Your liver as a viral filter - who, what, where, why?

The liver is a pretty amazing organ (see structure above) - especially when you take into account its often unrecognised immunological role. It's becoming more established that we can kind of think of the liver as a giant biological filter where it extracts all sorts of harmful molecules and pathogens from our blood. After all, it is a major component of the reticuloendothelial system - your bodies natural filtering system. From the minute any microbe reaches your blood stream, it is instantly taken to the liver on the back of our circulatory system. And, within an hour, nearly 100% of those pathogens are removed (see graph below) but what is doing this inside your liver? 

But, while this all sounds like a pretty good idea, removing those nasty viruses - when we move into the administration of viral gene therapy vectors, what we don't want is the liver to remove those helpful microbes. And it's becoming a pretty big research project, trying to engineer these vectors to evade our liver.

The system isn't perfect but how exactly does it work? - and if we understand how it works, can we subvert it?

26/09/11 Virology journal club #1 - is nucleolin the RSV receptor?

This week's journal club (Monday 26/09/2011) - the first of the coming academic year - was on the recently published paper identifying the molecule nucleolin as a functional receptor for respiratory synycitial virus (RSV) - a virus who's receptor has proved elusive to scientists; it was taken by a post-doc in our lab: Olivier Touzelot. This Nature Medicine study (get the paper here) was released in mid-August 2011 by a group at the The Hospital for Sick Children, Toronto, Ontario, Canada.

Abstract:

Human respiratory syncytial virus (RSV) causes a large burden of disease worldwide¹. There is no effective vaccine or therapy, and the use of passive immunoprophylaxis with RSV-specific antibodies is limited to high-risk patients^{2, 3, 4, 5}. The cellular receptor (or receptors) required for viral entry and replication has yet to be described; its identification will improve understanding of the pathogenesis of infection and provide a target for the development of novel antiviral interventions. Here we show that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro. We observed decreased RSV infection in vitro in neutralization experiments using nucleolin-specific antibodies before viral inoculation, in competition experiments in which virus was incubated with soluble nucleolin before inoculation of cells, and upon RNA interference (RNAi) to silence cellular nucleolin expression. Transfection of nonpermissive Spodoptera frugiperda Sf9 insect cells with human nucleolin conferred susceptibility to RSV infection. RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice (P = 0.0004), confirming that nucleolin is a functional RSV receptor in vivo.

RSV virus and nucleolin (Ncl) co-localisation in airway epithelial cells. Notice a lot of RSV without Ncl - yet they are both found in the same general distribution? Is something else playing a part, possiblu mediating the interaction between the two?

In our discussion, a number of points were raised regarding this paper and it's results:

• Why exactly was nucleolin focused on? If you look at the supplementary figures, a lot of proteins could have bound to the virus - especially as they were previously narrowing their search down cell surface proteins - what was wrong with the other humans ones (which had higher hits than nucleolin). 
• Sometimes data shown can be too good. Comments were given that in some of the figures (the vopa ones especially), the blots looked very neat with no spurious bands. Apparently this techniques has been carried out a lot before without so near as much luck as this case. The paper didn't say how theirs worked while others did not. Maybe these guys are just better at it than others...
• Their co-localisation data wasn't very convincing - a lot of RSV was seen attached to the cells without nucleolin - although, this might reflect the nature of nucleolin movement within the cell. And, in the mouse tissue - a lot of nucleolin positive staining was seen without viral infection.
• When transfecting in the nucleolin gene inside non-permissive insect cells - not a lot of protein was produced, and it was had a very strange distribution being only found in one single small area of the cell. Yet, this allowed near 100% infection by the virus. Maybe this does reflect how the receptor behaves naturally however - which may explain why only a little is required (also may explain the strange co-localisation data).
• Finally, this paper highlights how difficult it is to work with a gene/protein that is so necessary for cell-viability - how can you easily knock-down a gene that is expressed very highly in every cell and retain viability?

 While we all agreed it was a pretty good paper - even if all this data was taken as correct, it would still not explain the entire story of RSV infection of a cell, despite being completely effective in allowing insect cells to be infected. As nucleolin is expressed in every cell, you would expect RSV to infect all tissues of a human, but it only infects the respiratory epithelium. However, this may be an effect of viral release, with virus particles only being released into the space of the lung. Clearly this work needs repeated in a more appropriate model system.

Can we give natural selection a little boost...

