In vitro and in vivo translational efficiencies of the 5' untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates

We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of sequence identity in the 5' untranslated region (UTR), subtle differences in the primary and secondary structures, as well as differences in cell lines, influence translational efficiencies.     

Assessment of sequence diversity in the 5'-terminal region of Citrus tristeza virus from India

Twenty-one Citrus tristeza virus (CTV) isolates from India were characterized, using genotype-specific multiple molecular markers (MMM) from the 54'-terminal region and two other overlapping primer pairs (CN487/489 and CN488/491) from ORF1a (697-1484 nucleotides (nt)). The 5'-terminal genotype-specific primer pairs amplified about 500 bases from the 5'-end of the CTV genomic RNA (gRNA). With the three different MMM, the VT genotype-specific primers amplified 19 Indian CTV isolates. The T30-specific primers amplified five isolates, and the T36 primer amplified only one isolate T36. All isolates were amplified with CN488/491 primers; however, only 20 isolates were amplified with CN487/489 pair. A phylogenetic tree, derived from the sequences of the different MMM primer-amplified products, placed all the isolates into four distinct genogroups. Three of these four groups were typified by the reference isolates T30, T36, and VT. The fourth group, represented by the isolate BAN-2, was considered as a new genogroup. A phylogenetic tree based on sequences of the CN487/491 amplified products and other published sequences placed all of the isolates in eight genogroups. Phylogenetic correlation over the three different regions sequences of these CTV isolates showed more sequence variability between 1082 and 1484nt than between 1 and 500 or 697-1105 nt of the CTV gRNA. Based on three different 5' regions sequences and phylogenetic analysis, it is hypothesized that isolates BAN-1, BAN-2, and B165 are three naturally occurring variants that add to the complexity of the CTV populations in India.     

Enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain

An essential step for a productive infection by the dengue flavivirus (DEN) is translation of the m(7)G-capped, nonpolyadenylated positive-sense RNA genome. We have recently identified sequences within the DEN 3' untranslated region (UTR) that modulate viral translation. Here, we show that the DEN type 2 (DEN2) 3'UTR stimulated translation of m(7)G-capped DEN2 5'UTR-containing reporter mRNAs in baby hamster kidney (BHK) cells compared to a 3' vector sequence. Analogous to the 3' poly(A) tail, the DEN2 3'UTR also enhanced translation of reporter mRNAs containing (i) a nonfunctional A cap, (ii) the 5'UTR of human beta-globin, or (iii) a viral internal ribosome entry site (IRES). In all cases, approximately half of the translation efficiency was due to the terminal 3' stem-loop (3'SL) domain. In addition, the 3'SL domain increased the association of mRNAs with polysomes. Together, these results indicate that the DEN2 3'UTR, mediated in part by the 3'SL domain, enhances translation initiation, possibly after recognition of the 5' cap structure.     

A conserved secondary structure in the hypervariable region at the 5' end of Bamboo mosaic virus satellite RNA is functionally interchangeable

Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV.     

Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants

The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.     

Mutational analysis of the replication signals in the 3'-nontranslated region of citrus tristeza virus

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb messenger-sense RNA genome consisting of 12 open reading frames with nontranslated regions (NTR) at the 5' and 3' termini. The 273 nucleotide (nt) 3'-NTR is highly conserved ( approximately 95%) among the sequenced CTV isolates in contrast to the highly diverse 5'-NTR sequences. The 3' replication signals were mapped to the 3' 234 nts within the NTR. This region of CTV does not contain a poly-A tract nor does it appear to fold as a tRNA-mimic. Instead, a computer-predicted thermodynamically stable secondary structure comprised of 10 stem-and-loop (SL) structures, referred to as SL1 to SL10 (5' to 3'), was common to all CTV isolates. This putative structure was used as a guide to examine the 3' requirements for replication in vivo. The resulting data suggest that a complex 3' structure is required for those functions that provide for efficient replication of CTV in vivo such as minus-strand initiation, regulation of strand asymmetry, effective translation of the myriad of viral mRNAs, or stability of RNAs. Deletions into the 3'-NTR, up to 66 nts from the 5' direction and 11 nts from the 3' direction, deleting or disrupting putative SL1, SL2 and SL3, or SL10, resulted in continued replication, suggesting that these sequences are not essential for basal-level replication, but are required for efficient replication. Predicted stem loops 3 through 10 were examined by mutations designed to alter the primary structures while preserving the secondary structures. Mutations designed to disrupt the predicted stems of SL3, SL5, SL7, SL9, or SL10 resulted in substantially reduced levels of replication, while compensatory mutations resulted in partial restorations of replication, suggesting that these predicted secondary structures are involved in replication. Also, the putative loop sequences of SL5, SL6, SL7, and SL9 tolerated mutagenesis with continued but reduced levels of replication. In contrast, all mutations introduced into putative SL4, SL8, and the stem of SL6 prevented replication, suggesting that the primary structure of these regions make up the core of the 3' replication signal. The 3' triplet, CCA, was shown to be necessary for efficient replication, but deletion of eleven nts to expose an internal CCA resulted in continued replication.     

