Monday, November 28, 2016

Recent Discovery of CEACAM5 as a Middle East Respiratory Syndrome Coronavirus Attachment Factor

Almost all individuals are infected with a coronavirus sometime in their life whether they know it or not (1). Coronaviruses infect a variety of mammals and birds, commonly resulting in mild to moderate illnesses in the respiratory tract. Severe Acute Respiratory Syndrome virus is one of the most notorious members of the coronavirus family, responsible for causing about 775 deaths worldwide from 2002 to 2003 (1). A coronavirus virion contains positive-sense single-stranded RNA, an icosahedral protein capsid that surrounds the (+)-RNA, and a peripheral lipid envelope. The defining structures of coronavirus virions are their crown-shaped glycoprotein spikes that reside in the lipid envelope (1). There are six categories of coronaviruses, each with unique glycoprotein spikes that bind to specific cellular virus receptors.
Middle East respiratory syndrome coronavirus with
distinct glycoprotein spikes on exterior.

The attachment of a coronavirus glycoprotein spike to a cellular receptor is the first step in coronavirus replication and spread. The attachment of a viral coronavirus spike, more broadly termed a viral “adhesin,” to a cellular receptor is required for the virus to enter the “host” cell. Typically a coronavirus spike binds to a protein or carbohydrate receptor on the cell’s exterior membrane with a low affinity, meaning that the virus can unbind to the cellular receptor just as readily as it binds. In order to form a stable attachment to a cell, coronavirus spikes must bind to multiple cellular receptors at a time, and in some instances, also bind to coreceptors, which may be another protein or carbohydrate, in addition to the primary cellular receptor. Understanding how a virus binds to its host cells allows researchers to engineer vaccines that promote an immune response to certain regions of a virus adhesin, such the binding regions of the coronavirus glycoprotein spike. This immune response allows the body to produce “memory” T-lymphocytes, specific antibodies, and “memory” B-lymphocytes that function to readily recognize and destroy virions containing regions of the virus adhesin protein expressed by the vaccine.
Because coronaviruses’ are able to bind to a high diversity of cellular virus receptors, coronaviruses can infect a large variety of cell types. For example, SARS can enter a number of cells by binding to cells containing either angiotensin I converting enzyme 2 (ACE2) (2), liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) (3), or dendritic cell-specific intercellular adhesion molecule 3-grabbing non integrin (DC-SIGN) (4) as virus receptors on their membranes. Although the identity of many host cell receptors have been identified for a number of human coronaviruses, the complete set of virus receptors for the Middle East Respiratory Syndrome Coronavirus spike protein remains unknown.
Kissing a camel is a great way to acquire
 Middle East respiratory syndrome.
Since its discovery in Saudi Arabia in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has killed about 36% of the 1733 confirmed patients from the Arabian Peninsula and the Republic of Korea (5, 6). Although MERS-CoV infection results in cold-like symptoms for many fortunate victims, the virus has an alarming potential to cause severe acute respiratory disease and even death, especially among elderly, immunocompromised, diabetic, or cancer-suffering individuals (6). Common symptoms include fever, cough, shortening of breath, pneumonia, and diarrhea. MERS-CoV spreads by spillover events from camels to humans in the form of direct camel contact or consumption of camel meat and/or milk (6). Although not as common as camel-to-human transmission, the virus may also spread by close contact with an infected individual’s saliva or mucous (5). Due to the lack of vaccine and antiviral drug treatment for MERS-CoV, severe patients are treated by medical support and specialized care in order to maintain the function of their failing organs (6).
A recent study (October 2016) by Che-Man Chan et al. from the State Key Laboratory of Emerging Infectious Disease set out to discover MERS-CoV attachment and entry processes in an effort to provide direction for the development of vaccinations against MERS-CoV. Prior to Chan et al’s study, MERS-CoV has been shown to infect a large variety of tissues and cell types in humans and camels (7). DPP4, a ubiquitously expressed cell receptor, has been identified as the primary virus receptor of MERS-CoV (8). Furthermore, it is likely that a number of other unidentified host cell co-receptors are involved with DPP4 to either enhance or coordinate the attachment and entry of MERS-CoV into host cells.
