#openaccess: Exploitive prions versus your innocent immune system

Prion protein in red on dendritic cell beside neuron (green)

This blog focuses on trying to understand how viruses cause disease in their hosts - whether they be single cells or us, humans. Attempting to do so means that we must look at how viruses enter these hosts, survive within the hostile environment that is another organism and eventually make their way on to infect the next one. One thing this blog doesn't do, is look at how other kinds of pathogens complete this complex life cycle.

I'm going to change that now.

I'm going to take a look at prions - the dangerous proteins behind the fatal brain disorders such as: Kuru, mad-cow disease and scrapie, also known as 'Transmissible spongiform encephalopathies' or TSE's.

A paper published last week in PLoS Pathogens really highlights an issue that comes up a fair bit in this blog, that is: how pathogens - in this case prion proteins - exploit your immune system to promote their own survival. And in particular, how these molecular parasites make use of one cell: the dendritic cell. This work comes from the Medical Research Council's Prion Unit in the UK.

HIV restriction factor blocks respiratory viruses - but not how we thought

Measles virus - is it targeted by an HIV restriction factor?

Cellular organisms have evolved multiple defences to keep viruses and other genetic parasites at bay. One such shield is the development of an advanced adaptive immune system seen in vertebrates while another is the more evolutionary widespread 'innate' system made up of various expressed proteins and small RNAs. These molecules prevent particular stages of the viral replication cycle like entry, replication or exit. 

Another mechanism is to mutate the virus out of existence, i.e. change nucleotides throughout the viral genome so much so as to effectively knock-out that protein's function during infection and slow down replication so much so to allow your bodies' other defences to clear it. Since 2002 we have had tantalising clues that this is functioning during a viral infection, particular HIV and other retroviruses:

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.

Influenza - putting the Trojan into the horse but should you open it?

A trojan horse (dendritic cell) filled with virus

Inspired by a recent journal club article:

A number of pathogens infect via one organ but are able to move to another. Think of the likes of the initial HIV infection of cells within the reproductive tract followed by its transfer to the immune system (see my earlier post here). This transfer of viruses, bacteria or other parasites is responsible for the induction of an effective immune response but also it can lead to some of the more serious disease symptoms during infection, but how is it controlled?

Is there such a thing as a completely broad-spectrum antiviral?

I'm sure everyone is aware of the kind of effects virus infection and replication has on the health of humans and other animals (just scroll along my last blog posts and you'll see). 

It's really not good.

And, in most cases we don't have much to prevent or cure it: maybe a vaccine here, some antivirals there yet what we really would like is something that would act against ALL kinds of viruses, from influenzas to smallpox to ebola and even HIV. Most vaccines and antivirals target a very limited number of pathogens, usually only one. The search for this class of antiviral drugs has eluded us in the past yet a paper published this week in PLoS ONE reports (read paper here) the identification of an effective strategy termed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer or DRACO. See this MIT group's website here.

dsRNA - an important hallmark of viral infection

This approach takes advantage of chiefly two natural processes that occur within cells: the intracellular sensing of long double-stranded RNA molecules and the initiation of programmed cell death, known as apoptosis via what are called as caspase enzymes. Through linking these two pathways together via the construction of a novel protein molecule, this group aim to inhibit viral replication - and hence act as a broad-range antiviral as most known viruses generate dsRNAs.

How and why do cells 'see' ds-RNA?

ds-RNA binding domain from PKR protein

Our cells - and those of nearly every other organism - have a range of defenses to protect themselves from viral infection. One being, the ability to detect when they have been initially infected. For example: through the binding of double- stranded RNA molecules, which viruses produce a lot of. Think of it as a hallmark of virus replication and our cells naturally come equipped with a range of these detector modules that carry out this function using a double-stranded RNA binding motif found somewhere in their structure. Detection of ds-RNA sets off a serious of downstream reactions leading to the cell's normal antiviral response.

Examples:

PKR

RIG-1

MDA-5

TLR 3

ADAR

RNase L

What about apoptosis?

Apoptosis is a highly coordinated process leading to the self-destruction of the cell without any of the nasty inflammatory responses. The main players in this cellular suicide are the caspases - protease enzymes that specifically target and degrade hundreds of other proteins inside the cell in a well-defined cascade of activity. These proteins are activated through the construction of a larger complexes which themselves are produced as a response to certain noxious stimuli, one example is virus infection. One domain are the death effector domains (an example is a FADD domain) that recruit the initiator caspases into the death-induced signalling complex (DISC) thereby kick-starting the whole process of apoptosis and the cells' death.

