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helices alpha2 and alpha3 of west nile virus capsid protein are dispensable for assembly of infectious virions.the internal hydrophobic sequence within the flaviviral capsid protein (protein c) plays an important role in the assembly of infectious virions. here, this sequence was analyzed in a west nile virus lineage i isolate (crow v76/1). an infectious cdna clone was constructed and used to introduce deletions into the internal hydrophobic domain which comprises helix alpha2 and part of the loop intervening helices alpha2 and alpha3. in total, nine capsid deletion mutants (4 to 14 amino acids long) wer ...200919297470
the zoonotic flaviviruses of southern, south-eastern and eastern asia, and australasia: the potential for emergent viruses.the genus flaviviridae comprises about 70 members, of which about 30 are found in southern, south-eastern and eastern asia and australasia. these include major pathogens such as japanese encephalitis (je), west nile (wn), murray valley encephalitis (mve), tick-borne encephalitis, kyasanur forest disease virus, and the dengue viruses. other members are known to be associated with mild febrile disease in humans, or with no known disease. in addition, novel flaviviruses continue to be discovered, a ...200919486319
3' cis-acting elements that contribute to the competence and efficiency of japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions.the positive-strand rna genome of japanese encephalitis virus (jev) terminates in a highly conserved 3'-noncoding region (3'ncr) of six domains (v, x, i, ii-1, ii-2, and iii in the 5'-to-3' direction). by manipulating the jev genomic rna, we have identified important roles for rna elements present within the 574-nucleotide 3'ncr in viral replication. the two 3'-proximal domains (ii-2 and iii) were sufficient for rna replication and virus production, whereas the remaining four (v, x, i, and ii-1) ...200919494005
hepatitis c virus ns2 protein contributes to virus particle assembly via opposing epistatic interactions with the e1-e2 glycoprotein and ns3-ns4a enzyme complexes.the hepatitis c virus ns2 protein has been recently implicated in virus particle assembly. to further understand the role of ns2 in this process, we conducted a reverse genetic analysis of ns2 in the context of a chimeric genotype 2a infectious cell culture system. of 32 mutants tested, all were capable of rna replication and 25 had moderate-to-severe defects in virus assembly. through forward genetic selection for variants capable of virus spread, we identified second-site mutations in e1, e2, ...200919515772
trans-complementation of an ns2 defect in a late step in hepatitis c virus (hcv) particle assembly and maturation.recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis c virus (hcv) indicate that several nonstructural viral proteins, including ns2, ns3, and ns5a, are involved in the process of viral assembly and release. other recent work suggests that ser-168 of ns2 is a target of ck2 kinase-mediated phosphorylation, and that this controls the stability of the genotype 1a ns2 protein. here, we show that ser-168 is a critical determinant in the production o ...200919412343
progress on the development of therapeutics against west nile virus.a decade has passed since the appearance of west nile virus (wnv) in humans in the western hemisphere in new york city. during this interval, wnv spread inexorably throughout north and south america and caused millions of infections ranging from a sub-clinical illness, to a self-limiting febrile syndrome or lethal neuroinvasive disease. its entry into the united states triggered intensive research into the basic biology of wnv and the elements that comprise a protective host immune response. alt ...200919501622
theoretical study of the usutu virus helicase 3d structure, by means of computer-aided homology modelling.background: usutu virus belongs to the flaviviridae viral family and constitutes an important pathogen. the viral helicase is an ideal target for inhibitor design, since this enzyme is essential for the survival, proliferation and transmission of the virus. results: towards a drug-design approach, the 3d model of the usutu virus helicase structure has been designed, using conventional homology modelling techniques and the known 3d-structure of the murray valley encephalitis virus helicase, of th ...200919555498
production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.with the advent of subgenomic hepatitis c virus (hcv) replicons, studies of the intracellular steps of the viral replication cycle became possible. these rnas are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate jfh-1, efficient replication of these hcv rnas requires replication enhancing mutations (rems), previously also called cell culture adaptive mutations. these mutations cluster primarily in the central region of non-structural protein 5a ...200919521536
development of resistance to passive therapy with a potently neutralizing humanized monoclonal antibody against west nile virus.previous studies have established the therapeutic efficacy of humanized e16 (he16) monoclonal antibody against west nile virus in animals. here, we assess the potential for west nile virus strains encoding mutations in the he16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during he16 treatment. resistance to he16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect pr ...200919527169
cis-acting rna elements in human and animal plus-strand rna viruses.the rna genomes of plus-strand rna viruses have the ability to form secondary and higher-order structures that contribute to their stability and to their participation in inter- and intramolecular interactions. those structures that are functionally important are called cis-acting rna elements because their functions cannot be complemented in trans. they can be involved not only in rna/rna interactions but also in binding of viral and cellular proteins during the complex processes of translation ...200919781674
crystal structure of a novel conformational state of the flavivirus ns3 protein: implications for polyprotein processing and viral replication.the flavivirus genome comprises a single strand of positive-sense rna, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. one of these, nonstructural protein 3 (ns3), is an enzyme with both serine protease and ntpase/helicase activities. ns3 plays a central role in the flavivirus life cycle: the ns3 n-terminal serine protease together with its essential cofactor ns2b is involved in the processing of the polyprotein, where ...200919793813
identification of inhibitors of yellow fever virus replication using a replicon-based high-throughput assay.flaviviruses cause severe disease in humans and are a public health priority worldwide. however, no effective therapies or drugs are commercially available yet. several flavivirus replicon-based assays amenable to high-throughput screening of inhibitors have been reported recently. we developed and performed a replicon-based high-throughput assay for screening small-molecule inhibitors of yellow fever virus (yfv) replication. this assay utilized packaged pseudoinfectious particles containing a y ...200919651907
