RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.     

大麦黄矮病毒GAV株系ORF4基因在杆状病毒-昆虫细胞系统中的表达及亚细胞定位

根据已报道的大麦黄矮病毒GAV株系(BYDV-GAV)相关基因序列,利用RT-PCR方法获得ORF4基因。在杆状病毒-昆虫细胞系统中,成功表达了ORF4和GFP(绿色荧光蛋白)的融合蛋白(GFP:ORF4),Western blot检测到目的蛋白的表达。利用激光共聚焦显微镜观察其在细胞中的积累和亚细胞分布,发现ORF4基因编码的17kD蛋白(P4)能进入细胞核,并在细胞核膜上聚集。通过对ORF4基因编码的P4蛋白的N端和C端缺失突变结合蛋白质的结构预测分析,鉴定出N端α螺旋结构对于P4蛋白的核膜定位是必需的。这些结果为进一步研究ORF4基因在黄矮病毒GAV系统侵染中的生物学功能奠定了基础。     

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.     

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

A lipopeptide-encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.     

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.     

An orthopoxvirus serpinlike gene controls the ability of infected cells to fuse.

Most orthopoxviruses encode a functional hemagglutinin (HA), which is nonessential for virus growth in cell culture. However, inactivation of the HA gene leads to the formation of polykaryocytes (syncytia) by fusion of infected cells at neutral pH. Fusion is not observed when a functional HA gene is present. Deletion of open reading frames (ORFs) K2, K3, and K4 within the HindIII K fragment of the HA-positive (HA+) vaccinia virus strain WR also led to fusion of cells upon infection at neutral pH. A novel ORF inactivation procedure utilizing the polymerase chain reaction was used to specifically implicate the K2 ORF in this phenomenon. The K2 ORF (the viral SPI-3 gene) encodes a protein resembling serine protease inhibitors (serpins). Inactivation of the SPI-3 gene in any of the HA+ orthopoxviruses tested caused infected cells to fuse in a manner which appeared identical to that seen for HA- mutants, although fusion was most pronounced with cowpox virus. SPI-3-negative strains fused despite the fact that the HA was expressed and processed normally, i.e., cells infected with SPI-3 mutants remained functionally hemadsorption positive, and analysis of the HA protein by Western immunoblot suggested that posttranslational modifications of the HA protein appeared normal. Fusion triggered by SPI-3 mutants, like that for HA- mutants, was inhibited by the monoclonal antibody C3 directed against the vaccinia virus 14-kDa envelope protein. Therefore SPI-3- and HA-mediated fusion share a requirement for the 14-kDa protein, suggesting linkage of the seemingly disparate SPI-3 and HA genes through a common pathway which normally acts to prevent fusion of cells infected with wild-type virus.     

重叠延伸PCR法扩增EB病毒BZLF1N-BLRF2融合基因及生物信息学分析

利用重叠延伸PCR技术扩增EB病毒BZLF1N-BLRF2融合基因,构建原核表达载体pGEX-4T-1-BZLF1N-BLRF2,并进行了蛋白的诱导表达。通过序列测定和ORF软件分析,该融合基因含有1个1 005 bp的ORF,编码335个氨基酸。应用生物信息学方法,对该融合基因编码的蛋白ZtaN-p23从氨基酸组成、理化性质、信号肽、疏水性/亲水性、二级结构、亚细胞定位、功能域和高级结构等方面进行了预测和分析。结果表明,该蛋白的预测分子量为46.2 kD,理论等电点约为8.97,是亲水性蛋白,推测它定位于细胞质中。序列分析表明,该蛋白可能具有信号转导、转录调控、免疫应答等功能。预测的二级结构及三级结构都表明该蛋白含有较多的不规则卷曲和α-螺旋,三级结构上呈对称形状。     

Detection and differentiation of yellow head complex viruses using monoclonal antibodies

