Flavivirus Genome Research Articles

Identification and analysis of putative promoter motifs in Flavivirus genome

The genus Flavivirus comprises medically significant pathogenic virus; causing several infections in humans worldwide. Flavivirus genomes are 10-11 kb approximately and encode both structural and non structural region. The non structural region plays fundamental role in the stability, regulation and cell cycle of virus. The complete genomes of 26 Flavivirus were used for identification of promoter motifs through in silico approaches. The promoter sequences were encoded in merely 16 viruses and 10 viruses could not encode it. All these in silico identified promoter motifs was confirmed and verified with the known experimental data. This analysis suggests that presence of promoter may play a crucial role in the pattern of gene expression, regulation networks, cell specificity and development. It may also be useful for designing efficient expression vector and target specific delivery system in the gene therapy.

Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication

The 5' untranslated region (5'UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5' end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5' upstream AUG region (5'UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5'UAR, was necessary for efficient RNA synthesis using the viral 3'UTR as template.

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5′-terminal 190 nucleotides and the 3′-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5′CS/3′CSI and 5′UAR/3′UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3′-terminal nucleotides of the genome (position − 8 to − 16) to be single-stranded. Viral NS5 binds specifically to the 5′-terminal stem-loop (SL1) of the genomic RNA. The 5′SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5′SL1 RNA interaction facilitate NS5 binding to the 3′ end of the genome for the initiation of viral minus-strand RNA synthesis.

Genome cyclization as strategy for flavivirus RNA replication

Long-range and local RNA–RNA contacts in viral RNA genomes result in tertiary structures that modulate the function of enhancers, promoters, and silencers during translation, RNA replication, and encapsidation. In the case of flaviviruses, the presence of inverted complementary sequences at the 5′ and 3′ ends of the genome mediate long-range RNA interactions and RNA cyclization. The circular conformation of flavivirus genomes was demonstrated to be essential for RNA amplification. New ideas about the mechanisms by which circular genomes participate in flavivirus replication have emerged in the last few years. Here, we will describe the latest information about cis-acting elements involved in flavivirus genome cyclization, RNA promoter elements required for viral polymerase recognition, and how these elements together coordinate viral RNA synthesis.

Spatial and temporal organization of tick-borne encephalitis flavivirus replicated RNA in living cells

Flaviviruses are positive RNA viruses that assemble the replication complex in the cytoplasm of the infected cells. In order to get a dynamic view of the formation and distribution of flavivirus genomes in living cells we engineered a tick-borne encephalitis virus (TBEV) replicon with an array of binding sites for the phage MS2 core protein. The modified TBEV replicons were competent for RNA replication and allowed the visualization of replicated genomic RNA that accumulated in cytoplasmic structures with a distinct subcellular localization. Sites of TBEV replicated RNA accumulation were enriched in non-structural viral proteins and co-localized with the markers of the rough endoplasmic reticulum protein disulphide isomerase (PDI). In contrast no co-localization was observed with the markers CD-71 and EEA-1 for recycling vesicles, ERGIC53 for the intermediate compartment and TGN-46 for the trans-Golgi network. In human HOS cells, but not in hamster BHK21 cells, replicated TBEV RNA was found also associated with the marker Giantin for the Golgi indicating differences according to the cellular background. This study confirms and extends previous observations on the subcellular localization of flavivirus RNA and provides a useful tool to monitor the formation and distribution of flavivirus RNA genomes in living cells.

Analysis of the effects of alterations in the tick-borne encephalitis virus 3′-noncoding region on translation and RNA replication using reporter replicons

The 3′-noncoding region (3′-NCR) of the flavivirus genome includes a variable region that tolerates the insertion of heterologous genetic information. Natural isolates of tick-borne encephalitis virus (TBEV) have particularly long variable regions, which, for some strains, include an internal poly(A) tract. We constructed luciferase reporter replicons of TBEV to analyze the impact of various manipulations of the 3′-NCR on viral RNA translation and replication. The choice of the reporter gene, its position and processing within the viral polyprotein, and the choice of standards were found to be important for obtaining a sensitive and reliable test system. We observed that truncation or complete removal of the internal poly(A) tract, or even the entire variable region, had no significant impact on translation and replication of the RNA in mammalian cell culture. Substitution of the variable region with foreign genetic elements impaired RNA replication to various degrees but generally had no influence on viral translation. Expression cassettes driven by an IRES element inhibited RNA replication more strongly than did repetitive protein-binding elements derived from a bacteriophage, even when the ligand that binds these elements was co-expressed in the cells. Previously identified mutations in the IRES partially relieved this inhibition when introduced into the reporter replicon but provided no evidence for intramolecular competition for translation factors. Impairment of replication appeared to depend more on the type of foreign insert than on its length. These results provide a rational basis for the construction of TBEV-based vectors or vaccines as well as molecular tools for studying flavivirus replication.

