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According to several sources, Coronavirus is a helical virus, i.e. it should have a helical capsid. Although most sources did not labelled or pointed any kind of capsid. It was mysterious to watch diagrams of spherical envelops with no underneath capsids.
Cutaway view of coronavirus from Wikipedia
David Goodsell painting on PDB
No mention of capsid.
Initially I guessed the proteins around the RNA fibre formed the capsid, not labelled otherwise. Alternatively I was speculating whether coronavirus is lacking the capsid.
However a researchgate paper picture (heavily diagrammatic) shows a polygonal capsid, the picture is captioned as coronavirus.
It makes me think of whether the capsid is like a hollow spherical or polygonal thing spirally wrapped.
Now my questions are .
What is the actual/ realistic shape of coronavirus capsid?
Does it really contain a capsid?
(Just speculating) Is it SARS-CoV2 lacking a capsid, and thus special exception from g eneral description of coronavirus?
Does it really contain a capsid?
Yes. Coronaviruses have a capsid, but it's not reminiscent of the polygonal (icosahedral) capsid depicted in the Research Gate picture you referenced. Icosahedral capsids form a sort of shell around the viral genome, where helical capsids actually bind the viral nucleic acids, holding them in a more rigid shape.
Is it SARS-CoV2 lacking a capsid, and thus special exception from general description of coronavirus?
SARS-CoV-2 is not an exception. In fact, its nucleocapsid is a target for diagnostic tests.
What is the actual/ realistic shape of coronavirus capsid?
It is actually depicted in Goodsell animation you referenced. The nucleocapsid proteins are the squigly bits in and around the genomic RNA (labeled "N", for Nucleocapsid). The actual capsid is the complete complex of N proteins bound to genomic RNA, so it doesn't have that polygonal outline that maybe we're used to when we see depictions of viruses. If you want a more detailed look at the actual capsid protein structure, there's a depiction of its crystal structure in this New York Times piece co-authored by Carl Zimmer.
How seeing the molecular machinery of the coronavirus will help scientists design a treatment
In the race to develop a treatment for COVID-19, the disease threatening millions of lives around the world, scientists are studying every aspect of the SARS-CoV-2 coronavirus that causes it.
One vital step is understanding the precise shape and structure of the virus at the molecular level. Knowing the shape lets scientists identify targets and design drugs to hit them.
This approach was used to create the anti-influenza drugs oseltamivir and zanamivir (Tamiflu and Relenza) among others. It also shows great promise for the new coronavirus.
What is being done for COVID-19 treatment?
At present, scientists and clinicians are investigating the use of existing drugs such as chloroquine, which is used as an antimalarial and to treat autoimmune disorders, and the antiviral remdesvir. (Despite what you may read or hear, none of these are approved treatments for COVID-19 and should not be taken without medical instructions.)
Many pharmaceutical companies are also looking into therapies based on antibodies produced in people who have already been infected by the virus and recovered.
There are also several attempts in progress to create a vaccine which can be given to healthy people to make them immune. There are various approaches in play, including the use of genetic material or synthetic viral proteins to find ones that teach our immune systems to mount an effective defence against the virus.
Human trials are already under way for some of these experimental drugs and vaccines, and time will tell if they work. But almost certainly, an effective long-term strategy will also require significant research investment to allow the design of novel treatments that are specifically targeted for the SARS-CoV-2 virus.
How can you design new treatments without knowing what your target looks like?
Why structural biology?
If you want to design a key to a lock, it is much easier if you know what the lock looks like. In the same way, to design targeted treatments it is important to know what the target looks like.
In SARS-CoV-2 virus, the targets are its genetic material and proteins. These molecules let the virus invade humans and make multiple copies of itself.The main protease enzyme of the SARS-CoV-2 coronavirus, with a small chemical molecule in purple. Credit: Onisha Patel, Author provided
Structural biology is a field of science that allows us to see beyond what is visible to the naked eye, to see nanoscopic-sized molecules such as DNA, RNA and proteins. Structural biology methods such as X-ray crystallography and cryo-electronmicroscopy are currently being used at a rapid pace to visualise molecular components of the virus.
Within weeks from when the genetic sequence of the SARS-CoV-2 was made available, structural biologists have used these techniques to see proteins that make up the SARS-CoV-2 virus.
Some of these include the "spike" protein, a protein that helps the virus to gain entry into the host and enzymes that enable virus replication including the main protease, an attractive target for drug development. The inner molecular machinery of the SARS-CoV-2 virus is just beginning to be revealed and there is more to come.
How is structural biology helping with COVID-19 research?
Scientists are using structural biology data to look for special features within the viral proteins. One such feature is a cavity or space where a small chemical molecule (a potential drug) can fit.
Once identified, researchers work on the molecule to improve the fit and make it work better as a drug. Eventually the chemical molecule might fit tightly enough to stop the viral protein from doing its job, much like a spanner thrown into a set of gears.
This approach to the design of new and targeted drugs is called "structure-based drug design". It is more efficient, precise and time-saving. It has been used in the past to create anti-viral drugs such as oseltamivir (Tamiflu) and zanamivir (Relenza) that target the flu virus.
Scientists are now using this approach to discover drugs against SARS-CoV-2. For vaccine design and therapeutics development, knowing what the SARS-CoV-2 "spike" protein looks like is a major breakthrough. Scientists are already planning to use a stabilized version of this protein to screen for antibodies from people who have recovered from COVID-19 infection.
Where is structural biology data being shared?
Modern robotics, better instrumentation, faster data collection, better computing and software have revolutionized the speed with which structural biology information is being made available. This would not have been possible even five years ago.
The structural data collected is deposited at the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB), online open access repositories who make this data available worldwide free of charge. This open access policy makes it possible for scientists globally to answer some of the most intriguing questions on SARS-CoV-2 biology and find the best ways to design new treatments.
Whether for new drugs or vaccines, structural biology is at the frontline to understand the molecular machinery from viruses to humans.
This article is republished from The Conversation under a Creative Commons license. Read the original article.
New coronavirus variant: What is the spike protein and why are mutations on it important?
Say hello to Spike. Credit: National Institute of Allergy and Infectious Diseases, CC BY-SA
The emergence of a new variant of coronavirus has sparked renewed interest in the part of the virus known as the spike protein.
The new variant carries several peculiar changes to the spike protein when compared to other closely related variants—and that's one of the reasons why it's more concerning than other, harmless changes to the virus we have observed before. The new mutations may alter the biochemistry of the spike and could affect how transmissible the virus is.
The spike protein is also the basis of current COVID-19 vaccines, which seek to generate an immune response against it. But what exactly is the spike protein and why is it so important?
In the world of parasites, many bacterial or fungal pathogens can survive on their own without a host cell to infect. But viruses can't. Instead, they have to get inside cells in order to replicate, where they use the cell's own biochemical machinery to build new virus particles and spread to other cells or individuals.
Our cells have evolved to ward off such intrusions. One of the major defenses cellular life has against invaders is its outer coating, which is composed of a fatty layer that holds in all the enzymes, proteins and DNA that make up a cell. Due to the biochemical nature of fats, the outer surface is highly negatively charged and repellent. Viruses must traverse this barrier to gain access to the cell.The SARS-CoV-2 coronavirus molecule. Credit: Klerka/Shutterstock
Like cellular life, coronaviruses themselves are surrounded by a fatty membrane known as an envelope. In order to gain entry to the inside of the cell, enveloped viruses use proteins (or glycoproteins as they are frequently covered in slippery sugar molecules) to fuse their own membrane to that of cells' and take over the cell.
The spike protein of coronaviruses is one such viral glycoprotein. Ebola viruses have one, the influenza virus has two, and herpes simplex virus has five.
The architecture of the spike
The spike protein is composed of a linear chain of 1,273 amino acids, neatly folded into a structure, which is studded with up to 23 sugar molecules. Spike proteins like to stick together and three separate spike molecules bind to each other to form a functional "trimeric" unit.
The spike can be subdivided into distinct functional units, known as domains, which fulfill different biochemical functions of the protein, such as binding to the target cell, fusing with the membrane, and allowing the spike to sit on the viral envelope.The SARS-CoV-2 coronavirus molecule. Credit: Klerka/Shutterstock
The spike protein of SARS-CoV-2 is stuck on the roughly spherical viral particle, embedded within the envelope and projecting out into space, ready to cling on to unsuspecting cells. There are estimated to be roughly 26 spike trimers per virus.