focus on flaviviruses: current and future drug targets.infection by mosquito-borne flaviviruses (family flaviviridae) is increasing in prevalence worldwide. the vast global, social and economic impact due to the morbidity and mortality associated with the diseases caused by these viruses necessitates therapeutic intervention. there is currently no effective clinical treatment for any flaviviral infection. therefore, there is a great need for the identification of novel inhibitors to target the virus life cycle.200920165556
measure and countermeasure: type i ifn (ifn-alpha/beta) antiviral response against west nile virus.as a first line of defense after viral infection, host cells develop an intrinsic immune response to control virus dissemination and protect against serious infection. recent experiments have shown a dominant role of the ifn-alpha/beta response in protection against lethal west nile virus (wnv) by limiting the cellular and tissue tropism of infection. this review will focus on advances in identifying the host sensors that detect wnv and the adaptor molecules and signaling pathways that regulate ...200920375601
emergent infectious uveitis.infectious causes should always be considered in all patients with uveitis and it should be ruled out first. the differential diagnosis includes multiple well-known diseases including herpes, syphilis, toxoplasmosis, tuberculosis, bartonellosis, lyme disease, and others. however, clinicians should be aware of emerging infectious agents as potential causes of systemic illness and also intraocular inflammation. air travel, immigration, and globalization of business have overturned traditional patt ...200920404989
recombination in west nile virus: minimal contribution to genomic diversity.recombination is known to play a role in the ability of various viruses to acquire sequence diversity. we consequently examined all available west nile virus (wnv) whole genome sequences both phylogenetically and with a variety of computational recombination detection algorithms. we found that the number of distinct lineages present on a phylogenetic tree reconstruction to be identical to the 6 previously reported. statistically-significant evidence for recombination was only observed in one who ...200919821990
west nile virus infection alters midgut gene expression in culex pipiens quinquefasciatus say (diptera: culicidae).alterations in gene expression in the midgut of female culex pipiens quinquefasciatus exposed to blood meals containing 6.8 logs plaque-forming units/ml of west nile virus (wnv) were studied by fluorescent differential display. twenty-six different cdnas exhibited reproducible differences after feeding on infected blood. of these, 21 cdnas showed an increase in expression, and 5 showed a decrease in expression as a result of wnv presence in the blood meal. genbank database searches showed that o ...200919635880
west nile virus envelope protein glycosylation is required for efficient viral transmission by culex vectors.many, but not all, strains of west nile virus (wnv) contain a single n-linked glycosylation site on their envelope (e) proteins. previous studies have shown that e-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. e protein glycosylation also appears to play a role in attachment and entry of wnv into host cells in vitro; however, studies examining how e protein glycosylation affects the interactions of wnv with its mosquito vectors in vivo have not yet been perfo ...200919249803
ns5 of dengue virus mediates stat2 binding and degradation.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. hibber ...200919279106
ticks associated with macquarie island penguins carry arboviruses from four genera.macquarie island, a small subantarctic island, is home to rockhopper, royal and king penguins, which are often infested with the globally distributed seabird tick, ixodes uriae. a flavivirus, an orbivirus, a phlebovirus, and a nairovirus were isolated from these ticks and partial sequences obtained. the flavivirus was nearly identical to gadgets gully virus, isolated some 30 year previously, illustrating the remarkable genetic stability of this virus. the nearest relative to the orbivirus (for w ...200919194498
isolation of genotype v st. louis encephalitis virus in florida.we isolated and characterized st. louis encephalitis virus (slev) from cloacal swabs of naturally exposed adult sentinel chickens in 2006. phylogenetic analysis of slev strains isolated in florida indicated that brazilian slev circulated in 1972 and 2006; lineages were va and vb.200919331744
glyceraldehyde-3-phosphate dehydrogenase (gapdh) interaction with 3' ends of japanese encephalitis virus rna and colocalization with the viral ns5 protein.replication of the japanese encephalitis virus (jev) genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. glyceraldehyde-3-phosphate dehydrogenase (gapdh), a cellular metabolic protein, was found to colocalize with viral rna-dependent rna polymerase (ns5) in jev-infected cells. subcellular fractionation further indicated that gapdh remained relatively constant in the cytosol, ...200919368702
changing patterns of west nile virus transmission: altered vector competence and host susceptibility.west nile virus (wnv) is a flavivirus (flaviviridae) transmitted between culex spp. mosquitoes and avian hosts. the virus has dramatically expanded its geographic range in the past ten years. increases in global commerce, climate change, ecological factors and the emergence of novel viral genotypes likely play significant roles in the emergence of this virus; however, the exact mechanism and relative importance of each is uncertain. previously wnv was primarily associated with febrile illness of ...200919406093
molecular targets for flavivirus drug discovery.flaviviruses are a major cause of infectious disease in humans. dengue virus causes an estimated 50 million cases of febrile illness each year, including an increasing number of cases of hemorrhagic fever. west nile virus, which recently spread from the mediterranean basin to the western hemisphere, now causes thousands of sporadic cases of encephalitis annually. despite the existence of licensed vaccines, yellow fever, japanese encephalitis and tick-borne encephalitis also claim many thousands ...200918796313
extension of flavivirus protein c differentially affects early rna synthesis and growth in mammalian and arthropod host cells.the translation of flaviviral rna genomes yields a single polyprotein that is processed into the mature proteins by viral and host cell proteases. mature capsid protein c is freed from the polyprotein by the viral ns2b/3 protease, cleaving in the c-terminal region of protein c in front of the signal sequence for prm. protein c has been shown to be involved in viral assembly and rna packaging. to examine further the role of protein c and its production by proteolysis, we replaced the ns2b/3 capsi ...200919692461
