Molecular Characterization and Distribution of Cucumber green mottle mosaic virus in China

Cucumber green mottle mosaic virus (CGMMV) is an emerging virus on watermelon in China. We report here the almost complete nucleotide sequence and the characterization of the genome of a Chinese isolate (CGMMV-LN). Nucleotide sequence comparisons showed CGMMV-LN is closely related to CGMMV-KOM, with 99.4% identity. On the basis of the nucleotide sequence, a digoxigenin-labelled cDNA probe CG, complementary to the replicase gene region of CGMMV was synthesized. The specificity and sensitivity of the probe was tested. The detection limit of the method was equivalent to 0.8 μg fresh tissues infected by CGMMV. Two hundreds and eighteen watermelon samples collected from different regions in China during 2006–2007 were tested by this method. The distribution pattern of CGMMV in China during these years was revealed. The virus has spread in five provinces of China so far, including Liaoning, Hebei, Guangdong, Hubei and Shandong and might be an increasing tendency, which provides important information for CGMMV control in China.     

Production of Antiserum to Recombinant Coat Protein for Detecting Lily mottle virus in Yunnan, China

Lily mottle virus (LMoV), Potyvirus genus, is very difficult to purify for the preparation of diagnostic antisera. The coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants and cloned into the prokaryotic pET-30a vector to generate the recombinant plasmid pET-CP. The resulting carboxy-terminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by Isopropyl-β- d -thiogalactoside (IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to elicit a polyclonal antiserum in rabbits. The antiserum had a titre of 1 : 128 in double diffusion tests, and specifically recognized LMoV in Western blots, Enzyme-Linked Immunosorbent Assays and Immuno-Electron Microscopy. The CP antiserum was also used to evaluate LMoV infection of lily bulbs used for commercial production in Yunnan, China. Substantial levels of infection were found in both imported and native bulbs, and provide the basis to implement flexible, rapid and large-scale virus indexing of lily plants for use in propagation and to meet virus-free quarantine regulations.     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

非编码RNA在喉癌发生中的作用及其临床意义

喉癌是头颈部最常见的恶性肿瘤之一,喉癌患者吞咽、呼吸及发音功能均可受到严重影响,患者经受“有苦难言”的折磨,生活质量极差。因此,揭示喉癌发病的分子机制,以期提高喉癌早期诊断水平和防治效果一直是该研究领域的热点之一。近年来,随着基因测序技术、转录组学技术和生物信息学技术等分子生物学技术的应用,越来越多的与喉癌发生相关的非编码RNA先后被发现。实验证明,一些短链非编码RNA（如：上调的miR-16、miR-21、miR．106b和miR-1297,下调的let．7a、miR．1、miR．24、miR．34a／c、miR-137、miR-203和miR-206）以及长链非编码RNAf如：H19、HOTAIR和MALAT-1）均被发现参与了喉癌的发生、发展过程,它们发挥着促癌或者抑癌作用,影响细胞的增殖、浸润、转移和凋亡。这些非编码RNA将有可能为喉癌诊断和治疗分别提供新的标志物和治疗靶点。     

Identification of a therapeutic interfering particle—A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance

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Rootstock‐to‐scion transfer of transgene‐derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry

Small interfering RNAs (siRNAs) are silencing signals in plants. Virus‐resistant transgenic rootstocks developed through siRNA‐mediated gene silencing may enhance virus resistance of nontransgenic scions via siRNAs transported from the transgenic rootstocks. However, convincing evidence of rootstock‐to‐scion movement of siRNAs of exogenous genes in woody plants is still lacking. To determine whether exogenous siRNAs can be transferred, nontransgenic sweet cherry (scions) was grafted on transgenic cherry rootstocks (TRs), which was transformed with an RNA interference (RNAi) vector expressing short hairpin RNAs of the genomic RNA3 of Prunus necrotic ringspot virus (PNRSV‐hpRNA). Small RNA sequencing was conducted using bud tissues of TRs and those of grafted (rootstock/scion) trees, locating at about 1.2 m above the graft unions. Comparison of the siRNA profiles revealed that the PNRSV‐hpRNA was efficient in producing siRNAs and eliminating PNRSV in the TRs. Furthermore, our study confirmed, for the first time, the long‐distance (1.2 m) transfer of PNRSV‐hpRNA‐derived siRNAs from the transgenic rootstock to the nontransgenic scion in woody plants. Inoculation of nontransgenic scions with PNRSV revealed that the transferred siRNAs enhanced PNRSV resistance of the scions grafted on the TRs. Collectively, these findings provide the foundation for ‘using transgenic rootstocks to produce products of nontransgenic scions in fruit trees'.     

Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart-pathogenic coxsackievirus B3

This study describes the first application of unlocked nucleic acid (UNA)-modified small interfering RNAs (siRNAs) directed against a medically relevant target, the coxsackievirus B3. We systematically analyzed the impact of different siRNA modification patterns and observed good compatibility of the introduction of UNA with the maintenance of high antiviral activity. Additionally, the polarity of an siRNA was successfully reversed by modulating the relative stability of the termini with locked nucleic acid (LNA) and UNA as shown in a reporter assay. The potency of the reversed siRNA against the full-length target was, however, too low to inhibit the infectious virus. Altogether, combined modification of siRNAs with LNA und UNA provides a promising approach to alter and improve properties of an siRNA.     

Identification of maize lethal necrosis disease causal viruses in maize and suspected alternative hosts through small RNA profiling

Maize lethal necrosis disease (MLND) is a devastating viral disease of maize caused by double infection with Maize chlorotic mottle virus (MCMV) and any one of the Potyviridae family members. Management of MLND requires effective resistance screening and surveillance tools. In this study, we report the use of small RNA (sRNA) profiling to detect MLND causal viruses and further the development of alternative detection markers for use in routine surveillance of the disease-causing viruses. Small RNAs (sRNAs) originating from five viruses namely MCMV, Sugarcane mosaic virus (SCMV), Maize streak virus (MSV), Maize-associated totivirus (MATV) and Maize yellow mosaic virus (MYMV) were assembled from infected maize samples collected from MLND hot spots in Kenya. The expression of the identified viral domains was further validated using quantitative real-time PCR. New markers for the detection of some of the MLND causal viruses were also developed from the highly expressed domains and used to detect the MLND-causative viruses in maize and alternative hosts. These findings further demonstrate the potential of using sRNAs especially from highly expressed viral motifs in the detection of MLND causal viruses. We report the validation of new sets of primers for use in detection of the most common MLND causal viruses MCMV and SCMV in East Africa.     

The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites

There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure—often one or more stem-loops—either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these “fingers” of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.     

Argonaute 1 and 5 proteins play crucial roles in the defence against cucumber green mottle mosaic virus in watermelon

RNA silencing, a core part of plants' antiviral defence, requires the ARGONAUTE, DICER-like, and RNA-dependent RNA polymerase proteins. However, how these proteins contribute to watermelon's RNA interference (RNAi) pathway response to cucumber green mottle mosaic virus (CGMMV) has not been characterized. Here, we identify seven ClAGO, four ClDCL, and 11 ClRDR genes in watermelon and analyse their expression profiles when infected with CGMMV. ClAGO1 and ClAGO5 expression levels were highly induced by CGMMV infection. The results of ClAGO1 and ClAGO5 overexpression and silencing experiments suggest that these genes play central roles in watermelon's antiviral defence. Furthermore, co-immunoprecipitation and bimolecular fluorescence complementation experiments showed that ClAGO1 interacts with ClAGO5 in vivo, suggesting that ClAGO1 and ClAGO5 co-regulate watermelon defence against CGMMV infection. We also identified the ethylene response factor (ERF) binding site in the promoters of the ClAGO1 and ClAGO5 genes, and ethylene (ETH) treatment significantly increased ClAGO5 expression. Two ERF genes (Cla97C08G147180 and Cla97C06G122830) closely related to ClAGO5 expression were identified using co-expression analysis. Subcellular localization revealed that two ERFs and ClAGO5 predominantly localize at the nucleus, suggesting that enhancement of resistance to CGMMV by ETH is probably achieved through ClAGO5 but not ClAGO1. Our findings reveal aspects of the mechanisms underlying RNA silencing in watermelon against CGMMV.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

Interaction with capsid protein alters RNA structure and the pathway for in vitro assembly of cowpea chlorotic mottle virus

Viruses use sophisticated mechanisms to allow the specific packaging of their genome over that of host nucleic acids. We examined the in vitro assembly of the Cowpea chlorotic mottle virus (CCMV) and observed that assembly with viral RNA follows two different mechanisms. Initially, CCMV capsid protein (CP) dimers bind RNA with low cooperativity and form virus-like particles of 90 CP dimers and one copy of RNA. Longer incubation reveals a different assembly path. At a stoichiometry of about ten CP dimers per RNA, the CP slowly folds the RNA into a compact structure that can be bound with high cooperativity by additional CP dimers. This folding process is exclusively a function of CP quaternary structure and is independent of RNA sequence. CP-induced folding is distinct from RNA folding that depends on base-pairing to stabilize tertiary structure. We hypothesize that specific encapsidation of viral RNA is a three-step process: specific binding by a few copies of CP, RNA folding, and then cooperative binding of CP to the "labeled" nucleoprotein complex. This mechanism, observed in a plant virus, may be applicable to other viruses that do not halt synthesis of host nucleic acid, including HIV.     

