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Flaviviruses require complementarity between the 5' and 3' ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3' DAR sequence is also part of a hairpin (HP-3'SL), and both complementarity between 5' and 3' DAR motifs and formation of the HP-3'SL in the absence of the 5' end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5'-3' DAR complementarity and HP-3'SL formation are essential for viral RNA replication. [ABSTRACT FROM AUTHOR]

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCUOH-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i) The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii) The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CUOH, are absolutely essential for viral replication. (iii) The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv) The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v) Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis. [ABSTRACT FROM AUTHOR]

Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition. [ABSTRACT FROM AUTHOR]

The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1-/- mice, neither NS1-based vaccine provided protection against a lethal high dose (105 PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (10³ PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development. [ABSTRACT FROM AUTHOR]

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation. [ABSTRACT FROM AUTHOR]

Flavivirus nonstructural protein 5 (NS5) contains an N-terminal methyltransferase (MTase) domain and a C-terminal polymerase (RNA-dependent RNA polymerase [RdRp]) domain fused through a 9-amino-acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: the NS5 proteins from Japanese encephalitis virus (JEV) and Zika virus (ZIKV) adopt one conformation, while the NS5 protein from dengue virus serotype 3 (DENV3) adopts another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures. Replacement of the DENV2 NS5 linker with DENV1, DENV3, DENV4, JEV, and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, while the JEV and ZIKV NS5 linkers abolished replication. Thus, the JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design. [ABSTRACT FROM AUTHOR]

We previously produced a replication-defective West Nile virus lacking NS1 WNV-ΔNS1) that could propagate at low levels (105 IU/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-ΔNS1 as a non-infectious 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 on naïve Vero cells after continuously culturing of WNV-ΔNS1 on VeroNS1 cells for fifteen rounds. WNV-ΔNS1 was non-infectious in mice, even when IFNAR-/- mice were administrated with a high dose of WNV-ΔNS1. A single dose of WNV-ΔNS1 vaccination protected mice from a highly lethal challenge of 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. [ABSTRACT FROM AUTHOR]

Confirmed reports of Zika virus (ZIKV) in human seminal fluid for months after the clearance of viremia suggest the ability of ZIKV to establish persistent infection in the seminiferous tubules, an immune-privileged site in the testis protected by the blood-testis barrier, also called the Sertoli cell (SC) barrier (SCB). However, cellular targets of ZIKV in human testis and mechanisms by which the virus enters seminiferous tubules remain unclear. We demonstrate that primary human SCs were highly susceptible to ZIKV compared to the closely related dengue virus and induced the expression of alpha interferon (IFN-α), key cytokines, and cell adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1] and intracellular adhesion molecule 1 [ICAM-1]). Furthermore, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with a higher efficiency than in the blood-brain barrier model. ZIKV-infected SCs exhibited enhanced adhesion of leukocytes that correlated with decreases in SCB integrity. ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin, and JAM-A; however, exposure of SCs to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of the ZO-1 protein, which correlated with increased SCB permeability. Taken together, our data suggest that infection of SCs may be one of the crucial steps by which ZIKV gains access to the site of spermatozoon development and identify SCs as a therapeutic target to clear testicular infections. The SCB model opens up opportunities to assess interactions of SCs with other testicular cells and to test the ability of anti-ZIKV drugs to cross the barrier. IMPORTANCE Recent outbreaks of ZIKV, a neglected mosquito-borne flavivirus, have identified sexual transmission as a new route of disease spread, which has not been reported for other flaviviruses. To be able to sexually transmit for months after the clearance of viremia, ZIKV must establish infection in the seminiferous tubules, the site of spermatozoon development. However, little is known about the cell types that support ZIKV infection in the human testis. Currently, there are no models to study mechanisms of virus persistence in the seminiferous tubules. We provide evidence that ZIKV infection of human Sertoli cells, which are an important component of the seminiferous tubules, is robust and induces a strong antiviral response. The use of an in vitro Sertoli cell barrier to describe how ZIKV or inflammatory mediators derived from ZIKV-infected macrophages compromise barrier integrity will enable studies to explore the interactions of other testicular cells with Sertoli cells and to test novel antivirals for clearing testicular ZIKV infection. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I∙C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response. [ABSTRACT FROM AUTHOR]

The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation. [ABSTRACT FROM AUTHOR]

Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus. [ABSTRACT FROM AUTHOR]

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 ofKat 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 viralRNAreplication, as well as virion assembly. [ABSTRACT FROM AUTHOR]

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5's conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication. [ABSTRACT FROM AUTHOR]

Flavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1. trans complementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication. [ABSTRACT FROM AUTHOR]

Flavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and an in situ proximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn β-helix and two short β-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy. [ABSTRACT FROM AUTHOR]

Flavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimated Kd (dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach. [ABSTRACT FROM AUTHOR]