virus-specific cytolytic antibodies to nonstructural protein 1 of japanese encephalitis virus effect reduction of virus output from infected cells.we demonstrate the presence of nonstructural protein 1 (ns1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where japanese encephalitis virus (jev) is endemic. sera containing antibodies to ns1 but not those with antibodies to other jev proteins, such as envelope, brought about complement-mediated lysis of jev-infected bhk-21 cells. target cells infected with a recombinant poxvirus expressing jev ns1 on the cell surface ...200919264772
analysis of flavivirus ns5 methyltransferase cap binding.the flavivirus 2'-o-nucleoside n-terminal rna methyltransferase (mtase) enzyme is responsible for methylating the viral rna cap structure. to increase our understanding of the mechanism of viral rna cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (den) and yellow fever (yf) virus mtase enzymes. we solved an improved 2.1 a resolution crystal structure of den2 mtase, new 1.5 a resolution crystal structures of the yf ...200919101564
vector competence of australian mosquito species for a north american strain of west nile virus.since the establishment of west nile virus (wnv) into the united states, concern has arisen that this virus may also pose a serious threat to australian biosecurity. the vector competence of 19 australian mosquito species for a north american strain of wnv was evaluated. mosquitoes collected from cairns, brisbane, and sydney were exposed to blood containing 10(4.0+/-0.3) cell culture infectious dose(50)/mosquito wnv that was isolated from a crow during the 1999 new york outbreak. mosquitoes were ...200818973445
co-selection of west nile virus nucleotides that confer resistance to an antisense oligomer while maintaining long-distance rna/rna base pairings.west nile virus (wnv) genome cyclization is mediated by two pairs of long-distance rna/rna interactions: the 5'cs/3'csi (conserved sequence) and the 5'uar/3'uar (upstream aug region) base pairings. antisense peptide-conjugated phosphorodiamidate morpholino oligomers (ppmos), designed to interfere with the 5'cs/3'csi or 5'uar/3'uar base pairings, were previously shown to inhibit wnv. in this study, we selected and characterized wnvs resistant to a ppmo targeting the 3'uar (3'uar-ppmo). all resist ...200818842280
sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.positive-strand rna viruses, a large group including human pathogens such as sars-coronavirus (sars-cov), replicate in the cytoplasm of infected host cells. their replication complexes are commonly associated with modified host cell membranes. membrane structures supporting viral rna synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to b ...200818798692
detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase pcr-ligase detection reaction assay.the detection and successful typing of dengue virus (denv) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. a technique for the multiplex detection and typing of denv serotypes 1 to 4 (denv-1 to denv-4) from clinical samples by pcr-ligase detection reaction (ldr) has been developed. a serotype-specific pcr amplifies the regions of genes c and e simultaneously. the two amplicons are targeted ...200818685000
separate molecules of west nile virus methyltransferase can independently catalyze the n7 and 2'-o methylations of viral rna cap.west nile virus methyltransferase catalyzes n7 and 2'-o methylations of the viral rna cap (gpppa-rna-->m(7)gpppam-rna). the two methylation events are independent, as evidenced by efficient n7 methylation of gpppa-rna-->m(7)gpppa-rna and gpppam-rna-->m(7)gpppam-rna, and by the 2'-o methylation of gpppa-rna-->gpppam-rna and m(7)gpppa-rna-->m(7)gpppam-rna. however, the 2'-o methylation activity prefers substrate m(7)gpppa-rna to gpppa-rna, thereby determining the dominant methylation pathway as gp ...200818501946
ires-dependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells.the development of infectious cdna for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. moreover, the genomes of such alphaviruses can be engineered to contain rna elements that would be functional only in cells of vertebrate, but not insect, origin. in the present study, we developed a recombinant veev that is more attenuated than ...200818501401
west nile virus infection of drosophila melanogaster induces a protective rnai response.to determine if west nile virus (wnv) infection of insect cells induces a protective rnai response, drosophila melanogaster s2 and aedes albopictus c6/36 cells were infected with wnv, and the production of wnv-homologous small rnas was assayed as an indicator of rnai induction. a distinct population of approximately 25 nt wnv-homologous small rnas was detected in infected s2 cells but not c6/36 cells. rnai knockdown of argonaute 2 in s2 cells resulted in slightly increased susceptibility to wnv ...200818501400
efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles.recently, complete replication of hepatitis c virus (hcv) in tissue culture was established using the jfh1 isolate. to analyze determinants of hcv genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral rnas. using jfh1 helper viruses, we show that subgenomic jfh1 replicons lacking the entire core to ns2 coding region are efficiently encapsidated into infectious virus-like particles. similarly, chimeric helper vir ...200818480457
humanized monoclonal antibodies derived from chimpanzee fabs protect against japanese encephalitis virus in vitro and in vivo.japanese encephalitis virus (jev)-specific fab antibodies were recovered by repertoire cloning from chimpanzees initially immunized with inactivated je-vax and then boosted with attenuated jev sa14-14-2. from a panel of 11 fabs recovered by different panning strategies, three highly potent neutralizing antibodies, termed fabs a3, b2, and e3, which recognized spatially separated regions on the virion, were identified. these antibodies reacted with epitopes in different domains: the major determin ...200818480437
phosphorylation of yellow fever virus ns5 alters methyltransferase activity.serine/threonine phosphorylation of the nonstructural protein 5 (ns5) is conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. mass spectrometry was used to characterize phosphorylated residues of yellow fever virus (yfv) ns5 expressed in mammalian cells. multiple different phosphopeptides were detected. mutational and additional mass spectrometry data implicated serine 56 (s56), a conserved residue near the active site in the ns5 methyltransferase doma ...200818757072
genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia, 2007.zika virus (zikv) is a mosquito-borne flavivirus first isolated in uganda from a sentinel monkey in 1947. mosquito and sentinel animal surveillance studies have demonstrated that zikv is endemic to africa and southeast asia, yet reported human cases are rare, with <10 cases reported in the literature. in june 2007, an epidemic of fever and rash associated with zikv was detected in yap state, federated states of micronesia. we report the genetic and serologic properties of the zikv associated wit ...200818680646