Functional analysis of dengue virus cyclization sequences located at the 5' and 3'UTRs

Flavivirus RNA replication involves cyclization of the viral genome. A model for this process includes a promoter element at the 5' end of the genome and long-range RNA-RNA interactions. Two pairs of complementary sequences present at the ends of the viral RNA, known as 5'-3'CS and 5'-3'UAR, have been proposed to be involved in dengue virus genome cyclization. The requirement of 5'-3'CS complementarity for viral replication has been experimentally demonstrated for dengue and other mosquito borne flaviviruses. Here, we performed a functional analysis to study the role of 5'-3'UAR sequences using genomic and subgenomic dengue virus RNAs. We found that single mutations disrupting 5'-3' complementarity greatly compromised viral RNA synthesis. Although in most of the cases incorporation of compensatory mutations re-established viral RNA replication, certain nucleotides were found to be involved in alternative secondary structures also important for viral replication. In addition, mutations within 5' or 3'UAR in the context of an infectious dengue virus RNA resulted in spontaneous mutations that restored UAR base pairings. Together, we propose that specific UAR nucleotides as well as 5'-3'UAR complementarity constitute cis-acting signals involved in amplification of the dengue virus genome.     

HCV LNAzyme对病毒5'非编码区基因调控的特异性抑制作用

目的 探讨针对丙肝病毒5 '非编码区内源性核糖体进入位点的锁核酸核酶(LNAzyme)对病毒基因复制与表达的特异性抑制作用.方法 设计合成能切割HCV-5'-NCR-IRES位点的DNAzyme、硫代DNAzyme和LNAzyme.实验设对照组和实验组.对照组包括空白对照组、脂质体对照组和无关LNAzyme对照组.实验组包括DNAzyme、硫代DNAzyme组和LNAzyme组.以阳离子脂质体介导转染hepG2.9706细胞,用荧光定量PCR和化学发光技术分别监测24、48和96 h细胞培养上清液中HCV RNA含量及荧光素酶基因表达;四甲基偶氮唑蓝(MTT)法监测细胞活性.结果 加入核酶后,LNAzyme对HCV RNA复制和荧光素酶基因表达的抑制作用最强(P＜0.05),平均抑制率分别为48.02％和53.05％,且随用药时间延长,抑制率呈增高趋势,96 h后,平均抑制率分别为81.21％和84.25％.LNAzyme对细胞活性无影响.结论 LNAzyme能特异性抑制丙型肝炎病毒5 '非编码区的基因调控,且优于硫代修饰的DNAzyme.     

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5′-SL1, 5′-SL2, 5′-SL3 and 5′-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5′-SL2 is required for efficient RNA replication. 5′-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5′-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.     

The RNA encompassing the internal ribosome entry site in the poliovirus 5' nontranslated region enhances the encapsidation of genomic RNA

