Construction and characterization of subgenomic replicons of New York strain of West Nile virus

The lineage I strain of West Nile virus (WNV) frequently causes human epidemics, including the recent outbreak in North America (Lanciotti et al., 1999, Science 286:2333-2337). As an initial step in studying the replication and pathogenesis of WNV, we constructed several cDNA clones of a WNV replicon derived from an epidemic strain (lineage I) isolated from the epicenter of New York City in the year 2000. Replicon RNAs were in vitro transcribed from cDNA plasmids and transfected into BHK-21 cells. RNA replication in transfected cells was monitored by immunofluorescence analysis (IFA) and 5' nuclease real-time RT-PCR (TaqMan). The replicon RNAs contained large in-frame deletions (greater than 92%) of the C-prM-E structural region yet still replicated efficiently in BHK-21 cells. 5' nuclease real-time RT-PCR showed that a great excess of plus-sense replicon RNA over the minus-sense RNA was synthesized in transfected cells. Replication efficiency decreased upon insertion of a green fluorescent protein (GFP) reporter gene driven by an internal ribosomal entry site (IRES) in the upstream end of the 3' untranslated region of the replicon. Strong GFP expression was detected in cells transfected with a replicon containing IRES-GFP positioned in the plus-sense orientation. IFA showed that GFP and viral proteins were exclusively coexpressed in transfected cells. In contrast, no GFP fluorescence was observed in cells transfected with a replicon containing IRES-GFP positioned in the minus-sense orientation, despite high levels of synthesis of viral proteins and RNA in the cells. Substitution of the GFP gene in the plus-sense GFP replicon with the neomycin phosphotransferase gene allowed selection of geneticin-resistant cells in which WNV replicons persistently replicated without apparent cytopathic effect. These results suggest that WNV replicons may serve as a noncytopathic RNA virus expression system and should provide a valuable tool to study WNV replication.     

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.     

The structure and function of a cis-acting element located upstream of the IRES that influences Coxsackievirus B3 RNA translation

We have investigated the importance of a conserved hexa-nucleotide stretch in the apical loop within stem-loop C (SL C, nt 104-180), upstream of the ribosome landing site, on CVB3 IRES function. The deletion or substitution mutation at this apical loop resulted in significant decrease in IRES activity. Both the mutant IRES RNAs failed to interact with certain trans-acting factors. Furthermore, expression of CVB3 2A protease significantly enhanced IRES activity of the wild type, but the effect was not so pronounced on the mutant IRESs. It is possible that the mutant RNAs were unable to interact with some trans-acting factors critical for enhanced IRES function. Interestingly, the local structure of the IRES RNA was not significantly altered due to substitution mutation. Taken together, it appears that the SL C/c apical loop is critical for CVB3 IRES function.     

反义硫代寡核苷酸体外抑制柯萨奇病毒B3增殖的研究

目的 在ＣＶＢ３易感的Ｖｅｒｏ细胞系中观察与ＣＶＢ３ＲＮＡ５’ＮＣＲ的ｎｔ５８１－６０１区域区补的２１聚硫代ＡＯＤＮ抗病毒活性。方法 采用ＭＴＴ法测定细胞活性,直接观察ＣＰＥ,测定培养上清ＴＣＩＤ５０,并分别用ＥＬＩＳＡ和往点杂交法检测ＣＶＢ３抗原及其ＲＮＡ。结果 １．ＡＯＤＮ可推迟和减轻ＣＰＥ,且随ＡＯＤＮ浓度增加,对ＣＰＥ的抑制作用增强,感染细胞存活率也随ＡＯＤＮ浓度升高而升高;２．ＡＯＤＮ可     

Hepatitis C virus and the related bovine viral diarrhea virus considerably differ in the functional organization of the 5' non-translated region: implications for the viral life cycle

The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.     

Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells.     