Chan et al. began their study by identifying potential cell surface proteins that bind to MERS-CoV. Using VOPBA (Viral Overlay Protein Binding Assay) in order to identify potential cellular virus receptors, the authors discovered human Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5) as a host cell receptor for the MERS-CoV glycoprotein spike. CEACAM5 is a cellular membrane protein involved in cell proliferation, movement, apoptosis, attachment, and the innate immune response. Surprisingly, the authors did not identify DPP4 as a strong cell binding receptor for MERS-CoV. Next, using a technique called flow cytometry, the authors identified the presence of CEACAM5 on the membranes of a number of cells highly susceptible to MERS-CoV infection, strengthening the proposal that CEACAM5 serves a role in MERS-CoV attachment and/or entry. Furthermore, the authors used fluorescent antibodies to detect the presence DPP4 and CEACAM5 on human lung tissue epithelial cells. Because human epithelial lung tissue cells are highly prone to MERS-CoV infection, the presence of both DPP4 and CEACAM5 on the membranes of these cells suggests that the two proteins play a role in MERS-CoV cell attachment and/or entry.
In order to demonstrate CEACAM5’s ability to directly interact with the MERS-Cov glycoprotein spike, the researchers identified direct binding events between the MERS-CoV spike protein and cellular membrane protein CEACAM5. Using co-immunoprecipitation, which identifies protein-protein interactions, the authors detected binding of CEACAM5 to the MERS-CoV spike protein on cells infected with MERS-CoV. Next, the authors used a CEACAM5 antibody to block CEACAM5 and MERS-CoV interactions. The addition of this CEACAM5-blocking antibody decreased MERS-CoV entry into cells and viral propagation in cell culture, strengthening the notion that CEACAM5 serves as a host cell mediator of MERS-CoV attachment and/or entry. In addition to the antibody blocking assay, the researchers demonstrated that lowering levels of CEACAM5 on host cells (using siRNA treatment) declined levels of MERS-CoV entry. Finally, the authors used cells without either DPP4 or CEACAM5 to discover that MERS-CoV cannot infect cells with CEACAM5 alone, but can infect cells, albeit less efficiently, with DPP4 alone. In the presence of DPP4, higher levels of CEACAM5 led to higher levels of MERS-CoV entry. Therefore, CEACAM5 likely serves as a cell binding protein that facilitates MERS-CoV entry by acting as an “attachment factor” for MERS-CoV.
Overall, Chan et al.’s study discovered the role of CEACAM5 as a novel cell membrane protein that serves as an attachment factor for MERS-CoV. Although CEACAM5 does not directly mediate MERS-CoV entry into host cells, the membrane protein helps bind MERS-CoV to the outside of the cells in order to allow DPP4 to more easily initiate MERS-CoV entry. Chan et al.’s cell culture experiments (in vitro) studying CEACAM5 and MERS-CoV interactions provides a great preliminary model for MERS-CoV attachment and entry mechanisms; however, future study of CEACAM5 interactions with MERS-CoV in animal models (in vivo), such as mice (with exogenous human DPP4) (9), will provide a more realistic model of MERS-CoV attachment and entry in humans. Future studies of MERS-CoV and CEACAM5 may be difficult in mice owing to the numerous variables to control (age, weight, sex), and manipulating levels of CEACAM5 or DPP4 in mice involves more tedious and complex procedures than with cell culture. Despite the difficulties of research using animal models, the in vivo results will provide a more valid, and possibly even more developed, mechanism of the interactions between CEACAM5 and the MERS-CoV spike protein in humans.
Vaccine design for MERS-CoV is the most important future direction of this study. Earlier attempts of MERS-CoV vaccine design expressing the full MERS-CoV spike protein have produced adverse immune responses in camels (10). However, designing a subunit vaccine only containing the specific regions, also termed epitopes, of the MERS-CoV spike protein that bind to CEACAM5 and DPP4, instead of the whole MERS-CoV spike protein, may trigger a safer immune response that protects the organism from future MERS-CoV infection. Producing a subunit vaccine that only contains the CEACAM5 and DPP4 binding domains of the MERS-CoV spike could successfully allow the body to initiate the production of memory T lymphocytes, neutralizing antibodies, and memory B lymphocytes to readily defend against future MERS-CoV infections without the harm of a toxic immune response.

Chan CM, Chu H, Wang Y, Wong BH, Zhao X, Zhou J, Yang D, Leung SP, Chan JF, Yueng ML, Yan J, Lu G, Gao GF, Yuen KY. 2016. Carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of Middle East respiratory syndrome coronavirus. J. Virol. 90: 9114-9127. doi: 10.1128/JVI.01133-16.

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