黄瓜花叶病毒山东株（CMV—SD）RNA2全和cDNA克隆的构建及全序列分析

分别利用５’ＲＡＣＥ和３’ＲＡＣＥ确定了ＣＭＶ－ＳＤＲＮＡ２的５’和３’未端序列,在此基础上,利用ＲＴ－ＰＣＲ得到了ＲＮＡ２的５’端一半的ｃＤＮＡＤＱ ＢＴＧ Ｐｃ２５和３’端一半的ｃＤＮＡ克隆ＰＣ２３,并通过拼接构建了ＲＮＡ２全长ｃＤＮＡ克隆ＰＣ２Ｆ。     

Cloning and sequencing of RNA-1 cDNA from cucumber mosaic virus strain O

The complete nucleotide (nt) sequence (3369 nt) of RNA 1 of cucumber mosaic virus strain O (CMV-O) was determined. One open reading frame (ORF; 993 aa) could be deduced from the nt sequence. The homologies of the ORF between CMV-O and CMV-Q or CMV-Fny were calculated to be 85% or 97%, respectively. For CMV-O and CMV-Q, the first one-third of the ORF showed a higher degree of homology (89%), as compared with the other portions (82-85%); the first 224 aa showed more than 93% homology. A comparative study of the three viruses revealed that CMV-O is more homologous to CMV-Fny (subgroup I) [corrected]) than to CMV-Q (subgroup II) [corrected].     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。     

Helper virus-independent transcription and multimerization of a satellite RNA associated with cucumber mosaic virus

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.     

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.     

Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.     

A single-stranded loop in the 5' untranslated region of cucumber mosaic virus RNA 4 contributes to competitive translational activity

The 5' untranslated region (UTR) of cucumber mosaic virus (CMV) RNA 4 confers a highly competitive translational advantage on a heterologous luciferase open reading frame. Here we investigated whether secondary structure in the 5' UTR contributes to this translational advantage. Stabilization of the 5' UTR RNA secondary structure inhibited competitive translational activity. Alteration of a potential single-stranded loop to a stem by substitution mutations greatly inhibited the competitive translational activity. Tobacco plants infected with wild type virus showed a 2.5-fold higher accumulation of maximal coat protein than did plants infected with a loop-mutant virus. Amplification of viral RNA in these plants could not explain the difference in accumulation of coat protein. Phylogenetic comparison showed that potential single-stranded loops of 12-23 nucleotides in length exist widely in subgroups of CMV.     

Genetic variability and evolution of the satellite RNA of cucumber mosaic virus during natural epidemics.

The genetic structure of populations of cucumber mosaic virus (CMV) satellite RNA (satRNA) and its evolution were analyzed during the course of a CMV epidemic in tomatoes in eastern Spain. A total of 62 variants of CMV-satRNA from epidemic episodes in 1989, 1990, and 1991 were characterized by RNase protection assay (RPA); RPA patterns defined 60 haplotypes in the CMV-satRNA population. RPA of nine CMV-satRNAs of known sequences showed that numbers of nucleotide substitutions per site (dij) between different satRNAs can be estimated from RPA data. Thus, dij were estimated for any possible pair of field CMV-satRNA types, and nucleotide diversities within and between yearly subpopulations were calculated. Also, phylogenetic relationships among CMV-satRNAs were derived from RPA data (by parsimony) or from dij (by neighbor joining). From these analyses, a model for the evolution of CMV-satRNAs in field epidemics can be built. High genetic variability of CMV-satRNA results in very heterogeneous populations, even compared with those of other RNA genomes. The high diversity of the population is maintained through time by the continuous generation of variants by mutation, counterbalanced by negative selection; this results in a certain replacement of haplotypes from year to year. The sequential accumulation of mutations in CMV-satRNA leads to fast genetic divergence to reach what appears to be an upper permitted threshold.