We produced DRACOs with different dsRNA detection domains, apoptosis induction domains, and transduction tags (). The dsRNA detection domains included PKR1–181
with dsRBM 1 (NTE3L), dsRBM 2 (CTE3L), or dsRBM 1 and 2 (2×E3L) replaced by the dsRNA binding motif from poxvirus E3L, and RNaseL1–335
(which binds to 2–5A produced by endogenous cellular 2–5A synthetases in response to viral dsRNA). The apoptosis induction domains included FADD1–90
Death Effector Domain (DED, which binds to procaspase 8), Apaf-11–97
caspase recruitment domain (CARD, which binds to procaspase 9), and murine Apaf-11–97
(mApaf) CARD. Except for mApaf, all domains refer to the human sequence. Isolated dsRNA detection domains and apoptosis induction domains were produced as negative controls. Mutant DRACOs with deleterious K64E 
and homologous K154E mutations in the PKR domain were also produced as negative controls. Proteins were produced with TAT, PTD, or ARG tags on the N terminus, C terminus, or both termini. Proteins were expressed in BL21(DE3)pLysS Rosetta E. coli
. An empty expression vector was transformed into the E. coli
and the same purification protocol was followed, resulting in control extract without DRACOs.
A variety of DRACOs and controls were produced.
DRACO rapidly entered cells, persisted within cells for days, and mediated apoptosis in cells transfected with dsRNA. PKR-Apaf DRACO with PTD or TAT tags entered cells efficiently, whereas DRACO without a transduction tag did not (). DRACO entered cells within 10 minutes, reached a maximum after approximately 1.5 hours (, S1
), and persisted inside cells for at least 8 days (). L929 cells transfected with both DRACO and poly(I)
poly(C) dsRNA exhibited greatly increased apoptosis within 24 hours, whereas cells that received only DRACO did not (). Pan-caspase and caspase-9 inhibitors eliminated DRACO-mediated apoptosis in the presence of dsRNA.
DRACOs penetrated cells and persisted for days.
DRACOs mediated apoptosis in cells containing dsRNA.
We measured the viability of normal human lung fibroblast (NHLF) cells that had been treated with PKR-Apaf DRACOs or negative controls and then challenged with 130 plaque forming units (pfu) per well rhinovirus 1B (, S2
). Untreated cell populations succumbed to virus within days, indicating that any innate cellular responses were ineffective against the virus or blocked by the virus. DRACOs with PTD, TAT, and ARG tags prevented significant cytopathic effects (CPE) in virus-challenged cell populations by rapidly killing any initially infected cells, thereby terminating the infection in its earliest stages. DRACOs had no apparent toxicity in unchallenged cells. Isolated PKR1–181
domains were nontoxic but not antiviral, even when added simultaneously (but not covalently linked). DRACO with deleterious amino acid changes also had little efficacy. Likewise, an amount of purified bacterial extract (without DRACOs) approximately 10-fold greater than the average volume of DRACOs added to cells was nontoxic and not efficacious, demonstrating that any remaining bacterial contaminants such as lipopolysaccharide did not affect the cells or produce antiviral activity. Thus the antiviral efficacy appears to require intact functional DRACOs. Tests using DRACOs with protein transduction tags on the N terminus, C terminus, or both termini indicated that N-terminal tags generally worked the best (data not shown). DRACOs with transduction tags penetrated cells and were antiviral when administered by themselves (, S2A
), but efficacy was enhanced by co-administration with Roche FuGene 6 to maximize uptake (Figure S2B
), so FuGene was used in experiments unless otherwise noted. Cell viability measured 7 days post infection (dpi) showed little difference if DRACO-containing medium was removed 3 dpi after untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells after DRACOs were withdrawn ().
DRACOs were effective against rhinovirus 1B in NHLF cells.
DRACOs were added approximately 24 hours before virus unless otherwise noted, but other dosing times were tested (). One dose of PTD-PKR-Apaf DRACO was efficacious against rhinovirus 1B in NHLF cells when added up to 6 days before infection, supporting the western data () that DRACO persisted inside cells for at least 8 days. Up to 3 days after infection, one DRACO dose could still rescue a significant percentage of the cell population. After 3 days, virtually all of the cells had already been killed or at least infected by the virus.
Additional DRACO designs exhibited efficacy against rhinovirus (). Other effective dsRNA detection domains included NTE3L, CTE3L, 2×E3L, and RNaseL1–335
. Other effective apoptotic domains included FADD1–90
, and procaspases 
. Although the initial performance of these alternate DRACOs was generally inferior to that of PKR-Apaf human DRACO in these experiments, better performance might be achieved with further optimization. These results demonstrate that the alternate DRACO designs are nontoxic and efficacious against virus, and they support the DRACO mechanism of action.
