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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.

Mayaro virus (MAYV) is a neglected mosquito-borne alphavirus that causes illness similar to Chikungunya (CHIKV), Dengue (DENV) and Zika virus (ZIKV). Currently, there is no specific treatment or vaccine against MAYV infection. To develop an efficient antiviral screening assay for MAYV, we constructed the infectious clones of MAYV strain BeAr 20290 and its eGFP reporter virus. The reporter virus exhibited high replication capacity indistinguishable with the wild type MAYV, and was genetically stable within at least five rounds of passages in BHK-21 cell. The expression of eGFP correlated well with the viral replication. Using the known inhibitor ribavirin, we confirmed that the MAYV-eGFP reporter virus could be used for antiviral screening to identify the specific inhibitors against MAYV. Using the MAYV-eGFP based antiviral assay, we found that the compound 6-Azauridine which had antiviral activity against CHIKV and SFV, showed a significant inhibitory effect on MAYV replication.

Omsk hemorrhagic fever virus (OHFV) is a tick-borne flavivirus classified as a biosafety level-4 (BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion (OHFV-ΔNS1) and reporter virus replacing NS1 with the Gaussia luciferase (Gluc) (OHFV-ΔNS1-Gluc). Both the defective OHFV-ΔNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1 (BHK21(NS1)) but not in naïve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21(NS1) cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z′ value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.

Subgenomic flavivirus RNA (sfRNA) is essential for flavivirus-induced cytopathicity. sfRNA-deficient mutant dengue virus (DENV) M8 harboring a deleted domain II in the 3' untranslated region (UTR) has severely decreased cytopathicity and replication efficiency. Here, recovered viruses acquired from M8 by serial passages replicated similarly to the wild type (WT) but produced two types of sfRNA, whereas only one was detected in WT infected cells, resulting in mildly decreased cytopathicity. Sequencing and structure prediction showed that recovered viruses acquired a novel additional partial domain I sequence duplication and formed analogous RNA secondary structures. This was the first experimental evidence verifying that a longer precursor may exist in the 3' UTR of DENV. Additionally, the duplicated structures could be compensated by the homologous structures from other flaviviruses, rather than heterologous viruses, implying that homologous secondary structures or higher tertiary structures played more important roles in sfRNA production and viral replication.

N 6-methyladenosine (m6A) constitutes one of the most abundant internal RNA modifications and is critical for RNA metabolism and function. It has been previously reported that viral RNA contains internal m6A modifications; however, only recently the function of m6A modification in viral RNAs has been elucidated during infections of HIV, hepatitis C virus and Zika virus. In the present study, we found that enterovirus 71 (EV71) RNA undergoes m6A modification during viral infection, which alters the expression and localization of the methyltransferase and demethylase of m6A, and its binding proteins. Moreover, knockdown of m6A methyltransferase resulted in decreased EV71 replication, whereas knockdown of the demethylase had the opposite effect. Further study showed that the m6A binding proteins also participate in the regulation of viral replication. In particular, two m6A modification sites were identified in the viral genome, of which mutations resulted in decreased virus replication, suggesting that m6A modification plays an important role in EV71 replication. Notably, we found that METTL3 interacted with viral RNA-dependent RNA polymerase 3D and induced enhanced sumoylation and ubiquitination of the 3D polymerase that boosted viral replication. Taken together, our findings demonstrated that the host m6A modification complex interacts with viral proteins to modulate EV71 replication.