反义寡聚核苷酸对CVB3蛋白基因表达的抑制作用及量效关系的研究

目的 观察病毒基因组5′端非编码区内与翻译起始有关的位点和结构蛋白编码区的特异性反义核酸对病毒蛋白表达的影响及其量效关系。方法 应用病毒致细胞病变作用保护实验、空斑形成实验和空斑形成减少实验、Western blot实验等方法，观察特定的反义核酸抑制病毒感染的效果。结果 针对IRES位点、AUG区域和VPl区的3条反义核酸Scb561、Scb733、Scb2785，对病毒感染有明显的抑制作用。病毒的感染量为0．01MOI时，3种反义核酸在5Fmol／L时对病毒的抑制率均在90％以上，当病毒增加到10MOI时，抑制率仍在50％以上。Scb561和Scb733可明显的抑制病毒基因表达，当Scb561的浓度在0．625μmol／L时感染后的细胞内病毒蛋白量明显减少，增加到2．5／lmol／L时病毒蛋白表达几乎看不到。另外Scb561、Scb733剂量与抗病毒效果呈现正相关关系。随着Scb561和Scb733浓度的增加，其抗病毒活性也随之增加直到抑制率达到90％以上，有效剂量在0．6～5μmol／L之间，且对细胞无毒性作用。非特异寡聚核苷酸对照实验显示，5μmol／L浓度时对病毒感染无明显抑制作用。结论 针对核糖体进入位点和翻译起始位点的反义核酸，有明显的特异性抑制病毒基因表达的作用。为反义寡聚核苷酸成为一个潜在的治疗病毒感染的药物提供了重要的研究基础。     

5' terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart

Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5' non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5' viral genomic termini were detected, 5' terminal deletions ranged in size from 22 to 36 nucleotides. Sequence of the 5' third of this viral genome is of a modern strain, closely related to CVB2 strains isolated in Japan in 2002. A CVB3 chimera containing the 5' NTR with a 22 nt deletion produced progeny virus upon transfection of HeLa cells. When the 5' 22 nucleotide deletion was repaired, the virus induced myocarditis in mice and replicated like wild type virus in murine heart cells. This is the first report of these naturally-occurring defective enteroviral genomes in human myocarditis.     

Difference in susceptibility to MxA protein between a coxsackievirus B1 isolate and prototype,impact of serial cell culture passage

Human enteroviruses (EVs) cause a broad spectrum of acute and chronic diseases including meningitis and myocarditis. The type I interferon‐induced MxA protein has been shown to inhibit the replication of an EV, coxsackievirus B4 (CVB4), but not cardioviruses such as encephalomyocarditis virus and mengo virus, members of the Picornaviridae family. EVs consist of more than 60 distinct serotypes against which the antiviral activity of MxA was not investigated yet. The main aim of this study was to explore the antiviral activity of MxA protein against a clinical CVB1 isolate and other EV prototypes. Vero cells expressing constituvely MxA protein were infected with EVs, and the percentage of inhibiton of expression of enteroviral RNA and capsid VP1 protein was determined. Following infection of MxA‐transfected Vero cells with EVs, the expression of enteroviral RNA was inhibited by up to 99%, and that of VP1 protein by up to 85%. However, there was a difference in the percentage of MxA inhibition of EV replication between the different EV prototypes. This difference in MxA sensitivity was not due to a difference in the viral replication rates. The MxA protein was inactive against the clinical CVB1 isolate, and the replication rate of CVB1 isolate in MxA‐transfected Vero cells was higher than that in mock‐transfected Vero cells. A serial passage of the clinical CVB1 isolate and other EV prototypes resulted in an increase in their susceptibility to MxA protein. These results suggest the presence of MxA‐resistant EV variants that may escape innate immunity and cause disease. J. Med. Virol. 82:424–432, 2010. © 2010 Wiley‐Liss, Inc.     

Protein 2A of grapevine fanleaf nepovirus is implicated in RNA2 replication and colocalizes to the replication site.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.     

