Jul. 14, 2020).

[16] F. Ferron et al., “Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA,” Proc. Natl. Acad. Sci., vol. 115, no. 2, pp. E162–E171, Jan. 2018, doi: 10.1073/pnas.1718806115.

[17] C. J. Gordon, E. P. Tchesnokov, J. Y. Feng, D. P. Porter, and M. Gotte, “The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus,” J. Biol. Chem., Feb. 2020, doi: 10.1074/jbc.AC120.013056.

[18] L. Zhang et al., “Role of 1’-Ribose Cyano Substitution for Remdesivir to Effectively Inhibit both Nucleotide Addition and Proofreading in SARS-CoV-2 Viral RNA Replication,” bioRxiv, p. 2020.04.27.063859, Apr. 2020, doi: 10.1101/2020.04.27.063859.

Das Coronavirus unkompliziert selbst drucken und zusammenbauen – wir haben ein 3D-Modell dafür entworfen!
Abhängig vom User und dem jeweiligen 3D-Drucker sind die Details natürlich unterschiedlich. Die Methoden, die wir angewandt habenkönnen aber als Anhaltspunkt dienen. Nutzer ohne eigenen 3D-Drucker können die STL-Daten aber auch dafür verwenden, den Druck bei einem externen Dienstleister zu beauftragen. Wir hoffen, mit diesem Projekt nicht nur private Nutzer zu erreichen, sondern auch bessere Möglichkeiten für die Lehre und das öffentliche Verständis des Virus zu schaffen.

Unser Entwurf basiert auf aktuellesten wissenschaftlichen Erkenntnissen bezüglich der Proteinstrukutur und Größenverhältnisse. Mehr dazu hier.

Mit dem ausgedruckten und zusammengebauten Modell bekommt man eine Vorstellung, wie das Virion aussehen würde, wenn es um eine Million vergrößert wäre (1 mm des Models stellt 1 nm (10 Å) dar). Die RNA, das Erbgut des Virus, wäre dann zehn Meter lang und einen Millimeter dick.

Zusätzlich haben wir ein Modell eines menschlichen Antikörpers im selben Maßstab entworfen, welches zusätzlich zu den Strukuren des Virions gedruckt und je nach Wunsch an das Spike-Protein angehägt werden kann. Um das Drucken, Bemalen und Zusammenbauen zu erleichtern, haben wir die Virusstruktur in vier einzelne Komponenten zerlegt:

Bis jetzt wurden die Strukturen erfolgreich auf verschiedenen Schmelzschicht-Druckern (FDM), einem Rostok MAX v2 und einem Prusa I3 MK3 Drucker getestet. Mit anderen Methoden, wie Stereolithographie, wäre eine noch höhere Qualität durchaus möglich.

Jeder Teil ist im STL-Format verfügbar und sollte mit jeder geeigneten Slicer-Software druckbar sein.

Beim Zusammenbauen und Bemalen des fertigen Drucks geht man am besten nach eigenem Gutdünken vor. Die exakten Details unterschieden sich schließlich je nach Equipment und nach den Einstellungen.
Wir zeigen hier trotzdem unseren Aufbau in knapper Zusammenfassung.

Druck der Komponenten:

Der erste Schritt ist das Drucken der einzelnen Bestandteile. Die Virion-Kugel ist schnell gedruckt, da durch die flache Oberfläche keine weiteren Träger oder Verbindungen benötigt werden.
Dieser Teil kann mit einem Minimum an Füllung und Trägern gedruckt werden, aus Gründen der Stabilität empfehlen wir jedoch eine Füllung von mindestens 10%.

Die anderen Teile (Spikeproteine und Antikörper) stellen hierbei eine größere Herausforderung dar.
Das Spikeprotein muss für das fertige Model mindestens 95mal gedruckt werden. Hierzu können entweder individuelle Einstellungen genutzt oder die 25x STL-Datei 4mal gedruckt werden.
Es ist empfehlenswert das Spike-Protein mit dem Kopf in Richtung Druckbett zu drucken. Das erhöht die Stabilität und benötigt weniger Verbindungen und Vernetzungen zwischen den einzelnen Trägern.
Diese müssten sonst mit Fingerspritzengefühl vom sensiblen Stamm der Spikes entfernt werden. Wie viele Träger zusätzlich hinzugefügt werden, kann je nach Nutzer und der jeweiligen Situation entschieden werden.