H7N9 influenza A virus (IAV) is an emerged contagious pathogen that may cause severe human infections, even death. Understanding the precise cross talk between virus and host is vital for the development of effective vaccines and therapeutics. In the present study, we identified the nucleoprotein (NP) of H7N9 IAV as a positive regulator of RIG-I like receptor (RLR)-mediated signaling. Based on a loss-of-function strategy, we replaced H1N1 (mouse-adapted PR8 strain) NP with H7N9 NP, by using reverse genetics, and found that the replication and pathogenicity of recombinant PR8-H7N9NP (rPR8-H7N9NP) were significantly attenuated in cells and mice. Biochemical and cellular analyses revealed that H7N9 NP specifically interacts with tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) after viral infection. Subsequently, we identified a PXXQXS motif in the H7N9 NP that may be a determinant for the NP and TRAF3 interaction. Furthermore, H7N9 NP stabilized TRAF3 expression via competitively binding to TRAF3 with cellular inhibitor of apoptosis 2 (cIAP2), leading to the inhibition of the Lys48-linked polyubiquitination and degradation of TRAF3. Taken together, these data uncover a novel mechanism by which the NP of H7N9 IAV positively regulates TRAF3-mediated type I interferon signaling. Our findings provide insights into virus and host survival strategies that involve a specific viral protein that modulates an appropriate immune response in hosts. IMPORTANCE The NS1, PB2, PA-X, and PB1-F2 proteins of influenza A virus (IAV) are known to employ various strategies to counteract and evade host defenses. However, the viral components responsible for the activation of innate immune signaling remain elusive. Here, we demonstrate for the first time that the NP of H7N9 IAV specifically associates with and stabilizes the important adaptor molecule TRAF3, which potentiates RLR-mediated type I interferon induction. Moreover, we reveal that this H7N9 NP protein prevents the interaction between TRAF3 and cIAP2 that mediates Lys48-linked polyubiquitination of TRAF3 for degradation. The current study revealed a novel mechanism by which H7N9 NP upregulates TRAF3-mediated type I interferon production, leading to attenuation of viral replication and pathogenicity in cells and mice. Our finding provides a possible explanation for virus and host commensalism via viral manipulation of the host immune system.

Infections with zoonotic viruses, such as flaviviruses, influenza virus, and the SARS-CoV-2 pandemic coronavirus constitute an increasing global risk. Hence, an urgent need exists for the development of broad-spectrum antivirals to prevent such outbreaks. Here, we show that the maduramycin and CP-80,219 aglycone polyether ionophores exhibit effective broad-spectrum antiviral activity, against various viruses, including Japanese encephalitis virus (JEV), Dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), while also exhibiting promising activity against PR8 influenza virus and SARS-CoV-2. Moreover, liposome-encapsulated maduramycin and CP-80,219 provide full protection for mice from infection with JEV in vivo. Mechanistic studies suggest that aglycone polyether ionophores primarily inhibit the viral replication step without blocking endosome acidification to promote the fusion between viral and cellular membranes. The successful application of liposomes containing aglycone polyether ionophores in JEV-infected mice offers hope to the development of broad-spectrum antiviral drugs like penicillin back to 1940s.

International audience; Naturally occurring pentacyclic triterpenes, such as betulinic acid (BA) and its derivatives, exhibit various pharmaceutical activities and have been the subject of great interest, in particular for their antiviral properties. Here, we found a new anti-influenza virus conjugate, hexakis 6-deoxy-6-[4-N-(3β-hydroxy-lup-20(29)-en-28-oate)aminomethyl-1H-1,2,3-triazol-1-yl]-2,3-di-O-acetyl-α-cyclodextrin (CYY1-11, 1), in a mini library of pentacyclic triterpene–cyclodextrin conjugates by performing a cell-based screening assay and then exploring the underlying mechanisms. Our results showed that conjugate 1 possessed a high-level activity against the influenza virus A/WSN/33 with an IC50 value of 5.20 μM (SI > 38.4). The study of the mechanism of action indicated that conjugate 1 inhibited viral replication by directly targeting the influenza hemagglutinin protein (KD = 1.50 μM), thus efficiently preventing the attachment of the virion to its receptors on host cells and subsequent infection. This study suggests that multivalent BA derivatives have possible use as a new class of influenza virus entry inhibitors.

The emerging SARS-CoV-2 infection associated with the outbreak of viral pneumonia in China is ongoing worldwide. There are no approved antiviral therapies to treat this viral disease. Here we examined the antiviral abilities of three broad-spectrum antiviral compounds gemcitabine, lycorine and oxysophoridine against SARS-CoV-2 in cell culture. We found that all three tested compounds inhibited viral replication in Vero-E6 cells at noncytotoxic concentrations. The antiviral effect of gemcitabine was suppressed efficiently by the cytidine nucleosides. Additionally, combination of gemcitabine with oxysophoridine had an additive antiviral effect against SARS-CoV-2. Our results demonstrate that broad-spectrum antiviral compounds may have a priority for the screening of antiviral compounds against newly emerging viruses to control viral infection.