DRACOs were effective against rhinovirus 1B and other viruses.
In addition to improving survival of the cell population, DRACOs reduced viral titers from virus-challenged cells (, S4
). One dose of PKR-Apaf DRACO administered to NHLF cells 24 hours before 300 pfu/well rhinovirus 1B eliminated any measurable viral titer in cell supernatant samples collected 4 dpi.
The median effective concentration for DRACOs with PTD, TAT, and ARG tags against a variety of viruses was 2–3 nM, as illustrated for PTD-PKR-Apaf DRACO against rhinovirus 1B, murine encephalomyelitis, and murine adenovirus ().
DRACOs were effective against a broad spectrum of other viruses in a variety of cell types (–). DRACOs were effective against rhinoviruses 2 and 30 in NHLF cells (data not shown) and rhinovirus 14 in HeLa cells (Figure S4
). DRACOs were effective against murine adenovirus in L929 cells if added before or up to at least 72 hours after virus (, S5
), demonstrating efficacy against a DNA virus (, S5
), in murine cells (using human apoptotic DRACO domains to recruit endogenous murine procaspases), when treatment is delayed until significantly after infection (), and with a variety of DRACO designs (). DRACOs were effective against murine encephalomyelitis in L929 cells regardless of whether the DRACO-containing medium was removed 3 dpi (), whether DRACOs were added before or after infection (), and which DRACOs were used (, S6
). DRACOs were effective in Vero E6 cells against Amapari and Tacaribe, arenaviruses that are closely related to lymphocytic choriomeningitis virus (LCMV), Lassa, and Junin viruses (, S7
). Likewise, DRACOs were effective against Guama strain Be An 277 (, S9
); comparable results were obtained for Guama strain Be Ar 12590 (data not shown). Guama virus is a significant human pathogen and is closely related to other bunyaviruses such as Rift Valley fever, hantavirus, and Crimean-Congo virus. DRACOs were similarly effective against dengue type 2 (New Guinea C) hemorrhagic fever virus, a major human pathogen that is very closely related to other flaviviruses such as West Nile virus, Yellow fever virus, and Omsk virus (, S10
). DRACOs were also effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes (Figure S12
left), reovirus 3 in BALB/3T3 murine cells (Figure S12
center), and adenovirus 5 in AD293 cells (Figure S12
We have demonstrated DRACO efficacy against a broad spectrum of viruses.
We have demonstrated that DRACO is effective and nontoxic in a wide variety of cell types.
DRACOs were effective against murine adenovirus in L929 cells.
DRACOs were effective against murine encephalomyelitis in L929 cells.
DRACOs were effective against arenaviruses, bunyaviruses, and flaviviruses.
DRACOs appeared promising in proof-of-concept trials with adult BALB/c mice. Intraperitoneal (i.p.) PKR-Apaf DRACO penetrated the liver, kidney, and lungs and persisted at least 24–48 hours (). Live mice and harvested mouse organs showed no apparent toxicity. PTD-PKR-Apaf and TAT-PKR-Apaf DRACOs administered i.p. from day -1 through day 3 greatly reduced the morbidity in mice challenged intranasally (i.n.) with 1.3 LD50 influenza H1N1 A/PR/8/34 and reduced the day-2 lung viral titers by over an order of magnitude (). Similarly, PTD-RNaseL-Apaf, TAT-RNaseL-Apaf, and ARG-RNaseL-Apaf DRACOs administered i.p. from day -1 through day 3 prevented morbidity in mice challenged i.n. with 0.3 LD50 influenza and reduced the day-2 viral titers by an order of magnitude or more (). PKR-Apaf DRACO administered i.n. to mice penetrated the lungs and persisted over 24 hours (). PTD-PKR-Apaf, TAT-PKR-Apaf, and ARG-PKR-Apaf DRACOs administered i.n. on day 0 reduced the morbidity in mice challenged i.n. with 1 LD50 influenza ().
DRACOs appeared promising when administered via intraperitoneal (i.p.) injection in proof-of-concept trials with adult BALB/c mice.
DRACOs appeared promising when administered via intranasal (i.n.) injection in proof-of-concept trials with adult BALB/c mice.
Based on these encouraging initial animal trials, future work should be done to test and optimize antiviral efficacy, pharmacokinetics, and absence of toxicity in vitro and in vivo. Future experiments can further characterize and optimize dsRNA binding, apoptosis induction, cellular transduction, and other DRACO properties. More extensive trials are also needed to determine how long after infection DRACOs can be used successfully, or if DRACOs are useful against chronic viral infections without producing unacceptable levels of cell death in vivo.
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 ().