Ein Dual-Extruder-Drucker ist für das Herstellen der Spikes ideal, da die stabilisierenden Verbindungen zwischen den Spikes aus einem wasserlöslichen Plastik gedruckt und somit einfach zu entfernen sind. Auf jeden Fall erzeugt ein individueller Druck der Spikes oder zumindest eine geringere Anzahl pro Block ein besseres Ergebnis. Die Verarbeitung dieser Spikes ist dann einfacher, auch wenn der Druck zeitaufwändiger wird. Generell muss ein guter Kompromiss zwischen der Druckgenauigkeit, der Geschwindigkeit und dem Aufwand beim Aufarbeiten der Modelle gefunden werden.

Die Details dieses Prozesses hängen vor allem von der Art des Druckers, dem Aufbau und der Drucktechnik ab. Wir nutzten die bekannteste Technik: Schmelzschicht-Druck (FDM), als Plastik wurde Polylactide (PLA) verwendet, was die folgende Aufreinigung erleichterte.

Aufarbeitung

Um die Objekte möglichst sauber zusammensetzen und bemalen zu können, ist eine Aufarbeitung der Einzelteile notwendig. Die Stabilisierungsstücke können mit einer Zange entfernt werden, während kleinere Artefakte einen Abschliff benötigen. Auch ein Zahnstocher hat sich als hilfreich erwiesen.

Links die Virion- und Spike-Protein- Oberflächen nach dem Druck, mit erkennbaren Artefakten und Plastik-Fadenbildung . Auf der rechten Seite das mit Ethylacetat behandelte Virion mit einer glatten Oberfläche. Bilder von Ferdinand Kirsten, Matt Reeves.
Links die Virion- und Spike-Protein- Oberflächen nach dem Druck, mit erkennbaren Artefakten und Plastik-Fadenbildung . Auf der rechten Seite das mit Ethylacetat behandelte Virion mit einer glatten Oberfläche. Bilder von Ferdinand Kirsten, Matt Reeves.

Für PLA erwies sich Ethylacetat als die beste Reinigungsmethode um Oberflächen zu glätten und Überbleibsel der Träger zu entfernen. Das Ethylacetat löst das Plastik auf und zerstört somit kleine Unebenheiten auf den Oberflächen, wenn es bedacht angewendet wird. Hierbei kann unterschiedlich vorgegangen werden, wobei die schonendste Methode das Aussetzen in eine Ethylacetat-Dampf Umgebung in einem geschlossenen Gefäß ist. Es entsteht eine glatte Oberfläche mit genauen Details, der Prozess nimmt jedoch oft viele Stunden oder sogar einige Tage in Anspruch.

Die schnellere Methode , die ebenfalls zufriedenstellende Resultate liefert, ist das Eintunken der Objekte in Ethylacetat für 10-30 Sekunden. Anschließend werden sie abgetupft und zum Trocknen ausgelegt. Oft ist ein zweiter Reinigungsgang nötig. Für die größeren Virusteile kann es helfen ein Tuch, welches mit Ethylacetat getränkt ist, bis zum gewünschten Ergebnis über die Oberfläche zu reiben. Mit dieser Methode lassen sich die beiden Virionhälften auch hervorragend zusammenkleben. Eine kleine Menge Ethylacetat wird auf jeder Fläche der Hälften verteilt und die Hälften zusammengedrückt, bis sie zu einem einzigen Stück verschmolzen sind. Auch die Naht kann dann mit einem Ethylacetat-Tuch gut geglättet werden. Hierfür stellt Aceton-freier Nagellackentferner eine ausgezeichnete, frei käufliche Alternative dar, die die gleichen Ergebnisse erzielen dürfte. Bei der Handhabung dieser Chemikalien sollte immer geeignete Schutzausrüstung getragen werden ( Schutzbrille, Handschuhe etc.).

Spike-Proteine Frisch nach dem Druck (links) und nach der Aufarbeitung mit Ethylacetat (rechts), Bild von Ferdinand Kirsten.
Spike-Proteine frisch nach dem Druck (links) und nach der Aufarbeitung mit Ethylacetat (rechts), Bild von Ferdinand Kirsten.

Übrigens: Aceton erzielt für das andere häufig genutzte Druckmaterial, Acrylnitril-Butadien-Styrol (ABS) die gleiche Wirkung wie Ethylacetat für PLA.

