Sunday, 16 February 2014

Interferons - they're a cGAS (i)

I'm a typical virologist, I am always talking about interferons. A while ago,  under a slightly different guise, I wrote about the biological function of interferon proteins in stimulating the expression of myriad genes, many of which have proven antiviral activity. The fact that these genes and their protein products have antiviral functions makes them extremely interesting to researchers, like me, looking for new ways to treat human and animal infections with these viruses. Or even to anyone really who is interested in viral disease, evolution and medicine. These interferon stimulated proteins are especially interesting for studying viruses for which no vaccine exists. I'm thinking HIV, hepatitis C virus and even emerging infections like Ebola and rabies viruses. And until we develop good vaccines against these agents we're probably going to need antivirals. Note that even if we did have vaccines for these viruses they might not be economically viable to use and so we're back to making antivirals. Either that or we just screw those affected by the viruses. That's not going to happen.

Here's the issue though. Problem is, it takes years and years (major understatement) of research for humans to generate new antiviral drugs. So what if evolution has done the hard work for us? This is where the interferon proteins and their antiviral effectors come in. Turns out, evolution has done the hardwork for us. And this is where this paper, first author John Schoggins, with a host of other authors (many of which also carried out the experimental work) who worked in many labs, mainly across the US. Have a look at the paper for a list. These guys, along with an early paper featuring Sam Wilson and others (see my blog post linked to above), are pioneering the exploration of the - brace yourselves - the 'interferome' with a hope of generating novel antiviral drugs. My words not theirs. 

Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity (not open access, sorry)

Schoggins et al. 2013

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors123. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses45678910111213. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Now I have to come clean. This is now the area of science in which I find myself now operating so I might be a tad biased - or over zealous - about it's benefits. But the use interferon proteins as antiviral and immuno modulating drugs supports my claim. Although I cannot currently think of any drugs that have come from studying interferon induced proteins (apart from interferons themselves). But this is probably because our knowledge of them is currently so limited. Hence the current research focus. 

So now for the science (see the paper abstract above). You can read all about interferon proteins (IFNs) here as well as IFN stimulated genes (ISGs). Trust me, they are fascinating. Earlier work showed that IFNs, which themselves are induced by infection, induce the expression of hundreds of ISGs inside a cell. Each one of these ISGs work in concert and induce an antiviral state within the cell and surrounding cells in a given tissue, which functions to prevent or slow down the infection. And perhaps counter intuitively some event promoted infection when looked at independently of the other ISGs (hence probably an artefact). Each one of these genes might represent a novel target to tackle infectious diseases. For example: Maybe we could synthesise a recombinant protein version of these ISGs? or we could find a way to upregulate the expression of the ISG? or we could use it as a intellectual and physical scaffold to develop novel, next generation drugs based on ISGs? The list is endless. But now I'm getting a head of myself. 

To determine  if a gene or protein has antiviral or pro viral activity (which as I've explained above, is important for developing new drugs), these guys over expressed each ISG inside mammalian cells them infected a range of recombinant viruses expressing reporter genes, like GFP. Now two things make this particularly artificial: 1) the majority of cells used were deficient in STAT1 (an important IFN signalling molecule). This was probably done to limit the activity of each ISG to its direct antiviral effects and exclude any feedback loops operating. 2) the screen was carried using retroviral introduction and over-expression the genes. The fact that you are using viral infection to test antiviral function might screw with your results, whereby the very act of infecting could stimulate an IFN response. When you read the paper this was actually the case but all in all, this method of screening appears to work well, but of course requires much downstream work validating and characterising any 'hits'.

This screen allowed them to 'easily' prefer on a screen for good candidate ISGs for further characterisation. This screen importantly by using a range of biologically diverse viruses allowed them to find an ISG that inhibited a broad range of viruses. I say easily because on paper it might seem easy but I really don't think it is. It is a lot of work and each virus used comes with its own idiosyncrasies (e.g. cytopathic effects), which can be difficult to control for. But trying to find a broad antiviral is a bit like finding the holy grail of ISG (and indeed antiviral) research. Finding a broad spectrum antiviral (or antibiotic) is important not just on an economical basis but on a basis of rapidly developing new treatment for pathogens for which we have no information on. And as the paper describes, Schoggins and colleagues claim to have found just that.

After screening hundreds of these ISGs against a panel of viruses, Schoggins et al. focussed on only one, which is called cGAS. The reason why they picked this one is that through bioinformatic analsysis it appeared to have the same effect when over-expressed as IRF1. IRF1 is a very important molecule in the IFN pathway acting as a transcription factor up-regulating the expression of the ISGs and if cGAS clustered with IRF1, then maybe cGAS also had a very important role in antiviral immunity. At the time, cGAS was poorly characterised, thus the group focussed on cGAS as a broad spectrum antiviral. However, note that although they refer to cGAS as 'pan-viral', cGAS appears to predominantly inhibit positive sense RNA viruses as opposed to negative sense RNA viruses. The screened viruses also mainly included enveloped viruses, rather than non-enveloped viruses, which again might skew the results. For a mechanistic understanding of cGAS and antiviral immunity keep posted. Part (ii) will be up soon. But for now, be content that a 'broad spectrum' antiviral was discovered.

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