Tuesday, November 29, 2016

Prion Memory Potential in Plants

Prions first caught the attention of scientists when it was discovered these protein particles were responsible for infectious neurodegenerative diseases, like Mad-Cow Disease and CJD, in humans and other organismal groups.  Prions are self-perpetuating proteins with distinct functions regulated by their flexible structure (Newby & Lindquist, 2013).  Aside from the tissue degradation caused by infectious prion forms, normal prion proteins can act as “molecular memory” markers that allow for the inheritance of new traits across cell divisions (Shorter & Lindquist, 2005).  When a cell divides any changes made to the chromatin are typically erased, so prions that act as templates to save these changes enable external stimuli, like the environment, to impact the genetic information of the cell.

Memory conservation using prions has been observed in organisms from yeast to humans; however, plants were thought to record genetic changes through epigenetic marks, a method independent of prion proteins, until the work of Chakrabortee et al. (2016).  Their research aimed to identify prion-like domains (PrDs) within plant proteins and associate them with prion memory behavior.  Over 500 Arabidopsis plant proteins were identified as potential PrDs, but Chakrabortee et al. (2016) narrowed their focus to three PrDs that are involved in the flowering pathway.  Prion-like domains were identified and scored for similarity using a matching program that compared Arabidopsis protein sequences to known yeast prion sequences.  Out of the 8 proteins involved in the Arabidopsis flowering pathway, half were identified as PrDs - Luminidependens (LD), Flowering Locus PA (FPA), Flowering Locus CA (FCA), and Flowering Locus Y (FY).  Three of the Arabidopsis PrDs (LD, FPA, and PA) were expressed in yeast, and they displayed similar characteristics to yeast prions, such as foci fusion leading to one distinct foci for each protein.

Figure 1: Sup35 Assay with the flowering pathway
PrDs at low expression levels (left) and
over-expressed levels (right) on adenine-deficient 
medium. Figure taken from Chakrabortee et al. 2016.
A defining feature of prion proteins is their ability to continuously renew their structure to act as a template converting normal proteins of a similar type to the prion form.  To test this characteristic in the flowering pathway PrDs, a Sup35 protein was expressed in cells with each of the PrDs to determine if they influenced the function of this translational repressor (Sup35).  Normally Sup35 disrupts cell growth by causing the ribosomes to stop translation at the ending stop codon sequence; however, Sup35 bound to an active prion does not prevent ribosomes from reading through the stop codon to continue translation (Cai et al., 2014).  Only cells expressing the LD PrD were found to have template-like behavior, as over-expressed LD led to continued cell growth, when at lower levels of LD, Sup35 remained active and led to cell death in absence of adenine (Figure 1).  This data suggested LD PrDs have strong prion-like characteristics.

The effects of LD PrD were also observed to create heritable changes stabilized across several generations of cell growth.  Heritability is key as it allows information to be recorded and passed along from one generation to another.  The direct response of an organism to environmental changes can be retained using prion memory behavior, thus allowing faster paced phenotypic change.  With our environment changing more rapidly than ever, prions and their molecular memory may be essential for species survival (Figure 2).
Figure 2: Observed changes in flowering onset across the United States. Data from the Environmental Protection Agency marks advances and lapses in plant bloom time over two 9 year periods.  Sustained differences in bloom time may indicate the role of a heritable, molecular memory mechanism for this trait. 
The discovery of a plant protein with prion behavior is entirely new to the field of prion research.  The LD protein, which is part of the flowering pathway, clearly shows prion-like behavior in both structure and function.  The next step is to look for an association between changes in flowering patterns and LD structural changes to understand the role this PrD has in the Arabidopsis life cycle.  Plant memory mechanisms are still largely unknown, so these findings may help to explain how external stimuli can impact the chromatin in certain ways, following certain events.  This field therefore holds a great deal of potential for molecular biologists and conservation research focusing on phenotypic plant variation.