Bemalen und Zusammensetzen

Wie beim Druck, sind auch das Bemalen und die jeweiligen Malmethoden dem Nutzer individuell überlassen. Hier zeigen wir die Variante des Würzburger Modells, bei der wir versucht haben, der Illustration von Thomas Splettstösser möglichst treu zu bleiben.

Am Computer erstelltes Bild des Virusses von Thomas Splettstoesser (links) und das ferige 3D-Modell des Thorn Labs (rechts).
Am Computer erstelltes Bild des Virusses von Thomas Splettstoesser (links) und das ferige 3D-Modell des Thorn Labs (rechts).

Die Einzelteile wurden zu Beginn mit einem Primer überzogen, um die Farbe besser an das Modell zu binden. Außerdem wirkt dieser wie eine gleichmäßige Grundierung. Beim Auftragen des Primers und der Nutzung einer Airbrush muss auf die Sicherheit geachtet werden, um das Einatmen der schädlichen Substanzen zu vermeiden. Ein gut belüfteter Raum, ein Abzug und eine Zirkulation weg vom Körper sind zu empfehlen. Das Tragen von Handschuhen, einer Schutzbrille und einer Maske sollte für zusätzlichen Schutz sorgen.

In unserem Fall wurde das Modell hauptsächlich mit einer Airbrush bemalt und wir empfehlen diese Methode für die kleinen Oberflächendetails und komplexen Strukturen. Natürlich können auch alle Teile mit dem Pinsel angemalt werden, dies ist jedoch deutlich zeitaufwendiger und erfordert genaueres Arbeiten. Alle genutzen Farben, Verdünner, Primer und Lack sind von Citadel-Painting. Hier eine Liste der genutzten Farben und Amterisleien die für unser Modell verwendet wurden:

  • Grün: “Moot green”
  • Gelb: “Yriel Yellow”
  • Grau: “Dawnstone”
  • Braun: “Baneblade Brown”
  • Dunkelbraun: “Doombull Brown”
  • Hellblau (Aqua): “Gauss Blaster Green”
  • Türkis: “Kabalite Green”
Die Spike-Proteine Sortiert nach Farben (links oben), nur mit Grundierung (links unten) und nach dem Hervorheben mit Limettengrün (rechts). Bild von Kristopher Nolte.
Die Spike-Proteine Sortiert nach Farben (links oben), nur mit Grundierung (links unten) und nach dem Hervorheben mit Limettengrün (rechts). Bild von Kristopher Nolte.

Um den Effekt einer natürlichen Lichtquelle zu erzeugen wurden die Spikes in vier Gruppen unterteilt und unterschiedlich hell bemalt.
Wenn das Modell nicht für die feste Ausstellung auf einer Halterung oder Ähnlichem geplant ist, ist dieser Schritt nicht notwendig. Jedes Spike-Protein wurde mit einem helleren Limettengrün hervorgehoben (Highlighting), um einen stärkeren Kontrast zu erzeugen und die Oberfläche besser zu differenzieren. Anschließend wurde das Highlighting mit einem "Dry-brush" der hellblauen (Aqua) Farbe vollendet.

Virion-Kugel (oder auch liebevoll Kartoffel genannt) mit hervorgehobenen Hüllenproteinen (links) und nach der Grundierung (rechts). Bild von Kristopher Nolte.
Virion-Kugel (oder auch liebevoll Kartoffel genannt) mit hervorgehobenen Hüllenproteinen (links) und nach der Grundierung (rechts). Bild von Kristopher Nolte.

Nachdem das Virusmodell samt Spikes bemalt war, wurde die Farbe mit Glanzlack und einem matten Finish versiegelt. Dieser Schritt ist ebenfalls optional, aber zum Schutz gegen Abnutzung der Farben bei häufiger Handhabung des Modells zu empfehlen.

Nach all diesen Schritten kommt es endlich zum langersehnten Zusammensetzen der Einzelteile. Falls die Spike Proteine verschiedene Highlights bekommen haben, ist darauf zu achten, sich auf eine „Lichtquelle“ festzulegen und die Spikes dementsprechend anzuordnen und am Modell zu befestigen (Auf einem Ständer: Unten dunkler, nach oben heller). Um die Spikes an ihrer Position zu befestigen haben wir normalen Modellbaukleber verwendet. Starker Bastel-Kleber oder Ethylacetat können hierfür ebenfalls benutzt werden, sowie kleine Magnete für besondere Tüftlerinnen und Tüftler. Da unser Modell auf einer Halterung präsentiert werden soll, wurde hierfür ein Loch an der Unterseite des Modells freigelassen, in dem dann die Stange befestigt werden kann.

