The search of miRNA genes in Bombyx mori nuclear polyhedrosis virus genomes regions complementary to the latest genes

The search of miRNA genes in Bombyx mori nuclear polyhedrosis virus genome region complementary to very late genes has been carried out. The search miRNA algorithm in silico was developed by us. It was shown that NPV B. mori genome region containing orf4 gene complementary to ph gene encodes the potential miRNA. NPV B. mori genome region containing p74 gene complementary to p10 gene encodes mature miRNA and potential miRNA. The genome region containing orf1629 encodes two small non-coding RNAs complementary to orf 5'-end of polyhedrin miRNA. From obtained results it is proposed that two small noncoding RNAs complementary to regions of polyhedrin miRNA are included in polyhedra.     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

Expression of human papillomavirus type 6b E2 gene product with DNA-binding activity in insect (Bombyx mori) cells using a baculovirus expression vector

Human papillomavirus type 6b (HPV6b) has been shown to be a major etiologic agent of genital condylomas. The E2 gene, one of the early genes, has been shown to activate the enhancer element in trans in cells transformed with bovine papillomavirus type 1a (BPV1a) but the E2 gene product of any HPV has not been identified. The E2 gene of HPV6b was inserted into the polyhedrin gene of a Baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), 156 bp downstream from the translational start codon, and transferred into BmNPV by allelic replacement in a cotransfected Bombyx mori cell line, Bm-N. The predicted 46-kDa protein was produced by a recombinant virus in the infected Bm-N cells at a high level under the control of the polyhedrin promoter. We obtained the antibody against the putative E2-polyhedrin fusion protein by immunizing a rabbit with this protein. This protein reacted with the antibodies against polyhedrin and the fusion protein. This protein did specifically bind to the upstream regulatory region of the HPV6b and BPV1a genomes. This DNA binding activity was blocked by the antibody against this protein.     

Bombyx mori nuclear polyhedrosis virus-based vectors for expressing passenger genes in silkmoth cells under viral or cellular promoter control

The polyhedrin gene of the nuclear polyhedrosis virus of the silkmoth Bombyx mori (BmNPV) has been subjected to deletion mutagenesis. A number of clones containing partially deleted polyhedrin genes were characterized and four clones containing limited deletions of the 5'-untranslated or 5'-flanking sequences of the gene were further analyzed with respect to polyhedrin promoter activity. The functional characterization of the deletion mutants was achieved through the insertion of a chloramphenicol acetyl transferase (CAT) gene (cat) into each deletion junction. The resultant cat constructs were introduced into the genome of BmNPV through homologous recombination and the effect of each deletion on the activity of the polyhedrin promoter was evaluated by measurements of CAT enzymatic activity in extracts of tissue culture cells infected with the corresponding recombinant BmNPVs as well as by primer extension assays. Removal of the entire leader region and eleven adjacent residues of the 5'-flanking sequences of the polyhedrin gene results in a dramatic decrease in promoter activity, which, however, remains detectable through CAT activity measurements. Elimination of an additional 30 nucleotides (nt) of the upstream sequences results in the complete inactivation of the polyhedrin promoter. The functional characterization of a deletion mutant lacking 41 nt of the 5'-flanking sequences has demonstrated that no functions necessary for viral infectivity, replication or assembly are disrupted by this deletion, since the corresponding recombinant viruses propagate in the cells with the same kinetics and to the same extent as wild-type BmNPV. As a result of the deletion mutagenesis, two classes of transfer vectors have become available. The first class can be used for introducing into the viral genome foreign nucleotide sequences under polyhedrin promoter control, while the second one can be used for obtaining recombinant viruses harboring foreign genetic material in an environment which is devoid of polyhedrin promoter activity.     

Transfer vectors for maximal expression of passenger genes in the Bombyx mori nuclear polyhedrosis virus expression system

A series of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) transfer vectors has been developed containing various lengths of the polyhedrin promoter, including sequences 3' of the initiation codon. The ATG initiation codon was mutated in some of these vectors to allow for the production of authentic nonfusion proteins. The ability of the various polyhedrin promoter constructs to direct expression of foreign gene sequences was assessed using two test genes, chloramphenicol acetyl transferase (cat), and human metallothionein II. Accumulation of cat mRNA and nonfused protein was low when only polyhedrin promoter sequences to -8 (relative to the translational start site of polyhedrin mRNA) were included in the transfer vector, but cat expression was comparable with that of the wild-type polyhedrin gene when promoter sequences to +5 were present. Further addition of polyhedrin gene sequences to +26 or +94 resulted in no further increase in expression. Similar results were obtained for expression of human metallothionein II, where constructs encoding polyhedrin-metallothionein fusion proteins containing polyhedrin sequences to at least +5 resulted in high levels of mRNA and protein accumulation. The expression vectors containing the +5, +26, or +94 BmNPV polyhedrin promoter can thus be used to direct maximal levels of production of nonfused proteins (when the polyedrin ATG has been mutated) or of fusion proteins, depending on which is more suitable for a particular application. These new vectors are a useful addition to those presently available and should increase the utility of the BmNPV expression system for large-scale protein production. (c) 1993 John Wiley & Sons, Inc.     

丫纹夜蛾核多角体病毒转移载体质粒的组建

自从美国G.E.Smith等发展苜蓿丫纹夜蛾核多角体病毒(Autographa californica Nuclear Polyhedrosis Virus,简称Ac NPV)这一新的表达载体以来,先后已有几个外源性基因成功地获得了高效表达。如:人白细胞间素2(Interleukin 2)、人的c-myc基因产物、流感病毒血凝素等。与目前常用的原核和真核细胞表达系统比较,NPV载体     

A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus.     

家蚕NPV SOD基因序列和在大肠杆菌中表达

通过ＰＣＲ 克隆了家蚕核型多角体病毒（ＢｍＮＰＶ） ＳＯＤ基因, 并在大肠杆菌中进行表达, 证明了Ｂｍ ＮＰＶＳＯＤ基因产物确有ＳＯＤ活性, 其活力单位约为５７６ ｕ／ｍＬ 培养液。ＤＮＡ 测序结果表明Ｂｍ ＮＰＶ ＳＯＤ 基因编码１５１ 个氨基酸, 与人的ＳＯＤ１ 基因的核苷酸同源性为５６ ％ , 与ＡｃＮＰＶ 拟为的ＳＯＤ基因同源性为９７ ．２％ 。     

SINPV基因组酶切图谱及多角体基因的序列分析

用限制性内切酶ＥｃｏＲＩ、ＸａｂＩ、ＸｈｏＩ、ＢａｍＨＩ、ＰｓｔＩ、ＳａｃＩ、ＨｉｄｎⅢ、ＳｍａＩ酶解斜纹夜蛾核多角体病毒广州株基因组ＤＮＡ,分别得到２６、２６、２４、２０、１３、１７、９、１条片段,并算得基因组平均大小为１３６．０ｋｂｐ。以ＡｃＮＰＶ多角体基因的部分读码框为探针,经Ｓｏｕｔｈｅｍ杂交将ＳＩＮＰＶ多角体基因定位于ＸｂａＩＯ片段上。将此片段克隆并序列分析,结果表明ＳＩ     

Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra.

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.