Functional analysis of structural motifs in dicistroviruses

The family Dicistroviridae is composed of positive-stranded RNA viruses which have monopartite genomes. These viruses carry genome-linked virus proteins (VPg) and poly (A) tails. The 5' untranslated region (UTR) is approximately 500 nucleotides and contains an internal ribosome entry site (IRES). These features resemble those of vertebrate picornaviruses, but dicistroviruses have other distinct characteristics. Picornaviruses have a single large open reading frame (ORF) encoding the capsid proteins at the 5'-end and the replicases at the 3'-end. In contrast, dicistroviruses have two nonoverlapping ORFs. The 5'-proximal ORF encodes the replicases and the 3'-proximal ORF encodes the capsid proteins. Usually, positive-stranded viruses which have capsid protein genes in the 3' part of the genome produce subgenomic RNA for synthesis of the capsid proteins, because abundant quantities of the capsid proteins are required for the viral replication cycle. In dicistroviruses, translation of the capsid proteins is controlled by an additional IRES. This IRES is located in the intergenic region (IGR) between the replicase and capsid coding regions, and mediates the initiation of translation for the capsid proteins. The IGR-IRES has a multiple stem-loop structure containing three pseudoknots. We describe the characteristics of dicistroviruses, including the RNA elements and viral proteins.     

Specific interactions of HeLa cell proteins with Coxsackievirus B3 RNA: La autoantigen binds differentially to multiple sites within the 5' untranslated region

Translation initiation of the coxsackievirus B3 (CVB3) RNA occurs by internal ribosomal entry. The internal ribosomal entry site (IRES) of the virus has been mapped to the 5′ untranslated region (5′ UTR) of the genome. As well, the 5′ UTR has been suggested to play roles in determining the tissue tropism and infectivity of the virus. In this study, we investigated interactions between HeLa cell protein extracts and radiolabeled RNA of CVB3 5′ UTR by competitive UV cross-linking. We have observed a number of proteins that specifically interact with the three sub-cloned regions of the 5′ UTR. In particular, the molecular weights of five of these proteins resemble those of the eukaryotic translation initiation factors 4A, 4B and 4G, as well as the La autoantigen and the polypyrimidine tract binding protein. Based on this data, we focused on the interaction of the 5′ UTR with the La autoantigen, which was purified by the glutathione-S-transferase affinity method. We have confirmed the highly specific interaction of the La autoantigen with the 5′ UTR sequence nt 210–529. The core IRES (nt 530–630) and nt 1–209 also appear to bind to the La protein at moderate and weak affinities, respectively. A functional role of the La autoantigen in translation initiation is suggested.     

Subgenomic RNA as a riboregulator: negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2

Barley yellow dwarf virus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. Translation of BYDV genomic RNA (gRNA) and sgRNA1 is mediated by the BYDV cap-independent translation element (BTE) in the 3' untranslated region. sgRNAs 2 and 3 are unlikely to be mRNAs. We proposed that accumulation of sgRNA2, which contains the BTE in its 5' UTR, regulates BYDV replication by trans-inhibiting translation of the viral polymerase from genomic RNA (gRNA). Here, we tested this hypothesis and found that: (i) co-inoculation of the BTE or sgRNA2 with BYDV RNA inhibits BYDV RNA accumulation in protoplasts; (ii) Brome mosaic virus (BMV), engineered to contain the BTE, trans-inhibits BYDV replication; and (iii) sgRNA2 generated during BYDV infection trans-inhibits both GFP expression from BMV RNA and translation of a non-viral reporter mRNA. We conclude that sgRNA2, via its BTE, functions as a riboregulator to inhibit translation of gRNA. This may make gRNA available as a replicase template and for encapsidation. Thus, BYDV sgRNA2 joins a growing list of trans-acting regulatory RNAs.     

A Recombinant Classical Swine Fever Virus with a Marker Insertion in the Internal Ribosome Entry Site

Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.     

Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.     

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication.