Inside the Cell: The Biology of the Novel Coronavirus SARS-CoV-2

Though much of the world is hearing about them for the first time, coronaviruses are a large family of related viruses responsible for respiratory infections in vertebrates such as livestock, birds, bats and rodents. Coronaviruses are typically zoonotic, meaning they can be transmitted between species — often when individuals are in close proximity — allowing transmission via droplets produced through coughing or sneezing. In humans, illnesses can range from common cold-like symptoms to more severe diseases such as the Middle East Respiratory Syndrome (MERS-CoV), first reported in 2012 and Severe Acute Respiratory Syndrome (SARS-CoV) in 2003. A novel coronavirus, now known as SARS-CoV-2 has emerged as a major global threat to human health in 2020 and is responsible for the infectious disease COVID-19.

Worth noting is that members of the coronavirus family are all similar in structure. An outer glycoprotein coat surrounds the virus particle. A major component of this coat is the Spike or S-protein, which is critical for the interaction of the virus particle and the host cell. In fact, the virus family is so named because when viewed by electron microscopy, the S-protein coat appears to form a halo, or corona, around the particle that resembles a solar corona. The S-protein projects from the viral envelope, which is derived from the membrane of the host cell in which the virus replicates. The core of the virus particle contains the genetic material, a single RNA strand encoding the viral structural proteins as well as an RNA polymerase responsible for replication of the viral RNA and subsequent production of smaller RNAs that serve as templates for viral proteins. As is typical for all viruses, SARS-CoV-2 takes advantage of the cellular machinery normally used for secretion of proteins from the cell. Viral proteins are translated and assembled into virus particles in the lumen of the endoplasmic reticulum, along with host-cell surface proteins, then transported in membrane-bound vesicles to the cell surface before being released to infect adjacent cells.

The RNA based replication mechanism is also likely to be a key factor in the zoonotic nature of the coronaviruses; RNA replication lacks the error repair mechanism responsible for the proof reading of newly replicated DNA molecules that takes place during normal cell division.

This inherently error prone mechanism offers an evolutionary benefit: a small proportion of new virus particles released from an infected cell will contain S-proteins that differ slightly in their amino-acid sequences from the original protein. While many of these variants will of course have diminished ability to infect new host cells, some variants will allow cross-species infection to take place.

It is believed that SARS-CoV and MERS-CoV likely evolved from bat CoVs that were able to infect other host animals often found in close proximity to humans. SARS-CoV was detected in civets and raccoon dogs while MERS-CoV was identified in camels.¹ The most closely related bat coronavirus and SARS-CoV may have diverged as recently as 1986,² and in fact, the SARS-CoV-2 S-Protein retains 98% sequence identity with a bat coronavirus S protein.³

Given the critical role of the S-Protein in viral infection, much attention has been focused on elucidation of its three-dimensional structure. Upon contact with a specific protein on the surface of the host cell (Angiotensin-Converting Enzyme 2, ACE2), the S-protein undergoes a structural rearrangement, resulting in fusion of the viral membrane with that of the host cell. Quantitation of the S-Protein-ACE2 interaction by the Surface Plasmon Resonance (SPR) technique may also help explain the apparent ability of the SARS-CoV-2 to spread more readily from one infected individual to another compared with other coronaviruses: the SARS-CoV-2 S-Protein binds ACE2 with a 10 to 20-fold higher affinity than the SARS-CoV S-Protein^3.

Understanding of this step is the basis for efforts to develop vaccines and antiviral drugs that could work by binding tightly to the S-protein and locking it in a configuration that prevents fusion of the cellular and viral membranes.

The influenza virus family share some general similarities with the coronaviruses, also comprising a glycoprotein coat enclosing a genomic RNA core, which unusually is formed of separate RNA molecules each encoding one or two viral proteins. Two coat proteins are critical in the influenza viral life cycle: Hemagglutinin (HA) and Neuraminidase (NA). The HA protein allows the virus to bind to the host cell surface and subsequently enter the cell. While the coronavirus S-protein binds in a highly specific manner to the ACE2 cell surface protein, it appears the influenza HA protein targets a carbohydrate (sialic acid) present on a range of cell surface proteins. Although the mechanisms of viral internalization may be broadly similar, both result in fusion of the viral envelope membrane with the membrane of the host cell allowing release of the viral RNA into the cell.

A key difference between the two virus families, however, is the involvement of the NA protein in the release of influenza virus particles from infected cells. NA is the target of all four antiviral drugs currently approved by the FDA for the treatment of influenza virus infection. This set of NA inhibitors was included in a panel of 19 drugs screened by Tan et al.⁴ for their ability to inhibit the release of SARS-CoV virus particles from infected cells. In this study, cells were incubated with a virus preparation to allow infection, then the cells were cultured in a monolayer. An infected cell will release viral particles that infect surrounding cells resulting in the formation of a visible plaque in the cell monolayer, so the effects of the drugs may be quantified. Since the coronaviruses do not appear to utilize neuraminidase for viral release, it was unsurprising that the NA inhibitors, effective against influenza virus replication, did not appear to reduce SARS-CoV plaque formation.

Two other drugs, members of the Interferon family, did appear to reduce the number of viral plaques formed. The antiviral activity of interferons appears to result from their ability to stimulate production of enzymes that ultimately lead to the degradation of viral RNAs suggesting potential utility in the treatment of coronaviruses. However, to date no effective treatments for coronavirus infections have been identified, and approaches have focused on repurposing existing drugs. For example, a panel of five antiviral drugs was evaluated against SARS-CoV-2 in a cell culture system.⁵ As in the study by Tan et al., these drugs were developed to treat a range of viral as well as protozoal infections; this study included the widely used antimalarial drug chloroquine. While some of these drugs showed promise, this study highlighted the particular challenge of treating virus infections — targeting replication of the virus while not disrupting the machinery of the host cell. It’s likely that treatments will ultimately involve a cocktail of antiviral drugs necessitated by the ability of RNA based viruses to evolve rapidly and hence become resistant to individual drugs.

In parallel to the development of novel antiviral drugs, researchers are also focused on the development of vaccines to protect again infection. The elucidation of the SARS-CoV-2S protein structure should allow the identification of specific regions of the S protein critical for the mechanism of viral entry into the host cells, making them possible targets for vaccines. Traditional vaccine development often involves producing an inactivated (“killed”) form of a virus that must be manufactured rapidly and on a large enough scale to be used to immunize the at-risk population. For example, inactivated influenza virus is still commonly produced in a chicken egg system that has been in use for over 70 years. With the production system already established, adaptation to new seasonal influenza variants can be achieved relatively quickly.

It’s likely that in the case of 2019-nCoV, novel, faster approaches will be employed, using for example, cell-based systems to produce carefully chosen fragments of the S-protein to use as a vaccine. This approach may even be taken a step future by using an RNA encoding the appropriate viral protein as the vaccine. Now the process of vaccine manufacture can in-principle be eliminated as the protein production takes place in the body after vaccination. While the effectiveness of such approaches is still being studied, it is likely that their importance will only increase as novel infectious viruses continue to emerge.

References

1. Jie Cui, Fang Li & Zheng-Li Shi. Origin and evolution of pathogenic coronaviruses. Nature Reviews Microbiology volume 17, pages181-192(2019).
2. Vijaykrishna D, Smith GJ, Zhang JX, Peiris JS, Chen H, Guan Y (April 2007). Evolutionary insights into the ecology of coronaviruses. Journal of Virology. 81 (8): 4012-4020.
3. Wrapp D, Wang N, Kizzmekia S.C., Goldsmith, J.A., Hsieh C-L, Abiona, O., Graham B.. McLellan S.. Cryo-EM structure of the SARS-CoV-2 spike in the prefusion conformation. Inhibition of SARS Coronavirus Infection In Vitro with Clinically Approved Antiviral Drugs. Science 19 Feb 2020.
4. Emily L.C. Tan,* Eng Eong Ooi,† Chin-Yo Lin,* Hwee Cheng Tan,† Ai Ee Ling,‡ Bing Lim,* and Lawrence W. Emerg Infect Dis. 2004 Apr; 10(4): 581-586.
5. Manli Wang, Ruiyuan Cao, Leike Zhang, Xinglou Yang, Jia Liu, Mingyue Xu, Zhengli Shi, Zhihong Hu, Wu Zhong & Gengfu Xia. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Research (2020).