The FADD death effector domain for example was added to the end of the ds-RNA binding protein

I said above that virus infection itself activates apoptosis so why doesn't every virus-infected cell just die? Well, viruses have evolved neat tricks to circumvent this response as in most circumstances the death of the cell leads to the end of the infection. The hypothesis that this group are working under is that what if we could couple these two processes more-so than they are naturally as to sidestep the viruses' strategies at combating apoptosis, in a way, short-circuiting this whole pathway. Instead of going through long networks of protein-protein interactions, make it rely only on a single binding. 

So what did they do?

These DRACO proteins are chimeras of the two classes of molecules described above: one half ds-RNA binding and the other a death effector domain. And, finally, in order to get this protein into the cell, they added a 'transduction tag' - a short peptide sequence that is naturally taken up inside cells. So, does it work?

They initially made a whole range of these proteins, using different RNA binding motifs combined with a number of dead effector domains (see below). These recombinant proteins were then tested to see which was the best at getting into cells, binding ds-RNA, triggering apoptosis and most importantly, which was the safest.

The chimeric DRACO proteins (transduction tag in yellow, ds-RNA binding in red and apoptosis in green and blue)

Then they show that these proteins can indeed get into cells and in the presence of ds-RNA they can initiate apoptosis - see below. Also, these proteins were generally well tolerated by these particular cells used.

But does this actually inhibit virus replication?

To test this, they first added their proteins to the cells and then infected them with a virus - in this case human rhinovirus 1B (an important human pathogen) - see data below. Virus infection without administration of DRACO's resulted in 0% cell viability, that is all the cells were killed by the virus. When the proteins were added, viability increased to about 80%. The authors suggest that this because the proteins kill off any virus-infected cells very early thus preventing infection from spreading any further. They also test how effective the strategy is when we add the proteins at different times. It works best when added before infection and then decreases substantially (when added 1 day after infection, viability dropped to 50%).

The antiviral effectiveness of 5 different DRACO proteins in an in vitro rhinovirus infection. The higher the cell viability the better the approach.

Nicely, they extended this same analysis to a very wide range of viruses: dengue, influenza, arenaviruses, adenoviruses, reoviruses - anything they could their hands on. And they all worked much the same as the rhinovirus example above. These viruses are incredibly different from each other, showing just how broadly active this approach is. Different cell lines were also investigated opening the possibility of this approach in a veterinary perspective.

We know that sometimes what works in cell culture doesn't necessarily work in an animal, so the group tried their system in a mouse model of lethal influenza virus infection. When administered prior to challenge, the DRACOs had great results 0 at most 80% of the mice survived, compared to 0 without the proteins. They didn't check out what would happen if they added the DRACOs after infection, which would more closely mimic the situation in real-life.

So, is there such a thing as a completely broad-spectrum antiviral? If there wasn't one before this, there certainly is now after these promising initial results. Although further work will have to establish the exact mechanism of this approach and also apply it to other viruses in more model systems.

To sum up with what the author's say:

DRACOs should be effective against numerous clinical and NIAID priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered. We have demonstrated that DRACOs are effective against viruses with DNA, dsRNA, positive-sense ssRNA, and negative-sense ssRNA genomes; enveloped and non-enveloped viruses; viruses that replicate in the cytoplasm and viruses that replicate in the nucleus; human, bat, and rodent viruses; and viruses that use a variety of cellular receptors

 
Rider, T., Zook, C., Boettcher, T., Wick, S., Pancoast, J., & Zusman, B. (2011). Broad-Spectrum Antiviral Therapeutics PLoS ONE, 6 (7) DOI: 10.1371/journal.pone.0022572

Interferons, interferons, interferons - what exactly DO they do?

   The interferon (IFN) signalling pathway acts as a primary defense against all viruses through the induction of expression of hundreds of genes following infection; the exact functions of each are, at best, poorly understood. In order to gain a better insight into the antiviral mechanism of the induced genes, Schoggins, et al. (2011) performed a sensitive high-throughput screen of the effects of each one on infection with a range of RNA viruses.

Structure of the IFN-alpha protein. http://www.wikipedia.com/



Abstract:

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago^{1, 2, 3}, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.

