Isolation and reconstitution of the RNA replicase of the cytoplasmic polyhedrosis virus of silkworm,Bombyx mori

Summary After irradiation of the virus particles of CPV, the RNA replicase associated with the virion was isolated in the form of a genome-replicase complex with DEAE-Sephadex A-25 chromatography. This complex was then treated with Triton X-100 and purified by phosphocellulose column chromatography. The RNA replicase reconstituted with the doublestranded RNA of CPV showed both the enzyme activity of RNA polymerase and methyltransferase. The single-stranded RNA could not serve as the template for the RNA replicase. The role of the RNA replicase of CPV is discussed.     

"Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA

S-adenosyl-L-methionine (SAM) activated the virus-associated RNA polymerase of cytoplasmic polyhedrosis virus in vitro. Synthesis of single-stranded viral RNA (mRNA) proceeded depending on the presence of SAM.     

RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.     

Nucleoside triphosphate phosphohydrolase associated with cytoplasmic polyhedrosis virus.

Nucleoside triphosphate phosphohydrolase [EC 3.6.1.15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the gamma-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2.7.7.6] using calf thymus DNA as a template, and the phage Q beta RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.     

Characterization of 3''----5'' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.     

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.     

Microarray analysis of the gene expression profile in the midgut of silkworm infected with cytoplasmic polyhedrosis virus

In order to obtain an overall view on silkworm response to Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection, a microarray system comprising 22,987 oligonucluotide 70-mer probes was employed to compare differentially expressed genes in the midguts of BmCPV-infected and normal silkworm larvae. At 72 h post-inoculation, 258 genes exhibited at least 2.0-fold differences in expression level. Out of these, 135 genes were up-regulated, while 123 genes were down-regulated. According to gene ontology (GO), 140 genes were classified into GO categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates that 35 genes were involved in 10 significant (P < 0.05) KEGG pathways. The expressions of genes related to valine, leucine, and isoleucine degradation, retinol metabolism, and vitamin B6 metabolism were all down-regulated. The expressions of genes involved in ribosome and proteasome pathway were all up-regulated. Quantitative real-time polymerase chain reaction was performed to validate the expression patterns of 13 selected genes of interest. The results suggest that BmCPV infection resulted in the disturbance of protein and amino acid metabolism and a series of major physiological and pathological changes in silkworm. Our results provide new insights into the molecular mechanism of BmCPV infection and host cell response.     

昆虫质型多角体病毒的研究进展

质型多角体病毒(Cytoplasmic polyhedrosis virus,CPV)隶属呼肠孤病毒科Reoviridae质型多角体病毒属Cypovirus,通常基因组由10个节段双链RNA构成。RNA分子量为3～27u。根据病毒基因组dsRNA片段在聚丙烯酰胺或琼脂糖凝胶中电泳图谱的差异,目前CPV已被分为19个电泳型。不同于呼肠孤病毒科其它成员,CPV为单层衣壳,而不是常见的双层衣壳结构,衣壳蛋白主要由衣壳蛋白、大突起蛋白及塔式突起蛋白组成。大部分质型多角体病毒引起昆虫慢性疾病,造成寄主死亡或适应性降低。随着RNA病毒基因序列测定技术的成熟,质型多角体病毒的序列测定方面取得较大进展,目前GenBank核苷酸序列数据库中已经公布了家蚕Bombyx mori CPV电泳型1两个株系(H株和I株)、舞毒蛾Lymantria dispar CPV电泳型1、舞毒蛾CPV电泳型14及粉纹夜蛾Trichoplusia ni CPV电泳型全基因组序列,为该病毒的进化与起源的研究提供更多的遗传信息。本文从结构功能、侵染特点、基因组特点及应用前景等方面综述了昆虫质型多角体病毒的研究进展。