Chakrabortee, Sohini, Can Kayatekin, Greg A. Newby, Marc L. Mendillo, Alex Lancaster, and Susan Lindquist. "Luminidependens (LD) is an Arabidopsis protein with prion behavior." Proceedings of the National Academy of Sciences (2016): 201604478. 

Additional Sources:
Cai, Xin, Jueqi Chen, Hui Xu, Siqi Liu, Qiu-Xing Jiang, Randal Halfmann, and Zhijian J. Chen. "Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation." Cell 156.6 (2014): 1207-1222.

Newby, Gregory A., and Susan Lindquist. "Blessings in disguise: biological benefits of prion-like mechanisms." Trends in cell biology 23.6 (2013): 251-259.

Shorter, James, and Susan Lindquist. "Prions as adaptive conduits of memory and inheritance." Nature Reviews Genetics 6.6 (2005): 435-450.

Figure 1 - Data taken from figure 2A (Chakrabortee et al., 2016)

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.

  1. About Coronavirus. CDC. August 22, 2016. http://www.cdc.gov/coronavirus/about/
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  3. Jeffers SA, Tusell SM, Gillim-Ross L, Hemmila EM, Achenbach JE, Babcock GJ, Thomas WD, Jr, Thackray LB, Young MD, Mason RJ, Ambrosino DM, Wentworth DE, Demartini JC, Holmes KV. 2004. CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A. 101: 15748-15753. http://dx.doi.org/10.1073/pnas.0403812101.
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Understanding how viruses evade and deceive the immune system

We have all experienced firsthand the negative effects viruses can inflict on the human body at one point or another, but we rarely think about how our bodies react to and clear these viruses from our systems. The cells which are part of our immune system have paired receptors which control the intensity of our immune response to viruses in real time. There are two main types of receptors on our immune cells, activating receptors and inhibiting receptors, which increase and decrease the strength of our immune response respectively. This allows our bodies to respond to threats such as viral infection at an appropriate level and minimizes collateral damage to cells not involved in the immune response, which can become inflamed or damaged if the immune response is too strong. These receptor pairs also allow us to halt our immune response when the infectious threat has been ousted. Many prevalent viruses, however, have adapted to develop mechanisms which interrupt and confuse our immune response. Viruses are concerned mainly with the inhibiting receptors on our immune cells due to their potential to significantly hinder our body’s response to infection. As viruses have mutated, many have developed the ability to indirectly exploit the signalling capability of of these receptors, effectively persuading our immune system that there is no threat even as they infect and propagate.
So how exactly do viruses hijack and halt our antiviral response? The answer to this question varies as much as viruses themselves, but the cell types viruses target are consistent. Viruses interfere with Natural Killer (NK) cells and T cells, two of the major types of immune cells. NK cells, a type of white blood cell, have the potential to target cells to induce apoptosis - the programmed destruction of a cell - but only target cells which have viral proteins or cellular stress-induced proteins expressed on their surface, which bind to the activating receptors of the NK cell.
An NK Cell
NK cells also have inhibitory receptors; these receptors bind to molecules called Major Histocompatibility Complex I (MHC-I) molecules, which protects surrounding cells from immune response-related inflammation and damage and prevents NK cells from inducing programmed cell-destruction. However, this binding process also heightens the responsiveness of NK cells, which means that viruses must walk a thin line between expressing enough MHC-I to prevent the destruction of the infected cells they are replicating within, but not expressing so much that the NK cells become hyper-responsive and aware of the virus’s presence.

