Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

Identification and analysis of an alcelaphine herpesvirus 1 (AHV-1) cDNA clone expressing a fusion protein recognized by AHV-1-neutralizing antisera

Summary Rabbit antiserum to psoralen-inactivated alcelaphine herpesvirus 1 (AHV-1) virions was shown to react specifically with AHV-1-infected cells by indirect immunofluorescence. Western blot analysis using this antiserum identified a 15-kD virion protein that was also detected in infected-cell proteins between 12 and 144 h p.i., and a 37-kD protein present in infected cells between 24 and 120 h p.i. A cDNA library was constructed using mRNA obtained from AHV-1-infected fetal mouflon sheep kidney (FMSK) cells at 48 h p.i., when infected-cell proteins detected by antiserum were in abundance. Screening of the library with the rabbit anti-AHV-1 serum identified several positive clones. Southern blot analysis showed that one clone, designated 8'a, hybridized to a 4.4kbHindIII fragment of AHV-1 DNA. This AHV-1 cDNA clone expressed a fusion protein that was recognized by serum from a naturally and asymptomatically infected white-bearded wildebeest (Connochaetes taurinus albojubatus). The insert was sequenced and found to contain 833 bp. A search of the GenBank database for related sequences revealed greater than 40% homology to several other gammaherpesviruses: herpesvirus saimiri, cottontail herpesvirus, and Epstein-Barr virus.     

HearSNPV orf83 encodes a late, nonstructural protein with an active chitin-binding domain

The ORF83 (ha83) of Helicoverpa armigera nucleopolyhedrovirus (HearSNPV) was characterized during the present study. Sequence analysis and chitin-binding assay revealed that Ha83 contained an active chitin-binding domain. Northern blot and Western blot analyses demonstrated that ha83 was expressed as a late gene and encoded a nonstructural protein of HearSNPV. Ha83 gene was transcribed beginning at 12h post-infection in infected Helicoverpa zea cells (HzAM1). Western blot analysis using a rabbit derived polyclonal antibody showed the product of ha83 in infected cells was a 20 kDa protein, in tune with the theoretical size of 18.8 kDa. The protein was first detected in the cytoplasm of infected HzAM1 cells at 12h p.i., and was transported later into the nucleus during infection.     

Biosynthesis and localization of the Autographa californica nuclear polyhedrosis virus 25K gene product

Mutations of the AcMNPV 25K gene are associated with the "few polyhedra" phenotype (M. J. Fraser et al., 1983, J. Virol. 47, 287-300; B. Beames and M. D. Summers, 1989, Virology 168, 344-353). Polyclonal antisera was produced and used to investigate the time course of expression and localization of the 25K protein in infected cells. Western blot analysis detected 25K protein in both cytosolic and nuclear extracts from 18-24 hr p.i. through 96 hr p.i. and also in purified viral occlusions, but not in purified virions. Immunogold electron microscopy revealed that 25K protein was predominantly associated with amorphous cytoplasmic structures and to a lesser extent with a more electron-dense structure in the nucleus. Viral occlusions in cell sections were not specifically labeled by 25K antibody. Observations of purified viral occlusions and nuclei prepared for immunogold EM revealed the presence of contaminating amorphous material that was labeled with 25K antibody.     

Identification and characterization of odv-e25 of Spodoptera litura multicapsid nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) odv-e25 is 684 bp long, potentially encoding 227 amino acids with a predicted molecular weight of 24.9 kDa. Homology analysis indicated that SpltMNPV ODV-E25 has 35–65% amino acid identity with that of other known baculoviruses. RT-PCR results revealed that the odv-e25 is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (TTAAG). Western blot analysis of odv-e25 expression with an antiserum made against 6 × His tagged ODV-E25 expressed in Escherichia coli indicated that it was present as a doublet of approximately 27 kDa from 24 h through 96 h in SpltMNPV-infected Spli-221 cells. Similar results were seen on Western blots of Spodoptera exigua (Se)MNPV-infected Se301 cells. Immunofluorescence analysis showed that ODV-E25 was predominantly present in the cytoplasm of SpltMNPV-infected cells and localized to the envelopes of occlusion-derived virus.     

基于HepG2细胞胰岛素抵抗模型探讨miR-7-5p对Itch基因的靶向作用

目的:通过体外建立Hep G2细胞胰岛素抵抗模型,检测胰岛素抵抗状态下微小RNA-7-5p(miR-7-5p)及其预测靶基因Itch的差异表达,初步探讨miR-7-5p对Itch基因的靶向作用及其与胰岛素抵抗的关系。方法:采用适宜浓度的软脂酸诱导Hep G2细胞,建立体外胰岛素抵抗模型;基于生物信息学分析预测miR-7-5p可能作用的靶基因及其富集的相关信号通路;运用RT-q PCR和Western blot技术检测在胰岛素抵抗状态下miR-7-5p和Itch的表达变化。结果:0.25 mmol/L的软脂酸作用于Hep G2细胞24 h可诱导细胞产生胰岛素抵抗,RT-q PCR检测表明,与阴性对照组相比,胰岛素抵抗组的miR-7-5p表达下调(P0.01)。生物信息学分析结果表明,miR-7-5p有相当数量的靶基因富集于泛素-蛋白酶体系统,其中E3泛素连接酶Itch基因是与胰岛素抵抗最为相关的靶基因;Western blot结果揭示,在胰岛素抵抗状态下,Hep G2细胞中Itch蛋白表达上调(P0.01)。结论:miR-7-5p可能参与了胰岛素抵抗的病理生理过程,其机制可能通过靶向调控Itch基因的表达,进而直接或间接影响胰岛素信号通路的正常转导。