Zusammensetzen des Modells mit Kleber. Die Spike-Proteine werden in den dafür vorgesehenen Löchern befestigt. Bild von Kristopher Nolte.
Zusammensetzen des Modells mit Kleber. Die Spike-Proteine werden in den dafür vorgesehenen Löchern befestigt. Bild von Kristopher Nolte.

Hoffentlich hat euch unser kleines Abenteuer gefallen und inspiriert, euch an euer eigenes 3D-Coronamodell zu wagen. Die beschriebenen Arbeitsschritte haben insgesamt etwas mehr als eine Woche in Anspruch genommen. Das Drucken dauert etwa einen Tag.  Aufreinigung und Verfeinerung benötigten mehr als zwei Tage und das Bemalen des Modells ein ganzes Wochenende.

Die Dateien sind öffentlich auf Thingiverse verfügbar und das Modell ist lizensiert als Creative Commons BY-NC: Frei Verwendung und Veränderung für nicht-kommerzielle Zwecke und unter Nennung der "Coronavirus Structural Task Force" als Urheber.

3D-Druck Illustration von Thomas Splettstoesser (links) im Vergleich mit dem Modell von Dale Tonrud aus Oregon (mitte) und dem Thorn Lab aus Würzburg (rechts).
3D-Druck Illustration von Thomas Splettstösser (links) im Vergleich mit dem Modell von Dale Tonrud aus Oregon (mitte) und dem Thorn Lab aus Würzburg (rechts).

Wie bei jedem 3D-Modell, gibt es weit mehr als einen Weg, diese Aufgabe anzugehen und zu vollenden. Wir freuen uns, darauf, Eure Modelle zu sehen und mit Euch über Herangehensweisen und Techniken zu diskutieren - hier in den Kommentaren, auf Thingiverse oder Twitter!

Die Autoren:

Wir möchten hervorheben, dass dieser Artikel eine Zusammenarbeit mehrerer Leute ist:

Dale Tonrud und Thomas Splettstösser haben zusammen die STL Dateien für das 3D Modell erstellt und verfeinert. dale hatte die Idee, ein Modell zu drucken und diese wurde dann von Andrea Thorn aufgegriffen. Thomas und Dale sorgten dann dafür, das Modell möglichst realistisch und gleichzeitig gut für Handhabung und Druck in Einzelteilen zu gestalten. Dale druckte das erste Design des Modells aus.
Matt Reeves war für die Optimierung und den Druck des Würzburger Modells zuständig. Er fand heraus, wie das Modell am besten nachbearbeitet wird und trug zusammen mit dem Rest des Teams zur Verbessung des Modells bei.
Kristopher Nolte arbeitete zusammen mit Ferdinand Kirsten das gedruckte Modell auf und reinigte es. Kristopher war zudem für die filigrane Arbeit des Bemalens und Zusammensetzens des fertigen Virions verantwortlich.

Dieser Artikel ist übersetzt von Ferdinand Kirsten, Pairoh Seeliger und Kristopher Nolte, nach dem originalen Artikel in Englisch von Kristopher Nolte, Dale Tonrud und Matt Reeves.

Für die Bekämpfung der Lungenkrankheit COVID-19 und um eine weitere Ausbreitung der Pandemie zu stoppen, ist die Entwicklung neuer Medikamente essenziell. Ein vielversprechendes Angriffsziel für neue Wirkstoffe gegen den Erreger, SARS-CoV-2, ist die 3-Chymotrypsin-like Protease (3CL-protease), auch bekannt als Hauptprotease oder Mpro.

Damit neue Viren in der Wirtzelle gebildet werden können, muss die ssRNA des Virus in Proteine translatiert werden. Ein Großteil der Virusproteine werden als lange Polyproteine translatiert. Damit funktionale Proteine daraus entstehen können, müssen diese von Proteasen wie der Hauptprotease zerschnitten werden. Die Prozessierung der Polyproteine ppa1a und ppa1ab erfolgt an 11 Stellen in Richtung des C-terminalen Endes (abwärts von Nsp4). Für die Bildung von neuem Viruserbmaterial wird der RNA-abhängige RNA-Polymerasekomlex benötigt, der ein Teil dieser Polyproteine ist (nämlich Nsp7, Nsp8, Nsp12 und Nsp14). Seine Bildung kann durch eine gezielte Inhibition der Hauptprotease gestoppt werden, und damit die RNA-Replikation.