genetic variation of st. louis encephalitis virus.st. louis encephalitis virus (slev) has been regularly isolated throughout the americas since 1933. previous phylogenetic studies involving 62 isolates have defined seven major lineages (i-vii), further divided into 14 clades. in this study, 28 strains isolated in texas in 1991 and 2001-2003, and three older, previously unsequenced strains from jamaica and california were sequenced over the envelope protein gene. the inclusion of these new sequences, and others published since 2001, has allowed ...200818632961
architects of assembly: roles of flaviviridae non-structural proteins in virion morphogenesis.viruses of the flaviviridae family, including hepatitis c, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. we discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. ...200818587411
closing the door on flaviviruses: entry as a target for antiviral drug design.with the emergence and rapid spread of west nile virus in the united states since 1999, and the 50-100 million infections per year caused by dengue virus globally, the threat of flaviviruses as re-emerging human pathogens has become a reality. to support the efforts that are currently being pursued to develop effective vaccines against these viruses, researchers are also actively pursuing the development of small molecule compounds that target various aspects of the virus life cycle. recent adva ...200818585795
interaction of hepatitis c virus nonstructural protein 5a with core protein is critical for the production of infectious virus particles.nonstructural protein 5a (ns5a) of the hepatitis c virus (hcv) possesses multiple and diverse functions in rna replication, interferon resistance, and viral pathogenesis. recent studies suggest that ns5a is involved in the assembly and maturation of infectious viral particles; however, precisely how ns5a participates in virus production has not been fully elucidated. in the present study, we demonstrate that ns5a is a prerequisite for hcv particle production as a result of its interaction with t ...200818524832
a single n-linked glycosylation site in the japanese encephalitis virus prm protein is critical for cell type-specific prm protein biogenesis, virus particle release, and pathogenicity in mice.the prm protein of japanese encephalitis virus (jev) contains a single potential n-linked glycosylation site, n(15)-x(16)-t(17), which is highly conserved among jev strains and closely related flaviviruses. to investigate the role of this site in jev replication and pathogenesis, we manipulated the rna genome by using infectious jev cdna to generate three prm mutants (n15a, t17a, and n15a/t17a) with alanine substituting for n(15) and/or t(17) and one mutant with silent point mutations introduced ...200818524814
ns3 helicase domains involved in infectious intracellular hepatitis c virus particle assembly.a mutation within subdomain 1 of the hepatitis c virus (hcv) ns3 helicase (ns3-q221l) (m. yi, y. ma, j. yates, and s. m. lemon, j. virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric rna (hj3). although ns3-gln-221 is highly conserved across hcv genotypes, the leu-221 substitution had no effect on rna replication or ns3-associated enzymatic activities. however, while transfection of unmodified hj3 rna failed to produce either extracellular or ...200818508894
characterization of hepatitis c rna-containing particles from human liver by density and size.hepatitis c virus (hcv) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of hcv in human serum. previously, our group has found that hcv circulates in blood in association with very-low-density lipoprotein (vldl). our aim in this study was to characterize hcv rna-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with ...200818796720
a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping.nonsegmented negative-sense (nns) rna viruses cap their mrna by an unconventional mechanism. specifically, 5' monophosphate mrna is transferred to gdp derived from gtp through a reaction that involves a covalent intermediate between the large polymerase protein l and mrna. this polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an rna triphosphatase and guanylyltransferase. in these reactions, a 5' diphosphate mrna is capped by transfer of ...200818003731
replication-coupled packaging mechanism in positive-strand rna viruses: synchronized coexpression of functional multigenome rna components of an animal and a plant virus in nicotiana benthamiana cells by agroinfiltration.flock house virus (fhv), a bipartite rna virus of insects and a member of the nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (bmv), an rna virus that infects plants and is a member of the bromoviridae family. in bmv and fhv, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand rna viruses of diverse origin. to unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we h ...200818032497
dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells.dengue virus (denv) is an enveloped rna virus that causes the most common arthropod-borne infection worldwide. the mechanism by which denv infects the host cell remains unclear. in this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of denv. simultaneous tracking of denv particles and various endocytic markers revealed that denv enters cells exclusively via clathrin-mediated endocytosis. the virus particles move ...200819096510
crystal structure of the ns3 protease-helicase from dengue virus.several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. during infection, the flavivirus rna genome is translated into a polyprotein, which is cleaved into several components. nonstructural protein 3 (ns3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease n-terminal domain, with a segment of 40 residues ...200817942558
visualization of double-stranded rna in cells supporting hepatitis c virus rna replication.the mechanisms involved in hepatitis c virus (hcv) rna replication are unknown, and this aspect of the virus life cycle is not understood. it is thought that virus-encoded nonstructural proteins and rna genomes interact on rearranged endoplasmic reticulum (er) membranes to form replication complexes, which are believed to be sites of rna synthesis. we report that, through the use of an antibody specific for double-stranded rna (dsrna), dsrna is readily detectable in huh-7 cells that contain repl ...200818094154
functional analysis of potential carboxy-terminal cleavage sites of tick-borne encephalitis virus capsid protein.the mature capsid protein c of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral ns2b/3 protease. this cleavage is a prerequisite for the subsequent processing of the viral surface protein prm, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. protein c of tick-borne encephalitis virus (tbev) contains two amino acid sequence motifs within the carboxy-terminal region that match th ...200818160443