Identification of Mutated Cyclization Sequences That Permit Efficient Replication of West Nile Virus Genomes: Use in Safer Propagation of a Novel Vaccine Candidate

Existing live-attenuated flavivirus vaccines (LAV) could be improved by reducing their potential to recombine with naturally circulating viruses in the field. Since the highly conserved cyclization sequences (CS) found in the termini of flavivirus genomes must be complementary to each other to support genome replication, we set out to identify paired mutant CS that could support the efficient replication of LAV but would be unable to support replication in recombinant viruses harboring one wild-type (WT) CS. By systematic evaluation of paired mutated CS encoded in West Nile virus (WNV) replicons, we identified variants having single and double mutations in the 5'- and 3'-CS components that could support genome replication at WT levels. Replicons containing only the double-mutated CS in the 5' or the 3' ends of the genome were incapable of replication, indicating that mutated CS could be useful for constructing safer LAV. Despite the identity of the central portion of the CS in all mosquito-borne flaviviruses, viruses carrying complementary the double mutations in both the 5'- and the 3'-CS were indistinguishable from WT WNV in their replication in insect and mammalian cell lines. In addition to the utility of our novel CS pair in constructing safer LAV, we demonstrated that introduction of these mutated CS into one component of a recently described two-component genome system (A. V. Shustov, P. W. Mason, and I. Frolov, J. Virol. 81:11737-11748, 2007) enabled us to engineer a safer single-cycle WNV vaccine candidate with reduced potential for recombination during its propagation.

Usutu Virus Sequences inCulex pipiens(Diptera:Culicidae), Spain

To the Editor: Usutu virus (USUV) is an arbovirus and a member of the mosquito-borne cluster within the Flavivirus genus. USUV belongs to the Japanese encephalitis virus antigenic group, which is closely related to pathogens such as West Nile virus (WNV) (1). USUV has been isolated from a human in the Central African Republic and from several mosquito species from tropical and subtropical Africa (2). In late summer 2001, USUV emerged in central Europe and caused deaths in several species of resident birds in Austria (3). However, monitoring of USUV in dead birds from 2003 through 2005 showed that the absolute numbers of USUV–associated bird deaths declined, although USUV detection persisted in bird tissues (4). This decrease in USUV-associated bird deaths was attributed to herd immunity in the bird population (5). In the summer of 2005, USUV was detected in a blackbird in Hungary. The complete genomic sequence of the Hungarian USUV strain shared 99.9% identity with the strain circulating in Austria since 2001 (6). On the other hand, neutralizing antibodies against USUV have been detected in sera of resident and migrant birds in the United Kingdom without causing an obvious reduction in the bird population (7). From May through October 2006, monitoring of flaviviruses in mosquitoes was performed in the northeast region of Spain (Catalonia). This monitoring was implemented in the 3 main wetlands of the region: Aiguamolls de la Emporda (Girona Province) near France, where WNV was detected in dead horses in 2000 (8); Delta del Llobregat (Barcelona Province); and Delta de l’Ebre (Tarragona Province). Mosquitoes were collected by mosquito control services in these areas. Female mosquitoes were classified and grouped in pools according to date, species, and localization. During this period, 436 pools belonging to 9 mosquito species were collected. The most abundant species was Culex pipiens (n = 168). Viral RNA was recovered from mosquito pools by homogenization and viral RNA extraction with QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA), and then generic reverse transcription (RT)-nested PCR was used to identify flaviviruses (9). This procedure was used to amplify a specific fragment of the NS5 gene within the flavivirus genome. The 143-bp amplification product was detected by electrophoresis and purified by using QIAquick PCR Purification Kit (QIAGEN). Sequencing reactions were performed with ABI Prism BigDye Terminator Cycle Sequencing v.3.1 Ready Reaction (Applied Biosystems, Foster City, CA, USA), and analyzed by using an ABI PRISM model 3730 automated sequencer (Applied Biosystems). Assembly of the consensus sequences and translation into amino acid sequences was performed with Larsergene DNASTAR group of programs (DNASTAR Inc., Madison, WI, USA). Comparisons with published sequences of known flaviviruses were performed by searches with FASTA program in EMBL database (available from www.ebi.ac.uk/embl/) to identify the detected agent and to study the level of homology. One pool of Cx. pipiens captured in the middle of August 2006 from Delta del Llobregat, in a typical Mediterranean climate that contained 3 female mosquitoes, was positive for flaviviruses. That positive pool was obtained from the center of the village of Viladecans, where different common migratory and sedentary birds such as Passer domesticus, Hirundo rustica, or Delichon urbica feed and nest. The Spanish USUV sequence showed 97.97% homology to USUV strain SAAR-1776 from South Africa; it showed 94.94% similarity with USUV strain Vienna 2001 from Austria and USUV strain Budgapest from Hungary, with 2-nt and 5-nt differences, respectively (Figure). All of these were synonymous mutations and thus did not result in amino acids replacements. The homology data showed that the Spanish strain belongs to USUV species and is more related to the African USUV isolates than to central European isolates. Figure Comparison at nucleotide level of sequenced fragment among related Usutu virus (USUV). Dot indicates coincident nucleotide. The partial nucleotide sequence of detected Spanish USUV has been deposited in the GenBank database under accession no. {type:entrez-nucleotide,attrs:{text:AM909649,term_id:187453067,term_text:AM909649}} ... To date, no bird deaths observed in Barcelona Province have been associated with viral encephalitis. However, this region is where the USUV-specific RT-PCR–positive samples were obtained from Cx. pipiens mosquitoes. One possible explanation for these findings is that Spanish USUV could be naturally avirulent for birds because the African strains of USUV appear to be in Africa. Alternatively, USUV and other related viruses such as WNV may have been circulating in Spain for many years, as a result of regular reintroduction by birds migrating from Africa. Under such circumstances, natural genetic resistance, herd immunity, and cross-protective immunity caused by related viruses likely provided at least some protection against symptomatic infections. The discovery of USUV-specific RNA, most related to the African strains of USUV, in Cx. pipiens in Spain extends previous evidence (7,10) that USUV and related flaviviruses such as WNV are being introduced into western Europe from Africa, presumably by migratory birds.

