Structural and functional basis of low-affinity SAM/SAH-binding in the conserved MTase of the multi-segmented Alongshan virus distantly related to canonical unsegmented flaviviruses.

Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.

N-linked glycosylation of flavivirus E protein contributes to viral particle formation.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

ZDHHC11 Suppresses Zika Virus Infections by Palmitoylating the Envelope Protein.

Zika virus (ZIKV) is an RNA-enveloped virus that belongs to the Flavivirus genus, and ZIKV infections potentially induce severe neurodegenerative diseases and impair male fertility. Palmitoylation is an important post-translational modification of proteins that is mediated by a series of DHHC-palmitoyl transferases, which are implicated in various biological processes and viral infections. However, it remains to be investigated whether palmitoylation regulates ZIKV infections. In this study, we initially observed that the inhibition of palmitoylation by 2-bromopalmitate (2-BP) enhanced ZIKV infections, and determined that the envelope protein of ZIKV is palmitoylated at Cys308. ZDHHC11 was identified as the predominant enzyme that interacts with the ZIKV envelope protein and catalyzes its palmitoylation. Notably, ZDHHC11 suppressed ZIKV infections in an enzymatic activity-dependent manner and ZDHHC11 knockdown promoted ZIKV infection. In conclusion, we proposed that the envelope protein of ZIKV undergoes a novel post-translational modification and identified a distinct mechanism in which ZDHHC11 suppresses ZIKV infections via palmitoylation of the ZIKV envelope protein.

Structure and function of the NS5 methyltransferase domain from Usutu virus.

Usutu virus (USUV), is a mosquito-borne flavivirus currently spreading outside the African continent producing substantial avian mortality. In contrast, infected humans could exhibit mild neurological symptoms or remain asymptomatic. As in other flaviviruses, the capped USUV genome encodes three structural and seven non-structural (NS) proteins. Among the NS proteins, NS5 plays crucial roles in virus replication, harbouring the capping and methyltransferase (MTase) activities in its N-terminal domain and the RNA-dependent RNA polymerase (RdRP) activity at the C-terminus. In this work, we present the first structural and functional characterization of the USUV MTase domain. The first structure of the USUV MTase has been determined in complex with its natural ligands (S-adenosyl-L-methionine [SAM]) and S-adenosyl-L-homocysteine [SAH]) at 2.2 Å resolution, showing a molecular dimer in the crystal asymmetric unit. One molecule is bound to the methyl donor SAM while the second is bound to the reaction by-product SAH. Both molecules are almost identical and also show a high structural similarity to the MTase domains of other flaviviruses. The structure of the USUV MTase bound to the inhibitor sinefungin at 1.8 Å resolution is also described. Careful comparisons of the interactions in the SAM-binding cavity prompt us to hypothesize about the strength and weakness of the structure-based design of antivirals directed to the SAM/SAH binding site that could be effective to deal with this threat.

The ganglioside GM1a functions as a coreceptor/attachment factor for dengue virus during infection.

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.

Dynamic Interactions of Post Cleaved NS2B Cofactor and NS3 Protease Identified by Integrative Structural Approaches.

Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B47NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B47NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.

Precursors of Viral Proteases as Distinct Drug Targets.

Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called "stem" region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry.

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

From Capsids to Complexes: Expanding the Role of TRIM5α in the Restriction of Divergent RNA Viruses and Elements.

An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses.

Delayed by Design: Role of Suboptimal Signal Peptidase Processing of Viral Structural Protein Precursors in Flaviviridae Virus Assembly.

The Flaviviridae virus family is classified into four different genera, including flavivirus, hepacivirus, pegivirus, and pestivirus, which cause significant morbidity and mortality in humans and other mammals, including ruminants and pigs. These are enveloped, single-stranded RNA viruses sharing a similar genome organization and replication scheme with certain unique features that differentiate them. All viruses in this family express a single polyprotein that encodes structural and nonstructural proteins at the N- and C-terminal regions, respectively. In general, the host signal peptidase cleaves the structural protein junction sites, while virus-encoded proteases process the nonstructural polyprotein region. It is known that signal peptidase processing is a rapid, co-translational event. Interestingly, certain signal peptidase processing site(s) in different Flaviviridae viral structural protein precursors display suboptimal cleavage kinetics. This review focuses on the recent progress regarding the Flaviviridae virus genus-specific mechanisms to downregulate signal peptidase-mediated processing at particular viral polyprotein junction sites and the role of delayed processing at these sites in infectious virus particle assembly.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of duck MHC class I complexed with a TUMV viral peptide.

