苜蓿丫纹夜蛾核多角体病毒复制与宿主细胞核骨架关系初探

用选择性抽提方法和整装细胞电镜技术观察苜蓿丫纹夜蛾多粒包埋核型多角体病毒(AcMNPV)感染的Tn5B1细胞的核骨架结构体系,发现感染早期病毒的复制过程未对宿主细胞核骨架的形态结构产生明显影响,而感染晚期多角体的装配在核骨架网络中进行.以ie-1基因和多角体蛋白基因为探针,点杂交分析基因转录活性与宿主细胞核骨架的关系,结果表明在病毒感染早期,无论是正具转录活性的ie-1基因还是不具转录活性的多角体蛋白基因都可紧密结合在宿主细胞的核骨架上.     

Localization of human (pro)renin receptor lacking the transmembrane domain on budded baculovirus of <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus

Human (pro)renin receptor (hPRR), a construct with native transmembrane and cytoplasmic domains (hPRR-wTM), and hPRR lacking both (hPRR-w/oTM) were expressed using insect cells. The hPRR-wTM was expressed in the peripheral domains of the nucleus in infected Sf-9 cells, and its localization was observed in endoplasmic reticulum (ER). However, it could not be extracted from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by Triton X-100 treatment at 4°C. In contrast, hPRR-w/oTM was observed in punctate domains in the cytoplasm of infected Sf-9 cells, but intracellular hPRR-w/oTM did not co-localize in the Golgi apparatus and lysosomes. This indicates that hPRR-wTM and hPRR-w/oTM is localized in the ER and cytoplasmic organelles of Sf-9 cell, respectively. Moreover, the localization of hPRR-w/oTM in budded baculovirus of recombinant AcMNPV was confirmed by Western blotting. This is the first finding of the association of a foreign protein lacking a transmembrane domain with a baculovirus. If this finding is available for double displaying system, being capable of expression on the envelope and the capsid of baculovirus, it will lead to new methodology of baculovirus display system for tissue- and cell-specific targeting and intracellular targeting.     

Removal of 106 amino acids from the N-terminus of UDP-GlcNAc :alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I does not inactivate the enzyme

UDP-GlcNAc : -3-D-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal–anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnT I was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf 9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnT I activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnT I activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long.     

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.     

Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.     

Systematic Analysis of 42 Autographa Californica Multiple Nucleopolyhedrovirus Genes Identifies An Additional Six Genes Involved in the Production of Infectious Budded Virus

Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.     

Genome-based phylogeny of dsDNA viruses by a novel alignment-free method

Sequence alignment is not directly applicable to whole genome phylogeny since several events such as rearrangements make full length alignments impossible. Here, a novel alignment-free method derived from the standpoint of information theory is proposed and used to construct the whole-genome phylogeny for a population of viruses from 13 viral families comprising 218 dsDNA viruses. The method is based on information correlation (IC) and partial information correlation (PIC). We observe that (i) the IC-PIC tree segregates the population into clades, the membership of each is remarkably consistent with biologist's systematics only with little exceptions; (ii) the IC-PIC tree reveals potential evolutionary relationships among some viral families; and (iii) the IC-PIC tree predicts the taxonomic positions of certain “unclassified” viruses. Our approach provides a new way for recovering the phylogeny of viruses, and has practical applications in developing alignment-free methods for sequence classification.     

肌动蛋白在AcMNPV向细胞外运输过程中作用的初步研究

本实验利用AcMNPV(Autographa californica multiple nuclear polyhedrosis virus,AcMNPV)的bac-to-bac系统构建了两种重组病毒,即含GFP-actin融合基因的重组病毒AcMNPV-GFP-actin和含GFP基因的重组病毒AcMNPV-GFP。用这两种重组病毒分别感染Sf9细胞,以AcMNPV-GFP感染的Sf9细胞为对照,用共聚焦激光扫描显微镜观察了绿色荧光在病毒感染过程中的分布情况。由于肌动蛋白和绿色荧光蛋白是共定位的,所以绿色荧光的分布情况就是肌动蛋白的分布情况。实验中观察发现,AcMNPV-GFP感染的Sf9细胞中的绿色荧光,在整个感染过程中是弥散分布的,而AcMNPV-GFP-actin感染Sf9细胞后24172h这段时间内,肌动蛋白最初聚集在细胞核内,随后逐渐由细胞核向细胞质转移,最后完全聚集于细胞膜。根据实验结果,推测肌动蛋白可能参与了AcMNPV出芽型病毒粒子(BV)由细胞核向细胞质运输以及从细胞膜排出的过程。     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.