Genetic recombination between RNA components of a multipartite plant virus

Genetic recombination of DNA is one of the fundamental mechanisms underlying the evolution of DNA-based organisms and results in their diversity and adaptability. The importance of the role of recombination is far less evident for the RNA-based genomes that occur in most plant viruses and in many animal viruses. RNA recombination has been shown to promote the evolutionary variation of picornaviruses, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses and is implicated in the synthesis of the messenger RNAs of influenza virus and coronavirus. However, RNA recombination has not been found to date in viruses that infect plants. In fact, the lack of DI RNAs and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral RNAs. Here we provide the first molecular evidence for recombination of plant viral RNA. For brome mosaic virus (BMV), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3' end region of a single BMV RNA genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type BMV RNA components. This result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. Moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants.     

甜菜坏死黄脉病毒新疆分离物外壳蛋白基因的原核表达载体和植物表达载体的构建

将克隆入ｐＧＥＭ７ｚｆ（＋）的甜菜坏死黄脉病毒（ＢＮＹＶＶ）新疆分离物外壳蛋白（ＣＰ）基因的ｃＤＮＡ的ｐＧＥＢ３，用ＸｂａＩ切下，Ｋｌｅｎｏｗ补平，再用ＢａｍＨＩ切下ｃＤＮＡ片段。用ＮｄｅＩ切开原核表达载体ｐＪＷ２，用Ｋｌｅｎｏｗ补平，再用ＢａｍＨＩ切去小片段。将该ｃＤＮＡ与ｐＪＷ２大片段用Ｔ４连接酶连接，构建了ＢＮＹＶＶＣＰ基因的表达载体ｐＪＷＢ４，转化到大肠杆菌ＤＨ５α。经培养和高温诱导，ｐＪＷＢ４成功地表达出了ＢＮＹＶＶ的外壳蛋白。将ｐＧＥＢ３和ｐＢＩ１２１用Ｘｂａｌ和ＢａｍＨＩ酶切Ｔ４连接酶连接，构建了ＢＹＶＶＣＰ基因的植物表达载体，转化到ＤＨ５α，筛选出正向连结的阳性克隆ｐＢＩＢ３，转化入农杆菌ＬＢＡ４４０４（ｐＡＬ４４０４），经用ＰＣＲ扩增和γ３２Ｐ标记的探针杂交约证实为阳性克隆。往甜菜植株中转化工作正在进行中。     

斜带石斑鱼神经坏死病毒基因组RNA1和RNA2序列测定及分析

根据GenBank数据库公布的鱼类神经坏死病毒(nervous necrosis virus,NNV)同源序列设计了7对特异性引物,采用RT-PCR方法扩增出目的片断,将PCR产物测序和分析.斜带石斑鱼Epinephelus coioides神经坏死病毒(orange-spotted NNV,OGNNV)基因组由两个片断(RNA1和RNA2)组成,RNA1由3 103个核苷酸组成,含有一个开放阅读框,编码982个氨基酸;RNA2由1 433个核苷酸组成,含有一个开放阅读框,编码338个氨基酸.OGNNV基因组与新加坡GGNNV(greasy grouper NNV)的基因组有高度的相似性.分析病毒的RNA2序列发现:OGNNV与DGNNV(dragon grouper NNV)、RGNNV(redspotted NNV)和GGNNV的亲缘关系很近,并且具有相同的中和位点;分析病毒的RNA1序列,发现在OGNNV的RNA1序列中同样可以找到依赖RNA的RNA聚合酶的6个模序(motif).根据同源性比较和系统进化分析,OGNNV属于RGNNV血清型的成员.     

Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus

The 9,213-nucleotide structure of the AIDS/lymphadenopathy virus has been determined from molecular clones representing the integrated provirus and viral RNA. The sequence reveals that the virus is highly polymorphic and lacks significant nucleotide homology with type C retroviruses characterized previously. Together with an analysis of the two major viral subgenomic RNAs, these studies establish the coding frames for the gag, pol and env genes and predict the expression of a novel gene at the 3' end of the genome unrelated to the X genes of HTLV-1 and -II.     

植物中RNA沉默机制的研究进展

近年来,人们对植物中RNA沉默机制的的认识日渐清晰,小RNAs在其中发挥重要作用.文章综述了植物中RNA沉默的机制、RNA沉默的主要途径及其在防御外源DNA序列入侵过程中的主要功能.并简要介绍了由DNA病毒编码的基因沉默抑制子在对抗宿主沉默反应的作用.文章最后阐述了对RNA沉默进行深入研究的必要性,对需要研究的问题进行了分析,为抗病毒作物育种提供了有力依据.     

Functional analysis of DNA 4 coding region from a Chinese Zhangzhou isolate of banana bunchy top virus

The DNA 4 coding region of banana bunchy top virus from a Chinese Zhangzhou isolate (BBTV-ZZ) is cloned by PCR. The sequencing analysis shows that it is 351 nucleotides long and it putatively encodes a protein of 116 amino acids. On the basis of a plant binary vector pBin438, the plant expression vector pBBTV-4B harboring the BBTV-ZZ DNA 4 coding region has been constructed and then transferred to tobacco (Nicotiana tobacum cv. Xanthi nc) by a Agrobacterium-mediated procedure. Under insect-free condition, movement-defective mutant of CMV-Fny strain (CMV-Fny-△MP) is mechanically inoculated on the lower leaves of transgenic plants. Systemic symptoms with different degrees of severity are developed in the upper uninoculated leaves of transgenic plants at 12 days postinoculation (dpi), while no symptoms can be seen in the uninoculated leaves of untransformed plants at any time. Accumulation of CMV-Fny is detected on the upper uninoculated leaves of transgenic plants, but is not on that of untransformed plants by indirect double antibody sandwich enzyme-link immunosorbent assay (DAS-ELISA). The results reveal that transgenic plants have acquired the property of cell-to-cell movement and systemic spread of CMV-Fny-△MP. This suggests that the protein encoded by BBTV-ZZ DNA 4 might have function of viral movement protein.     

An intron with a constitutive transport element is retained in a Tap messenger RNA

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.     

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.     

Transposon-induced deletions in unc-22 of C. elegans associated with almost normal gene activity

The unc-22 gene of Caenorhabolitis elegans encodes a protein which is a component of the myosin-containing A-band of the worm's striated body-wall muscle. Among 51 revertants of a transposon-induced mutant, we have identified four which retain a barely detectable mutant phenotype. Molecular analysis shows that three of these have in-frame deletions of 1.0, 1.3 and 2.0 kilobases, whereas the fourth partial revertant and two other apparently complete revertants have small insertions. All these rearrangements involve coding sequence and, in the case of the deletions, result in polypeptides that are shorter than the wild-type protein. The region of the gene containing these rearrangements contains 10 copies of a motif recognized in other regions of the gene (our unpublished data). We suggest that one explanation for the minimally mutant phenotype associated with the deletions is that the size and the repeated nature of the unc-22 protein structure make it relatively tolerant of substitutions or deletions involving one or a small number of repeated motifs. These results could explain why in some human genetic diseases, such as Duchenne's muscular dystrophy, deletions can be associated with only mild forms of the disease.     

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).