Todd H. Rider*, Christina E. Zook, Tara L. Boettcher, Scott T. Wick, Jennifer S. Pancoast, Benjamin D. Zusman
Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, Massachusetts, United States of America
Abstract Top
Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO) that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and 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.
Citation: Rider TH, Zook CE, Boettcher TL, Wick ST, Pancoast JS, et al. (2011) Broad-Spectrum Antiviral Therapeutics. PLoS ONE 6(7): e22572. doi:10.1371/journal.pone.0022572
Editor: Suryaprakash Sambhara, Center for Disease Control and Prevention, United States of America
Received: May 20, 2011; Accepted: June 24, 2011; Published: July 27, 2011
Copyright: © 2011 Rider et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is funded by grant AI057159 (http://www.niaid.nih.gov/Pages/default.aspx) from the National Institute of Allergy and Infectious Diseases and the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, with previous funding from the Defense Advanced Research Projects Agency, Defense Threat Reduction Agency, and Director of Defense Research & Engineering. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the United States government.
Competing interests: THR is the inventor on patents and patent applications covering DRACOs: Rider TH (issued October 24, 2006) Anti-pathogen treatments. U.S. Patent 7,125,839; Rider TH (issued July 28, 2009) Anti-pathogen treatments. U.S. Patent 7,566,694; Rider TH (filed June 18, 2009) Anti-Pathogen Treatments. U.S. Patent Application 20100098680; Rider TH (filed February 7, 2003) Anti-Pathogen Treatments. European Patent Application 03716001.7; Rider TH (filed February 7, 2003) Anti-Pathogen Treatments. Canadian Patent Application 2,475,247; Rider TH (filed February 7, 2003) Anti-Pathogen Treatments. Patent Cooperation Treaty Serial No. US03/03978; Rider TH (filed February 7, 2003) Anti-Pathogen Treatments. Japanese Patent Application 2003565429; Rider TH (filed November 19, 2009) Anti-Pathogen Treatments. Japanese Patent Application 2009262426. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: thor@LL.MIT.EDU
Introduction Top
A serious threat is posed by viral pathogens, including clinical viruses (HIV, hepatitis viruses, etc.), natural emerging viruses (avian and swine influenza strains, SARS, etc.), and viruses relevant to potential bioterrorism (Ebola, smallpox, etc.). Unfortunately, there are relatively few prophylactics or therapeutics for these viruses, and most which do exist can be divided into three broad categories [1]–[3]: (1) Specific inhibitors of a virus-associated target (e.g., HIV protease inhibitors, RNAi) generally must be developed for each virus or viral strain, are prone to resistance if a virus mutates the drug target, are not immediately available for emerging or engineered viral threats, and can have unforeseen adverse effects. (2) Vaccines also require a new vaccine to be developed for each virus or viral strain, must be administered before or in some cases soon after exposure to be effective, are not immediately available for emerging or engineered viral threats, can have unforeseen adverse effects, and are difficult to produce for certain pathogens (e.g., HIV). (3) Interferons and other pro- or anti-inflammatories are less virus-specific, but still are only useful against certain viruses, and they can have serious adverse effects through their interactions with the immune and endocrine systems.
To overcome these shortcomings of existing approaches, we have developed and demonstrated a novel antiviral approach that is effective against a very broad spectrum of viruses, nontoxic in vitro and in vivo, and potentially suitable for either prophylactic or therapeutic administration. Our approach, which we call a Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO), is designed to selectively and rapidly kill virus-infected cells while not harming uninfected cells.
Our DRACO approach combines two natural cellular processes. The first process involves dsRNA detection in the interferon pathway. Most viruses have double- or single-stranded RNA (ssRNA) genomes and produce long dsRNA helices during transcription and replication; the remainder of viruses have DNA genomes and typically produce long dsRNA via symmetrical transcription [4]–[5]. In contrast, uninfected mammalian cells generally do not produce long dsRNA (greater than ~21–23 base pairs) [4]–[5]. Natural cellular defenses exploit this difference in order to detect and to attempt to counter viral infections [6]–[7]. For example, protein kinase R (PKR) contains an N-terminal domain with two dsRNA binding motifs (dsRBM 1 and 2) and a C-terminal kinase domain [8]–[9]. Binding of multiple PKR proteins to dsRNA with a length of at least 30–50 base pairs [5] activates the PKRs via trans-autophosphorylation; activated PKR then phosphorylates eIF-2α, thereby inhibiting translation of viral (and cellular) proteins. Other examples of proteins that detect viral dsRNA include 2′,5′-oligoadenylate (2–5A) synthetases [10], RNase L (activated via dimerization by 2–5A produced by 2–5A synthetases in response to dsRNA [11]), TLR 3 [12], interferon-inducible ADAR1 [13], and RIG-I and Mda-5 [6]–[7].
The second natural process used by our approach is one of the last steps in the apoptosis pathway [14], in which complexes containing intracellular apoptosis signaling molecules, such as apoptotic protease activating factor 1 (Apaf-1) [15]–[16] or FLICE-activated death domain (FADD) [17]–[18], simultaneously bind multiple procaspases. The procaspases transactivate via cleavage, activate additional caspases in the cascade, and cleave a variety of cellular proteins [14], thereby killing the cell.
Many viruses attempt to counter these defenses. A wide variety of viruses target dsRNA-induced signaling proteins, including IPS-1, interferon response factors (IRFs), interferons and interferon receptors, JAK/STAT proteins, and eIF-2α [19]–[20]. Some viral products attempt to sequester dsRNA (e.g., poxvirus E3L [21]) or to directly interfere with cellular dsRNA binding domains (e.g., HIV TAR RNA [19]–[20]). Virtually all viruses that inhibit apoptosis do so by targeting early steps in the pathway, for example by inhibiting p53, mimicking anti-apoptotic Bcl-2, or interfering with death receptor signaling [22]–[23]. Among the few viral proteins that directly inhibit one or more caspases are African swine fever virus A224L (which inhibits caspase 3) [24], poxvirus CrmA (which inhibits caspases 1, 8, and 10 but not others) [25], and baculovirus p35 (which inhibits several caspases but is relatively ineffective against caspase 9) [25].
Because PKR activation and caspase activation function in similar ways and involve proteins that have separate domains with well-defined functions, these two processes can be combined to circumvent most viral blockades [26]–[27]. In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA. If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.
For delivery into cells in vitro or in vivo, DRACOs can be fused with proven protein transduction tags, including a sequence from the HIV TAT protein [28], the related protein transduction domain 4 (PTD) [29], and polyarginine (ARG) [30]. These tags have been shown to carry large cargo molecules into both the cytoplasm and the nucleus of all cell types in vitro and in vivo, even across the blood-brain barrier.
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