Viruses in the Flaviviridae family such as Zika virus (ZIKV), dengue virus (DENV), and Japanese encephalitis virus (JEV) are major public health concerns. The development of antiviral agents against these viruses is urgently needed. We have previously discovered that the Keggin structured polyoxometalate POM-12 has potent inhibitory activity against hepatitis C virus, another member of the Flaviviridae family. In this study, we tested its antiviral activity of DENV, JEV and ZIKV, and found that POM-12 dramatically inhibited their infection with IC50 value of 1.16 μM, 1.9 μM and 0.64 μM, respectively. Mechanistic studies indicated that POM-12 directly disrupted the integrity of these virions. Moreover, POM-12 also targeted the post-entry steps of viral replication of JEV, but having no similar activities on ZIKV and DENV. The differential actions of POM-12 on these viruses suggest that surface topology and charge of virion may have influence on its drug effect, and thus POM-12 may be modified to more efficiently inhibit these and other similar viruses.

Zika virus (ZIKV) is an important emerging human pathogen associated with microcephaly, Guillain-Barre syndrome and meningoencephalitis. Developing rapid and reliable HTS assay is important for ZIKV drug discovery. Here, we constructed a dicistronic ZIKV replicon (ZIKV-Pac-Rluc-Rep) that contained the Renilla luciferase (Rluc) reporter gene separated from the puromycin N-acetyl-transferase (Pac) selectable marker by a short peptide cleavage site. A clonal replicon cell line stably expressing high level of ZIKV replicon was established by selection with puromycin. By optimizing cell number, compound concentration and incubation time, a robust replicon cell-based HTS assay was developed with a calculated Z' value of >0.5. The fully optimized assay was further validated using several known flavivirus replication inhibitors. Altogether, the replicon cell-based HTS assay developed in this study will facilitate the discovery of antiviral compounds against ZIKV.

In this study, an in vitro ligation method was developed to assemble a full-length infectious cDNA clone of the Zika virus (ZIKV). Four contiguous cDNA subclones covering the complete ZIKV genome were constructed with unique BglI restriction sites at the ends of each fragment. The BglI restriction sites only allow in vitro ligation to happen between interconnecting restriction sites from adjacent cDNA fragments, resulting in an intact full-length cDNA of ZIKV. RNA transcripts derived from the full-length cDNA were infectious. The recombinant virus replicated as efficiently as the wild-type virus with similar growth kinetics and plaque morphologies in Vero and C6/36 cells. Both viruses were inhibited by NITD008 treatment. This in vitro ligation method will facilitate manipulation of the viral genome through genetic modifications of four separated subclones of ZIKV for the rapid and rational development of candidate vaccines and viral replication study.

We previously produced a replication-defective West Nile virus (WNV) lacking NS1 (WNV-ΔNS1) that could propagate at low levels (105 infectious units [IU]/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-ΔNS1 as a noninfectious vaccine. To explore this idea, we developed an NS1-expressing Vero cell line (VeroNS1) that significantly improved the yield of WNV-ΔNS1 (108 IU/ml). We evaluated the safety and efficacy of WNV-ΔNS1 in mice. WNV-ΔNS1 appeared to be safe, as no replicative virus was found in naive Vero cells after continuous culturing of WNV-ΔNS1 in VeroNS1 cells for 15 rounds. WNV-ΔNS1 was noninfectious in mice, even when IFNAR−/− mice were administered a high dose of WNV-ΔNS1. Vaccination with a single dose of WNV-ΔNS1 protected mice from a highly lethal challenge with WT WNV. The antibody response against WNV correlated well with the protection of vaccinated mice. Our study demonstrates the potential of the NS1 trans complementation system as a new platform for flavivirus vaccine development. IMPORTANCE Many flaviviruses are significant human pathogens that frequently cause outbreaks and epidemics around the world. Development of novel vaccine platforms against these pathogens is a public health priority. Using WNV as a model, we developed a new vaccine platform for flaviviruses. WNV containing a NS1 deletion (WNV-ΔNS1) could be efficiently trans complemented in Vero cells that constitutively expressed WT NS1 protein. A single-dose immunization with WNV-ΔNS1 elicited robust immune responses in mice. The immunized animals were fully protected against pathogenic WNV infection. No adverse effects related to the WNV-ΔNS1 vaccination were observed. The results have demonstrated the potential of the NS1 complementation system as an alternative platform for flavivirus vaccine development, especially for highly pathogenic flaviviruses.

Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When trans-complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIV(NS2A)) that could initiate single-round infection. The trans-complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIV(NS2A) production, with titers of >10(7) infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIV(NS2A) elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIV(NS2A) vaccine. The technology could be applicable to vaccine development for other viral pathogens. IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens.

Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1,443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection., Highlights • The eGFP-JEV reporter virus was established. • The eGFP-JEV -based high throughput screening (HTS) assay was developed with a calculated Z′ factor of >0.5. • A library of 1,443 FDA-approved drugs was screened to determine their ability to inhibit JEV infection using this HTS assay. • 5 novel hit drugs were identified to be active against JEV infection.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae . During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo . Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo . Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients.

Japanese encephalitis virus (JEV), an important pathogen in Eastern and Southern Asia and the Pacific, has spread to Australia and other territories in recent years. Although the vaccine for JEV has been used in some countries, development of efficient antiviral drugs is still an urgent requirement. Replicon systems have been widely used in the research of viral replication and antiviral screening for West Nile virus (WNV), yellow fever virus (YFV) and dengue virus (DENV). Here, a novel JEV replicon harboring the Rluc and Pac gene (JEV-Pac-Rluc-Rep) was constructed. Furthermore, we established a BHK-21 cell line harboring JEV-Pac-Rluc-Rep (BHK-21/PAC/Rluc cell line) through continuous puromycin selection. Characterization of cell line stability showed that the replicon RNA could persistently replicate in this cell line for at least up to 10 rounds of passage. Using a known flavivirus inhibitor, the JEV replicon cell line was validated for antiviral screening. The JEV replicon cell line will be a valuable tool for both compound screening and viral replication studies.

West Nile virus (WNV), a mosquito-borne flavivirus, is an important neurotropic human pathogen. As a biosafety level-3 (BSL-3) agent, WNV is strictly to BSL-3 laboratories for experimentations, thus greatly hindering the development of vaccine and antiviral drug. Here, we developed a novel pseudo-infectious WNV reporter virus expressing the Gaussia luciferase (Gluc). A stable 293TNS1 cell line expressing NS1 was selected for trans-supplying NS1 protein to support the replication of WNV-ΔNS1 virus and WNV-ΔNS1-Gluc reporter virus with large-fragment deletion of NS1. WNV-ΔNS1 virus and WNV-Gluc-ΔNS1 reporter virus were confined to complete their replication cycle in this 293TNS1 cell line, displaying nearly identical growth kinetics to WT WNV although the viral titers were lower than those of WT WNV. The reporter gene was stably maintained in virus genome at least within three rounds of passage in 293TNS1 cell line. Using a known flaviviruses inhibitor, NITD008, we demonstrated that the pseudo-infectious WNV-Gluc-ΔNS1 could be used for antiviral screening. Furthermore, a high-throughput screening (HTS) assay in a 96-well format was optimized and validated using several known WNV inhibitors, indicating that the optimized HTS assay was suitable for high-throughput screening WNV inhibitors. Our work provides a stable and safe tool to handle WNV outside of a BSL-3 facility and facilitates high throughput screening for anti-WNV drugs.

Flavivirus includes a large group of human pathogens with medical importance. Especially, neurotropic flaviviruses capable of invading central and peripheral nervous system, e.g. Japanese encephalitis virus (JEV) and Zika virus (ZIKV), are highly pathogenic to human and constitute major global health problems. However, the dynamic dissemination and pathogenesis of neurotropic flavivirus infections remain largely unknown. Here, using JEV as a model, we rationally designed and constructed a recombinant reporter virus that stably expressed Renilla luciferase (Rluc). The resulting JEV reporter virus (named Rluc-JEV) and parental JEV exhibited similar replication and infection characteristics, and the magnitude of Rluc activity correlated well with progeny viral production in vitro and in vivo. By using in vivo bioluminescence imaging (BLI) technology, we dissected the replication and dissemination dynamics of JEV infection in mice upon different inoculation routes. Interestingly, besides replicating in mouse brain, Rluc-JEV predominantly invaded the abdominal organs in mice with typical viscerotropism. Further tests in mice deficient in type I interferon (IFN) receptors demonstrated robust and prolonged viral replication in the intestine, spleen, liver, kidney and other abdominal organs. Combined with histopathological and immunohistochemical results, the host type I IFN signaling was evidenced as the major barrier to the viscerotropism and pathogenicity of this neurotropic flavivirus. Additionally, the Rluc-JEV platform was readily adapted for efficacy assay of known antiviral compounds and a live JE vaccine. Collectively, our study revealed abdominal organs as important targets of JEV infection in mice and profiled the unique viscerotropism trait controlled by the host type I IFN signaling. This in vivo visualization technology described here provides a powerful tool for testing antiviral agents and vaccine candidates for flaviviral infection.