West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

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

Universal primers that amplify RNA from all three flavivirus subgroups

BackgroundSpecies 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 new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups.ResultsBioinformatic analysis of 257 published full-length Flavivirus genomes revealed conserved regions not previously targeted by primers. Two degenerate primers, Flav100F and Flav200R were designed from these regions and used to generate an 800 base pair cDNA product. The region amplified encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing was successful using standard conditions with RNA from over 60 different flavivirus strains representing about 50 species. The cDNA from each virus isolate was sequenced then used in phylogenetic analyses and database searches to confirm the identity of the template RNA.ConclusionComprehensive testing has revealed the broad specificity of these primers. We briefly discuss the advantages and uses of these universal primers.

Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct

Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3' noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.

Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication

The pentanucleotide (PN) sequence 5'-CACAG-3' at the top of the 3' stem-loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.

Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly

The West Nile virus minus-strand 3' terminal stem loop (SL) RNA was previously shown to bind specifically to cellular stress granule (SG) components, T cell intracellular antigen-1 (TIA-1) and the related protein TIAR. In vitro TIAR binding was 10 times more efficient than TIA-1. The 3'(-)SL functions as the promoter for genomic RNA synthesis. Colocalization of TIAR and TIA-1 with the viral replication complex components dsRNA and NS3 was observed in the perinuclear regions of West Nile virus- and dengue virus-infected cells. The kinetics of accumulation of TIAR in the perinuclear region was similar to those of genomic RNA synthesis. In contrast, relocation of TIA-1 to the perinuclear region began only after maximal levels of RNA synthesis had been achieved, except when TIAR was absent. Virus infection did not induce SGs and progressive resistance to SG induction by arsenite developed coincident with TIAR relocation. A progressive decrease in the number of processing bodies was secondarily observed in infected cells. These data suggest that the interaction of TIAR with viral components facilitates flavivirus genome RNA synthesis and inhibits SG formation, which prevents the shutoff of host translation.

Structure and Function of Flavivirus NS5 Methyltransferase

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

Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

BackgroundThe 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 took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.ResultsYF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.ConclusionThis system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.

Construction and Mutagenesis of an Artificial Bicistronic Tick-Borne Encephalitis Virus Genome Reveals an Essential Function of the Second Transmembrane Region of Protein E in Flavivirus Assembly

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3' noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.

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, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.

Inhibition of Flavivirus Infections by Antisense Oligomers Specifically Suppressing Viral Translation and RNA Replication

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

The Topology of Bulges in the Long Stem of the Flavivirus 3′ Stem-Loop Is a Major Determinant of RNA Replication Competence

All flavivirus genomes contain a 3'terminal stem-loop secondary structure (3'SL) formed by the most downstream approximately 100 nucleotides (nt) of the viral RNA. The 3'SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3'SL nucleotide sequence could not substitute for that of the WN 3'SL to support WN genome replication. To determine what WN virus-specific 3'SL nucleotide sequences were required for WN virus replication, WN virus 3'SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3'SL nucleotide sequence such that the overall 3'SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3'SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3'SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3'SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3'SL are primary determinants of replication competence for flavivirus genomes.

The majority of the nucleotides in the top loop of the genomic 3′ terminal stem loop structure are cis-acting in a West Nile virus infectious clone

The flavivirus genome RNA terminates with a conserved 3′ stem loop (SL) structure that was shown to be essential for virus replication. A stretch of conserved nts is located in the top loop (TL) of this structure. Mutation of the TL nts (5′ ACAGUGC 3′) in a WNV infectious clone indicated that 3 of the 7 TL nts (5′ A CA GUG C 3′) are critical for virus replication. Mutation of 3 of the other nts reduced the efficiency of virus replication. The four 5′ TL nts are conserved in both mosquito- and tick-borne flavivirus genomes, while the TL 3′ C is conserved in mosquito-borne viruses. The conservation of two or three G–C base pairs in the TL flanking sequences suggests that a stable stem is necessary for precise presentation of the TL sequence. The TL may participate in RNA as well as protein interactions.