yellow fever virus ns3 plays an essential role in virus assembly independent of its known enzymatic functions.in flaviviruses it has been proposed that there is a coupling between genome replication and virion assembly and that nonstructural proteins are involved in this process. it was previously reported that mutations in yellow fever virus (yfv) nonstructural protein ns2a blocked production of infectious virus and that this block could be released by a suppressor mutation in ns3. here, based on studies using a yfv replicon-based trans-packaging system as well as full-length yfv cdna, we report that m ...200818199634
defining the levels of secreted non-structural protein ns1 after west nile virus infection in cell culture and mice.infection with west nile virus (wnv) causes a febrile illness that can progress to meningitis or encephalitis, primarily in humans that are immunocompromised or elderly. for successful treatment of wnv infection, accurate and timely diagnosis is essential. previous studies have suggested that the flavivirus non-structural protein ns1, a highly conserved and secreted glycoprotein, is a candidate protein for rapid diagnosis. herein, we developed a capture enzyme-linked immunosorbent assay (elisa) ...200818205232
universal primers that amplify rna from all three flavivirus subgroups.species within the flavivirus genus pose public health problems around the world. increasing cases of dengue and japanese encephalitis virus in asia, frequent outbreaks of yellow fever virus in africa and south america, and the ongoing spread of west nile virus throughout the americas, show the geographical burden of flavivirus diseases. flavivirus infections are often indistinct from and confused with other febrile illnesses. here we review the specificity of published primers, and describe a n ...200818218114
genetic determinants of virulence in pathogenic lineage 2 west nile virus strains.we determined complete genome sequences of lineage 2 west nile virus (wnv) strains isolated from patients in south africa who had mild or severe wnv infections. these strains had previously been shown to produce either highly or less neuroinvasive infection and induced genes similar to corresponding highly or less neuroinvasive lineage 1 strains in mice. phylogenetic and amino acid comparison of highly and less neuroinvasive lineage 2 strains demonstrated that the nonstructural genes, especially ...200818258114
formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes.the replication/transcription complex of the arterivirus equine arteritis virus (eav) is associated with paired membranes and/or double-membrane vesicles (dmvs) that are thought to originate from the endoplasmic reticulum. previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. here, site-directed mutagenesis was used to investigate the role of ns ...200818305048
genetic variability of west nile virus in us blood donors, 2002-2005.west nile virus (wnv) was detected in the united states in 1999, has reoccurred every summer since, and has become endemic. transfusion transmission was documented in 2002, and screening of blood donations for wnv began in 2003. we investigated genetic variation of wnv in human isolates obtained from specimens collected from 30 infected blood donors who tested positive for wnv rna during 2002-2005. complete genomic sequences of 8 isolates and structural gene sequences from 22 additional isolates ...200818325259
role of nonstructural protein ns2a in flavivirus assembly.flavivirus nonstructural (ns) proteins are involved in rna replication and modulation of the host antiviral response; however, evidence is mounting that some ns proteins also have essential roles in virus assembly. kunjin virus (kun) ns2a is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. previously, we have shown that an isoleucine (i)-to-asparagine (n) substitution at position 59 of the ns2a protein blocked the production o ...200818337583
the relative contribution of antibody and cd8+ t cells to vaccine immunity against west nile encephalitis virus.west nile virus (wnv) is a mosquito borne, neurotropic flavivirus that causes a severe central nervous system (cns) infection in humans and animals. although commercial vaccines are available for horses, none is currently approved for human use. in this study, we evaluated the efficacy and mechanism of immune protection of two candidate wnv vaccines in mice. a formalin-inactivated wnv vaccine induced higher levels of specific and neutralizing antibodies compared to a dna plasmid vaccine that pro ...200818339459
antigen-specific immunotherapy of cervical and ovarian cancer.we contrast the efforts to treat ovarian cancer and cervical cancer through vaccination because of their different pathobiology. a plethora of approaches have been developed for therapeutic vaccination against cancer, many of which target defined tumor-associated antigens (taas). persistent infection with oncogenic human papillomavirus (hpv) types causes cervical cancer. furthermore, cervical cancer patients frequently mount both humoral and t-cell immune responses to the hpv e6 and e7 oncoprote ...200818363994
west nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin.west nile virus (wnv) has been the leading cause of viral encephalitis in the united states since 1999. the endocytic processes involved in the internalization of infectious wnv by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of wnv has not been investigated previously. in this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-beta-cyclodextrin resulte ...200818385233
the two-component ns2b-ns3 proteinase represses dna unwinding activity of the west nile virus ns3 helicase.similar to many flavivirus types including dengue and yellow fever viruses, the nonstructural ns3 multifunctional protein of west nile virus (wnv) with an n-terminal serine proteinase domain and an rna triphosphatase, an ntpase domain, and an rna helicase in the c-terminal domain is implicated in both polyprotein processing and rna replication and is therefore a promising drug target. to exhibit its proteolytic activity, ns3 proteinase requires the presence of the cofactor encoded by the upstrea ...200818442976
maturation of west nile virus modulates sensitivity to antibody-mediated neutralization.west nile virions incorporate 180 envelope (e) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. the e proteins of newly synthesized west nile virus (wnv) are organized into trimeric spikes composed of pre-membrane (prm) and e protein heterodimers. during egress, immature virions undergo a protease-mediated cleavage of prm that results in a reorganization of e protein into the pseudo-icosahedral arrangement characteristic of mature virion ...200818464894