One of these functional units binds to a protein on the surface of our cells called ACE2, triggering uptake of the virus particle and eventually membrane fusion. The spike is also involved in other processes like assembly, structural stability and immune evasion.
Vaccine vs spike protein
Given how crucial the spike protein is to the virus, many antiviral vaccines or drugs are targeted to viral glycoproteins.
For SARS-CoV-2, the vaccines produced by Pfizer/BioNTech and Moderna give instructions to our immune system to make our own version of the spike protein, which happens shortly following immunization. Production of the spike inside our cells then starts the process of protective antibody and T cell production.
- The spike protein is made up of different sections that perform different functions. Credit: Rohan Bir Singh, CC BY
- The SARS-CoV-2 virus is changing over time. Credit: NIAID-RML, CC BY
One of the most concerning features of the spike protein of SARS-CoV-2 is how it moves or changes over time during the evolution of the virus. Encoded within the viral genome, the protein can mutate and changes its biochemical properties as the virus evolves.
Most mutations will not be beneficial and either stop the spike protein from working or have no effect on its function. But some may cause changes that give the new version of the virus a selective advantage by making it more transmissible or infectious.
One way this could occur is through a mutation on a part of the spike protein that prevents protective antibodies from binding to it. Another way would be to make the spikes "stickier" for our cells.
This is why new mutations that alter how the spike functions are of particular concern—they may impact how we control the spread of SARS-CoV-2. The new variants found in the UK and elsewhere have mutations across spike and in parts of the protein involved in getting inside your cells.
Experiments will have to be conducted in the lab to ascertain if—and how—these mutations significantly change the spike, and whether our current control measures remain effective.
This article is republished from The Conversation under a Creative Commons license. Read the original article.
Roles of Nucleocapsid in Immunity
Nucleocapsid protein is a most abundant protein of coronavirus. During virion assembly, N protein binds to viral RNA and leads to formation of the helical nucleocapsid. The abundance and high hydrophilicity of N protein are supposed to contribute to potent immunity after SARS infection. About a week after SARS onset, N protein-specific antibody may be detected and sustains for long time. The corresponding epitopes in N protein were summarized as Figure 2. N371-390 and N385-407 have a potent ability to react with the serum of 94-97% patients, suggesting the epitope site at the C-terminus of the N protein is likely to be located at codons 371-407. As other coronavirus, N protein of SARS virus is able to induce specific CTL by use of DNA vaccine. The epitopes for CTL induction remain unknown.
Figure 1. Nucleocapsid protein (N protein) in coronavirus virion structure. The genome RNA is complexed with the N protein to form a helical cased within the viral membrane, HE, hemagglutinin-esterase S, spike E, small membrane envelope M, membrane are all transmembrane proteins.
Figure 2. Antigenic motifs of SARS nucleocapsid protein. Blank bar indicates stronger ability to induce antibody production. The number indicates amino acids of nucleocapsid protein.
The TMPRSS2 protease
The interaction between spike proteins and the ACE2 receptor is clearly more complicated than a simple lock-and-key relationship. Many more molecules may be involved in the process allowing SARS-CoV-2 to invade cells. At the moment, we know of at least one other key player: TMPRSS2 (transmembrane serine protease 2). Adding it to the burglary analogy becomes a bit forced, but you can think of TMPRSS2 as an inside man working for the factory that the burglar wants to turn into a robot-manufacturing plant: TMPRSS2 meets the burglar outside the building to prepare or 'prime' the lock pick (spike) so it will properly fit the factory's locks.
The spike protein of SARS-CoV and SARS-CoV-2 is activated by the protease TMPRSS2 before it binds to . [+] the ACE2 receptor.
Markus Hoffmann / German Primate Centre
TMPRSS2 is a protease enzyme, a type of protein that cuts other proteins, and is another potential target for drugs. Biologists at the German Primate Centre in Göttingen found that SARS-CoV-2 depends on TMPRSS2 protease to invade cells and more importantly from a therapeutic perspective, showed that a protease inhibitor previously approved for clinical use, camostat mesylate, can block the virus from entering cells. In the analogy, the inhibitor is a security guard who intercepts the inside man before they prepare the burglar's lock pick.
The Göttingen team also found that antibodies isolated from SARS patients, which surround and neutralize the SARS-CoV spike protein at high efficiency, can also 'cross-neutralize' SARS-CoV-2's spikes to a moderate extent. This would be like the factory learning from a prior break-in and changing the locks, and suggests that a SARS vaccine that prompts the body to produce antibodies against SARS-CoV could partially protect against SARS-CoV-2 and possibly prevent COVID-19. This result seems to support the University of Washington's study, but could also be explained by antibody neutralization of other parts ( ' epitopes ' ) of antigens from SARS-CoV-2, not the receptor-binding domain of its spike protein.
All the studies I've described above were done in lab-grown cells, not humans or even animal models. Due to the phenomenal speed at which COVID-19 is spreading, however, clinical trials are progressing at a rapid pace in order to determine which approaches and treatments might work, and are safe. Armed with that knowledge, it's only a matter of time before scientists find a way to fight back against SARS-CoV-2.
Coronavirus capsid missing? - Biology
a Biomedical Research Institute (BIOMED), Catholic University of Argentina (UCA) – National Scientific and Technical Research Council (CONICET), C1107AFF Buenos Aires, Argentina
* Correspondence e-mail: [email protected]
Viral infection compromises specific organelles of the cell and readdresses its functional resources to satisfy the needs of the invading body. Around 70% of the coronavirus positive-sense single-stranded RNA encodes proteins involved in replication, and these viruses essentially take over the biosynthetic and transport mechanisms to ensure the efficient replication of their genome and trafficking of their virions. Some coronaviruses encode genes for ion-channel proteins – the envelope protein E (orf4a), orf3a and orf8 – which they successfully employ to take control of the endoplasmic reticulum–Golgi complex intermediate compartment or ERGIC. The E protein, which is one of the four structural proteins of SARS-CoV-2 and other coronaviruses, assembles its transmembrane protomers into homopentameric channels with mild cationic selectivity. Orf3a forms homodimers and homotetramers. Both carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Orf8 is a very short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers. This review addresses the contribution of biophysical and structural biology approaches that unravel different facets of coronavirus ion channels, their effects on the cellular machinery of infected cells and some structure–functional correlations with ion channels of higher organisms.
1. Introduction and background
Only a few weeks after the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, biophysical studies produced atomic scale data on key structures of the causative agent, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This attests to a very positive reaction by the scientific community in tackling a biomedical problem of unprecedented proportions. Structural biology was the first scientific discipline to apply powerful biophysical methods and produce solid data in attempts to understand the pathogenesis of the disease and search for its biomedical remediation (Barrantes, 2021 ).
From a genomic perspective, SARS-CoV-2 belongs to the same category of positive-strand RNA [(+)RNA] viruses as hepatitis C, Chikingunya and Zika viruses. From a taxonomic viewpoint, SARS-CoV-2 belongs to the subfamily Coronavirinae in the Coronaviridae family that comprises four genera: Alphacoronavirus , Betacoronavirus , Gammacoronavirus and Deltacoronavirus (González et al. , 2003 Letko et al. , 2018 ). Coronaviruses (CoVs) pack between 26 and 32 kilobases of single-stranded positive-sense RNA inside their envelope. CoVs were first identified in the late 1970s in electron microscopy (EM) studies of negatively stained specimens and their name was coined due to the fuzzy solar corona appearance surrounding the spherical virion envelope (Almeida & Tyrrell, 1967 ), which we now know stems from the coverage of the envelope with copies of the spike (S) protein, one of the four structural proteins in these viruses. The highly pathogenic avian bronchitis virus was discovered in the early 1930s (Estola, 1970 ), but it was not known until decades later that it belonged to the Gammacoronavirus genus of the Coronaviridae (González et al. , 2003 ). CoVs cause mild to severe respiratory, enteric and neurological diseases in species ranging from avians to mammals (Lai & Cavanagh, 1997 Cui et al. , 2019 Woo et al. , 2014 ).
The first study to identify a CoV infection in humans is attributed to Hartley and coworkers, who found antibodies to Mouse hepatitis virus (MHV) in the serum of affected patients (Hartley et al. , 1964 ). A total of seven human CoVs (HCoVs) have since been identified. HCoV-OC43, HCoV-293, HCoV-NL63 and HKU1-CoV generally cause mild respiratory diseases, mainly forms of the common cold, along with other viruses with tropism for the nasal and upper respiratory tract mucosae. A second category of HCoVs comprises the highly pathogenic SARS-CoV and MERS-CoV, the etiological agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), responsible for the epidemics in 2003 and 2012, respectively, and SARS-CoV-2, the causative agent of the current COVID-19 pandemic.
