The innate immune response provides the first line of defense against viral pathogens. Innate immune activation occurs in an antigen-independent fashion and relies on the ability of the host to recognize pathogens through specific pattern recognition receptors (e.g. Toll-like receptors, RIG-I-like receptors, NOD-like receptors). Engagement of these molecules activates signaling pathways that lead to the production of cytokines, chemokines, and interferons (IFNs), the latter of which bind their cognate receptors, signal through the JAK-STAT pathway, and transcriptionally induce hundreds of interferon-stimulated genes (ISGs). Type I (IFNalpha/beta) and type III (IL28A, IL28B, IL29) IFNs are often considered the antiviral classes, although type II IFN (IFNgamma) also has well-described antiviral properties. Each of these IFNs induces a unique and partially overlapping set of ISGs. In addition, some ISGs are directly induced by viral infection in the absence of IFN production. This leads to ISG induction through multiple mechanisms that are intersecting and often self-reinforcing.
The first ISGs were discovered more than 25 years ago, and the past decade has seen genome-scale cataloging of these molecules. Depending on cell type, IFN dose, and time of treatment, microarray studies identify 50-1000 ISGs, with 200-500 genes typical of many cell types. Insight into the effector functions of ISG-encoded proteins, however, has been limited primarily to a handful of molecules, including the "classical ISGs" PKR, MX1, OAS1 and more recently effectors such as APOBEC3G, TRIM5, ZAP, ISG15, ADAR, IFITM1/2/3, tetherin, and viperin.
Major outstanding questions of interest are:
- Which ISGs, among the many hundreds, have antiviral activity?
- Which virus(es) do antiviral ISGs target?
- What are the mechanisms of action of antiviral effectors?
High throughput screens to identify novel antiviral ISGs
We recently established a platform to rapidly screen a panel of more than 350 ISGs for antiviral activity. The screen relies on lentiviral delivery of a bicistronic mRNA consisting of an ISG and the red fluorescent protein, TagRFP. ISG-expressing cells are challenged with a green fluorescent protein (GFP)-expressing virus and replication is quantified by high-throughput FACS. We have used this screening technology to uncover antiviral ISGs targeting a panel of more than 20 human and animal viruses, including: hepatitis C virus, HIV-1, West Nile virus, chikungunya virus, yellow fever virus, dengue virues, influenza A virus, measles virus, respiratory syncytial virus, vaccinia virus, and many others. We have an ongoing interest in screening the ISG library against additional viruses, re-screening previously screened viruses in unique cellular environments, and extending the ISG screens to other non-viral pathogens.
Mechanisms of action of IFN effectors
We have uncovered a number of interesting interferon effectors, some of which target multiple viruses and others that exhibit more restricted specificity. We are interested in uncovering their antiviral mechanism of action from the perspective of the virus and the host.
Using molecular virological tools, we can dissect where in the viral life cycle a particular antiviral ISG is acting. For example, some of the IFN effectors targeting hepatitis C virus inhibit translation of the incoming viral RNA genome, while others target genome amplification. Theortically, an ISG could target other life cycle steps, including entry, uncoating, assembly, and egress. We have the techniques in place to study each of these stages, depending on the virus.
With respect to the host, we are interested in uncovering cell biological, biochemical, and genetic mechanisms of ISG action. This includes determining subcellular localization, interacting partners, and functional protein domains to help uncover antiviral mechanisms. Some ISGs are putative transcription factors, and we use genomics approaches to characterize their transcriptional targets.
Lastly, our ISG screens also uncovered a small, but very interesting, number of ISGs that enhanced virus replication. These particular effectors are largely uncharacterized in the literature and we hypothesize that they may be host factors that are hijacked during viral infections to promote the virus' own agenda.