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Can we give natural selection a little boost? On the way to breeding fungus-resistant frogs - Identify MHC alleles that confer protection and immunity - then breed. Sound like a good idea? Frog killer immune genes revealed Scientists take a big step toward understanding why some frogs are able to survive the fungal disease chytridiomycosis while others quickly die. View or comment on Connor Bamford's post »

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Targeting antivirals to the host, part 2 - success?

Viral pneumonia (from influenza possibly) results from an accumulation of immune cells and fluid in the lung tissue. But how can we stop it?

  In the northern hemisphere it's coming up to 'flu season - the time when many of us will be getting our yearly influenza vaccinations. In the coming months during the thousands of potential viral infections, some form of disease/clinical symptoms may set in (maybe not if you're vaccinated) - think of that headache, cough, runny nose, etc.  We can think of these as a function of both the virus replicating and your response (i.e the host) to that same infection.  And, in infections such as influenza, the ramping up of the cellular immune response in an organ like the lung is responsible for much of the clinical course of the disease (the pneumonia - see above).

  The strange thing is that a lot of our therapies are only targeted to the virus aspect (antivirals) of this: blocking particular enzymes, receptor binding molecules etc. But, what if we could target the host response? And through doing this eliminate disease, while potentially allowing the virus to infect, replicate and ultimately use us without inflicting damage. 

How dangerous are viral quasispecies?

Chikungunya virus particles emerging from an infected cell - is genetic diversity important to this virus?

  

   At around 1 mutation per 1,000 - 100,000 nucleotides per round of replication, RNA viruses have the highest mutation rate of anything seen in nature to date. During an infection of a single cell, thousands of new genomes are produced that will go on to make new virus particles; each genome will differ from another at most maybe 10 nucleotides (given an average virus of 10 kilobases in length). 

Uncovering a missing link in viral evolution - how did some get so big?

Vietnamese jungle.

Credit: FLETCHER & BAYLIS/SCIENCE PHOTO LIBRARY

  Out of the dense, tropical rainforest of Northern Vietnam, researchers have discovered a missing link in viral evolution. Through the large-scale screening of trapped mosquitoes, a joint Dutch and Japanese group may have identified the secrets of how one group of viruses - the nidoviruses - got really big. 

  This work potentially answers one of the more prevailing mysteries in viral evolution: how can RNA viruses escape the evolutionary restrictions placed on them by their very high mutation rates and get more complex? If you look closer though, more questions are thrown back than are answered.

  The positive-sense RNA viruses are an extremely large and diverse group of viruses, housing many known - and unknown - human pathogens and indeed many non-pathogenic, environmentally influential microbes. On the whole, these viruses don't get very big (see the graph below); having a genome made of RNA isn't particularly a good thing if you want to have a long genome, encoding lots of complex genes. The enzymes these viruses use to copy their genomes are nowhere near as accurate as their cellular counterparts. And, introducing mutations every few thousands nucleotides is bound to impact on your evolutionary potential and fitness.

Transgenic cats shed (green) light on HIV immunity - but is it any use?

Transgenic - GFP cats

There are currently two AIDS pandemics raging across the world: the well-known one in humans and the less recognised one in domestic cats. Feline AIDS, caused by the feline immunodeficiency virus (FIV) - a close relative to HIV - is pretty similar to that afflicting millions of humans and could potentially be used as a model system for studying HIV infection and disease. One major scientific barrier to HIV research in primates is the difficulty in generating transgenic animals efficiently. This, however, could be sidestepped through employing FIV/domestic cat as the experimental system.

The 9,000 year-long relationship between the human species and domestic cats has brought a lot to each respective species: we get rid of nasty pests and enjoy the whole 'cat experience' while they feast on the human-associated rodents and gain some shelter and people love. But what some people might not realise is that the domestic cat has - and will probably continue - to help us in biomedical research. And, for them, the increased research into cat biology may also spill over into non-domestic species, who are on the brink of extinction and might be aided by this work.

Mendeley Virology group

Mendeley - (get it here) the relatively recently released .pdf manager for both desktop and onlin archiving also allows you to start online social groups where any participant can upload a paper or a link to be viewed by anyone else (see their blog post).

Obviously, you can see how this could vastly speed up the transmission of new ideas around the world via this kind of global scientist (and non-scientist alike) network. But what it also lets you do is comment - and discuss -  the paper or even just update each other on how they are getting on.

For example: see the synthetic biology group

The ability for anyone (provided you sign-up to Mendeley, which is very easy, fast and extremely useful) to rapidly upload scientific papers may allow for greater understanding, post-peer review discussion of papers and the ability to generate a 'community-driven' bank of literature on a particular subject. It is also a great way to collaborate scientifically on a project.