The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of>70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on vesicular stomatitis virus (VSV), a rhabdovirus. Yet the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype 2 (DENV2), and human coronavirus virus (HCoV) OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrated that TRIM56's antiflavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2, and HCoV-OC43 through overlapping and distinct molecular determinants. [ABSTRACT FROM AUTHOR]

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 μMand 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. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and type II interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR-/- mice) are susceptible to infection with mouseadapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limited use for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to generate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lacking IFNAR expression on CD11c+ dendritic cells and LysM+ macrophages succumbed completely to DENV infection, while mice deficient in the receptor on either CD11c+ or LysM+ cells were susceptible to infection but often resolved viremia and recovered fully from infection. Conditional IFNAR mice responded with a swift and strong CD8+ T-cell response to viral infection, compared to a weak response in IFNAR-/- mice. Furthermore, mice lacking IFNAR on either CD11c+ or LysM+ cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates. [ABSTRACT FROM AUTHOR]

Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to>95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. [ABSTRACT FROM AUTHOR]

Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to>95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. IMPORTANCE The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication. [ABSTRACT FROM AUTHOR]

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC50). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC50 was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans. [ABSTRACT FROM AUTHOR]

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral meth-yltransferase(s) to methylate N-7 and 2'-0 cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-0 methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune re-striction. A cytoplasmic virus defective in 2'-0 methylation is replicative, but its viral RNA lacks 2'-0 methylation and is recog-nized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vac-cine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-0 cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-0 methylation was stable in cell culture after being pas-saged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and re-tained the engineered mutation iti vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-0 methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-0 methyltransferases. [ABSTRACT FROM AUTHOR]

We report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery. [ABSTRACT FROM AUTHOR]

Flavivirus nonstructural protein 2A (NS2A) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. Although flavivirus NS2A is known to associate with the endoplasmic reticulum (ER) membrane, the detailed topology of this protein has not been determined. Here we report the first topology model of flavivirus NS2A on the ER membrane. Using dengue virus (DENV) NS2A as a model, we show that (i) the N-terminal 68 amino acids are located in the ER lumen, with one segment (amino acids 30 to 52) that interacts with ER membrane without traversing the lipid bilayer; (ii) amino acids 69 to 209 form five transmembrane segments, each of which integrally spans the ER membrane; and (iii) the C-terminal tail (amino acids 210 to 218) is located in the cytosol. Nuclear magnetic resonance (NMR) structural analysis showed that the first membrane-spanning segment (amino acids 69 to 93) consists of two helices separated by a "helix breaker." The helix breaker is formed by amino acid P85 and one positively charged residue, R84. Functional analysis using replicon and genome-length RNAs of DENV-2 indicates that P85 is not important for viral replication. However, when R84 was replaced with E, the mutation attenuated both viral RNA synthesis and virus production. Remarkably, an R84A mutation did not affect viral RNA synthesis but blocked intracellular formation of infectious virions. Collectively, the mutagenesis results demonstrate that NS2A functions in both DENV RNA synthesis and virion assembly/maturation. The topology model of DENV NS2A provides a good starting point for studying how flavivirus NS2A modulates viral replication and evasion of host immune response. [ABSTRACT FROM AUTHOR]

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3DpoL) and the transfer of UMP by 3DpoL 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 3DpoL) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and 1317) at Site-311 reduced the VPg uridylylation activity of 3DpoL by > 90%. None of the Site-311 mutations affected the RNA elongation activity of 3DpoL, 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 3DpoL, which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3DpoL. Mutation of a polymerase active site in 3DpoL and Site-311 in 3DpoL 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 3DpoL during EV71 VPg uridylylation, such that one 3DpoL presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3DpoL, 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. [ABSTRACT FROM AUTHOR]

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic β-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease. [ABSTRACT FROM AUTHOR]

The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic a-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) replicates in the skin; however, cell targets in the skin have not been identified. In the current studies, WNV infected the epidermis and adnexal glands of mouse skin, and the epidermal cells were identified as keratinocytes by double labeling for WNV antigen and keratin 10. Inoculation of mice with WNV replicon particles resulted in high levels of replication in the skin, suggesting that keratinocytes are an initial target of WNV. In addition, primary keratinocytes produced infectious virus in vitro. In conclusion, keratinocytes are cell targets of WNV in vivo and may play an important role in pathogenesis. [ABSTRACT FROM AUTHOR]

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3' untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5'-3' exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3' UTR. Mutations or deletions of various secondary structures in the 3' UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNVKUN) 3' UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFNstimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles. [ABSTRACT FROM AUTHOR]

Flavivirus methyltransferase catalyzes both guanine N7 and ribose 2′-OH methylations of the viral RNA cap (GpppA-RNA→m7GpppAm-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. [ABSTRACT FROM AUTHOR]

RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5′- and 3′-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5′-terminal 20 nucleotides (5′End) or targeting the 3′-terminal element involved in a potential genome cyclizing interaction (3′CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5′End or 3′CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 µM concentration without apparent cytotoxicity. The 3′CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3′CSI sequences of specific viruses. Mode-of-action analyses showed that the 5′End and 3′CSI PMOs suppressed viral infection through two distinct mechanisms. The 5′End PMO inhibited viral translation, whereas the 3′CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3′ untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle. [ABSTRACT FROM AUTHOR]

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-α/β) and gamma IFN (IFN-γ) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-α. IFN-α signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition. [ABSTRACT FROM AUTHOR]

Recent outbreaks of West Nile Virus (WNV) have been associated with an increase in morbidity and mortality in humans, birds, and many other species. We have initiated studies to define the molecular mechanisms by which a recent pathogenic isolate of WNV evades the host cell innate antiviral response. Biochemical and microarray analyses demonstrated that WNV induced the expression of beta interferon (IFN-β) and several IFN-stimulated genes late in infection of cultured human cells. The late expression of these antiviral genes was due to the delayed activation of the transcription factor IFN regulatory factor 3 (IRF-3). Despite this host response, WNV was still able to replicate efficiently. The effect of the IRF-3 pathway on WNV replication was assessed by examining virus replication and spread in cultures of wild-type or IRF-3-null mouse embryo fibroblasts. The absence of IRF-3 was marked by a significant increase in plaque size and a sustained production of infectious particles. Although the activation of the IRF-3 pathway was not sufficient to block virus replication, our results suggest that IRF-3 target genes function to constrain WNV infection and limit cell-to-cell virus spread. [ABSTRACT FROM AUTHOR]

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10[sup 9] VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10[sup 10] VLPs per 10[sup 6] transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus, tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8[sup +]-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 10[sup 7] VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10[sup 6] splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses. [ABSTRACT FROM AUTHOR]

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A Renilla luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NSSmt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3' untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5' region of the capsid gene (5'CS) abolished RNA replication. Restoration of the 5'CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5'CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication. [ABSTRACT FROM AUTHOR]

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response. As such, viral pathogens have devised multiple mechanisms to antagonize this pathway and thus facilitate infection. Dengue virus (DENV) encodes several proteins (NS2a, NS4a, and NS4b) that have been shown individually to inhibit the IFN response. In addition, DENV infection results in reduced levels of expression of STAT2, which is required for IFN signaling (M. Jones, A. Davidson, L. Hibbert, P. Gruenwald, J. Schlaak, S. Ball, G. R. Foster, and M. Jacobs, J. Virol. 79:5414-5420, 2005). Translation of the DENV genome results in a single polypeptide, which is processed by viral and host proteases into at least 10 separate proteins. To date, no single DENV protein has been implicated in the targeting of STAT2 for decreased levels of expression. We demonstrate here that the polymerase of the virus, NS5, binds to STAT2 and is necessary and sufficient for its reduced level of expression. The decrease in protein level observed requires ubiquitination and proteasome activity, strongly suggesting an active degradation process. Furthermore, we show that the degradation of but not binding to STAT2 is dependent on the expression of the polymerase in the context of a polyprotein that undergoes proteolytic processing for NS5 maturation. Thus, the mature form of NS5, when not expressed as a precursor, was able to bind to STAT2 but was unable to target it for degradation, establishing a unique role for viral polyprotein processing in providing an additional function to a viral polypeptide. Therefore, we have identified both a novel mechanism by which DENV evades the innate immune response and a potential target for antiviral therapeutics. [ABSTRACT FROM AUTHOR]

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses. [ABSTRACT FROM AUTHOR]

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC50) of 2.4 nM and a 50% cytotoxic concentration (CC50) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC90s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV. [ABSTRACT FROM AUTHOR]

An abstract of the article "The Helical Domains of the Stem Region of Dengue Virus Envelope Protein Are Involved in both Virus Assembly and Entry," by Su-Ru Lin and colleagues is presented.

An abstract of the article "Keratinocytes Are Cell Targets of West Nile Virus In Vivo," by Pei-Yin Lim and colleagues.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial "hit" improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 µM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 µM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 µM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA → m7GpppA-RNA → m7GpppAm-RNA, by using S-adenosyl-L-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2'-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2'-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2'-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m7GpppA-RNA, after which the m7G moiety is repositioned to the GTP-binding pocket to register the 2'-OH of the adenosine with SAM, generating m7GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy. [ABSTRACT FROM AUTHOR]

The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m7 GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m7 GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m7 Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery. [ABSTRACT FROM AUTHOR]

The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA →m7GpppA →m7GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2′-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2′-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy. [ABSTRACT FROM AUTHOR]

Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m7GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m7GpppA-capped RNAs to m7GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m7GpppA- and m7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K61-D146-K182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy. [ABSTRACT FROM AUTHOR]