The CTL immune response mediated by MHC I plays an important role in duck anti-TMUV infection. This study reports the expression, purification and crystallization of a complex of duck MHC class I molecules Anpl-UAA*SD, duck ß2-microglobulin (Anpl-ß2m) and the polypeptide LRKRQLTVL (LRK9) derived from Tembusu virus (TMUV) NS3. The crystal diffraction resolution is 1.50 Å and belongs to the P62 space group, and the unit cell parameters are a = 82.468, b = 82.468, c = 112.507. The Matthew's constant is calculated to be 2.32 Å3 Da -1, and an asymmetric unit contains a complex molecule with a solvent content of 47%. The research lays the foundation for the structure of immune molecules about duck anti-TMUV research.

Determinants of duck Tembusu virus NS2A/2B polyprotein procession attenuated viral replication and proliferation in vitro.

Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, has caused serious economic losses for the Chinese poultry industry. The genome is translated into a polyprotein that is cleaved to mature protein by host and viral proteases in the host cell, and this proteolytic process is important for the viral life cycle. However, the cleavage mechanism of DTMUV polyprotein is still unclear. In this study, we identified that several amino acids (P1-R, P1'-G, P2-R, P3-T, and P4-V) were vital for NS2A/2B cleavage. Meanwhile, both NS2A and NS2B were essential in cis for polyprotein NS2A/2B intramolecular cleavage. Subsequently, a DTMUV replicon and an infectious clone showed that the P1 site is essential to viral replication, while a mutation in P1' could boost viral RNA replication. Furthermore, a recombinant virus with P1 and P1' site mutations named rDTMUV-NS2A/2B-P1P1'(AA) was rescued from transfected BHK21 cells. The maximum viral titers and viral genome copies of rDTMUV-NS2A/2B-P1P1'(AA) were much lower than those of rDTMUV-WT both in the intracellular and extracellular samples of transfected and infected BHK21 cells. Taken together, the NS2A/2B cleavage sites processed by the NS2B3 protease are vital for DTMUV proliferation and virulence.

Proteomics Computational Analyses Suggest That the Envelope Glycoproteins of Segmented Jingmen Flavi-Like Viruses are Class II Viral Fusion Proteins (b-Penetrenes) with Mucin-Like Domains.

Jingmen viruses are newly described segmented flavi-like viruses that have a worldwide distribution in ticks and have been associated with febrile illnesses in humans. Computational analyses were used to predict that Jingmen flavi-like virus glycoproteins have structural features of class II viral fusion proteins, including an ectodomain consisting of beta-sheets and short alpha-helices, a fusion peptide with interfacial hydrophobicity and a three-domain architecture. Jingmen flavi-like virus glycoproteins have a sequence enriched in serine, threonine, and proline at the amino terminus, which is a feature of mucin-like domains. Several of the serines and threonines are predicted be modified by the addition of O-linked glycans. Some of the glycoproteins are predicted to have an additional mucin-like domain located prior to the transmembrane anchor, whereas others are predicted to have a stem consisting of two alpha-helices. The flavivirus envelope protein and Jingmen flavi-virus glycoproteins may have diverged from a common class II precursor glycoprotein with a mucin-like domain or domains acquired after divergence.

Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction.

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-ß and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-ß transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-ß inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro.

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

RNase L Antiviral Activity Is Not a Critical Component of the Oas1b-Mediated Flavivirus Resistance Phenotype.

