The complete sequence of a sugarcane mosaic virus isolate causing maize dwarf mosaic disease in China

The complete sequence of a potyvirus from maize in Zhejiang Province was determined. The RNA was 9596 nucleotides long, excluding the 3'-poly (A) tail, and there was a single long open reading frame (ORF) of 9192 nts encoding a 346.1 ku polyprotein. The polyprotein had substantial amino acid sequence homology with those encoded by the RNAs of a Chinese isolate of sorghum mosaic virus (SrMV-C) and a Bulgarian isolate of maize dwarf mosaic virus, but it was most closely related to sugarcane mosaic virus (SCMV) isolates, for which only partial sequences have been published. According to the published criteria for distinguishing potyviruses, the sequence reported here is clearly a strain of SCMV, but it also showed a surprisingly high amino acid homology with SrMV-C in the HC-Pro, P3 and Cl proteins.     

Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences

To investigate whether a 12 nucleotide DNA-based miniEGSs can silence the expression of human cytomegalovirus （HCMV） UL49 gene efficiently, A HeLa cell line stably expressing UL49 gene was constructed and the putative miniEGSs （UL49-miniEGSs） were assayed in the stable cell line. Quantitative RT-PCR and western blot results showed a reduction of 67% in UL49 expression level in HeLa cells that were transfected with UL49-miniEGSs. It was significantly different from that of mock and control miniEGSs （TK-miniEGSs） which were 1 and 7%, respectively. To further confirm the gene silence directed by UL49-miniEGSs with human RNase P, a mutant of UL49-miniEGSs was constructed and a modified 51 RACE was carried out. Data showed that the inhibition of UL49 gene expression directed by UL49-miniEGSs was RNase P-dependent and the cleavage of UL49 mRNA by RNase P was site specific. As a result, the length of DNA-based miniEGSs that could silence gene expression efficiently was only 12 nt. That is significantly less than any other oligonucleotide-based method of gene inactivation known so far. MiniEGSs may represent novel gene-targeting agents for the inhibition of viral genes and other human disease related gene expression.     

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus （BTV） have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue （BT）. We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 （VP7） and the non-structural protein （NS1） gene from different serotypes of BTV by DNAstar showed that the 5＇end of the NS1 gene is the most conserved region. The primer pairs （P1 and P2） were designed based on the highly conserved region of NS 1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus （EHDV） serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes     

RNase MRP RNA and human genetic diseases

RNase MRP RNA is the RNA subunit of the RNase mitochondrial RNA processing （MRP） enzyme complex that is involved in multiple cellular RNA processing events. Mutations on RNase MRP RNA gene （RMRP） cause a recessively inherited developmental disorder, cartilage-hair hypoplasia （CHH）. The relationship of the genotype （RMRP mutation）, RNA processing deficiency of the RNase MRP complex, and the phenotype of CHH and other skeletal dysplasias is yet to be explored.     

Analysis of nucleotide sequence of wheat yellow mosaic virus genomic RNAs

Wheat yellow mosaic virus (WYMV) isolate HC was used for viral cDNA synthesis and sequencing. The results show that the viral RNA1 is 7629 nueleotides encoding a polyprotein with 2407 amino acids, from which seven putative proteins may be produced by an autolytie cleavage processing besides the viral coat protein. The RNA2 is 3639 nueleotides and codes for a polypretein of 903 amino acids, which may contain two putative non-structural proteins. Although WYMV shares a similarity in genetic organization to wheat spindle streak mosaic virus (WSSMV), the identities in their nucleotide sequences or deduced amino acid sequences are as low as 70% and 75 % respectively. Based on this result, it is confirmed that WYMV and WSSMV are different species within Bymovirus.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses

lacZa-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome （BAC） by homologous recombination in E. coli. The resulting recombinant BAC （pBeckerZF1） was confirmed by PCR and sequencing. Green fluorescent protein （GFP） gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect （CPE） observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3, vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains,field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeⅠ site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into nonimmunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.     

VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

A cDNA corresponding to the coding region of VP1, the putative RNA-dependent RNA polymerase, of infectious bursal disease virus (IBDV) was cloned and inserted into the genome of a vaccinia virus inducible expression vector. The molecular mass and antigenic reactivity of VP1 expressed in mammalian cells are identical to those of its counterpart expressed in IBDV-infected cells. The results presented here demonstrate that VP1 is efficiently incorporated into IBDV virus-like particles (VLPs) produced in mammalian cells coexpressing the IBDV polyprotein and VP1. Incorporation of VP1 into VLPs requires neither the presence of IBDV RNAs nor that of the nonstructural polypeptide VP5. Immunofluorescence, confocal laser scanning microscopy, and immunoprecipitation analyses conclusively showed that VP1 forms complexes with the structural polypeptide VP3. Formation of VP1-VP3 complexes is likely to be a key step for the morphogenesis of IBDV particles.     

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.     

一步法扩增克隆IBDV上海超强毒VP2—4—3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV)上海株SH95的病毒核酸dsRNA ,应用随机引物将RNA反转录成cDNA ,以此为模板一步扩增出A片段前体融合蛋白基因即VP2 4 3基因 ,将其克隆入 pGEM T载体 ,并进行序列分析 ,其与超强毒株HK4 6的核苷酸序列的同源性达 98% ,整个基因有 5个氨基酸差异 ,同源性达99.5 1% (10 0 7/ 10 12 )。     

Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide.

The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.     

传染性法氏囊病病毒多聚蛋白基因在家蚕中的表达

将传染性法氏囊病病毒(IBDV)细胞致弱株(JD1株)的基因组A节段基因重组于家蚕杆状病毒转移载体pAcHLT-C中,获得的重组转移载体pAcHLT-C-A与线性化病毒Bm-BacPAK6 DNA共转染家蚕培养细胞,获得重组病毒BacPAK-A。DIG标记的DNA点杂交证实重组病毒基因组中含有A节段基因,重组病毒感染家蚕5龄幼虫进行表达, ELISA和Western blotting等结果表明多聚蛋白基因在蚕体内得到了表达,表达产物具有免疫反应性,表达量在感染后5～6 d达到最高。家蚕生物反应器表达IBDV多聚蛋白具有我国的资源优势,为今后研制低成本、实用化的IBDV基因工程疫苗打下基础。     

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.     

传染性法氏囊病毒的抗原及分子特征

用鸡胚成纤维细胞对来自野外的 5 个传染性法氏囊病毒株 (IBDV-JD1 、 JD2 、 NB 、 HZ1 、 HZ2) 进行分离,测定理化特性、致病性,同时进行血清亚型测定及 A 片段基因组的克隆分析 . 试验所用 5 个法氏囊组织悬液在鸡胚成纤维细胞盲传 2~14 代后适应细胞并产生细胞病变 . 细胞适应的 IBDV 毒株的理化和形态特征与经典传染性法氏囊病毒株一致 . 除 IBDV-HZ1 、 HZ2 属经典 IBDV 血清型外, IBDV-JD1 、 JD2 和 NB 毒株分属不同的血清亚型 . 人工感染实验结果显示,分离的 IBDV 毒株产生与野外病例相似的临床症状和病变,出现法氏囊滤泡髓质的淋巴细胞变性、坏死和消失 . 基因组序列分析显示, IBDV-NB 毒株 A 片段由 3 264 个核苷酸组成,编码由 145 个氨基酸残基组成的 VP5 和由 1 012 个氨基酸残基组成的多聚蛋白 . 与来自 GenBank 的 IBDV Ⅰ型毒株比较, NB 毒株 A 片段编码的多聚蛋白与 JD1 毒株的同源性最高,达 99.5% , VP2 与 JD1 、 CEF94 、 D78 的同源性为 99.8% , VP3 与 JD1 的同源性为 99.2% , VP4 与 JD1 的同源性为 100% , VP5 与 JD1 , HZ2 , P2 , CEF94 , CT , Cu-1 和 D78 毒株的同源性为 99.3%. NB 毒株 VP2 蛋白的第 253 、 280 、 284 位氨基酸残基与 IBDV 变异毒株和经典毒株一致,但不同于 IBDV 超强毒株 . 这些结果暗示 IBDV 的抗原表位是构象依赖性表位, IBDV 血清亚型的形成与 IBDV 弱毒疫苗病毒株密切相关 .     

Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans.

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.     

Genomic structure of the large RNA segment of infectious bursal disease virus.

The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis. We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments. The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins. The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E. coli. These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates. We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo. The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus.