Knock-Knock, it’s CoV-2

A discussion on how the coronavirus infiltrates our cells to establish infections

We’re living in truly unprecedented times – brought to our knees by an imp of a virus, with seemingly no way out. And while there’s a ton of news (and fake news) flying around, one of the most heartening trends has been the global research community’s rapid response to this situation. Indeed, torrents of papers are being released at whirlwind speed, as research teams race to unearth the mysteries of the latest menace, the COVID-19, caused by the SARS-Coronavirus-2 (referred to as COV-2 henceforth). 

With the entire world tottering on the edge of a crisis (and what a multi-faceted, Lernaean Hydra-like crisis at that!) and most of us shut away indefinitely, just about now seems the best time to break the year-long hiatus and restart the blog. 

In this piece, I’ll be putting out some super basic facts about the COV-2, as well as an extended discussion on how it manages to enter and infect cells in our body. I’m also taking this as an opportunity to elucidate some of the fundamentals of virology (which should ideally be a separate article, but maybe at a later point in time). Also, I’m hopeful of posting a follow-up piece covering some potential vaccine strategies against COV-2 sometime soon, so do share your feedback for any other areas which could be incorporated.   

The Basics

It’s always difficult to explain what a virus really is. Cartoons sometimes do a wonderful job by depicting them as threateningly colored spiked balls with devilish faces (sometimes horns too!). School textbooks conveniently brush them aside as the last – and often briefest – section in a chapter on microbes, along with the most commonplace of all descriptions – “viruses are neither living nor non-living”. Well, at least it’s probably true. 

The view I would like to take of viruses involves neither horns nor philosophical ponderings on life and death. I think the simplest way to visualize a virus is as an unbelievably intelligent amalgam of chemicals capable of deftly manipulating even the most complex cellular systems to do its bidding, all while suavely (and ruthlessly) outsmarting some of Nature’s most exquisitely engineered defence mechanisms. Sounds like it’s non-living? Well, trust a few billion years of evolution to make even the puniest entity appear infinitely superior in its forethought and organization. 

The bare-bones description of a virus would be in terms of its constituent molecules. Typically, a virus consists of its genetic material along with accompanying proteins. The genetic material of a virus can be either DNA (DNA viruses) or RNA (RNA viruses). This would usually be encased within a protein coat (called the capsid). Such a virus is called “naked” as it possesses only the viral genes and an encasing protein shell. Some viruses go a step further by incorporating an outer layer made up of lipids (an important biomolecule which makes up substances like fats and waxes), and proteins and these are commonly called as “enveloped” viruses. For the purpose of this piece, that’s about all the jargon you’ll need.

The “coronavirus” as the media seems to have christened the current virus at large (despite WHO’s best efforts) is but one of the many members of a family of viruses called coronaviruses. The term “corona” (from the Latin for Crown) is a reference to the spiky projections commonly visible on the surface of these viral particles. A species within this family are the Severe acute respiratory syndrome-related coronavirus. Even within the species, several strains commonly exist, out of which two have been the prime newsmakers:

  1. SARS-COV / SARS-COV-1, which caused the 2002-2004 SARS epidemic (henceforth referred to as COV)
  2. SARS-COV-2, the agent behind the current COVID-19 pandemic (henceforth referred to as COV-2)
An electron micrograph of representative coronavirus particles. The spike-like projections are characteristic of this species. Image source: Centers for Disease Control and Prevention’s Public Health Image Library (PHIL), with identification number #4814.

Genetically, they are both very similar and are distinguishable only as two different strains of coronavirus. They’re both enveloped RNA viruses and have extremely similar mechanisms of infection. The current COV-2 pandemic is suspected to have kicked-off as a zoonosis event (when a disease is transmitted to man from an animal), originating in the bats and transmitted through an intermediate host to humans in Wuhan province of China. While an exact description of the trajectory is still controversial, this appears to be the most reasonable event history. An extended elaboration on this, as well as controversy theories of bioterrorism, are quite an enticing prospect, but one which we’ll have to save for an upcoming article.

The viral particles may spread via droplets or from contaminated surfaces (the reason why “social distancing” is the need of the hour), and the most severe afflictions appear to be targeted at the respiratory system. What makes it even more difficult to manage than the earlier SARS epidemic is that even apparently asymptomatic individuals are capable of shedding the viral particles, making containment efforts complicated to effectively implement.

The prime focus of this article is on how the COV-2 manages to infiltrate the cells of our body, which has been very recently established by leading investigations over the last few months. An interesting observation is how heavily the current research has borrowed from the prior work on COV during the SARS epidemic – a sentiment that will make its pertinence felt multiple times throughout our discussion.

Viral Structure and Mode of Attachment to Cell Surfaces

The COV-2’s structural composition has been of great interest, particularly from a vaccine development point-of-view. Based on the available literature about the virus family (coronaviruses), we already know that the COV-2 is an enveloped virus, which also has a pretty interesting feature called “spike protein”. These “spikes” are a special class of proteins that are embedded in the viral envelope and extend outwards like a protrusion (the reason why all those images of the COV-2 look like a rather prickly ball). The coronavirus family’s spike proteins have long since been implicated in a crucial step of the infection cycle – mediating the successful entry of viral material into the host/target cell. 

Schematic illustrating the basic organization of a coronavirus viral particle. The legend table on the right summarily explains each of the components. Image source: Seah, I. et. al. Revisiting the dangers of the coronavirus in the ophthalmology practice. Eye (2020) 

Under normal circumstances, our cells are completely surrounded by a plasma membrane layer, which is only selectively permeable (i.e., allows only select substances like useful hormones, proteins, etc. through). This represents a significant barrier for any house-hunting virus, as our cell membranes can often be ridiculously choosy about not letting in suspicious entities. A loophole, however, is for the virus to directly fuse its own outer membrane with that of the cell’s and pour all its contents into the now vulnerable host. While other mechanisms of entry do exist (briefly described in the next section), let’s stick with this one for our current discussion. 

The COV-2, hence, comes prepared for the task on hand. And the spike protein is exactly what helps it do so, by binding to a particular protein on the host cell’s surface (called a receptor, because, you guessed it, it “receives” the viral spike protein), and setting the stage for the subsequent fusion of the two membranes. This step is critical because without stably attaching onto a host cell surface, the virus would never be able to initiate the infection. Hence, a reasonably strong interaction between the receptor and the viral spike protein is a mandatory prerequisite.

Now, identifying which particular proteins are involved is not the most straightforward of tasks. Our cells have a few thousand different types of surface proteins (which also have a diversity of distributions depending on which cell type we’re considering). Fortunately, extensive research carried out during the previous SARS-COV epidemic proved useful. Researchers quickly narrowed down on a human protein called ACE2 which has now been shown to act as a receptor for the COV-2 spike protein. 

To be fair, ACE2’s (which stands for Angiotensin-converting enzyme 2) role in life isn’t exactly to bind viruses and enable worldwide lockdowns. Its main job in physiological scenarios is to act as a counter to the ACE enzyme (yes, they really didn’t invest much creativity in naming these proteins!). ACE is important for vasoconstriction (narrowing of blood vessels) which increases bodily blood pressure, thereby regulating the amount of fluids in the body. It achieves this by converting the inactive hormone angiotensin to angiotensin II. ACE2, our main focus, is a counterweight to this process, by cleaving angiotensin II (a vasoconstrictor) to angiotensin 1-7 (a vasodilator) thereby lowering blood pressure and relaxing the blood vessels. The balance between these two is what regulates the body’s fluid content and overall blood pressure. 

Unfortunately enough, ACE2 is also a liability in many ways. Members of the coronavirus family have been shown to have an especial liking for the ACE2, and use it as a convenient hook to attach themselves to the surface of human cells, thereby initiating the steps leading up to the viral infection. Extensive studies implicating this were carried out in the aftermath of the SARS-COV epidemic, and insights from the same proved instrumental in guiding efforts over the past few months which have determined that once again, the receptor at fault is ACE2, which allows COV-2 to attach and initiate the steps for entry into the cell.

Illustration showing how the COV-2 spike protein binds to the ACE2 receptor on cell surfaces. The anti-ACE2 antibody-based strategy has been explained below. Image source: https://www.rndsystems.com/resources/articles/ace-2-sars-receptor-identified 

Using advanced microscopy and structure prediction tools (most notably, cryo-EM microscopy – something we may cover in a future article!), researchers have been able to rapidly determine not only the structure of the viral spike protein but also of the ACE2 in complex with the spike protein. This is pretty much groundbreaking work because a precise determination of these structures can provide valuable information for designing drugs / therapeutic strategies against the COV-2. For instance, if we can make antibodies (large, immunologically active proteins which attach to specific surfaces) which are able to target and bind the ACE2/viral spike proteins, we can interfere with this attachment process, and thereby prevent any further progress of the viral infection. While this promises to be an effective strategy, work is still ongoing on this front.  

Membrane Fusion between Virus and Cells

Viruses employ a variety of mechanisms to enter host cells (most of which have fancy names!). These are all essentially an effort to breach the barrier imposed by the cell’s membrane and access the bountiful resources on the other side. Which is sort of a spoiler for the main question here – Why on earth do viruses seem so desperate to enter our cells?

Recall that a virus is essentially classified as a non-living entity until it’s outside the cell. Well, that pretty much explains the question above! The ultimate objective for a virus, to replicate (make multiple copies of itself), can only be achieved using resources and machinery available to living cells. 

A given viral particle is nothing but a collection of DNA/RNA and proteins (if its a naked virus), or proteins and lipids (if its an enveloped virus). And generating copies of itself is an expensive and complicated affair indeed – tons of proteins have to be generated, the genetic material has to be copied and amplified, and everything needs to be precisely packaged together in exactly the right conformation. To do so, not only are raw materials in terms of molecules like amino acids and nucleotide bases (the building blocks of proteins and DNA/RNA respectively) required but also specific enzymes required for this manufacturing process. Cells represent a literal warehouse in terms of these requirements, with plenty of molecules to serve as raw materials as well as ready-made machinery just floating around – all ready to be hijacked by a smart enough invader. And viruses are a perfect fit for the job, as they bring in their own genetic material, plus some cunningly selected special enzymes of their own to help take over the cell’s setup. For instance, some RNA viruses carry along an enzyme called reverse transcriptase. This is because our own cells have DNA as the primary mode of storing genetic information, and thereby use a DNA->RNA->Protein workflow in order to generate useful proteins. Hence, these RNA viruses achieve a unique workaround by bringing in their own reverse transcriptase, which converts their RNA to DNA, which is now compatible with the cell’s machinery. 

Now that we’ve cleared up the objectives, let’s get back to the modus operandi of how exactly viruses manage to enter our cells. There are three ways by which the actual entry occurs:

  1. Membrane Fusion – The membrane of the virus fuses with the membrane of the cell, thereby releasing all the contents inside the cell. The viral envelope may remain on the cell surface. 
Viral entry by membrane fusion. Blue bars are the cell surface receptors to which the viral envelope’s spike proteins (orange) bind. The nucleocapsid is in green, and the virus’ genetic material is in red. Image source: By Nossedotti (Anderson Brito) – Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=14114853

  1. Injection – Some viruses prefer to keep it simple! Attach to the cell surface, and just inject their genetic material into the cells. While risky, it’s a remarkably straightforward strategy that requires little investment on behalf of the virus. 
  1. Endocytosis – A classic example of how viruses hijack regular cellular processes. Most cells have a well-regulated system wherein nutrients and important molecules from the outside may be taken up for consumption at the membrane using special sacs called vesicles (or endosomal vesicles), and using which the cell also expels waste products to the outside. Think of these vesicles as small bubble-like delivery cabs running from the surface of the cell to the interior. The essential trick the virus needs to perfect is fooling the cells into misidentifying it as a useful substance, gaining acceptance by covertly entering one of these vesicles, and breaking out of these once they’re inside the cell. Devious, yet so wickedly delightful!
Viral entry by endocytosis. The grey outlined bubble is the vesicle. Image source: By Nossedotti (Anderson Brito) – Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=14114788

Knowing how the virus manages to enter is pretty much a big deal because knowledge of this enables us to tailor-make drugs to interfere with this process (like the example of the ACE2/viral spike targeting antibodies above).  

Suspicions that COV-2 possibly employed more than one step for mediating its entry were once again based on insights gained from pioneering research on the original SARS-COV virus. Even with the SARS-COV, scientists were able to discern that the ACE2 wasn’t the only player involved. While actual motivations and rationale leading to this realization may have been numerous, two, in particular, are quite logically appealing. 

This first piece of logic was that even though ACE2 had been identified as crucial for the attachment of COV, the infection hotspots for COV within the body did not correlate completely with those cell types/tissue where ACE2 expression was also high. Hence, it was suspected that other factors could have a say in determining the actual choice of location. Another line of reasoning involved the observation that when ACE2 was purified, a second protein seemed to accompany it, given the context of a COV infection. Based on these observations, a second culprit was soon identified – an enzyme called TMPRSS2, which is a protease (capable of slicing up specific proteins to pieces). 