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.     

CT23 knockdown attenuating malignant behaviors of hepatocellular carcinoma cell is associated with upregulation of metallothionein 1

The cancer-testis antigen 23 (CT23) gene has been reported in association with the pathogenesis and progress of hepatocellular carcinoma (HCC). However, the alterations of gene expression profiling induced by CT23 knockdown in HCC cells remains largely unknown. In this study, the RNA interfering (RNAi) method was used to silence CT23 expression in BEL-7404 cells. Microarray analysis was performed on mRNA extracted from the CT23 knockdown cells and the control cells to determine the alterations of gene expression profiles. The result showed a total of 1051 genes expressed differentially (two-fold change), including 470 genes upregulated and 581 gene downregulated in the CT23 knockdown cells. A bioinformatic analysis showed that the functional differentially expressed genes (DEGs) were linked to cell proliferation, migration, and apoptosis, and metallothionein 1 (MT1) attained the maximum enrichment scores in functional annotation, classification, and pathway analysis of DEGs. Furthermore, Western blot analysis and cell behaviors assays verified that CT23 modulates cell proliferation, migration, and apoptosis by regulating MT1 expression in HCC cells and non-neoplastic hepatocytes. In summary, downregulated CT23 gene in BEL-7404 cells might change the expressions of carcinogenesis and progression related genes in HCC by upregulating MT1 expression, which would provide insight into searching for a novel therapeutic target for HCC.     

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem‐associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23‐kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N‐terminal 157‐amino‐acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV‐like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23‐derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem‐specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV‐specific symptoms (vein clearing and necrosis, and stem pitting), but not the non‐specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem‐specific accumulation of the p23Δ158–209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N‐terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.     

Identification of messenger and long noncoding RNAs associated with gallbladder cancer via gene expression profile analysis

The present study aimed to investigate the long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) involved in the progression of gallbladder cancer and explore the potential physiopathologic mechanisms of gallbladder cancer in terms of competing endogenous RNAs (ceRNAs). The original lncRNA and mRNA expression profile data (nine gallbladder cancer tissues samples and nine normal gallbladder samples) in GSE76633 was downloaded from the Gene Expression Omnibus database. Differentially expressed mRNAs and lncRNAs between gallbladder cancer tissue and normal control were selected and the pathways in which they are involved were analyzed using bioinformatics analyses. MicroRNAs (miRNAs) were also predicted based on the differentially expressed mRNAs. Finally, the co-expression relation between lncRNA and mRNA was analyzed and the ceRNA network was constructed by combining the lncRNA-miRNA, miRNA-mRNA, and lncRNA-mRNA pairs. Overall, 373 significantly differentially expressed mRNAs and 47 lncRNAs were identified between cancer and normal tissue samples. The upregulated genes were significantly enriched in the extracellular matrix (ECM)-receptor interaction pathway, while the downregulated genes were involved in the complement and coagulation cascades. Altogether, 128 co-expression relations between lncRNA and mRNA were obtained. In addition, 196 miRNA-mRNA regulatory relations and 145 miRNA-lncRNA relation pairs were predicted. Finally, the lncRNA-miRNA-gene ceRNA network was constructed by combining the three types of relation pairs, such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6. mRNAs and lncRNAs may be involved in gallbladder cancer progression via ECM-receptor interaction pathways and the complement and coagulation cascades. Moreover, ceRNAs such as XLOC_011309-miR-548c-3p-SPOCK1 and XLOC_012588-miR-765-CEACAM6 can also be implicated in the pathogenesis of gallbladder cancer.     

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12

Summary Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1^intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1^intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1^intB indicated that this element was integrated at the same attP site as previously identified for SSV1^intA. A comparison of the DNA sequences at the left attachment sites of SSV1^intA and SSV1^intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 by inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed. Offprint requests to: W. Zillig