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. IMPORTANCE Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.

Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis. Furthermore, we show that WNV superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV superinfection exclusion is a result of competition for intracellular host factors that are required for viral RNA synthesis.

Lycorine potently inhibits flaviviruses in cell culture. At 1.2-microM concentration, lycorine reduced viral titers of West Nile (WNV), dengue, and yellow fever viruses by 10(2)- to 10(4)-fold. However, the compound did not inhibit an alphavirus (Western equine encephalitis virus) or a rhabdovirus (vesicular stomatitis virus), indicating a selective antiviral spectrum. The compound exerts its antiviral activity mainly through suppression of viral RNA replication. A Val-->Met substitution at the 9th amino acid position of the viral 2K peptide (spanning the endoplasmic reticulum membrane between NS4A and NS4B proteins) confers WNV resistance to lycorine, through enhancement of viral RNA replication. Initial chemistry synthesis demonstrated that modifications of the two hydroxyl groups of lycorine can increase the compound's potency, while reducing its cytotoxicity. Taken together, the results have established lycorine as a flavivirus inhibitor for antiviral development. The lycorine-resistance results demonstrate a direct role of the 2K peptide in flavivirus RNA synthesis.

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5′CS/3′CSI (conserved sequence) interaction, a 5′UAR/3′UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5′UARs capable of forming the 5′/3′ viral RNA interaction were replicative. In contrast, WNV containing a 5′UAR mutation that abolished the 5′/3′ viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5′/3′ RNA interaction. The 5′UAR/3′UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5′UAR/3′UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3′UAR could alternate between pairing with the 5′UAR or with the 3′ end of the flaviviral genome. Therefore, the 5′UAR/3′UAR pairing may release the 3′ end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.

Coxsackievirus A16 (CA16) is one of the major causes of hand, foot, and mouth disease (HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious cDNA clone of an epidemic strain of Coxsackievirus A16 (CA16) was assembled, and subsequently a reporter virus (eGFP-CA16) was constructed by inserting the eGFP gene between the 5′-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site (ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the eGFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC(50) of NITD008. In summary, this stable infectious cDNA clone should provide a valuable experimental system to study CA16 infection and host response. The eGFP-CA16 is expected to provide a powerful tool to monitor eGFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections. [Image: see text]

Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I 0 , ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity. IMPORTANCE Recently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

Flavivirus NS4A and NS4B are important membrane proteins for viral replication that are assumed to serve as the scaffold for the formation of replication complexes. We previously demonstrated that a single Lys-to-Arg mutation at position 79 in NS4A (NS4A-K79R) significantly impaired Japanese encephalitis virus (JEV) replication. In this study, the mutant virus was subject to genetic selection to search for the potential interaction between NS4A and other viral components. Sequencing of the recovered viruses revealed that, in addition to an A97E change in NS4A itself, a Y3N compensatory mutation located in NS4B had emerged from independent selections. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that both adaptive mutations greatly restored the replication defect caused by NS4A-K79R. Our results, for the first time to our knowledge, clearly showed the genetic interaction between NS4A and NS4B, although the mechanism underlying their interaction is unknown.

Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 μM and a 50% cytotoxic concentration of 119.97 μM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment. IMPORTANCE Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.

Molecular epidemiologic studies of North American (NA) West Nile virus (WNV; Flaviviridae, Flavivirus) have documented the displacement of the introduced NY99 genotype with WN02. In addition, these studies have shown that particular substitutions are under positive selection. One occurs in the C-terminus of the NS4A coding sequence and results in a valine to methionine substitution at position nine of the 2K peptide. 2K-V9M confers the ability to overcome superinfection exclusion in vitro. We hypothesized that WNV strains bearing 2K-V9M have higher fitness than wildtype in Culex quinquefasciatus mosquitoes. Although infection rates and viral titers were not significantly different, virus dissemination rates were significantly higher with WNV 2K-V9M. As a super-infecting virus, WNV 2K-V9M was more successful than wildtype, however, in a mixed infection, 2K-V9M was not. These data support observations that 2K-V9M confers a context-specific selective advantage in mosquitoes and provides an in vivo mechanism for its positive selection.

The flavivirus NS5 harbors both a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Both enzyme activities of NS5 are critical for viral replication. Recently, the full-length NS5 crystal structure of Japanese encephalitis virus reveals a conserved MTase-RdRP interface that features two conserved components: a six-residue hydrophobic network and a GTR sequence. Here we showed for the first time that these key interface components are essential for flavivirus replication by various reverse genetics approaches. Interestingly, some replication-impaired variants generated a common compensatory NS5 mutation outside the interface (L322F), providing novel routes to further explore the crosstalk between MTase and RdRP., Author Summary Flaviviruses, such as Japanese encephalitis virus (JEV) and dengue virus 1-4 (DENV1-4), are important arthropod-borne pathogens causing major public health threats worldwide. For example, JEV is the most common cause of viral encephalitis in eastern and southern Asia, affecting over 50,000 patients, and leads to 15,000 deaths annually. DENV is responsible for an estimated 50 million cases of dengue fever and over 450,000 cases of life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) each year. Currently, there is no effective antiviral therapeutics available for flaviviruses. Non-structural protein 5 (NS5) is a multifunctional, viral protein that contains a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Both MTase and RdRP are required for replication of the viral genome. A conserved interface between MTase and RdRP was first identified in the full-length NS5 crystal structure of JEV. In this study we used the infectious clones of both JEV and DENV-2 to perform functional mutagenesis analyses and demonstrated for the first time that the recently identified conserved interface between MTase and RdRP is critical for viral replication. In a replicon system, we also confirmed that mutations within the conserved interface greatly affect viral RNA replication during viral infection. Our functional validation of the conserved MTase-RdRP interface consolidated its potential as a novel target for anti-flavivirus drug development.

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102Min vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. The NS5 protein of JEV is a key component of the viral replicase complex, which plays a crucial role in viral pathogenesis. In this study, tandem affinity purification (TAP) followed by mass spectrometry analysis was performed to identify novel host proteins that interact with NS5. Heat shock protein 70 (Hsp70), eukaryotic elongation factor 1-alpha (eEF-1α) and ras-related nuclear protein (Ran) were demonstrated to interact with NS5. In addition to NS5, Hsp70 was also found to interact with NS3 which is another important member of the replicase complex. It was observed that the cytoplasmic Hsp70 partially colocalizes with the components of viral replicase complex including NS3, NS5 and viral dsRNA during JEV infection. Knockdown of Hsp70 resulted in a significantly reduced JEV genome replication. Further analysis reveals that Hsp70 enhances the stability of viral proteins in JEV replicase complex. These results suggest an important role for Hsp70 in regulating JEV replication, which provides a potential target for the development of anti-JEV therapies.

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D(80)DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D(80)DDG in NS2B on protease activity and viral replication, the negatively charged region D(80)DD and the conserved residue G83 of NS2B were mutated (D(80)DD/E(80)EE, D(80)DD/K(80)KK, D(80)DD/A(80)AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D(80)DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D(80)DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D pol ) and the transfer of UMP by 3D pol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D pol ) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D pol by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D pol , which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D pol , which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D pol . Mutation of a polymerase active site in 3D pol and Site-311 in 3D pol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro . These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D pol during EV71 VPg uridylylation, such that one 3D pol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D pol , which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

Although flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of flavivirus replication complex and could be targeted for potential antiviral development.

Many, but not all, strains of West Nile virus (WNV) contain a single N-linked glycosylation site on their envelope (E) proteins. Previous studies have shown that E-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. E protein glycosylation also appears to play a role in attachment and entry of WNV into host cells in vitro; however, studies examining how E protein glycosylation affects the interactions of WNV with its mosquito vectors in vivo have not yet been performed. We mutated the E protein glycosylation site from NYS to IYS in a previously described full-length clone of the NY99 genotype of WNV (WT), resulting in a virus that lacked the glycan at aa154. WNV-N154I replicated less efficiently than WNV-WT in Culex mosquito tissues, although the extent of the decrease was greater in Cx. pipiens than in Cx. tarsalis. Following peroral infection, mosquitoes infected with WNV-N154I were less likely to transmit virus than those infected with WNV-WT. Interestingly, all but one of the mosquitoes infected with WNV-N154I transmitted a revertant virus, suggesting that there is strong selective pressure toward E protein glycosylation. Together these data suggest that loss of the glycan at aa154 on the WNV E protein can severely restrict viral spread in the mosquito vector.