Die Hauptprotease ist eine Cysteinprotease, für die eine katalytische Dyade, gebildet aus den Aminosäuren Cystein und Histidin, typisch ist. Die Hauptprotease besteht aus zwei identischen senkrecht zueinander stehenden Protomeren, welche sich aus je drei Domänen zusammensetzen. An ihrem N-terminalen Ende liegen die Domänen I und II. Sie formen je eine antiparallele, chymotrypsinartige β-Tonnenstruktur, in der sich die Substratbindungsstelle befindet (siehe Bild weiter unten). Die Domäne III am C-terminalen Ende des Protomers besteht aus fünf α-Helices, die sich zu einem Cluster zusammenlagern und durch Ausbildung einer Salzbrücke zwischen Glu-290 des einen und Arg-4 des anderen Protomers zur Dimerisierung beitragen.

Die beiden Protomere werden auch durch den sogenannten "N-Finger" verbunden. Er besteht aus den letzten 7 Aminosäureresten am N-Terminus und stellt einen Kontakt zur Domäne II des jeweils anderen Protomers her, wodurch eine Kontaktfläche von ~1394 Å2 entsteht. Die Bildung des Homodimers ist unerlässlich für die Protease-Aktivität, da der Ser-1 Rest am N-terminalen Ende mit dem Glu-166 Rest des anderen Protomers interagiert und so die Substratbindungsstelle in Form hält. Diese Substratbindungsstelle besteht aus der katalytischen Dyade Cys-145 und His-41. Direkt daneben befindet sich die Substratbindungstasche S1, die aus den aus den Aminosäureresten von Phe-140 und His-163, sowie den Hauptkettenatomen von Glu-166, Asn-142, Gly-143 und His-172, besteht. Die Substratbindungsstelle bindet spezifisch Glutamin im Motiv Leu-Gln↓(Ser,Ala,Gly) des zu schneidenden Protein-Substrats, da das Carbonyl-Sauerstoffatom des Glutamins durch die Aminosäuren Gly-143 und Cys-145 stabilisiert wird.

Picture of corona virus main protease structure with labels.
Das Bild zeigt die Struktur der Hauptprotease des Coronavirus. Das eine Protomer, dargestellt als Bändermodell in Gelb, ist unterteilt in die Domänen I, II und III. Das andere (identische) Protomer ist als Stabmodell dargestellt und mit der experimentellen Elektronendichte des PDB-Eintrags 6y2e überlagert. Bild erstellt von Sam Horrell.

Durch die Entwicklung von Inhibitoren, die gezielt an die Substratbindungsstelle der Protease binden, könnte die Spaltung der Polyproteine gehemmt, und somit die Virus RNA-Replikation gestoppt werden, so dass keine neuen Viren entstehen. Der Vorteil hierbei ist, dass bis heute keine menschlichen Proteasen mit ähnlicher Spezifität bekannt sind, weshalb eine Toxizität der Inhibitoren unwahrscheinlich ist. Potenzielle Inhibitoren lassen sich aufgrund ihrer chemischen Struktur in zwei Klassen unterteilen. Die erste Klasse umfasst Peptide, die spezifisch an die katalytische Stelle des Enzyms binden und durch kovalente Bindung mit Cys-145 die Substratbindung verhindern. Die zweite Klasse besteht aus kleinen organischen Molekülen, welche an Gruppen in der Substratbindungsstelle des Enzyms binden und so kompetitiv den Substrateintritt in den Hohlraum des aktiven Zentrums verhindern. Ein solcher Stoff, der als Wirkstoff zur Therapie von COVID-19 eingesetzt werden könnte, ist der HIV1-Protease-Inhibitor Lopinavir. Sollte sich die Wirksamkeit von Lopinavir gegen SARS-CoV-2 bestätigen, hätte das den Vorteil, dass Lopinavir bereits als HIV-Medikament für den Menschen zugelassen ist - und somit schneller zum Einsatz kommen könnte.

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