Essential Role of Cyclization Sequences in Flavivirus RNA Replication

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.     

Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 5' stem-loop of genomic RNA

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

昆津病毒复制子——一种新型病毒载体

病毒复制子 (Replicon) 是指来源于病毒基因组的能够自主复制的RNA分子,保留了病毒非结构蛋白基因,而结构蛋白基因缺失或由外源基因替代。昆津病毒 (Kunjun virus) 为黄病毒科黄病毒属成员,其复制子具有表达效率高、细胞毒性低、遗传稳定等特点,在病毒基因组复制调控机制、外源蛋白表达、新型疫苗和基因治疗等领域得到了广泛应用。以下就昆津病毒复制子系统的构建、特性及应用方面的研究进展作一综述。     

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 domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.     

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.     

Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.     

Subgenomic replicons of the flavivirus Kunjin: construction and applications.

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.     

Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.     

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.     

Functional analysis of two cavities in flavivirus NS5 polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.     

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 of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.     

Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus

Replication of the segmented double-stranded (ds) RNA genome of viruses belonging to the Reoviridae family requires the RNA-dependent RNA polymerase (RdRP) to use 10-12 different mRNAs as templates for (-) strand synthesis. Rotavirus serves as a model system for study of this process, since its RdRP (VP1) is catalytically active and can specifically recognize template mRNAs in vitro. Here, we have analyzed the requirements for template recognition by the rotavirus RdRP and compared those to the requirements for formation of (-) strand initiation complexes. The results show that multiple functionally independent recognition signals are present at the 3'-end of viral mRNAs, some positioned in nonconserved regions upstream of the highly conserved 3'-terminal consensus sequence. We also found that RdRP recognition signals are distinct from cis-acting signals that promote (-) strand synthesis, because deletions of portions of the 3'-consensus sequence that caused viral mRNAs to be poorly replicated in vitro did not necessarily prevent efficient recognition of the RNA by the RdRP. Although the RdRP alone can specifically bind to viral mRNAs, our analysis reveals that this interaction is not sufficient to generate initiation complexes, even in the presence of nucleotides and divalent cations. Rather, the formation of initiation complexes also requires the core lattice protein (VP2), a virion component that forms a T = 1 icosahedral shell that encapsidates the segmented dsRNA genome. The essential role that the core lattice protein has in (-) strand initiation provides a mechanism for the coordination of genome replication and virion assembly.     

Dissecting RNA recombination in vitro: role of RNA sequences and the viral replicase.

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.     

Requirements for West Nile virus (-)- and (+)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (-)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (-)- and (+)-strand template RNAs containing 5'- and 3'-terminal regions (TR) with the conserved sequence elements. Our results show that both 5'- and 3'-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3'-TR of the (-)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5'-CYC motif is critical for (-)-strand RNA synthesis but neither the (-)-strand 5'- nor 3'-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5'-cap inhibits the (-)-strand RNA synthesis from the 3' fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that "cyclization" of the viral RNA play a role for (-)-strand RNA synthesis but not for (+)-strand RNA synthesis.     

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.     

Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.