A T Cell (in white)
T cells are similar to NK cells in that they are also a type of white blood cell; however, T cells mature in the thymus and can be identified by the T-cell receptors on their surface, which can recognize antigen fragments which are bound to MHC molecules.
Viruses interfere with the function of our immune cells and dampen antiviral response within our bodies in a number of ways. They can interact with inhibitory receptors on immune cells to downregulate antiviral activity, evade NK cell-based immune responses, and influence inhibitory signaling to impair T cell activation and cause T cell exhaustion, leading to the formation of viral reservoirs (a type of cell or anatomical location where the virus consistently accumulates and replicates with increased stability) and establishment of persistent infection (Blankson et al, 2002).
A number of viruses interact with inhibitory receptors on immune cells to prevent regulatory function of the immune cell. Dengue virus (DENV), for example, interacts with an inhibitory receptor found on a number of immune cells such as NK and T cells. This results in a decrease in function of interferon-stimulated genes within these cells which code for proteins which help the cell halt viral replication. Viruses such as HIV-1 also engage inhibitory signalling on immune cells in order to affect their response to the presence of entities which are deadly to cells, such as viruses. HIV-1, for example, causes the expression of a receptor protein known as DCIR on CD4+ T cells. DCIR suppresses the creation of certain interferons - signalling proteins which regulate immune activity - within the T cell, which allows HIV-1 to replicate without interference from immune cells. DCIR’s presence can also lead to the release of a protein which induces programmed cell death (known as apoptosis) in CD4 T cells. As these immune cells are destroyed, the body’s immune response to the infection is weakened.
Figure 1: Methods by which viruses manipulate inhibitory receptor signaling in order to avoid immune response
Other viruses have adapted to specifically avoid the immune response of our Natural Killer cells. Human cytomegalovirus (HCMV), for example, infects cells and expresses a protein called UL18 which is a homologue of MHC-I. This means that, for all intents and purposes, UL18 appears the same as MHC-I to our NK cells. However, NK cells bind to UL18 at a rate 1000 times greater than they bind to MHC-I cells, meaning that HCMV has a powerful influence over NK cell activity when it expresses this protein. When UL18 binds to NK cells, it inhibits their ability to influence programmed destruction of infected cells, allowing HCMV to replicate safely. Epstein Barr virus (EBV), the most well-known cause of mononucleosis, takes a similar approach to downregulating NK cell activity. EBV expresses a protein called EBNA-3A. This protein binds a human leukocyte antigen, HLA-A, which is a type of MHC-1 molecule. This binding forming a complex which is recognized by an NK cell inhibitory receptor, downregulating the response of the NK cell. Viruses such as HIV-1 don’t just control the level of MHC-I levels present on cells, but can influence the type of epitope - the part of the cell that the NK-cell binds to to influence its response, which suggests that viruses are developing alternative methods to thwart our body’s defenses.
A major benefit viruses receive from manipulating inhibitory signaling is the establishment of viral persistence: preventing the virus from being cleared from the body and developing chronic infection. CD4 and CD8 positive T cell responses are necessary to clear a virus from the human body, and a number of viruses have developed mechanisms to inhibit these responses. For example, LILRB1, a T cell inhibiting receptor protein which binds HCMV glycoprotein UL18, is expressed to a greater degree on CD8+ cytotoxic T cells in individuals with persistent HCMV infections; this exacerbates downregulation of T cell activity within the infected host and allows the virus to remain and replicate for extended periods. Additionally, during Hepatitis C virus (HCV) infection, a HCV core protein causes a protein called PD-L1 to be expressed. PD-L1 binds a T cell protein called PD-1 which inhibits the T cell’s activity. This exhausts T cells, causing them to progressively lose their function and preventing them from mediating viral infection (Yi et al, 2010).
We know a good deal about how viruses interrupt and influence cell response, but how is this information useful to us? Uncovering the mechanics of antiviral suppression is the first step in developing drugs which prevent viruses from inhibiting our immune response. If we know, for example, that a particular virus encodes a higher-affinity homolog of a protein which binds to NK cells and downregulates their activity, a drug could be developed that represses expression of this viral gene or mutates it to render the homolog ineffective. If we understand the signalling pathway or receptor which a viral protein tampers with, we can begin to develop counter-inhibitory vaccines which may prevent the virus from interacting with host inhibitory pathways and evading innate antiviral response.

Ong EZ, Chan KR, Ooi EE (2016) Vira Manipulation of Host Inhibitory Receptor Signaling for Immune Evasion. PLoS Pathog 12(9): e1005776. doi:10.1371/journal.ppat.1005776

Additional Sources

Picture Sources:
  1. Figure 1, from paper