So, lets see how it works for virology, join the Mendeley group (link to the right of my blog). Upload a paper and comment on it.

Molecular virology is a group in Biological Sciences on Mendeley.

What explains HIV -induced pain syndrome?

Does HIV-1 Tat protein induce pain in HIV/AIDS sufferers? Notice a Tat induced increase in activity when compared to control or inactivated protein. (Taken from: Chi, et al, 2011)

Pain is an extremely complicated symptom - just see the TED talk below on chronic pain, yet of the 33.4 million people currently living with HIV/AIDS worldwide, 90% will experience peripheral neuropathy - otherwise known as pain. This a general unrecognised and under-diagnosed (and under-treated) outcome of HIV infection, yet the reasons how and why the viral infection induces this are unknown, but probably are pretty complex.

Dengue versus the epigenome

Dengue virus (DENV) particles. Does dengue interact with the host epigenome? And, why?

Epigenetic modification of chromosomal structure has the ability to rapidly and stably alter gene expression and function within cells, tissues and whole organisms. And, changes have been found to induce a number of diseases in humans, such as cancer and others.

The ability of infecting viruses to influence this process has now been realized with a number of large DNA viruses being shown to take advantage of host epigenetic modifications, including: the histone proteins that are necessary for the structure and modulation of chromatin.

But what hasn't been seen is whether RNA viruses, which replicate within the cytoplasm are able to at least interact with the host chromosome structures. And, more importantly, why would they do so? Researchers have just published evidence suggesting that dengue virus interacts directly with host histone proteins and requires such activity for replication (see PLoS paper here). The reasons why are not understood.

DENV-C specific histone binding (binds to H2A, B, 3 and 4) but not GFP. Does this has an affect on host function?

Dengue Virus (DENV) is a small, single-stranded, positive-sensed RNA virus and belongs within the Flavivirus genus, alongside yellow fever virus and west Nile fever virus. It is spread between humans via the bite of the female Aedes mosquito and can lead to the development of very high fever (41 degrees Celsius), headache and in some cases, a lethal hemorrhagic fever.

It is estimated that two-fifths of the worlds' population are at risk of DENV infection and it is endemic in over 100 countries within tropical or sub-tropical climates. There is currently no clinically proven treatment for dengue fever nor are there any vaccines available, making any research into the basic biology of the virus all the more important.

Using a 'tagged' protein, this group showed that a recombinant DENV C protein (capsid protein forming the outer layers of the virus particle - yellow in the above picture) interacts with the four core histones, H2A, H2B, H3 and H4 found within the nucleus of huh-7 cells, a human hepatocellular carcinoma cell line used a lot in DENV research. See the figure above for the specific binding. DENV is known to infect human liver cells, especially in fatal cases.

GFP-tagged DENV-C co-localizes with huh-7 nuclear and cytoplasmic histones (H2A and H2B)

When over-expressed within these cells, a GFP-tagged C localised to the same areas of the nucleus as each of the histones did and this was extended to DENV-infected cells (see above). C also interacted with the histones while in the cytoplasm as well. In vitro C and histones formed dimers together in the absence of nucleic acids and it was subsequently shown to bind DNA with - or without - histone proteins. See below.
 
This interaction was shown to disrupt the normal oligomerization of histones but not that of histone-DNA binding. Infection up-regulated the expression of the core histones and was essential for normal DENV replication.

DENV-C binds to huh-7 H2A, B, 3 and 4 and causes formation of oligomers (the bands higher up on the gel - compared to lower and smaller bands).

The physical basis of this interaction is currently not understood but is believed to result from the structural similarities between the histones and DENV-C. A common folding pattern - important for oligermization - is shared by both and the authors state they are in the process of crystallizing the two together. The biological significance of this observation was not really addressed here, apart from the fact that histones were required for DENV replication (see graph below). The authors hypothesize the reasons as to why DENV might need this process, but provide no evidence to back it up.

DENV replication requires H2A and H3 proteins. Shown here by siRNA knockowns of the two (columns 1 - 4) when compared to non-specific controls (5 and 6).

It would be interesting to see if this interaction applied to other human cells, including primary cell lines and maybe in vivo. And, if it extended to mosquito cells as well? But, where in the genome does DENV-C bind? Is it specific or more general? What is the basis of the requirement for these histone proteins? Finally, is it possible to inhibit this relationship to develop new anti-DENV therapies.


Colpitts, T., Barthel, S., Wang, P., & Fikrig, E. (2011). Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells PLoS ONE, 6 (9) DOI: 10.1371/journal.pone.0024365