The structural–functional correlations of SARS-CoV-2 ion-channel proteins and their comparison with those of other pathogenic CoVs have still not been fully characterized. This is an area that needs investigation, particularly because these proteins are purported to fulfil a role in infected cells, with possible implications for interventions that interfere with viral replication. This short review discusses the topic of ion-channel-forming protein structures in CoVs in general and in SARS-CoV-2 in particular, in an attempt to put the subject in perspective both from molecular biology and phylogenetic standpoints, and to draw attention to their potential as targets for prophylactic and/or therapeutic interventions.
Comprehensive reviews of the biological and evolutionary (Li et al. , 2020 ), epidemiological (Su et al. , 2016 ), clinical (Richardson et al. , 2020 Guan et al. , 2020 ), microbiological (Fung & Liu, 2019 ) and physicochemical (Scheller et al. , 2020 ) aspects and structure (Harrison, 2015 Tortorici & Veesler, 2019 ) of CoVs have appeared. The reader is also referred to the reviews on the recent contribution of biophysics and structural biology to current advances in COVID-19 (Barrantes, 2021 ) and the new possibilities for repurposed drugs (Barrantes, 2020 Cavasotto & Di Filippo, 2021 Cavasotto et al. , 2021 ) in handling the current pandemic.
2. Overall structure of CoVs
A common characteristic of members of the Coronaviridae is the spiky appearance of the capsid (Neuman et al. , 2006 Neuman & Buchmeier, 2016 Wrapp et al. , 2020 ). Contemporary cryo-EM images of the SARS-CoV-2 virion isolated from the supernatant of infected cells show roughly spherical bodies with a diameter of 91 ± 11 nm (Ke et al. , 2020 ), i.e. very similar to other CoVs. Around 70% of the SARS-CoV-2 genome encodes proteins involved in RNA replication, with the rest of the genome coding for structural and nonstructural proteins. Besides the aforementioned S protein, CoVs have three additional structural proteins, the nucleocapsid (N), envelope (E) and membrane (M) proteins, 16 nonstructural proteins and eight open reading frame (ORF) accessory proteins (Díaz, 2020 ).
N, the nucleocapsid protein, resides together with the RNA genome in the ribonucleoprotein (RNP) core inside the envelope. The N protein chaperones and protects the genomic RNA. The M protein is an integral membrane glycoprotein that contributes to adapting a region of the endoplasmic reticulum–Golgi complex intermediate compartment (ERGIC) membrane for virus assembly, thus defining the shape of the viral envelope. When expressed alone, M accumulates in the Golgi complex, but when expressed together with the E protein, virus-like particles that are akin to authentic virions in size and shape are assembled. This observation has led to the idea that the M and E proteins constitute the minimal building blocks for envelope formation (de Haan et al. , 1999 ).
3. Viral ion-channel-forming proteins (viroporins)
In recent years, X-ray crystallography, transmission EM and cryo-EM and nuclear magnetic resonance (NMR) techniques have been applied to study the structures of some viral ion-channel-forming proteins, also termed `viroporins' (Liao et al. , 2006 Fischer & Hsu, 2011 ). The name, partly borrowed from the bacterial β -barrel porins, alludes to their ability to act as ion-conducting pores in membrane bilayers, but in fact they are more elaborate than this, exhibiting, for example, ion selectivity. In silico studies using sequence-based molecular modelling and homology modelling have provided complementary insights into these structures, finding common architectures as well as diversity (see, for example, the review by OuYang & Chou, 2014 ). Viroporins exhibit a low degree of homology with ion channels of prokaryotic or eukaryotic origin if one considers their overall structure, although their transmembrane (TM) regions do bear some resemblance to the corresponding regions of ion channels of higher organisms (Fischer & Hsu, 2011 ), as analysed in the section on the evolution of these proteins.
One of the first descriptions of ion-channel proteins in viruses dates back to the early 1990s, when the matrix M2 protein of influenza virus was shown to confer ion permeability on monovalent cations upon heterologous transfection of Xenopus oocytes (Pinto et al. , 1992 ). Subsequently, it was demonstrated that this applies to influenza A and B viruses, which also display permeability for protons, whereas the M2 proteins of influenza C and D viruses exhibit selectivity for chloride ions, with some permeability for protons (see the review by To & Torres, 2019 ).
Between the genes coding for the S protein and those for other viral envelope genes, the CoV RNA genome contains a locus that is conserved throughout the entire family. In the SARS-CoV genome, this region includes a complete or truncated ORF (Zhang et al. , 2014 ) containing the gene encoding the E protein (also termed orf4a), orf3a and orf8a, three proteins that form ion channels (Castaño-Rodriguez et al. , 2018 ). The CoVs MERS-CoV, HCoV-229E, HCoV-OC43 and Porcine epidemic diarrhoea virus (PEDV) encode two such ion channel-forming proteins (To et al. , 2016 Castaño-Rodriguez et al. , 2018 ).
4. The small E protein (orf4a): pentameric structure and membrane topology
At only 76 amino acids long, E is the smallest of the four structural proteins of CoVs (Pervushin et al. , 2009 ). It is an integral membrane protein that is present in substoichiometric quantities relative to other proteins embedded in the envelope bilayer membrane its precise functions are still not fully known, except that its TM domain possesses ion-channel properties and is probably involved in virion assembly (Siu et al. , 2008 ) and virion release from infected cells (reviewed in Schoeman & Fielding, 2019 ). This has been documented for MHV its expression in Escherichia coli leads to increased permeability, growth arrest and ultimately cell lysis (Madan et al. , 2005 ). The genomes of SARS-CoV (Liao et al. , 2013 ), MERS-CoV (Surya et al. , 2015 ) and Infectious bronchitis virus (IBV), a highly pathogenic avian infectious bronchitis virus from the Gammacoronavirus genus (To et al. , 2017 ), also code for E proteins. E possesses a short hydrophilic amino-terminal domain that is exposed to the cytoplasmic compartment of the host cell (Maeda et al. , 2001 Raamsman et al. , 2000 ) and a relatively long (25-amino-acid) TM domain. The TM domain of the E protein is highly conserved among CoVs, with % sequence identity and 98% sequence similarity (Cao et al. , 2020 ).
The exact topology of E relative to the membrane is still an ongoing debate. Two amino acids are the main contributors to the hydrophobicity of the TM domain: valine and leucine (Wu et al. , 2003 ). The TM domain is followed by a long hydrophilic carboxy-terminus (Ye & Hogue, 2007 ) containing three cysteine residues that have been suggested to play a role in the association of E with the spike glycoprotein S (Wu et al. , 2003 ). Expression of the SARS-CoV E protein in Vero E6 cells showed that it is N-glycosylated and that the two membrane-spanning domains comprise amino-acid sequences 11 and 37, i.e. with a short loop between the two (Chen et al. , 2009 ). When analysing the TM region of E, one should consider the two possible loci inhabited by this protein: (i) its native viral envelope bilayer lipid membrane and (ii) the host-cell intracellular membranes. In the former case, the E protein has been proposed to traverse the viral lipid bilayer as a single-passage helix (Raamsman et al. , 2000 ) or a double-passage helix (Raamsman et al. , 2000 Maeda et al. , 2001 Chen et al. , 2009 ). During the life cycle in the host cell, E is mainly localized at the sites of viral replication, i.e. ER, Golgi and ERGIC membranes. Recombinant CoVs lacking the E protein show significantly lower viral titres/propagation-incompetent progeny, suggesting the importance of E in virion production and maturation (Schoeman & Fielding, 2019 ). E from IBV has been reported to cross the Golgi membranes just once, with the N-terminus facing the Golgi lumen and the C-terminus facing the cytoplasm (Corse & Machamer, 2000 ), whereas E from MHV is purported to traverse the lipid bilayer twice, with both the N- and C-termini exposed to the cytoplasmic compartment, which is topologically equivalent to the interior of the virion (Maeda et al. , 2001 ). A further proposal suggests that the TM region of SARS-CoV E contains a 12-amino-acid hairpin, which the authors propose is capable of deforming lipid bilayers by increasing their curvature, a process that would occur during virion budding from infected cells (Arbely et al. , 2004 ). The NMR data of Torres and coworkers (Li et al. , 2014 ) appeared to confirm the suggestion of a hairpin-like structure formed by two helices joined by a less ordered segment in the SARS-CoV E monomer. Subsequent work from the same group indicated that E monomers possess only one membrane-embedded α -helical segment.
In terms of their oligomeric organization, a molecular-modelling exercise led Torres and coworkers to suggest that the TM segments of SARS-CoV E protein monomers adopt a pentameric structure (Torres et al. , 2005 ). NMR later led to experimentally supported models of the SARS-CoV E protein structure (Pervushin et al. , 2009 Surya et al. , 2018 Li et al. , 2014 ).
Fig. 1 ( a ) shows a model of the SARS-CoV E protein monomer obtained by solid-state NMR spectroscopy in detergent–lipid micelles. The recombinant E protein was expressed in bacteria and the lowest-energy structure was calculated (Li et al. , 2014 ). A long straight α -helix, the purported channel-forming domain, is joined through a flexible linker domain (residues 46) to a short peripheral C-terminal helix (residues 55) that bends obliquely with respect to the longer helix and to the main axis of the ion-channel pore (see Fig. 2 ) at the level of residue Tyr42. Subsequent NMR studies in lauryl–myristoyl–phosphatidyl glycerol (LMPG) micelles showed the monomer (residues 8) to consist of three segments, with the two α -helices penetrating the bilayer and an overall shape resembling a fishing hook (Fig. 1 b ).
| ||Figure 1 |
NMR work by the Torres group led to early models of the monomeric form of the SARS-CoV E protein in detergent–lipid micelles (Li et al. , 2014 ), showing two helical segments joined by a more disordered flexible region flanked by amino-acid residues Tyr42 and Thr55 ( a ). The N-terminal portion of the TM region [indicated by residue Val14 in ( a )] is purported to protrude into the lumenal side of the Golgi membranes, and the C-terminal portion (here indicated by Asn64) to be partly exposed to the cytoplasm. ( b ) Subsequent work showed the calculated E monomers to consist of three α -helical segments in LMPG micelles. The model shown corresponds to an ensemble of ten calculated monomeric structures, with the backbone rendered as a line representation. Reproduced from Surya et al. (2018 ) with permission from Elsevier Masson SAS under the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0).
| ||Figure 2 |
Comparative views of SARS-CoV and SARS-CoV-2 E protein models derived from NMR data. The top panel shows the lowest-energy NMR-derived structural model of the SARS-CoV E protein homopentamer (PDB entry 5x29 Surya et al. , 2018 ) in a lateral view ( a ) and an end-on view ( b ). The bottom panel shows an NMR-derived model of the SARS-CoV-2 E protein (PDB entry 7k3g Mandala et al. , 2020 ) in lateral ( c ) and end-on ( d ) views. Notice that the ion-channel-lining helices of the SARS-CoV E protein are more perpendicular to the membrane plane compared with those of the SARS-CoV E protein. Images were produced using CCP 4 mg (McNicholas et al. , 2011 ).
The homo-oligomer of the E protein is apparently self-generated through specific amino-acid linear motifs of five copies of the monomer, as suggested for the human coronavirus HCoV-OC43 (Stodola et al. , 2018 ). Fig. 2 draws a comparison between the E proteins of the two related viruses SARS-CoV and SARS-CoV-2. The model of the SARS-CoV protein is derived from the NMR study in LMPG micelles (Surya et al. , 2018 ) and that of the SARS-CoV-2 E structure is derived from NMR data at 2.4 Å resolution of the protein reconstituted in ERGIC-mimetic liposomes containing phosphocholine, phosphoethanolamine, phosphatidylinositol, phosphoserine and cholesterol (Mandala et al. , 2020 ). For comparative purposes, liposomes made up of dimyristoylphosphocholine (DMPC):dimyristoylphosphoglycerol (DMPG) membranes were employed. The NMR structures depart from an ideal α -helical geometry, apparently due to deformations produced by three phenylalanine residues stacked between the helical chains lining the narrow pore. The blocker hexamethylene amiloride binds to polar amino-acid residues in the amino-terminal lumen of the pore.
The ring of TM segments forming the SARS-CoV-2 protein E ion channel proper is also a pentameric bundle, but the helices run perpendicular to the membrane plane, whereas in the SARS-CoV E protein the homologous chains of the five monomers are assembled into a pentameric body with an inner ring of TM α -helical segments (residues 15) tilted by ° relative to the axis of the central ion-permeation pathway (Fig. 2 , top panel). The most striking difference between the SARS-CoV and SARS-CoV-2 ion channels is the simpler architecture of the latter, with a bundle of straight α -helical chains forming a single ring, contrasting with the two helical rings in SARS-CoV. Since the two proteins share % sequence homology, the possibility arises that the different structures of their membrane-associated regions are partly due to the different media used in the NMR studies. A pentameric structure has also been proposed for the E protein of MERS-CoV (Surya et al. , 2018 ).
In functional terms, early studies using planar lipid bilayers showed that synthetic peptides corresponding to the SARS-CoV E protein form ion channels in planar lipid bilayers with selectivity for permeating monovalent cations over monovalent anions (Wilson et al. , 2004 ). The E protein of SARS-CoV was also found to modify the permeability of E. coli membranes when expressed under reducing conditions, under which E adopts a monomeric state, whereas nonreducing conditions rendered the E protein in dimeric and homotrimeric forms (Liao et al. , 2004 ). Reduction exposed two cysteine residues essential for S—S bond-mediated oligomerization the changes in permeability were therefore attributed to the exposure of the cysteine residues, although no distinction could be established between the direct and indirect nature of the permeability changes using planar lipid bilayers (Verdiá-Báguena et al. , 2012 ). The E channel displays monovalent cation selectivity (Westerbeck & Machamer, 2019 ), although it has also been reported to permeate Ca 2+ (Nieto-Torres et al. , 2015 ). Ion conductance can be blocked by hexamethylene amiloride, but not by amiloride, a drug that inhibits the viral replication of some synthetic E proteins from CoV (Pervushin et al. , 2009 ). Based on the conductance properties of E in planar bilayers (Wilson et al. , 2004 ) or upon transient expression in Xenopus oocytes and yeast systems, E was suggested to form nonselective channels for monovalent cations, although in the latter case Li + partially reduced the inward currents (Zhang et al. , 2014 ). As observed with orf3a, viral production diminished when protein E expression was abrogated by applying siRNA to infected cells (Zhang et al. , 2014 ). The permeability properties of the new SARS-CoV-2 E protein reconstituted in lipid bilayers with a composition reported to mimic that of the ERGIC membrane has recently appeared, showing that the ion channel displays a mild cationic permeability. This can be blocked by the binding of hexamethylene amiloride and amantadine to polar residues shallowly located at the N-terminal lumen (Mandala et al. , 2020 ), thus confirming earlier work on the inhibitory action of this drug on the viral channel (Pervushin et al. , 2009 ).
In vitro experiments have shown that the lipid composition of the host planar bilayer modulates the ion conductance of the SARS-CoV E protein channel (Verdiá-Báguena et al. , 2012 ). Lipid charge was also found to play a role: the E protein showed no cation selectivity in uncharged lipid membranes, whereas negatively charged lipids resulted in mild cationic selectivity (Verdiá-Báguena et al. , 2012 ). The charge of the ionizable groups of the E protein, as well as those from host lipids such as diphytanoyl phosphatidylserine, was found to modify channel conductance (Verdiá-Báguena et al. , 2013 ).
The mutagenesis of amino-acid residues in the hydrophobic TM domain of the E protein alters virus replication, which is restored upon re-establishing the α -helical structure (Ye & Hogue, 2007 ). Helix-restored E protein is more sensitive to treatment with hexamethylene amiloride, a drug that inhibits the HIV ion channel Vpu and the E protein channel from human HCoV-229 and rodent MHV, but not avian IBV (Wilson et al. , 2006 ). N15A and V25F mutations in the TM region of the SARS-CoV E protein have been reported to abrogate ion conductance (Verdiá-Báguena et al. , 2012 ).
SARS-CoV orf3a is a much larger (274 amino acids) viral ion-channel protein it possesses three transmembrane domains. Initially described as a structural protein unique to SARS-CoV (Shen et al. , 2005 ), it was subsequently reported that the orf3a protein from the same virus, named U274 by these authors, was a nonstructural protein that interacted with the M, E and S structural proteins and orf7/U122 (Tan et al. , 2004 ). Recombinant SARS-CoV orf3a protein can form a homotetrameric complex in orf3a-transfected cells (Lu et al. , 2006 Marquez-Miranda et al. , 2020 ). A tetramer consisting of four TM segments each has also been proposed using computational structure-prediction approaches (Wang et al. , 2012 ). When expressed in Xenopus oocytes, SARS-CoV orf3a is a K + -sensitive channel that can efficiently be inhibited by Ba 2+ . Similarly, ion-channel activity is generated upon transfection of PEDV into Xenopus oocytes or yeast cells (Wang et al. , 2012 ). If cells are transfected with siRNA, thus suppressing orf3a expression, infection with SARS-CoV replication is not affected, but virion release is (Lu et al. , 2006 ). Synthetic peptides corresponding to each of the constituent TM segments of orf3a were reconstituted into artificial lipid bilayers. When the three peptides were assembled in a 1:1:1 mixture, ion-channel activity was observed, but either TM2 or TM3 was required to induce currents TM1 failed to do so. Full-length orf3a expression showed weak cationic selectivity and rectification (Chien et al. , 2013 ).
Recently, SARS-CoV-2 orf3a was heterologously expressed in Spodoptera frugiperda , reconstituted in liposomes, and single-channel currents were recorded from excised patches (Kern et al. , 2020 ). Electrophysiologically, orf3a behaved as a cation channel with a large single-channel conductance (375 pA) with modest selectivity for Ca 2+ and K + over Na + . The channel was not blocked by Ba 2+ as was the case for the SARS-CoV channel (Lu et al. , 2006 ), nor was it inhibited by the small drug emodin.
Kern and coworkers also employed cryo-EM to image the apo form of the dimeric and tetrameric structures of orf3a reconstituted in lipid nanodiscs. SARS-CoV-2 appears as a homotetramer in which each monomer of the dimers contributes three TM segments arranged in clockwise fashion, making a total of six membrane-spanning domains per dimer (Figs. 3 a and 3 b ). Dimers are joined by a covalent S—S bond distended between homologous Cys133 residues in each dimer in the in silico model produced by Marquez-Miranda et al. (2020 ) (Fig. 3 b ) or residues Trp131, Arg134, Lys136, His150, Thr151, Asn152, Cys153 and Asp155 in the recent molecular-modelling studies of Cavasotto and coworkers (Cavasotto & Di Filippo, 2021 Cavasotto et al. , 2021 ), bringing the total number of TM helices to 12 and thus making this the largest and most elaborate viral channel protein known to date. The .9 Å resolution cryo-EM structure of SARS-CoV-2 orf3a (Kern et al. , 2020 ) is very similar to that of the orf3a channel structure of SARS-CoV (Lu et al. , 2006 ), corroborating the structural homology between several of the molecular constituents of these two human pathogenic viruses from the seven known to date. The all-helical TM region of the protein, with a length of Å, adopts a peculiar novel topography in the lipid bilayer, whereas the cytosolic domain of the dimer ( Å) is formed by two β -sandwiches (Fig. 3 c ). The novel ion-channel structure possesses two potential ion pores, one in each dimer the walls of the ion channel proper are lined by TM1 and TM2 of each monomer (Fig. 3 c ). The extracellular-facing end of the ion channel exhibits a narrow bifurcated pore that is reminiscent of the structure of vestibules in ion channels of higher organisms. This outer portion of the channel leads to a large polar cavity that is open to the cytosol. The orf3a structure was captured by cryo-EM in a conformation tentatively corresponding to a tetramer, and ascribed by the authors to the closed or inactivated state (Kern et al. , 2020 ). A tubular electron-dense region potentially corresponding to lipid acyl chains was partially resolved in between the TM helices.
| ||Figure 3 |
( a ) Cryo-EM map at 2.9 Å resolution of the full-length SARS-CoV-2 orf3a tetramer expressed in S. frugiperda and reconstituted in lipid nanodiscs (Kern et al. , 2020 EMDB entry EMD-22136). The backbone chain of one of the protomers docked into the cryo-EM map is outlined in burgundy. The horizontal blue lines mark the limit between the upper TM domain and the lower cytoplasmic domain of the tetramer formed by multiple β -sheets. ( b ) Ribbon model of the SARS-CoV-2 orf3a tetramer derived from cryo-EM studies of the protein reconstituted in lipid nanodiscs. Each dimer possesses six TM helices and a cytoplasmic domain with predominantly β secondary structure. The two dimers in the tetramer are purported to be covalently joined by a disulfide bond formed by homologous Cys133 residues in each monomer (Marquez-Miranda et al. , 2020 ). The molecular-modelling study by these authors revealed the presence of a chloride ion site in the channel lumen. ( c ) Ribbon models of the SARS-CoV-2 orf3a dimer: an Å cylinder in a lateral view (left) and an end-on perspective as viewed from the extracellular side (right) derived from cryo-EM studies of the protein reconstituted in lipid nanodiscs (Kern et al. , 2020 ). Each protomer of the dimer has three helices that can fully traverse a lipid bilayer ( Å) and a 30 Å-long cytoplasmic domain with predominantly β secondary structure. The projection of the dimer onto the membrane plane is elliptic, with a major axis of Å in width. The central ion path is flanked by TM segments 1 and 2, as seen in the centre of the end-on view. Images were produced using CCP 4 mg .
Interestingly, the molecular model from the cryo-EM data of the orf 3a protein (PDB entry 6xdc Marquez-Miranda et al. , 2020 ) disclosed the presence of chloride-binding sites inside the ion-permeation pathway of SARS-CoV-2 orf3a.
Information on Orf8a is still very scanty. It is a cysteine-rich 29-amino-acid single-passage TM peptide present at least in SARS-CoV. Orf8a forms cation-selective ion channels with a conductance close to 9 pS when assembled in lipid bilayers in several putative oligomeric forms from tetramers to hexamers (Chen et al. , 2011 ). In silico calculations suggested that the first 22 amino acids of the single-TM domain of orf8a form a homopentameric bundle (Hsu et al. , 2015 ). The pentameric bundle was also purported to exhibit weak cation selectivity attributable to hydrophilic and hydrophobic stretches of amino acids in the channel lumen.
7. The contribution of viral ion-channel proteins to pathogenesis
SARS-CoV orf3a has been implicated in viral release, inflammasome activation and cell death, and its deletion reduces viral titres and morbidity in model systems (Freundt et al. , 2010 ). E downregulates the type-1 interferon (IFN) receptor by inducing serine phosphorylation of the IFN α -receptor subunit 1 degradation motif and increasing receptor ubiquitination (Minakshi et al. , 2009 ).
The fact that a large proportion (>70%) of the CoV genome, including that of SARS-CoV-2, is devoted to RNA replication dictates the preferential intracellular tropism of the virus to the biosynthetic machinery of the cell. Viral proteins are initially synthesized in the endoplasmic reticulum (ER), but most of the post-translational modifications occur at the overlapping interface of the ER and the Golgi complex: the ERGIC zone. Indeed, labelling the E protein with Rab-1, a cytochemical marker of the intermediate compartment and the ER, showed the accumulation of MHV CoV-A59 E protein in the ERGIC region of the cell electron microscopy provided further evidence that E induces the formation of tubular structures and induces curvature of the pre-Golgi membranes, subsequently altering the Golgi-complex membranes (Raamsman et al. , 2000 Nieto-Torres et al. , 2015 ). E is purported to confer stability to the viral membrane and to contribute to the later stages of the virion cycle in the infected cell: the assembly and budding processes (Neuman et al. , 2011 ). E assembles by budding into the lumen of the early Golgi compartment (Westerbeck & Machamer, 2019 ). Alanine-scanning mutagenesis studies of the extramembrane domain of E have shown that certain mutations impair viral assembly and maturation, i.e. morphogenesis of MHV virions (Fischer et al. , 1998 Siu et al. , 2008 ). Although according to some authors (Venkatagopalan et al. , 2015 ) E does not traffic to the cell surface, avian IBV E protein interacts physically with the M protein and is able to retain M in the compartment that it resides in (Lim et al. , 2001 ). In SARS-CoV this interaction is postulated to take place through the hydrophobic TM domains (Chen et al. , 2009 ). Propagation and shedding of SARS-CoV virus-like particles requires the co-expression of E and N proteins together with the M protein (Siu et al. , 2008 ).
In addition to these roles in the assembly, trafficking and shedding of virions, the E protein is also involved in the stimulation of the immune response in the infected organism. The evolutionary conservation of the E protein among CoVs (Cao et al. , 2020 ) makes it an interesting candidate for vaccine development, and therefore knowledge of its surface epitopes is of biological and biotechnological importance. A step in this direction is the tentative mapping of the surface epitopes of the SARS-CoV-2 E protein based on the structure from SARS-CoV (Tilocca et al. , 2020 Fig. 4 ). Polyclonal antibodies that recognize the N-terminal 19 amino-acid residues of the SARS-CoV E protein inhibit its ion-current ability (Wilson et al. , 2004 ).
| ||Figure 4 |
Putative surface epitopes of the SARS-CoV and SARS-CoV-2 E proteins. ( a ) Model of the homopentameric SARS-CoV E protein (PDB entry 5x29). Selected epitope sequences are mapped in each monomer as displayed in ( b ). Epitope sequences are coloured as follows: blue, LIVNSVLLFLAFVVFLLVTLAILTALRLCAY cyan, LLVTLAILTALRLCA green, LTALRLCAY olive green, CNIVNVSLVKPSFYV red, SLVKPSFYV orange, LVKPSFYVYSRVKNL yellow, LVKPSFYVY magenta, KPSFYVYSRVKNLNS. Reproduced from Figs.ق( a ) and 2( b ) of Tilocca et al. (2020 ) with permission from Elsevier Masson SAS under the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0).
Ion-channel activity resulting from the expression of CoV viroporins induces stress responses and activates pro-inflammatory pathways, and can lead to cell death (Minakshi et al. , 2009 ). In vitro , expression of the SARS-CoV orf3a protein in the pulmonary epithelial cell line A549 upregulates the expression of intracellular and secreted levels of the three subunits of fibrinogen (Tan et al. , 2005 ). Infection of Vero E6 cells with SARS-CoV leads to apoptosis, which in turn triggers a virus-initiated cytopathic effect (Yan et al. , 2004 ). Other pathogenic changes include rearrangement of the membrane (accumulation of intracellular vesicles), Golgi fragmentation and cell death induced by SARS-CoV orf3a, which is reduced upon deletion of orf3a (Freundt et al. , 2010 ). The virus also downregulates the inositol-requiring enzyme 1 (IRE-1) signalling pathway in the unfolded protein response (DeDiego et al. , 2011 ). Most recently, the orf3a protein of SARS-CoV-2 has also been shown to induce apoptosis in various cell lines in vitro . The apoptotic process involves the activity of caspase-8, i.e. following the so-called extrinsic pathway, which induces the release of mitochondrial cytochrome c and caspase-9 activation the levels of apoptosis were, however, lower than those induced in Vero E6 cells by SARS-CoV (Ren et al. , 2020 ).
Another expression of the pathogenic effects induced by the SARS-CoV E protein is the increase in permeability of the ERGIC/Golgi membrane, leading to the cytosolic release of Ca 2+ with concomitant activation of the NLRP3 inflammasome and induction of interleukin 1 β (IL-1 β ) production (Nieto-Torres et al. , 2015 ). When (+)RNA viruses such as the CoVs utilize the host-cell ERGIC membranes to reproduce their genomes, they modify this membrane complex to produce a structurally different organelle: the replication complex or replication organelle (Snijder et al. , 2020 ). This modified intermediate membrane compartment is characterized by the appearance of double-membrane vesicles (DMVs) nm in diameter where the double-stranded RNA is copied into new (+)RNA genomes (Fig.م ). How is the nascent genomic (+)RNA released from inside the DMVs into the cytosol? A recent cryo-EM tomography study of cells infected with SARS-CoV-2 and other CoVs such as MHV identified a pore traversing the two adjacent lipid bilayers of the DMVs. The pore is formed by six copies of the nonstructural protein nsp3 essential for viral replication (Wolff et al. , 2020 ).
| ||Figure 5 |
CoV-induced DMVs revealed by cryo-EM tomography. ( a ) Tomographic slice (7 nm thick) of a cryo-lamella milled through an MHV-infected cell at a middle stage of infection. ( b ) 3D model of the tomogram, with the segmented content annotated. ERGIC, ER-to-Golgi intermediate compartment. Reproduced from Fig. 1 of Wolff et al. (2020 ) with permission from the publisher under the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0).
Analogous structures called necked spherules can be observed in cells infected with other (+)RNA viruses such as Zika virus (a flavivirus), Chikungunya virus (an alphavirus) and nodaviruses. In the latter case, RNA-replication organelles were imaged by cryo-EM tomography in the outer mitochondrial membrane opening towards the cell cytoplasm. The spherule neck appears as a ring containing 12 copies of the nodavirus RNA-replication protein A (Unchwaniwala et al. , 2020 ).
CoV pathogenesis is intimately related to the ability of the infective viruses to hijack the various molecular effectors required to bind to and enter the host cell, replicate their RNA, assemble and be released from the host cell. Viral assembly and intracellular migration is tightly coupled to the ERGIC cellular machinery and its vesicle-mediated transport. Expression of the E protein from avian IBV has recently been discovered to induce neutralization of the Golgi pH, altering the secretory pathway through interaction with host-cell factors, thereby protecting the IBV spike protein S from premature cleavage and increasing the efficacy of infective virion release from the cell (Westerbeck & Machamer, 2019 ).
Successful expression of the E protein thus appears to be an essential requisite for pathogenesis in fact, attenuated SARS-CoV virions lacking E proteins have been suggested as vaccine candidates (Netland et al. , 2010 ). Viruses lacking both E and orf3a are not viable, and full-length E and orf3a proteins are required for maximal SARS-CoV replication and virulence. In contrast, the viroporin orf8a has only a minor impact on these activities (Castaño-Rodriguez et al. , 2018 ).
E-protein-mediated channel activity has been correlated with enhanced pulmonary damage following accumulation of liquid and electrolytes in pulmonary oedema observed in SARS, driven by the inflammasome NLRP3 and IL-1 β overexpression (Nieto-Torres et al. , 2015 ). Porcine reproductive and respiratory syndrome virus infection also involves inflammasomes and IL-1 β -induced inflammation and injury (Zhang et al. , 2013 ). This pathology could be related to disruption of the epithelial apicobasal integrity in alveolar cells. In fact, SARS-CoV E protein has been shown to interact with PALS1, a tight-junction-associated protein in mammalian cells (Teoh et al. , 2010 ). The interaction is mediated by a PDZ-binding motif at the carboxy-terminus of E that binds to a PDZ domain in PALS1. The authors speculate that SARS-CoV E hijacks PALS1 through this mechanism, abrogating epithelial cell differentiation, a phenomenon that could also occur in the alveolar cells in the pulmonary affectation of SARS (Teoh et al. , 2010 ) or SARS-CoV-2. In the case of MHV, expression of E results in cellular apoptosis (Maeda et al. , 2001 ). The PDZ-binding motif in E, which is also present in the orf3a channel-forming protein, lends it the capacity to bind to more than 400 target proteins in the infected host cells (Castaño-Rodriguez et al. , 2018 ), thus giving these viroporins sufficient versatility to perturb multiple aspects of normal cell function.
The E protein is probably involved in the neurotropism of CoVs. The human CoV HCoV-OC43 requires full expression of its E protein for efficient replication and propagation in neuronal cells in culture and for neurovirulence in the central nervous system (Stodola et al. , 2018 ).
8. Therapeutic potential of channel blockers acting on CoV ion channels
The COVID-19 pandemic has given new impetus to research on the E proteins of Betacoronavirus , the genus to which SARS-CoV-2 belongs. A genomic analysis of the entire database of betacoronaviruses showed that the gene coding for the E protein is segregated into three different clusters, one of which includes only SARS-CoV, SARS-CoV-2 and two bat CoVs. SARS-CoV-2 E and the two bat CoVs are 100% identical, whereas E from SARS-CoV and SARS-CoV-2 shows 95% homology (Alam et al. , 2020 ). The five C-termini of the homopentamer protrude into the cytoplasmic compartment, where three point mutations (The55Ser, Val56Phe and Glu69Arg) and a deletion (Gly70) mark the difference between the latter two viruses. The extremes of the C-terminal regions also harbour the loci of the key tetrapeptide segment (DLLV) involved in PDZ-domain recognition (Alam et al. , 2020 ).
