[Show abstract][Hide abstract] ABSTRACT: Several systems-level datasets designed to dissect host-pathogen interactions during influenza A infection have been reported. However, apparent discordance among these data has hampered their full utility toward advancing mechanistic and therapeutic knowledge. To collectively reconcile these datasets, we performed a meta-analysis of data from eight published RNAi screens and integrated these data with three protein interaction datasets, including one generated within the context of this study. Further integration of these data with global virus-host interaction analyses revealed a functionally validated biochemical landscape of the influenza-host interface, which can be queried through a simplified and customizable web portal (http://www.metascape.org/IAV). Follow-up studies revealed that the putative ubiquitin ligase UBR4 associates with the viral M2 protein and promotes apical transport of viral proteins. Taken together, the integrative analysis of influenza OMICs datasets illuminates a viral-host network of high-confidence human proteins that are essential for influenza A virus replication.
[Show abstract][Hide abstract] ABSTRACT: Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
Full-text · Article · Mar 2014 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Dengue virus has emerged as a global health threat for over one third of mankind. As a positive strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis and Vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate, and by thymidine in the presence of floxuridine, suggesting an unexpected role of thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated anti-flavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for anti-flaviviral drug development.
Full-text · Article · Jul 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited
by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers
(PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both.
However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary
to sequences that are highly conserved across the arenaviruses and located at the 5′ termini of both genomic segments were
effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell
cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5′ genomic
termini represent promising targets for pan-arenavirus antiviral therapeutic development.
Full-text · Article · Aug 2011 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5' cyclization sequence (5'CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an "in vivo-ready" version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
Full-text · Article · Jul 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the swine industry in many countries, and current control strategies are inadequate. Previously, we explored the strategy of using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to inhibit PRRS virus (PRRSV) replication. PPMOs are nuclease-resistant and single-stranded DNA analogs containing a modified backbone conjugated to a cell-penetrating peptide and can act as antisense agents through steric blockade of complementary messenger RNA. A PPMO (designated 5UP2) targeting highly conserved sequence in the 5'-terminal region of the PRRSV genome was found to produce multi-log10 inhibition of PRRSV replication in cultured cells. To evaluate 5UP2 in vivo, we here administrated the PPMO to 3-week-old piglets via intranasal instillation at 24h before, and 2 and 24h after infection with PRRSV (strain VR2385). Blood samples were collected at 6, 10 and 14 days post-infection (dpi) for detection of PRRSV RNA and antibodies. Necropsy was performed at 14 dpi. Monitoring weight gain in all piglet groups throughout the experiment indicated that PPMO was well tolerated at the doses used. PPMO 5UP2 treatment significantly reduced PRRSV viremia at 6 dpi. On day 14, piglets receiving 5UP2 had significantly less interstitial pneumonia and lower level of anti-PRRSV antibodies than untreated piglets. In alveolar macrophages isolated at the time of necropsy, the expression of antiviral genes in PPMO-treated piglets was elevated in comparison with untreated. This study provides further data indicating that the 5UP2 PPMO can be considered a candidate component for a novel PRRS control strategy.
No preview · Article · Jul 2011 · Antiviral research
[Show abstract][Hide abstract] ABSTRACT: Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log(10) units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.
Full-text · Article · Feb 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration-approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC₅₀ potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
Full-text · Article · Oct 2010 · Assay and Drug Development Technologies
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58(IPK), which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58(IPK) was associated with the activation of PKR (PERK) and the phosphorylation of eIF2alpha, suggesting that p58(IPK), a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58(IPK) and induction/activation of CHOP, SREBP1, and caspase-12.
Preview · Article · Sep 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV) has a significant impact on public health throughout Asia, and there is a pressing need for development of new therapeutics against it.
Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense agents that enter cells readily and interfere with gene expression. Four PPMOs, targeting various locations in the JEV genome, were evaluated for antiviral activity against JEV in cultured cells and the mouse model of JEV infection.
A PPMO (P10882) targeting the JEV 3' cyclization sequence (3'CSI) had significant antiviral activity in Vero (epithelial), Neuro2A (neuronal) and J774E (macrophage) cells at concentrations that were not cytotoxic. P10882 added before infection suppressed JEV replication to an undetectable level in Vero cells and produced a 93% and 66% reduction in titre in J774E and Neuro2A cells, respectively, when measured at 24 h post-infection. In uninfected cells, fluorescein-labelled PPMOs entered J774E cells most efficiently, followed by Vero and Neuro2A cells. The antiviral effect of P10882 was also demonstrated in vivo, where 60%-80% of 1-week-old mice treated intracerebrally with a 20 mg/kg dose of P10882 every 12 h for 5 days were protected from a lethal dose of JEV and showed an undetectable level of virus in brain tissue at 2 days post-infection.