In mice, resistance to central nervous system (CNS) disease induced by members of the genus Flavivirus is conferred by an allele of the 2'-5' oligoadenylate synthetase 1b gene that encodes the inactive full-length protein (Oas1b-FL). The susceptibility allele encodes a C-terminally truncated protein (Oas1b-tr). We show that the efficiency of neuron infection in the brains of resistant and susceptible mice is similar after an intracranial inoculation of two flaviviruses, but amplification of viral proteins and double-stranded RNA (dsRNA) is inhibited in infected neurons in resistant mouse brains at later times. Active OAS proteins detect cytoplasmic dsRNA and synthesize short 2'-5'-linked oligoadenylates (2'-5'A) that interact with the latent endonuclease RNase L, causing it to dimerize and cleave single-stranded RNAs. To evaluate the contribution of RNase L to the resistance phenotype in vivo, we created a line of resistant RNase L-/- mice. Evidence of RNase L activation in infected RNase L+/+ mice was indicated by higher levels of viral RNA in the brains of infected RNase L-/- mice. Activation of type I interferon (IFN) signaling was detected in both resistant and susceptible brains, but Oas1a and Oas1b mRNA levels were lower in RNase L+/+ mice of both types, suggesting that activated RNase L also has a proflaviviral effect. Inhibition of virus replication was robust in resistant RNase L-/- mice, indicating that activated RNase L is not a critical factor in mediating this phenotype.IMPORTANCE The mouse genome encodes a family of Oas proteins that synthesize 2'-5'A in response to dsRNA. 2'-5'A activates the endonuclease RNase L to cleave single-stranded viral and cellular RNAs. The inactive, full-length Oas1b protein confers flavivirus-specific disease resistance. Although similar numbers of neurons were infected in resistant and susceptible brains after an intracranial virus infection, viral components amplified only in susceptible brains at later times. A line of resistant RNase L-/- mice was used to evaluate the contribution of RNase L to the resistance phenotype in vivo Activation of RNase L antiviral activity by flavivirus infection was indicated by increased viral RNA levels in the brains of RNase L-/- mice. Oas1a and Oas1b mRNA levels were higher in infected RNase L-/- mice, indicating that activated RNase L also have a proflaviviral affect. However, the resistance phenotype was equally robust in RNase L-/- and RNase L+/+ mice.

Avian Flavivirus Infection of Monocytes/Macrophages by Extensive Subversion of Host Antiviral Innate Immune Responses.

Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus in China and Southeast Asia with features of rapid spread, an expanding host range, and cross-species transmission. The mechanisms of its infection and pathogenesis remain largely unclear. Here, we investigated the tropism of this arbovirus in peripheral blood mononuclear cells of specific-pathogen-free (SPF) ducks and SPF chickens and identified monocytes/macrophages as the key targets of TMUV infection. In vivo studies in SPF ducks and SPF chickens with monocyte/macrophage clearance demonstrated that the infection of monocytes/macrophages was crucial for viral replication, transmission, and pathogenesis. Further genome-wide transcriptome analyses of TMUV-infected chicken macrophages revealed that host antiviral innate immune barriers were the major targets of TMUV in macrophages. Despite the activation of major pattern recognition receptor signaling, the inductions of alpha interferon (IFN-α) and IFN-ß were blocked by TMUV infection on transcription and translation levels, respectively. Meanwhile, TMUV inhibited host redox responses by repressing the transcription of genes encoding NADPH oxidase subunits and promoting Nrf2-mediated antioxidant responses. The recovery of either of the above-mentioned innate immune barriers was sufficient to suppress TMUV infection. Collectively, we identify an essential step of TMUV infection and reveal extensive subversion of host antiviral innate immune responses.IMPORTANCE Mosquito-borne flaviviruses include a group of pathogenic viruses that cause serious diseases in humans and animals, including dengue, West Nile, and Japanese encephalitis viruses. These flaviviruses are zoonotic and use animals, including birds, as amplifying and reservoir hosts. Avian Tembusu virus (TMUV) is an emerging mosquito-borne flavivirus that is pathogenic for many avian species and can infect cells derived from mammals and humans in vitro Although not currently pathogenic for primates, the infection of duck industry workers and the potential risk of TMUV infection in immunocompromised individuals have been highlighted. Thus, the prevention of TMUV in flocks is important for both avian and mammalian health. Our study reveals the escape of TMUV from the first line of the host defense system in the arthropod-borne transmission route of arboviruses, possibly helping to extend our understanding of flavivirus infection in birds and refine the design of anti-TMUV therapeutics.