The ion-channel function of the SARS-CoV-2 E protein has recently been explored experimentally in bacteria using three indirect assays of channel activity: growth deficiency upon overexpression, growth recovery in a K + uptake-deficient E. coli strain and cytoplasmic acidification in acidic growth media (Singh Tomar & Arkin, 2020 ). Exploring a library of repurposed drugs, these authors find that glicazide, which is of therapeutic application in type-2 diabetes mellitus, apparently blocks channel activity, as does the drug memantine. Memantine is a low-affinity, voltage-dependent, noncompetitive antagonist of the glutamatergic NMDA receptor, the 5-HT3 receptor and the α 7 nicotinic acetylcholine receptor, which are three members of the pentameric ligand-gated ion channels (pLGICs), and is also an agonist of the dopamine D2 receptor. Memantine is used as a drug in Alzheimer's disease, with its therapeutic ability apparently residing in its channel-blocking activity. Using a prokaryotic model system, the proton- and GABA-gated pentameric ion-channel protein GLIC from the bacterium Gloeobacter violaceus , Ulens and coworkers showed that memantine blocks channel activity by obstructing the channel vestibule facing the extracellular milieu (Ulens et al. , 2014 ).
The drug 5-( N , N -hexamethylene)amiloride has been shown to block the SARS-CoV E protein channel in a manner similar to the mechanism operating on the HIV-1 Vpu channel (Wilson et al. , 2006 ). Other viroporins, albeit from non-CoV viruses, have also been found to be potential targets of blocking compounds that interfere with the assembly and release of mature virions (Behmard et al. , 2018 ).
Second to the large superfamily of G-protein coupled receptors (GPCRs), ion channels are among the most sought-after membrane-protein targets by the United States Federal Drug Administration. Ion-channel modulators in particular have shown therapeutic potential and successes, for example as blockers of influenza M2 channels (Moorthy et al. , 2014 Sakai et al. , 2018 Niu et al. , 2019 ). It is expected that the current pandemic will prompt further research into this important area, with obvious therapeutic opportunities.
9. Viral ion-channel proteins, lipid domains and evolution
As analysed in the preceding section, several drugs with pharmacological activity as ion-channel blockers also inhibit ion fluxes mediated by viral ion-channel proteins. Here, I speculate on other possible common features between the two types of channels. It is estimated that about 4000 million years ago planet Earth witnessed the appearance of protein molecules with the capacity to selectively permeate ions through the plasma membranes of prokaryotic organisms such as the cyanobacterium G. violaceus or the bacillus Erwinia chrysanthemi . Comparison of the crystal structures of these proteins in prokaryotes and their homologs in eukaryotes has led to the notion that they belong to the same superfamily of pLGICs (Tasneem et al. , 2004 ) that share a high degree of structural homology and phylogenetic conservation (Barrantes, 2015 ). Furthermore, scrutiny of the ion-channel protein phylogeny disclosed interesting points of contact with the evolution of the machineries involved in lipid and, in particular, sterol biosynthesis. This observation led to the proposal of the possible co-evolution of the hopanoids (sterol surrogates) with ion channels in prokaryotes and the appearance of sterols with ion-channel proteins of eukaryotes (Barrantes & Fantini, 2016 ).
The timing of the appearance of viruses is still a controversial issue. Some evolutionists contend that viruses originated from `ancient' cells that existed before the last universal cellular (common) ancestor (LUCA) gave rise to modern cells, i.e. to the three superkingdoms of Archaea, Bacteria and Eukarya (Forterre, 2005 ), from ancient RNA cells that predated the LUCA (Nasir et al. , 2020 ). In contrast, other theories support the idea that viruses evolved by recombinational reassortment of genes in a co-evolutionary process with cells rather than being ancestral to them (Hendrix et al. , 2000 Adachi et al. , 2020 Cui et al. , 2019 ). A third line of thought conveys the idea that viruses stem from cells via a process of reductive evolution, as hypothesized for giant DNA viruses (Koonin & Yutin, 2018 ). In the case of invertebrate viruses, co-evolution is supported by the large-effect polymorphisms for host resistance and viral evasion, which may have been favoured by virus-mediated selection (Obbard & Dudas, 2014 ).
Against this backdrop, an intriguing question is whether the cross-talk between channel-protein motifs and microenvironmental lipids observed in higher cells also occurs in viral ion-channel proteins. Indeed, the envelope lipid bilayer of influenza virus type A harbours cholesterol-rich, ordered lipid domains (`lipid rafts' To & Torres, 2019 ), the characteristic lateral heterogeneities that are employed by eukaryotic cells as signalling platforms. Moreover, the cytoplasmic tail of influenza virus M2 binds human annexin A6, a Ca 2+ /lipid-scaffold protein that interacts with ordered lipid domains and regulates the homeostatic equilibrium of cholesterol, while it negatively modulates viral infection (Ma et al. , 2012 ). Similarly, a short linear motif in the cytoplasmic tail of influenza A M2 establishes interactions with another constitutive ordered lipid domain-resident protein, the cholesterol-binding protein caveolin-1 inhibition of caveolin-1 expression diminishes H1N1 influenza viral titres by hindering virus replication (Sun et al. , 2010 ). These may represent examples of molecular mimicry, where the virus appropriates cellular elements that enable it to interact with other endogenous partners of the host cell that are normally involved in physiological mechanisms, for example, facilitating biogenesis, membrane association or trafficking of the virus, among multiple other processes.
In the case of CoVs, lipid domains have been reported to serve as entry sites for SARS-CoV in Vero E6 cells (Lu et al. , 2008 ), possibly due to the enrichment of its receptor, ACE2, in these platforms cholesterol depletion of ACE2-expressing cells by acute treatment with methyl- β -cyclodextrin reduced the binding of the S protein by 50% (Glende et al. , 2008 ). The alpha- and betacoronaviruses infect only mammals (Yu et al. , 2020 ) and may have appeared as variants of bat coronaviruses much more recently in evolutionary terms (Letko et al. , 2018 Cui et al. , 2019 ). All CoVs having orf3a structural homologs evolved from the bat gene pool, whereas all those without orf3a structural homologs derive from rodent, avian or pig gene pools (Kern et al. , 2020 ), suggesting co-evolution of the ion-channel protein orf3a in CoVs whose reservoir is the bat.
Another interesting case is provided by the similar structure and pharmacological sensitivity of the two pentameric viral ion channels orf8a and E and the proton- and GABA-sensitive prokaryotic channel GLIC. The pentameric bundle formed by the first 22 residues of the 39-amino-acid-long SARS-CoV orf8a (Chen et al. , 2011 ) provides a structural framework on which to model the homologous pentameric M2 helical array of the bacterial GLIC protein from the cyanobacterium G. violaceus (Hsu et al. , 2015 ), a representative member of the superfamily of pGLICs (Nys et al. , 2013 ). Both orf8a and GLIC permeate chloride ions, and the chloride flux is voltage-sensitive (Hsu et al. , 2015 ). More remarkably, the GLIC prokaryotic ion channel is blocked by memantine (Ulens et al. , 2014 ), the same drug recently shown to inhibit the ion-channel function of the SARS-CoV-2 E protein (Singh Tomar & Arkin, 2020 ). Thus, the reported lack of homology between viral and prokaryotic/eukaryotic ion channels (McClenaghan et al. , 2020 ) may relate only to their primary sequence their folding in space may provide the required 3D structure to constitute a binding site for small organic molecules acting as inhibitory drugs.
Knowledge of the phylogenetic kindredness between ion-channel proteins of human pathogenic viruses and those of animal reservoirs may contribute not only to our understanding of the biology of the virus ion channels per se, but also to the development of therapeutic strategies to combat them. There are still many open avenues that could contribute to these endeavours and thus help to prevent future pandemics. Understanding the mechanisms of coronavirus ion channels is among these opportunities.
10. Future directions in viral ion-channel research
During recent months, structural biology has made an unparalleled contribution to our understanding of the current pandemic. Biophysical approaches, exploiting previous methodological knowhow and information accrued over the last two decades on other CoVs, have produced new detailed data on the components of SARS-CoV-2 (Barrantes, 2021 ). These help to shed light on the mechanisms involved in viral recognition by host-cell receptors and possible interventions to hinder this and other steps of the infective cycle of the virus. In comparison with the massive amount of data accrued on the S glycoprotein, viral ion-channel proteins are still relatively unexplored from both the purely structural and mechanistic points of view. New structural data are needed to understand how viruses can alter the morphology of cellular components, modify the secretory vesicle transport system to subserve viral RNA replication, protect the spike protein from premature cleavage and efficiently assemble it into new virions through interaction with hijacked host-cell factors. A wide spectrum of techniques, in particular the powerful cryo-EM, cryo-tomography and super-resolution optical microscopies, offer new possibilities to investigate viral ion channels with unprecedented resolution: atomic level in the case of cryo-EM and nanometric level (the mesoscale) in the case of live specimen studies with optical nanoscopy (Barrantes, 2021 ).
In addition, the very nature of viral ion channels makes it inherently possible to apply a bifrontal approach combining the strengths of the structural cryo-imaging biophysical techniques (for example serial cryo-focused ion beam/scanning EM volume imaging) with single-molecule electrophysiology, in the form of single-channel patch-clamp recording of subcellular organelles, to characterize the structure–function correlations that are still missing in order to understand the pathogenic effects of CoVs on cellular function.
The author declares no conflicts of interest.
Funding for this research was provided by: Consejo Nacional de Investigaciones Científicas y Técnicas (grant No. PIP 857/2015).
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The lipoprotein envelope
Surrounding viruses of either helical or icosahedral symmetry are lipoprotein envelopes, unit membranes of two lipid layers interspersed with protein molecules (lipoprotein bilayer). These viral membranes are composed of phospholipids and neutral lipids (largely cholesterol) derived from cell membranes during the process known as budding. Virtually all proteins of the cell membrane, however, are replaced by proteins of viral origin during budding. Although all the viral envelope lipids originate from the cell, their relative proportions vary from those in the cell membrane because the viral proteins select only certain lipids during budding.
Associated with the virion membrane are “integral” glycoproteins, which completely traverse the lipid bilayer, and “peripheral” matrix proteins, which line the inner surface. The glycoproteins contain regions of amino acids that, in the first step of viral infection, recognize host-cell receptors. Matrix proteins appear to function in the selection of regions of the cell membrane to be used for the viral membrane, as well as to aid the virion in entering cells.
Solve the problem that took viruses millions of years to conquer by efficiently fitting nucleic acid and proteins into a small package.
COVID-19 Learning Note: Without complete cellular machinery, coronaviruses cannot reproduce on their own. Unlike bacteria, which can multiply inside or outside your body, a virus has to infect a living cell to make more viruses. This means any coronaviruses that happen to get on a surface will inactivate over time and that the most likely way to get infected is through direct contact with an infected person.
Tools and Materials
For each model, you'll need:
- Card stock or other heavy paper
- Two to three feet (60–90 centimeters) of yarn
- Six extra-large cotton balls
- Printable triangle template provided in the Assembly section below (contains two strips, each with 20 equilateral triangles whose sides are 1.5 inches [3.8 cm] long)
- Optional: small, plastic zip-top bag (not shown)
Print or copy the triangle template onto the cardstock (the two strips of 20 triangles each should fit onto one standard 8.5 x 11-inch [A4] sheet). Only one strip is required for each model.
To Do and Notice
Cut out the individual equilateral triangles from the template. Experiment with different ways of taping up to 20 of the triangles together so that they completely enclose the yarn and cotton balls.
What’s Going On?
Viruses are composed of nucleic acid genomes and interior proteins that are surrounded by a protective protein shell called a capsid.
Instead of making the capsid out of one giant protein, viruses typically utilize many identical copies of the same protein that combine together to form this outer shell. This way, the virus can be economical, using one gene repetitively to make many small proteins instead of devoting a large portion of its genome to making a large protein coat.
When making your paper container, you may have found a shape that uses several triangles to enclose the yarn and cotton balls, which represent a virus's nucleic acid and interior proteins, respectively. The majority of viruses are composed of triangular protein sub-units that associate to form an icosahedron—a 20-sided shape. This shape helps the virus to minimize its surface-area-to-volume ratio, which allows it to carry the most genetic material and internal proteins inside a given protein shell.
One way to categorize viruses is by whether or not they are surrounded by a membrane, called a viral envelope. A large number of viruses that infect humans have envelopes, including HIV, the virus that causes AIDS. You can place your capsid into the plastic bag to model these enveloped viruses.
Coronaviruses, such as the one that causes COVID-19, also have envelopes. Unlike HIV, they have helical interiors, which is another common shape for viruses.
Icosahedrons have three axes of rotational symmetry—two-fold (180°), three-fold (120°) and five-fold (72°). At the two-fold axis of symmetry, the shape will look the same if it’s rotated on this axis 180°. Can you find all three axes of symmetry?
This model can also be used to illustrate the concept of gene therapy. Gene therapy seeks to treat diseases caused by defective or deficient proteins by introducing genetic material as medicine. Instead of manufacturing and injecting a functional version of the protein, gene therapists modify viruses so that their genomes contain a copy of the gene that encodes for the correct form of the diseased protein. Viral genes are removed from the genome, so that when the virus—now a viral vector—enters the target cell, it produces correct copies of the therapeutic protein instead of creating new virions (complete virus particles).
Gene therapy has been used in clinical trials to treat a wide range of diseases, including HIV, hemophilia, cancer, and “bubble-boy disease” (Severe Combined Immune Deficiency).
If you are a teacher making these models with your class, we recommend one model for every 1–2 students.
Cowpea Mosaic Virus Capsid: A Promising Carrier for the Development of Carbohydrate Based Antitumor Vaccines
Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.
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Scientists discover genetic and immunologic underpinnings of some cases of severe COVID-19
New findings by scientists at the National Institutes of Health and their collaborators help explain why some people with COVID-19 develop severe disease. The findings also may provide the first molecular explanation for why more men than women die from COVID-19.
The researchers found that more than 10% of people who develop severe COVID-19 have misguided antibodies―autoantibodies―that attack the immune system rather than the virus that causes the disease. Another 3.5% or more of people who develop severe COVID-19 carry a specific kind of genetic mutation that impacts immunity. Consequently, both groups lack effective immune responses that depend on type I interferon, a set of 17 proteins crucial for protecting cells and the body from viruses. Whether these proteins have been neutralized by autoantibodies or―because of a faulty gene―were produced in insufficient amounts or induced an inadequate antiviral response, their absence appears to be a commonality among a subgroup of people who suffer from life-threatening COVID-19 pneumonia.
These findings are the first published results from the COVID Human Genetic Effort, an international project spanning more than 50 genetic sequencing hubs and hundreds of hospitals. The effort is co-led by Helen Su, M.D., Ph.D., a senior investigator at the National Institute of Allergy and Infectious Diseases (NIAID), part of NIH and Jean-Laurent Casanova, M.D., Ph.D., head of the St. Giles Laboratory of Human Genetics of Infectious Diseases at The Rockefeller University in New York. Major contributions were made by Luigi Notarangelo, M.D., chief of the NIAID Laboratory of Clinical Immunology and Microbiology (LCIM) Steven Holland, M.D., director of the NIAID Division of Intramural Research and senior investigator in the NIAID LCIM clinicians and investigators in hospitals in the Italian cities of Brescia, Monza and Pavia, which were heavily hit by COVID-19 and researchers at the Uniformed Services University of the Health Sciences in Bethesda, Maryland.
The wide variation in the severity of disease caused by SARS-CoV-2, the virus behind COVID-19, has puzzled scientists and clinicians. SARS-CoV-2 can cause anything from a symptom-free infection to death, with many different outcomes in between. Since February 2020, Drs. Su and Casanova and their collaborators have enrolled thousands of COVID-19 patients to find out whether a genetic factor drives these disparate clinical outcomes.
The researchers discovered that among nearly 660 people with severe COVID-19, a significant number carried rare genetic variants in 13 genes known to be critical in the body’s defense against influenza virus, and more than 3.5% were completely missing a functioning gene. Further experiments showed that immune cells from those 3.5% did not produce any detectable type I interferons in response to SARS-CoV-2.
Examining nearly 1,000 patients with life-threatening COVID-19 pneumonia, the researchers also found that more than 10% had autoantibodies against interferons at the onset of their infection, and 95% of those patients were men. Biochemical experiments confirmed that the autoantibodies block the activity of interferon type I.
Q Zhang et al. Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science DOI: 10.1126/science.abd4570 (2020).
P Bastard et al. Auto-antibodies against type I IFNs in patients with life-threatening COVID-19. Science DOI: 10.1126/science.abd4585 (2020).
NIAID Director Anthony S. Fauci, M.D., NIAID Senior Investigator Helen C. Su, M.D., Ph.D., and Luigi Notarangelo, M.D., chief of the NIAID Laboratory of Clinical Immunology and Microbiology, are available for interviews.
To schedule interviews, please contact NIAID Office of Communications, (301) 402-1663, [email protected]
NIAID conducts and supports research — at NIH, throughout the United States, and worldwide — to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID website.