  
   The interferons are a multifunctional family of around 20 cell-signaling proteins that are secreted from cells following viral infection (as reviewed here). Our cells expend a lot of energy attempting to detect infection and following this, they express high concentrations of IFN proteins. Following secretion, they bind to receptors - and activate - nearby cells alerting them to the viral assault. 

   Through a complex signaling network a range of genes are actively expressed across the genome that alter the cell in such a way that it becomes harder for viruses to infect them. A rapid antiviral defence system is set-up within the host's tissues and organs. As shown in mice lacking STAT1 a key IFN signal mediator, this IFN signaling network is required to limit viral replication and disease yet also bide time for the development of an adaptive immune response. IFNs are extremely important in our fight against viruses. Only a handful of these IFN-stimulated genes (ISGs) have been characterised while hundreds still sit untouched.

 
What did they do?
 
   Using previously published gene expression data the group chose 389 ISGs for characterisation. To determine what function these ISGs have on virus replication, Schoggins, et al. developed an intracellular assay in which a retroviral vector expressing high levels of both the individual ISG alongside a red fluorescent protein was used to infect IFN pathway-deficient cells in vitro and  then 48 - 72 hours following ISG expression, these exact cells were again infected with a range of RNA viruses expressing a green-fluorescent protein (see figure below). Viruses used included: hepatitis C virus, HIV, yellow fever virus, west Nile virus, Venezuelan equine encephalitis virus and chikungunya virus. 

A) ISG/RFP-expressing retrovirus, B) experimental outline

   

Example results

  Using basic Fluorescent Activated Cell Sorting (FACs) they were able to sort the cells based upon what colour they were, for example: red and the ISG inhibited the virus; green and it did not. More importantly they were able to specifically quantify the levels of green and red to assess the exact levels of virus replication in the cell population. The strongest inhibitors were extensively validated. On top of being expressed individually, some ISGs were expressed in combination to determine whether their antiviral effects were additive. The group were finally able to pinpoint where in the virus replication cycle inhibition took place: entry, transcription, translation, replication or exit.

 The major findings included:

• Most ISGs inhibit virus replication

   As would be expected for an antiviral response, and one which has been shown to already inhibit a wide range of viruses, the majority of the tested ISGs inhibited replication to some degree across a range of viruses looked at. This is a good thing. This means your assay is doing what is supposed to do.

• There are two types of antiviral genes: modest and strong inhibitors

   Owing to how the IFN system has evolved, we may categorise the ISGs into two antiviral classes: modest and strong inhibitors. The modest ones act specifically, targeting limited aspects of the virus replication cycle; the stronger ones function as a positive feedback, increasing the expression of key IFN signaling genes

• Inhibition is additive - more ISGs equals more inhibition

   Upon the activation of the IFN pathway, hundreds of genes are upregulated resulting in an antiviral state within the cell. The defence system is not set up so that protection lies in the hands of a single gene/protein but in the hands of many - it is a truly cooperative process. In this system, following the expression of different combinations of ISGs together within a single cell, the author's noted that inhibition of viral replication increased. 

• Translational inhibition is the most common antiviral mechanism

   The group asked, using a number of assays, at what point do these ISG's inhibit hepatitis C virus replication - is it: entry or translation of HCV mRNA/genome. They found that, in this case, it was not entry but translation. Although, this effect is probably particular to positive-sense RNA viruses. 

• Some ISGs enhanced virus infection

   Interestingly, they found that following expression of a number of ISG's, virus replication increased. Something of a surprise for an antiviral pathway. The mechanisms of why/how these genes did this was not addressed but we can assume that in a real-life infection, the other 380-odd inhibitory genes also upregulated would cancel these out. 

HCV-induced hepatocellular carcinoma. WIll this IFN research aid in potential HCV antivirals? http://www.stanford.edu

Some caveats exist, however:

• Over-expression systems may not reflect in vivo situation

   The results from this work are extremely interesting, important and useful yet the way it was done may not accurately represent what happens when you are infected. Although, I feel that this was not what was set out to be determined by the investigators, which I think were stimulated by the need of novel antiviral candidates. This research will of course aid in that field through the identification of potentially life-saving targets. 