molecular mechanisms of antibody-mediated neutralisation of flavivirus infection.flaviviruses are a group of positive-stranded rna viruses that cause a spectrum of severe illnesses globally in more than 50 million individuals each year. while effective vaccines exist for three members of this group (yellow fever, japanese encephalitis, and tick-borne encephalitis viruses), safe and effective vaccines for several other flaviviruses of clinical importance, including west nile and dengue viruses, remain in development. an effective humoral immune response is critical for protec ...200818471342
association of hepatitis c virus replication complexes with microtubules and actin filaments is dependent on the interaction of ns3 and ns5a.the hepatitis c virus (hcv) rna replication complex (rc), which is composed of viral nonstructural (ns) proteins and host cellular proteins, replicates the viral rna genome in association with intracellular membranes. two viral ns proteins, ns3 and ns5a, are essential elements of the rc. here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that ns3 and ns5a interact with tubulin and actin. furthermore, immunofluorescence microscopy and electron mi ...200818562541
a complex rna motif defined by three discontinuous 5-nucleotide-long strands is essential for flavivirus rna replication.tertiary or higher-order rna motifs that regulate replication of positive-strand rna viruses are as yet poorly understood. using japanese encephalitis virus (jev), we now show that a key element in jev rna replication is a complex rna motif that includes a string of three discontinuous complementary sequences (tdcs). the tdcs consists of three 5-nt-long strands, the left (l) strand upstream of the translation initiator aug adjacent to the 5'-end of the genome, and the middle (m) and right (r) st ...200818669441
complement and its role in protection and pathogenesis of flavivirus infections.the complement system is a family of serum and cell surface proteins that recognize pathogen-associated molecular patterns, altered-self ligands, and immune complexes. activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. in this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by flaviviruses, with an emphasis on experiments wit ...200819388173
allelic variation in the hepatitis c virus ns4b protein dramatically influences rna replication.in the huh-7.5 hepatoma cell line, replication of the genotype 1a h77 strain of hepatitis c virus (hcv) is attenuated compared to that of the genotype 1b con1 strain. this study identifies the poorly characterized integral membrane protein, ns4b, as a major determinant for this replication difference. chimeric h77 subgenomic replicons containing the entire ns4b gene from con1 in place of the h77 ns4b sequence replicated approximately 10-fold better than the h77 parent and to levels similar to th ...200717360748
west nile virus strain kunjin ns5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral rna synthesis.using west nile virus strain kunjin virus (wnv(kun)) as a model system for flavivirus replication, we showed that the virus replication complex (rc) is associated with the dsrna template located in induced membranes only in the cytoplasm. in this report we established for the first time that the rna-dependent rna polymerase ns5 is located in flavivirus-induced membranes, including the site of viral rna replication. we found no evidence for nuclear localization of the essential rc components ns5 ...200717374759
in situ reactions of monoclonal antibodies with a viable mutant of murray valley encephalitis virus reveal an absence of dimeric ns1 protein.studies on the ns1 protein of flaviviruses have concluded that formation of a stable homodimer is required for virus replication. however, previous work has reported that substitution of a conserved proline by leucine at residue 250 in ns1 of kunjin virus (kunv) eliminated dimerization, but allowed virus replication to continue. to assess whether this substitution has similar effects on other flaviviruses, it was introduced into an infectious clone of murray valley encephalitis virus (mvev). con ...200717374761
structural evidence for regulation and specificity of flaviviral proteases and evolution of the flaviviridae fold.pathogenic members of the flavivirus family, including west nile virus (wnv) and dengue virus (dv), are growing global threats for which there are no specific treatments. the two-component flaviviral enzyme ns2b-ns3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. here, we report the crystal structure of wnv ns2b-ns3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/bpti. we show that aprotinin bind ...200717400917
high levels of subgenomic hcv plasma rna in immunosilent infections.a genetic analysis of hepatitis c virus (hcv) in rare blood donors who remained hcv seronegative despite long-term high-level viremia revealed the chronic presence of hcv genomes with large in frame deletions in their structural genes. full-length hcv genomes were only detected as minority variants. in one immunodeficiency virus (hiv) co-infected donor the truncated hcv genome transiently decreased in frequency concomitant with delayed seroconversion and re-emerged following partial seroreversio ...200717493654
functional requirements of the yellow fever virus capsid protein.although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. by use of a trans-packaging system that involved packaging a yellow fever virus (yfv) replicon into pseudo-infectious particles by supplying the yfv structural proteins using a sindbis virus helper construct, the functional elements within the yfv capsid p ...200717526891
processing of capsid protein by cathepsin l plays a crucial role in replication of japanese encephalitis virus in neural and macrophage cells.the flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. in this study, we found a small capsid protein in cells infected with japanese encephalitis virus (jev) but not in the viral particles. the small capsid protein was shown to be generated by processing with host cysteine protease cathepsin l. an in vitro cleavage assay revealed that cathepsin l cleaves the capsid protein between amino acid residues lys(18) and arg(19), which are well ...200717553875
rapid fixation of a distinctive sequence motif in the 3' noncoding region of the clade of west nile virus invading north america.phylogenetic analysis of complete genomes of west nile virus (wnv) by a variety of methods supported the hypothesis that north american isolates of wnv constitute a monophyletic group, together with an isolate from israel and one from hungary. we used ancestral sequence reconstruction in order to obtain evidence for evolutionary changes that might be correlated with increased virulence in this clade (designated the n.a. clade). there was one amino acid change (i-->t at residue 356 of the ns3 pro ...200717587514
epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from japanese encephalitis virus infections in equine sera.west nile virus (wnv) is now widely distributed worldwide, except in most areas of asia where japanese encephalitis virus (jev) is distributed. considering the movement and migration of reservoir birds, there is concern that wnv may be introduced in asian countries. although manuals and guidelines for serological tests have been created in japan in preparedness for the introduction of wnv, differential diagnosis between wnv and jev may be complicated by antigenic cross-reactivities between these ...200717596430
a curious coincidence: mosquito biodiversity and the limits of the japanese encephalitis virus in australasia.the mosquito culex annulirostris skuse (diptera: culicidae) is the major vector of endemic arboviruses in australia and is also responsible for the establishment of the japanese encephalitis virus (jev) in southern papua new guinea (png) as well as its incursions into northern australia. papua new guinea and mainland australia are separated by a small stretch of water, the torres strait, and its islands. while there has been regular jev activity on these islands, jev has not established on mainl ...200717598922
biochemical characterization of a recombinant japanese encephalitis virus rna-dependent rna polymerase.japanese encephalitis virus (jev) ns5 is a viral nonstructural protein that carries both methyltransferase and rna-dependent rna polymerase (rdrp) domains. it is a key component of the viral rna replicase complex that presumably includes other viral nonstructural and cellular proteins. the biochemical properties of jev ns5 have not been characterized due to the lack of a robust in vitro rdrp assay system, and the molecular mechanisms for the initiation of rna synthesis by jev ns5 remain to be el ...200717623110
crystal structure and activity of kunjin virus ns3 helicase; protease and helicase domain assembly in the full length ns3 protein.flaviviral ns3 is a multifunctional protein displaying n-terminal protease activity in addition to c-terminal helicase, nucleoside 5'-triphosphatase (ntpase), and 5'-terminal rna triphosphatase (rtpase) activities. ns3 is held to support the separation of rna daughter and template strands during viral replication. in addition, ns3 assists the initiation of replication by unwinding the rna secondary structure in the 3' non-translated region (ntr). we report here the three-dimensional structure (a ...200717658551
a single positively selected west nile viral mutation confers increased virogenesis in american crows.west nile virus (wnv), first recognized in north america in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. despite the well-described epidemiological patterns of wnv in north america, the basis for the emergence of wnv-associated avian pathology, particularly in the american crow (amcr) sentinel species, and the large scale of the north american epidemic and epiornitic is uncertain. we report here that the introduction of a ...200717694056
three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle.positive-strand rna viruses are the largest genetic class of viruses and include many serious human pathogens. all positive-strand rna viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. however, the exact sites of rna synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. we applied electron microscope tomograp ...200717696647
identification of novel small-molecule inhibitors of west nile virus infection.west nile virus (wnv) has spread throughout the united states and canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. no therapy or vaccine is currently approved for use in humans. using high-throughput screening assays that included a luciferase expressing wnv subgenomic replicon and an ns1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,00 ...200717715228
differential effects of mutations in ns4b on west nile virus replication and inhibition of interferon signaling.west nile virus (wnv) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. previously, we demonstrated that replication of wnv inhibits the interferon (ifn) signal transduction pathway by preventing the accumulation of phosphorylated janus kinase 1 (jak1) and tyrosine kinase 2 (tyk2) (j. t. guo et al., j. virol. 79:1343-1350, 2005). through a genetic analysis, we have now identified a determinant on the nonstructural protein 4b (ns4b) that contro ...200717715229
differential expression of interferon (ifn) regulatory factors and ifn-stimulated genes at early times after west nile virus infection of mouse embryo fibroblasts.although lineage i west nile virus (wnv) strain eg101 induced beta interferon (ifn-beta) production as early as 12 h after infection in primary mouse embryo fibroblasts and did not inhibit the jak-stat signaling pathway, it was still able to replicate efficiently. to gain insights about possible viral countermeasures used by this virus to suppress the host response, the cell transcriptional profile and the kinetics of ifn regulatory factor (irf) expression and activation were examined at early t ...200717804507
dengue virus serotype infection specifies the activation of the unfolded protein response.dengue and dengue hemorrhagic fever have emerged as some of the most important mosquito-borne viral diseases in the tropics. the mechanisms of pathogenesis of dengue remain elusive. recently, virus-induced apoptosis mediated by the unfolded protein response (upr) has been hypothesised to represent a crucial pathogenic event in viral infection. in an attempt to evaluate the contribution of the upr to virus replication, we have characterized each component of this signalling pathway following deng ...200717888185
structure of the murray valley encephalitis virus rna helicase at 1.9 angstrom resolution.murray valley encephalitis virus (mvev), a mosquito-borne flavivirus endemic to australia, is closely related to japanese encephalitis virus and west nile virus. nonstructural protein 3 (ns3) is a multifunctional enzyme with serine protease and dexh/d-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. cloning, purification, and crystal structure determination to 1.9 angstrom resolution of the ns3 helicase of ...200717893366
construction and characterization of recombinant flaviviruses bearing insertions between e and ns1 genes.the yellow fever virus, a member of the genus flavivirus, is an arthropod-borne pathogen causing severe disease in humans. the attenuated yellow fever 17d virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. we described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17d virus expressing a green fluorescent protein variant (egfp). this approach too ...200717971212
mathematical modeling of subgenomic hepatitis c virus replication in huh-7 cells.cell-based hepatitis c virus (hcv) replicon systems have provided a means for understanding hcv replication mechanisms and for testing new antiviral agents. we describe here a mathematical model of hcv replication that assumes that the translation of the hcv polyprotein occurs in the cytoplasm, that hcv rna synthesis occurs in vesicular-membrane structures, and that the strategy of replication involves a double-stranded rna intermediate. our results shed light on the intracellular dynamics of su ...200717035310