P10882, which targets sequence that is highly conserved across members of the JEV serocomplex, was previously shown to be effective in a mouse model of West Nile disease, and represents a candidate antiviral agent against members of the JEV serocomplex.
Full-text · Article · Mar 2010 · Journal of Antimicrobial Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5-10 microM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed.
[Show abstract][Hide abstract] ABSTRACT: Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA).
We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39.
In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 μM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection.
These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.
No preview · Article · Jan 2010 · Antiviral therapy
[Show abstract][Hide abstract] ABSTRACT: Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70-98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70-90%, while an equivalent dose of ACV produced only 40-50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100microg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10microg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5-50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.
Full-text · Article · Sep 2009 · Antiviral research
[Show abstract][Hide abstract] ABSTRACT: Measles virus (MeV) is a highly contagious human pathogen. Despite the success of measles vaccination programs, measles is still responsible for an estimated 245,000 deaths each year. There are currently no antiviral compounds available for the treatment of measles. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are antisense compounds that enter cells readily and can interfere with mRNA function by steric blocking. A panel of PPMO was designed to target various sequences of MeV RNA that are known to be important for viral replication. Five PPMO, targeting MeV genomic RNA or mRNA, inhibited the replication of MeV, in a dose-responsive and sequence-specific manner in cultured cells. One of the highly active PPMO (PPMO 454), targeting a conserved sequence in the translation start site of the mRNA coding for the nucleocapsid protein, inhibited multiple genotypes of MeV. This report provides evidence that PPMO treatment represents a promising approach for developing antiviral agents against measles and other paramyxoviruses.
[Show abstract][Hide abstract] ABSTRACT: In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2alpha (eIF2alpha). In addition, two of the three cellular eIF2alpha kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2alpha phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2alpha phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2alpha phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2alpha on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2alpha phosphorylation.
Full-text · Article · Mar 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS) causes extensive economic losses in the swine industry. Current strategies and vaccines to control the disease are inadequate. We previously demonstrated that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) could potently inhibit PRRS virus (PRRSV) replication in cell cultures. PPMOs are single-stranded DNA analogues containing a modified backbone and cell-penetrating peptide. PPMOs are nuclease-resistant, water-soluble, can enter cells readily and exhibit highly specific binding to complementary RNA. In this study, we examined PPMO-mediated inhibition of PRRSV replication in a primary culture of porcine pulmonary alveolar macrophages (PAMs).
PAMs were collected from piglets, pre-incubated in culture and infected with PRRSV. Viability, cytopathic effects, virus yield and apoptosis of PAMs in the presence or absence of a PPMO (5UP2) were examined. The 5UP2 PPMO is complementary to a conserved sequence in the 5'-terminal region of the PRRSV genome. The level of several interferon-associated gene products and activity of caspases were monitored.
PRRSV infection induced the activity of caspases-3/7, -8 and -9 significantly. Treatment of PAMs with 5UP2 resulted in protection of the cells from PRRSV-induced cell death for at least 7 days and avoided the activation of the caspases evaluated. 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-beta and chemokines observed in infected and mock-treated PAMs.
PPMO-mediated suppression of PRRSV replication in PAMs was associated with a reduction of apoptotic and inflammatory responses. These results provide further rationale for the development of PPMO 5UP2 as an antiviral to control PRRSV infection.
No preview · Article · Jan 2009 · Antiviral therapy
[Show abstract][Hide abstract] ABSTRACT: West Nile virus (WNV) genome cyclization is mediated by two pairs of long-distance RNA/RNA interactions: the 5'CS/3'CSI (conserved sequence) and the 5'UAR/3'UAR (upstream AUG region) base pairings. Antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), designed to interfere with the 5'CS/3'CSI or 5'UAR/3'UAR base pairings, were previously shown to inhibit WNV. In this study, we selected and characterized WNVs resistant to a PPMO targeting the 3'UAR (3'UAR-PPMO). All resistant viruses accumulated one-nucleotide mutations within the 3'UAR, leading to a single-nucleotide mismatch or a weakened base-pairing interaction with the 3'UAR-PPMO. Remarkably, a one-nucleotide mutation within the 5'UAR was correspondingly co-selected; the 5'UAR mutation restored the base pairing with the 3'UAR mutation. Mutagenesis of WNV demonstrated that the single-nucleotide change within the 3'UAR-PPMO-target site conferred the resistance. RNA binding analysis indicated that the single-nucleotide change reduced the ability of 3'UAR-PPMO to block the RNA/RNA interaction required for genome cyclization. The results suggest a mechanism by which WNV develops resistance to 3'UAR-PPMO, through co-selection of the 5'UAR and 3'UAR, to create a mismatch or a weakened base-pairing interaction with the PPMO, while maintaining the 5'UAR/3'UAR base pairings.