This post has been reviewed by one of our subject matter experts, Dr. Phil Lange.
For the original article, please follow this link: https://doi.org/10.1016/j.bbrc.2020.03.044. The research was conducted by the National Clinical Research Center for Infectious Disease.
Viruses spread within our bodies by using the components of our cells to replicate themselves. Different viruses differ greatly in which cell types they invade, how they invade them, and the receptors they use to gain entry. Receptors are proteins on the surface of our cells that are often targets of messenger molecules. Messenger molecules allow cells to communicate from different parts of our body. Our body cells differ vastly in the types of functions they perform, thus, each type of cell has to react to specific messenger molecule signals to coordinate their functions. Therefore, each cell type is unique in the types of receptors they express and the levels at which they express them. This is why viruses tend to only infect limited cell types with specific receptors.
So which cell types do coronaviruses infect?
Angiotensin I converting enzyme 2 (ACE2) is a cellular receptor involved in the regulation of blood pressure. It also serves as the entry receptor for coronaviruses like SARS-CoV (SARS) and has been identified as a receptor for SARS-CoV-2, the virus that causes Coronavirus disease-19 (COVID-19). When SARS-CoV-2 comes into contact with the ACE2 receptor, the viral membrane (a fatty fluid, semi-permeable, sphere that surrounds the virus) fuses with the membrane of our cells, which have a similar chemical composition. During the fusion process, SARS-CoV-2 is released into the interior of our cells. ACE2 is found on the surface of many cells in the human body including those in the lung, liver, stomach, ileum (small intestine), colon and kidney. This suggests that SARS-CoV-2 may be able to affect multiple cell types across multiple organ systems.
The researchers in this study used RNA sequencing data from 119 cell types found across 13 different tissues. When a human cell makes proteins, it first produces a genetic material called RNA. RNA, like DNA, is comprised of 4 different of bases in a unique sequence. When DNA is transcribed (copied) into RNA, its sequence is retained (with some exceptions). The sequence of RNA, and thus the DNA it was produced from, acts as an instruction manual to make particular proteins. By this logic, the amounts of specific RNA sequences found in a cell can help predict the amount of corresponding protein that is being produced. Based on this principle, these researchers looked at the quantity of ACE2 RNA in all the different human cell types to predict the amount of ACE2 protein receptor on their surfaces. The results are shown in the figure below (black dots at higher expression levels represent higher expression). Despite the cellular RNA quantity being used in this research article as an indication of the protein levels, it must be known that even if RNA is present the corresponding protein is not necessarily produced. Keep this limitation in mind when interpreting these computerized results; more laboratory studies should be conducted to determine the quantity at which different cell types express ACE2 proteins (see “Limitations of the Study” section for a more in depth explanation).
Furong Qia, Shen Qiana, Shuye Zhang, and Zheng Zhang. Biochem Biophys Res Commun. 2020. https://doi.org/10.1016/j.bbrc.2020.03.044 (Fig 1.)
Contrary to what they expected, ACE2 RNA was present relatively low amounts in the lung and only in Alveolar Type 2 (AT2) cells. AT2 cells are a known SARS-CoV-2 target cell. AT2 cells produce pulmonary surfactant, a substance that lines the interior of the lungs to help them get oxygen into the body and carbon dioxide out. When AT2 cells are infected and damaged by SARS-CoV-2, the amount of surfactant they make is reduced. This is why patients experience breathing difficulties upon SARS-CoV-2 infection. Additionally, ACE2 RNA expression was found in certain cells of the gastrointestinal tract (the path food takes from the mouth to the toilet), liver and kidneys. Considering that typical COVID-19 cases present mostly as lung-related symptoms, it was surprising that many of these other areas showed higher ACE2 RNA expression. To explain this, these researchers suggested the presence of other membrane protein receptors on the surface of AT2 lung cells that SARS-CoV-2 also binds to in addition to ACE2. The researchers suggested that these other receptors (co-receptors) allow SARS-CoV-2 to better target AT2 cells (over the other ACE2-expressing cells).