a mouse cell-adapted ns4b mutation attenuates west nile virus rna synthesis.an adaptive mutation (e249g) within west nile virus (wnv) ns4b gene was consistently recovered from replicon rnas in c3h/he mouse cells. the e249g is located at the c-terminal tail of ns4b predicted to be on the cytoplasmic side of the endoplasmic reticulum membrane. the e249g substitution reduced replicon rna synthesis. compared with the wild-type ns4b, the e249g mutant protein exhibited a similar efficiency in evasion of interferon-beta response. recombinant e249g virus exhibited smaller plaqu ...200717178141
cost-effective real-time reverse transcriptase pcr (rt-pcr) to screen for dengue virus followed by rapid single-tube multiplex rt-pcr for serotyping of the virus.virus detection methodology provides detection of dengue virus in the early phase of the disease. pcr, targeting cdna derived from viral rna, has been used as a laboratory-based molecular tool for the detection of dengue virus. we report the development and use of three real-time one-step reverse transcriptase pcr (rt-pcr) assays to detect dengue cases and serotype the virus involved. the first rt-pcr assay uses sybr green i as the reporting dye for the purpose of cost-effective screening for de ...200717215345
construction of an infectious cdna clone for a brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in ns1.to help understand the mechanism of pathogenesis of dengue virus (dv), we set out to create an infectious cdna of the brazilian prototype strain of dv serotype 1 (dv1-br/90). pcr-amplified fragments of dv1-br/90 cdna were readily assembled into a subgenomic cdna that could be used to produce replicating rnas (replicons), lacking the structural protein-encoding regions of the genome. however, assembly of a cdna capable of producing infectious virus was only possible using a bacterial artificial c ...200717289102
participation of rab5, an early endosome protein, in hepatitis c virus rna replication machinery.like most positive-strand rna viruses, hepatitis c virus (hcv) is believed to replicate its genome on the surface of rearranged membranes. we have shown previously that hcv ns4ab, but not the product ns4b, inhibits endoplasmic reticulum (er)-to-golgi protein traffic (k. v. konan, t. h. giddings, jr., m. ikeda, k. li, s. m. lemon, and k. kirkegaard, j. virol. 77:7843-7855). however, both ns4ab and ns4b can induce "membranous web" formation, first reported by egger et al. (d. b egger, r. gosert, l ...200717301141
crystal structure of the dengue virus rna-dependent rna polymerase catalytic domain at 1.85-angstrom resolution.dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the flavivirus genus, which includes several important human pathogens, such as yellow fever and west nile viruses. the ns5 protein from dengue virus is bifunctional and contains 900 amino acids. the s-adenosyl methionine transferase activity resides within its n-terminal domain, and residues 270 to 900 form the rna-dependent rna polymerase (rdrp) catalytic ...200717301146
long-term storage of dna-free rna for use in vaccine studies.rna replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. this study examined the recovery, integrity and function of in vitro transcribed replicon rna encoding hepatitis c virus (hcv) proteins. to remove residual template dna, the rna was digested with turbo dnase followed by rneasy dnase set and purified through an rneasy column. the rna was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed ...200718072597
communicable diseases network australia national arbovirus and malaria advisory committee annual report, 2005-06.this report describes the epidemiology of mosquito-borne disease in australia for the mosquito-borne disease season 1 july 2005 to 30 june 2006, in which the second largest number of notifications since 1995-96 was reported. ross river virus (rrv) infections (66%), barmah forest virus (bfv) infections (23%) and malaria (9%) were the most common mosquito-borne diseases reported in 2005-06. national rrv notifications were the fifth largest on record. the northern territory had the highest rate of ...200617330382
kunjin virus replicons: an rna-based, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications.the application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. however, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. a number of replicon-based vectors derived from positive-strand rna viruses have recently been developed, and this paper reviews the current knowledge on the first flavivirus replicon system, which is based on the ...200616436039
cleavage targets and the d-arginine-based inhibitors of the west nile virus ns3 processing proteinase.mosquito-borne wnv (west nile virus) is an emerging global threat. the ns3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. we have isolated and biochemically characterized the recombinant, highly active ns3 proteinase. we have determined that the ns3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. we determined that aprotinin and d-arginine-based 9-12- ...200616229682
rna secondary structure in the coding region of dengue virus type 2 directs translation start codon selection and is required for viral replication.dengue virus is a positive-strand rna virus and a member of the genus flavivirus, which includes west nile, yellow fever, and tick-borne encephalitis viruses. flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (c) protein. dengue virus type 2 (denv2) and other mosquito-borne flaviviruses initiate translation of c from a start codon in a suboptimal context and have multiple in-frame augs downstream. here, ...200616474125
a single amino acid substitution in the west nile virus nonstructural protein ns2a disables its ability to inhibit alpha/beta interferon induction and attenuates virus virulence in mice.alpha/beta interferons (ifn-alpha/beta) are key mediators of the innate immune response against viral infection. the ability of viruses to circumvent ifn-alpha/beta responses plays a crucial role in determining the outcome of infection. in a previous study using subgenomic replicons of the kunjin subtype of west nile virus (wnv(kun)), we demonstrated that the nonstructural protein ns2a is a major inhibitor of ifn-beta promoter-driven transcription and that a single amino acid substitution in ns2 ...200616474146
dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited.viruses have developed numerous mechanisms to usurp the host cell translation apparatus. dengue virus (den) and other flaviviruses, such as west nile and yellow fever viruses, contain a 5' m7gpppn-capped positive-sense rna genome with a nonpolyadenylated 3' untranslated region (utr) that has been presumed to undergo translation in a cap-dependent manner. however, the means by which the den genome is translated effectively in the presence of capped, polyadenylated cellular mrnas is unknown. this ...200616501107
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