Poliovirus replicons were constructed which contain the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) substituted for the poliovirus IRES. To monitor gene expression and encapsidation, the gene encoding firefly luciferase was substituted for the P1 gene. Replicons can be encapsidated following serial passage in the presence of a recombinant vaccinia virus, VV-P1, which expresses the poliovirus P1 protein following infection. Encapsidation of the wild-type replicon (PV-Luc) was accomplished at either 33 or 37 degrees C; the lower temperature actually resulted in greater amounts of encapsidated replicon. In contrast, the replicon with the EMCV IRES element (EMCV-Luc) was not efficiently encapsidated at 37 degrees C and, following serial passage with VV-P1 at 37 degrees C, was not amplified. EMCV-Luc was efficiently encapsidated, however, following serial passage with VV-P1 at 33 degrees C. Using the encapsidated EMCV-Luc obtained at 33 degrees C, we found that cells infected with EMCV-Luc at 33 or 37 degrees C produced similar amounts of luciferase. Encapsidated EMCV-Luc and PV-Luc had similar thermal stability at 33 and 37 degrees C. A single-round encapsidation analysis revealed that less EMCV-Luc was encapsidated at 37 than at 33 degrees C; less EMCV-Luc was encapsidated at 33 degrees C compared to PV-Luc at either 37 or 33 degrees C. The results of our studies suggest that in addition to influencing translation/replication, the IRES region of poliovirus can function to enhance encapsidation.     

Non-genotype-specific role of the hepatitis C virus 5' untranslated region in virus production and in inhibition by interferon

The 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) is structured into four domains (I-IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5′UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1-7 recombinants with highly diverse 5′UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5′UTR of genotypes 1-6 or with heterotypic chimeric (1a/3a and 3a/1a) 5′UTR domains I, II or III, and 1a recombinants with 5′UTR domain I of genotypes 1-6. All were fully functional in Huh7.5 cells; therefore, the 5′UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5′-terminus was required. We demonstrated that J6/JFH1 with 5′UTR of genotypes 1-6 responded similarly to interferon-α2b. This study provides novel insight into the role of the 5′UTR in the HCV life cycle and facilitates HCV basic research and testing of 5′UTR-targeting antivirals.     

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.     

Downregulation of Bamboo mosaic virus replication requires the 5' apical hairpin stem loop structure and sequence of satellite RNA

Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.     

Nineteen nucleotides in the variable region of 3' non-translated region are dispensable for the replication of dengue type 1 virus in vitro

In many flaviruses, first 50-400 nucleotides of 3' non-translated region (3' NTR) exhibit lower conservation level than other regions and are called "variable region". Two dengue type 1 virus (DENV-1) strains, which have 17- and 29-nt deletion in the variable region, were recently isolated from Japanese dengue fever patients. The effect of a small deletion in the 3' NTR was analyzed using two DENV-1 viruses which were prepared from a newly developed infectious cDNA clone. These included a recombinant virus rDENV-1(02-20), without any deletion in 3' NTR, and rDENV-1m10, with 19-nt deletion in the variable region of rDENV-1(02-20). These two viruses were compared for growth kinetics and plaque morphology in Vero, Huh-7 and C6/36 cells. No apparent difference was detected between rDENV-1(02-20) and rDENV-1m10 in replication efficiency and plaque size in these cell lines. The results suggest that the complete variable region of DENV-1 is dispensable for virus replication and propagation in vitro.     

Incidence and epidemiology of Citrus tristeza virus in the Valencian community of Spain

The first outbreak of citrus tristeza disease in Spain caused by Citrus tristeza virus (CTV) was recorded in 1957 in the Valencian Community (VC). In total c. 40 million trees, mainly of sweet orange and mandarin grafted on sour orange rootstocks, declined due to CTV. Large-scale surveys in different municipalities of the VC indicated that the disease spread very fast. Incidence increased from 11% in 1989 to 53% in 1998. Toxoptera aurantii and Aphis spiraecola (inefficient aphid vectors of CTV) predominated before 1985-87. Since then the relatively efficient vector Aphis gossypii has become dominant and induced an epidemic that has been modelled. The large number of A.gossypii that visited each clementine tree (estimated to exceed 97000 per year) explained the difference between the temporal pattern of spread of CTV in clementine which followed the Gompertz model and that in sweet orange (logistic model). The susceptibility of the different citrus species to CTV infection by aphids seems to depend on the number of young, succulent shoots produced. The epidemiological data allowed specific recommendations to be made to growers in order to facilitate a change to a modern citrus industry based on the use of selected varieties grafted on tristeza-tolerant rootstocks produced within a certification scheme. This has been done already in almost 90% of the VC citrus-growing area. The tristeza problem has been solved unless more aggressive isolates are introduced and become prevalent.     

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.