The researchers pulled data from the Viral Receptor database of known receptors that ssRNA (single-stranded RNA) viruses bind (ssRNA receptors). A ssRNA virus is a virus that carries its genetic information on RNA in a single strand. As previously mentioned, human cells pass on their information as double-stranded DNA, which is transcribed into single-stranded RNA and further processed into specific proteins. A ssRNA virus skips that DNA transcription step and hijacks the host cell’s components to directly make its own viral proteins from its ssRNA. The newly produced viral proteins then assemble into more viruses that are released from the cell. Other ssRNA coronaviruses like SARS-CoV (SARS) are known to bind to multiple ssRNA receptors. The authors predict that SARS-CoV-2 does the same.
These researchers used this data to correlate expression of 51 known ssRNA viral receptors and an additional 400 membrane proteins (protein with a variety of functions on the surface of cells). Correlating expression means that they looked at how dependant the expression of one receptor/membrane protein is on the expression of another receptor. In simpler terms, if the correlation is high between two receptors, you can predict with a certain level of certainty that they are either both present or both absent on the same cell. They represented this correlation as a heat map shown below. Each little box represents the degree of correlation between two receptors/membrane proteins, with the red demonstrating high correlation.
Furong Qia, Shen Qiana, Shuye Zhang, and Zheng Zhang. Biochem Biophys Res Commun. 2020. https://doi.org/10.1016/j.bbrc.2020.03.044 (Fig 2.)
They found that 94 genes are expressed alongside the ACE2 gene. The genes with the highest correlation to ACE2 were ANPEP, ENPEP, and DPP4 membrane proteins. ANPEP and DPP4 are known coronaviruses receptors (feline coronaviruses and MERS-CoV). ENPEP is a membrane protein also involved in the regulation of blood pressure like ACE2, but it is not a known viral receptor.
These results indicate that SARS-CoV-2 (COVID-19) may bind to these peptidases alongside ACE2 to gain entry to cells. These researchers suggest that SARS-CoV-2 may bind to known receptors of other coronaviruses. If coronaviruses share receptors, this may explain why all known coronaviruses tend to target the same tissues and present similar symptoms in the respiratory tract.
ACE2 and these co-receptors were found in the lung, liver, stomach, ileum, rectum, colon, blood, bone marrow, spleen, esophagus, kidney, skin and eye. This suggests that SARS-CoV-2 could also infect these tissues and cause damage; therefore, much more research has to be done to understand the effect COVID-19 has across the body. Moreover, binding of SARS-CoV-2 to DPP4, ANPEP, and ENPEP should be studied in the laboratory to better understand COVID-19 disease dynamics and find possible avenues for the development of antiviral medications.
Limitations of the Study
As mentioned in the Research Re-Hashed section, this a bioinformatics study (on the computer, using previously generated data) and with that comes major limitations. For instance, these researchers looked at RNA expression to determine which cell types have the most ACE2 protein expression. Although RNA expression can provide some insight, the corresponding proteins aren’t necessarily expressed. Determining the level of protein expression from the expression of RNA is difficult as there are many factors that determine how much protein a single RNA molecule produces. Therefore, more laboratory studies that look directly at protein expression must be conducted to truly determine the level of ACE2 expression on the surface of different cell types. In other words, laboratory studies must be conducted to understand what cell types SARS-CoV-2 infects.
Another limitation in the study design is that these researchers looked at which genes are expressed in high correlation in ACE2 to predict what other receptors are involved in SARS-CoV-2. Firstly, ACE-2 expression alone does not mean the cell is susceptible to SARS-CoV-2 even if it is a known receptor used for entry. For instance, once inside the cell, certain cell types may have better mechanisms to kill the virus and evade infection. Certain tissues may be better at evading infection as well. For instance, even if stomach tissue has ACE2 receptors, the stomach acid may kill the virus before it has the chance to infect those ACE2-containing cells. Looking at which genes are expressed alongside ACE2 can help predict what other receptors are involved in SARS-CoV-2 but still provides no concrete evidence.
In conclusion, this is a useful study to narrow down the cell types SARS-CoV-2 infects and which receptors (other than ACE2) it can use to gain entry into the cell. However, this is a preliminary computer-based study and should only be used to guide future laboratory-based studies which can provide more conclusive evidence.