Three monoclonal antibodies (MAbs) raised against pathogenic yellow head virus (YHV) from Thailand were tested against tissues of shrimp from Thailand, Australia, Ecuador and India that were purported to be infected with yellow head complex viruses. MAbs V-3-2B and Y-18 were specific to gp116 and gp64 envelope proteins, respectively, while Y-19 was specific to a 20 kDa putative nucleoprotein p20. As a preliminary step, the site of reactivity of the 3 MAbs in YHV was determined by immuno-electron microscopy using ultra-thin sections of YHV-infected shrimp tissue and negatively stained, semi-purified YHV particles. As expected, MAb Y-19 reacted with viral nucleocapsids in ultra-thin sections but not with negatively stained, whole virions; MAb V-3-2B did react with negatively stained, whole virions, but not with virions or nucleocapsids in ultra-thin sections. Unexpectedly, MAb Y-18 did not react with whole or sectioned virions. By immunohistochemistry, MAbs Y-19 and Y-18 reacted with Penaeus monodon tissues infected with either YHV or with gill-associated virus (GAV) from Australia, while MAb V-3-2B reacted with YHV only. In addition, all the YHV and GAV tissue samples gave positive in situ hybridization reactions with a cDNA probe specific to the ORF1b gene of YHV. They also gave expected differential RT-PCR results for YHV and GAV. By contrast, 2 natural Thai shrimp specimens with no gross signs of disease gave similar immunohistochemical reactions and RT-PCR reactions to GAV. However, sequencing of their RT-PCR products showed that they shared 92.7% identity with GAV, but only 79.0% identity with YHV. Although specimens from Ecuador and India displayed histopathology suggestive of YHV infection, they gave negative immunohistochemical reactions with all 3 Mabs, and negative in situ hybridization results. Additional work is required to determine whether a virus from the yellow head complex was responsible for their observed histopathology. These data show that the 3 YHV MAbs could be used in diagnostic situations to differentiate some viruses in the yellow head virus complex.     

Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames.

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.     

Characterization of a unique chromosomal copper resistance gene cluster from Xanthomonas campestris pv. vesicatoria

We characterized the copper resistance genes in strain XvP26 of Xanthomonas campestris pv. vesicatoria, which was originally isolated from a pepper plant in Taiwan. The copper resistance genes were localized to a 7,652-bp region which, based on pulsed-field gel electrophoresis and Southern hybridization, was determined to be located on the chromosome. These genes hybridized only weakly, as determined by Southern analysis, to other copper resistance genes in Xanthomonas and Pseudomonas strains. We identified five open reading frames (ORFs) whose products exhibited high levels of amino acid sequence identity to the products of previously reported copper genes. Mutations in ORF1, ORF3, and ORF4 removed copper resistance, whereas mutations in ORF5 resulted in an intermediate copper resistance phenotype and insertions in ORF2 had no effect on resistance conferred to a copper-sensitive recipient in transconjugant tests. Based on sequence analysis, ORF1 was determined to have high levels of identity with the CopR (66%) and PcoR (63%) genes in Pseudomonas syringae pv. tomato and Escherichia coli, respectively. ORF2 and ORF5 had high levels of identity with the PcoS gene in E. coli and the gene encoding a putative copper-containing oxidoreductase signal peptide protein in Sinorhizobium meliloti, respectively. ORF3 and ORF4 exhibited 23% identity to the gene encoding a cation efflux system membrane protein, CzcC, and 62% identity to the gene encoding a putative copper-containing oxidoreductase protein, respectively. The latter two ORFs were determined to be induced following exposure to low concentrations of copper, while addition of Co, Cd, or Zn resulted in no significant induction. PCR analysis of 51 pepper and 34 tomato copper-resistant X. campestris pv. vesicatoria strains collected from several regions in Taiwan between 1987 and 2000 and nine copper-resistant strains from the United States and South America showed that successful amplification of DNA was obtained only for strain XvP26. The organization of this set of copper resistance genes appears to be uncommon, and the set appears to occur rarely in X. campestris pv. vesicatoria.     

TT病毒ORF2在Cos7细胞中的表达及蛋白质定位

应用PCR方法从含有1TrVirusORF2的质粒pET-His-TTV2中扩增出606bp的蛋白质编码区,并将其克隆到真核表达载体pEGFP．NI中以表达成GFP—VP2融合蛋白。构建出的重组质粒pEGFPTFV2经过酶切分析和PCR鉴定。用脂质体介导法将pEGFPTTV2质粒DNA转染Cos7细胞,通过RT-PCR分析,证实细胞中存在ORF2基因的转录产物。用共聚焦显微镜结合PI染色技术研究1TTV P2蛋白在细胞中的分布情况。结果表明,1TrVVP2分布在细胞质中和细胞核膜内侧。因此推测VP2作为一种非结构蛋白,功能可能是参与病毒DNA的复制或转录。