The existence of a second step is interesting because this now suggests a more complicated mechanism of entry. The role of TMPRSS2 was identified as being critical in cleaving a subunit of the viral spike protein, thereby exposing and activating a second hidden protein, called a fusion peptide. Peptides are short stretches of amino acids that aren’t quite fully functional proteins but do have some capabilities. The fusion peptide is the key element that enables both the viral and the cell membrane to finally fuse together, thereby enabling entry of the virus into the cell. Hence, in the absence of a protease like TMPRSS2, the virus would be able to attach to the cell surface, but unable to proceed, since the membrane fusion step wouldn’t take place unless the fusion peptide gets activated. This was further validated in experiments where a chemical inhibitor of TMPRSS2 (called camostat mesylate) was used, which successfully managed to reduce COV’s infectivity by preventing the activation of the fusion peptide, hence interfering with the entry of the virus into the cells.

A generalized model for coronavirus undergoing membrane fusion. The host membrane is the long grey colored band looping around the entire figure. The viral spike protein depicted in (A) is in the pre-fusion form. The large grey top half is what binds the receptor (aqua-green color circle) on the host membrane. Following this, a protease (say TMPRSS2) will cleave the large top half from the bottom subunit, as shown in (B). The bottom subunit is the fusion peptide, made up of three strands depicted in the colors yellow, blue, and pink for each of the constituent strands. Once the cleavage and activation occur the fusion peptide inserts into the host membrane and proceeds towards fusion of both membranes. Dotted lines in (A) and (B) indicate the relative position into which each strand of the fusion peptide would insert, solid lines in (D) indicate how the fusion peptide strands actually insert themselves. Image source: Walls et. al. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion, PNAS 2017.

Concluding Overview

Recent investigations over the past few months, which have benefited tremendously from key insights developed by research on the previous SARS-COV epidemic, have achieved quite a few breakthroughs. 

We now have a handle on how the COV-2 manages to gain access to our cells. An initial attachment to the cell surface by binding of the viral spike protein with the cell surface protein ACE2, followed by cleavage of the spike protein by the cellular enzyme TMPRSS2 to expose the COV-2 fusion peptide, finally leading to fusion of the viral and cellular membranes. Furthermore, recent results also seem to indicate that the COV-2 spike protein has a much higher affinity to bind the ACE2 – touted as a probable reason for the increased pathogenicity and damage inflicted by the CoVID-19 as compared to the SARS. 

These are extremely helpful signposts from a vaccine/drug design point of view. For instance, if we could develop a vaccine based on the viral spike protein, and thereby train our immune system to remember (and recognize in the future) the viral spike protein, we essentially manage to ensure that upon an actual attack by the CoV-2 our body would pour out antibodies against the spike protein to mask it and prevent attachment, thereby eliminating any chance of an infection. Or, we could develop drugs in the laboratory that bind to and mask either the viral spike protein, or the ACE2, and administer it to patients (as well as prophylactically to health workers), thereby eliminating the possibility of successful attachment by preemptively blocking such an interaction. Furthermore, studies may also be conducted to find out effective (more importantly, safe to use) chemical inhibitors of TMPRSS2, the temporary activation of which may be beneficial in halting the spread of the infection.  

Reiterating is my favorite way to wrap up, so allow me to end on a hopeful note. In the coming weeks, it would be interesting to explore the fundamentals and experimental rationale driving some emerging strategies for developing vaccines and treatments to combat the spread of COVID-19. Additionally, we may also fleetingly delve into a recent study which claims to conclusively discredit the conspiracy theories linking the current pandemic to a bioterrorism plot. Also, as always, do get back to me with feedback and suggestions on related topics that could be covered!

References for further reading:

1. SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients – N Engl J Med 2020

2. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor – Hoffman et. al., Cell 2020

3. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus – Li et. al., Nature 2003

4. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry – Shulla et. al., Journal of Virology, 2011

5. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2 – Matsuyama et. al.; Journal of Virology, 2010

6. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor – Li et. al., Science 2005

7. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein – Walls et. al., Cell 2020

8. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 – Yan et. al., Science 2020

9. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion – Walls et. al., PNAS 2017

Featured cover image credits: CDC Public Health Image Library; CDC/ Alissa Eckert, MS; Dan Higgins, MAMS. https://phil.cdc.gov/Details.aspx?pid=2871

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