• Only a relatively small number of diverse viruses were screened

   The viruses tested in this system included only a relatively small sample of virus diversity - mainly focusing upon small positive sense RNA viruses (HCV etc.), and one retrovirus: HIV-1. We saw that a number of these ISG's inhibited these viruses but does this reflect what may happen with other viruses? What effect would they have on large DNA viruses, negative-sense RNA viruses or double-stranded RNA viruses? While the fact that the majority of the genes inhibited replication probably wouldn't change, the specifics most likely would. A number of these genes would specifically target pathways and systems that are preferentially used by these small positive sense RNA viruses while some would target those used by other viruses.This experiment would only 'see' those affecting these small positive sense RNA viruses. Although, this may not be a bad thing. These viruses are major causes of morbidity and mortality in both human and other animal populations worldwide.

• Doesn't take into consideration virus IFN modulation

   Every virus probably has different ways of inhibited the IFN response themselves before they are eliminated from the host following ISG expression. This system would not pick up of the majority of these modulatory mechanisms as they occur prior to the expression of ISGs. So, again, as I mentioned before, this work does not reflect what would happen during infection in vivo and nor does it really matter.

Schoggins, J., Wilson, S., Panis, M., Murphy, M., Jones, C., Bieniasz, P., & Rice, C. (2011). A diverse range of gene products are effectors of the type I interferon antiviral response Nature DOI: 10.1038/nature09907

Uncovering the biomedical and evolutionary importance of primate innate immunity

Primates such as chimpanzees differ in the diseases that affect them - especially when we compare them to those that affect us. Progression to AIDs, cancer incidence, Alzheimers disease and malaria either do not affect or cause less severe diseases in non-human primates than they do in humans. So why are our primate cousins so differently affected by these particular diseases than we are? What causes these differences? Are they environmentally mediated? Behavioural? Molecular?

What does the innate immune system have to say about it?

The pathogenesis of disease is an important area of research when trying to understand the origins and progression of diseases. It depends on many variables - host specific, environmental or pathogen specific. A recent publication has sought to understand these differences from the point of view of host factors mainly the innate immune system. By comparing genome-wide gene expression patterns in primary immune cells (monocytes) cultured in vitro from groups of humans, chimpanzees and rhesus macaques and applying bioinformatic analysis to the results, the investigators were able to detect those genes whose expression is altered upon stimulation with lipopolysacharide (LPS), an important activator of the innate immune system. From this data set they could ask, functionally, what makes the human immune system different?



3,170 genes were seen to be differentially expressed with 793 changing in all three species indicating a conserved function. Other genes showed species-specific changes allowing researchers to ask what makes each species unique? More importantly what makes non-human primates different when it comes to diseases? There were 335 genes in the human monocytes that were expressed differently and these were divided between particular pathways.

What does this data have to say about specific diseases?

Those genes listed as being involved in viral infection were those most likely to be species-specific indicating the rapid adaptation of host immunity to fast-evolving viral pathogens. The data sheds light on the possible lo incidence of cancer in non-human primates by detecting the difference in apoptosis/cancer related genes - although the signifigance of monocyte gene expression when considering the whole-organism in diseases such as cancer is difficult to say. Most interestingly seen are those genes involved in HIV infection and AIDs  whose expression may explain why chimpanzees do not progress to AIDs or do so slowly.

Despite the problems in culturing non-human primate immune cells in vitro and the difficulties in controlling for environmental effects (different diets of primates), this study goes some way to understanding at a functional level what makes humans human from an immunological point of view. Although focussing on a single cell type, the monocyte, other cells may prove useful in investigating innate differences - as other cells also function in innate immunity - mucosal epithelium for one. This work paves the way for more detailed molecular analysis but also of more genome-wide work looking at other cells, other activators and pathogens (not LPS but HIV?). Understanding what makes humans different in a pathogenic light should focus not just on immunology at the gene expression level but also differences in epigenomics, behaviour, anatomy and cell biology.

 

 

Varki, Aijit. 2000. A Chimpanzee Genome Project is a Biomedical Imperitive. Genome Res. 2000. 10: 1065-1070. doi: 10.1101/gr.10.8.1065

Barreiro, Luis B., John C. Marioni, Ran Blekhman, Matthew Stephens, and Yoav Gilad. 2010. Functional Comparison of Innate Immune Signaling Pathways in Primates. Ed. Greg Gibson PLoS Genetics 6, no. 12 (December): e1001249. doi:10.1371/journal.pgen.1001249. http://dx.plos.org/10.1371/journal.pgen.1001249.