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m 7 GpppAm-RNA). The methyltransferase is physically linked to an RNA-dependent RNA polymerase (RdRp) in the flaviviral NS5 protein. Here, we report genetic interactions of West Nile virus (WNV) methyltransferase with the RdRp and the 5′-terminal stem-loop of viral genomic RNA. Genome-length RNAs, containing amino acid substitutions of D146 (a residue essential for both cap methylations) in the methyltransferase, were transfected into BHK-21 cells. Among the four mutant RNAs (D146L, D146P, D146R, and D146S), only D146S RNA generated viruses in transfected cells. Sequencing of the recovered viruses revealed that, besides the D146S change in the methyltransferase, two classes of compensatory mutations had reproducibly emerged. Class 1 mutations were located in the 5′-terminal stem-loop of the genomic RNA (a G35U substitution or U38 insertion). Class 2 mutations resided in NS5 (K61Q in methyltransferase and W751R in RdRp). Mutagenesis analysis, using a genome-length RNA and a replicon of WNV, demonstrated that the D146S substitution alone was lethal for viral replication; however, the compensatory mutations rescued replication, with the highest rescuing efficiency occurring when both classes of mutations were present. Biochemical analysis showed that a low level of N7 methylation of the D146S methyltransferase is essential for the recovery of adaptive viruses. The methyltransferase K61Q mutation facilitates viral replication through improved N7 methylation activity. The RdRp W751R mutation improves viral replication through an enhanced polymerase activity. Our results have clearly established genetic interactions among flaviviral methyltransferase, RdRp, and the 5′ stem-loop of the genomic RNA.

Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2′-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of nonstructural protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5′ end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m 7 GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA → m 7 GpppA-RNA → m 7 GpppAm-RNA, using S -adenosyl- l -methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2′-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2′-O methylation. In the context of virus, West Nile viruses (WNVs) defective in N-7 methylation are non-replicative; however, WNVs defective in 2′-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.

Adenoviruses are common pathogens. The localization of their receptors coxsackievirus and adenovirus receptor, and desmoglein-2 in cell-cell junction complexes between polarized epithelial cells represents a major challenge for adenovirus infection from the apical surface. Structural proteins including hexon, penton base and fiber are excessively produced in serotype 5 adenovirus (Ad5)-infected cells. We have characterized the composition of structural protein complexes released from Ad5 infected cells and their capacity in remodeling cell-cell junction complexes. Using T84 cells as a model for polarized epithelium, we have studied the effect of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. The initial Ad5 infection in T84 cell culture was inefficient. However, progressive distortion of cell-cell junction in association with fiber release was evident during progression of Ad5 infection. Incubation of T84 cell cultures with virion-free supernatant from Ad5 infected culture resulted in distortion of cell-cell junctions and decreased infectivity of Ad5-GFP vector. We used gel filtration chromatography to fractionate fiber containing virion–free supernatant from Ad5 infected culture supernatant. Fiber containing fractions were further characterized for their capacity to inhibit the infection of Ad5-GFP vector, their composition in adenovirus structural proteins using western blot and LC-MS/MS and their capacity in remolding cell-cell junctions. Fiber molecules in complexes containing penton base and hexon, or mainly hexon were identified. Only the fiber complexes with relatively high content of penton base, but not the fiber-hexon complexes with low penton base, were able to penetrate into T84 cells and cause distortion of cell-cell junctions. Our findings suggest that these two types of fiber complexes may play different roles in adenoviral infection.

We report a genetic interplay among three pairs of long-distance RNA interactions that are involved in West Nile virus (WNV) genome cyclization and replication: 5′CS/3′CSI ( c onserved s equence), 5′UAR/3′UAR ( u pstream A UG r egion), and 5′DAR/3′DAR ( d ownstream A UG r egion). Deletion of the complete 3′CSI element is lethal for WNV replication, but the replication of the 3′CSI deletion virus could be rescued by second site mutations. Functional analysis, using a genome-length RNA and replicon, mapped the compensatory mutations to the 5′UAR/3′UAR and 5′DAR/3′DAR regions. Biochemical analysis showed that the 3′CSI deletion abolished the 5′ and 3′ RNA interaction of the genome; the compensatory mutations could partially restore the 5′ and 3′ genome cyclization. These results demonstrate, for the first time, that a flavivirus without 3′CSI could restore genome cyclization and viral replication through enhancement of the 5′UAR/3′UAR and 5′DAR/3′DAR interactions.