ABSTRACT
The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells.Bovine Herpesvirus 1(BHV-1),which is a viral pathogen of cattle,can infect FBL cells and induce cytopathic effects.Real-time PCR assays showed that BHV- 1 's infection could repress the basal or inducible transcription of bISG15 in FBL cells.It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis.Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG 15 in FBL cells,so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed.The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3.Taken together,our work suggested that BHV-I had some molecular mechanism to resist the cellular bISG15'santiviral functions.
ABSTRACT
In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.
ABSTRACT
Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Subject(s)
Animals , Cattle , Humans , Catalytic Domain , Genetics , DNA , Genetics , DNA, Viral , HIV-1 , Genetics , Metabolism , Immunodeficiency Virus, Bovine , Genetics , Integrases , Chemistry , Genetics , MetabolismABSTRACT
Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.
ABSTRACT
In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.
ABSTRACT
Objective To develop prototype foamy virus (PFV) detection method by loop-mediated isothermal amplification. Methods Three pairs of primers targeting core region of PFV integrase were designed in this study and Bst DNA polymerase was used to amplify target sequence at 63℃. The system was established with all the conditions optimized. Results The method was established with the plasmid containing target sequence as the template. This method could specifically detect PFV infectious clone, no crossreaction was observed with human immunodeficieney virus infectious clone, bovine immunodefieiency virus infectious clone and bovine foamy virus infectious clone as templates. The detection capability of this system was 50 copy, one order more sensitive than PCR. The amplification could be finished in 15 min and human genomic DNA did not adversely affect the amplification efficiency. Conclusion The PFV detection method by loop-mediated isothermal amplification was established and it had potential usefulness in PFV detection.
ABSTRACT
Besides its function as a pathogenicity determinant, the Tombusvirus P19 also serves as a suppressor of RNA interference (RNAi) by sequestering intracellular small RNAs such as the small interfering RNAs (siRNAs) and microRNAs (miRNAs). However, the effect of P19 on mammalian cells has not been evaluated before. A human embryonic kidney 293 cell line that stably expressed p19 (HEK293-p19) was generated. Flow cytometric analysis revealed that over-expression of P19 caused a significant accumulation of G2/M phase cells. Cell proliferation assays demonstrated a reduced DNA replication and cell growth in HEK293-p19 cells. Moreover, p19 altered the expression profiles of a number of cell cycle regulators in HEK293 cells, such as upregulafion of cyclin A1, CDK2, CDK4, CDK6, p18, cyclin D2, p19INK4d and E2F1, and downregulation of p15, cyclin A2, cyclin B1 and cyclin E1. Thus, the data strongly indicate that p19 might influence multiple G2/M regulators to cause G2/M arrest.
ABSTRACT
ISG15, the first ubiquitin-like molecule identified two decades ago, is encoded by interferon stimulated gene 15 ( ISG 15), where its robust expression can be induced by viral infections or interferon treatments. ISG 15 conjugate to other proteins as the ubiquitin and was found to be involved in innate immune response. However, the functions of ISG15 modification remained unclear. We cloned bovine ISG15(bisg15) into a prokaryotic expression vector pET28a( + ) with a His-tag to generate a soluble form of bISG15 fusion protein, and purified with Ni-NTA Sepharose chromatography. The purified protein was concentrated and used to immune Balb/c mice to raise the antiserum, which could specifically recognize bISG15 expressed in eukaryotic cells by Western blot analysis. The concentrated bISG15 protein and its antiserum were then used to establish an in vitro bISG15 modification system. Our studies have demonstrated that cellular proteins could be conjugated to bISG15 with this system.
ABSTRACT
The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.
ABSTRACT
Virus infection or interferon can stimulate robust expression of the protein ISG15 that is encoded by interferon stimulated gene 15, which was the first unbiquitin-like molecule identified two decades ago. While ubiquitin and its many important functions have been well established, the functions of ISG15 and its post-translational conjugation are still largely unknown . Recently, some specific enzymes have been identified to be involved in the ISG15 modification system, suggests that ISG15 and its modification system play important roles in the innate immune response and regulation of interferon signaling. The history of ISG15 discovery and its biochemical characterization were briefly introduced. Then such topics as the ISG15 gene expression and the ISG15 modification will be focued on, and finally summarize new findings which have implications for ISG15 and its modification system in immunology and interferon signal transduction were summarized.
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Objective To study the chemical constituents from the exocarp of Juglans mandshurica.Methods The compounds were isolated by Diaion HP-20,Sephadex LH-20,MCI CHP-20,and silica gel column chromatography.Their structures were identified by physicochemical properties and spectroscopic analysis.Results Twelve compounds were elucidated as pinostrobin (Ⅰ),quercetin (Ⅱ),quercetin-3-O-?-D-glucoside (Ⅲ),chrysophanol (Ⅳ),gallic acid (Ⅴ),4-hydroxyl-cinnamic acid methyl ester (Ⅵ),vanillin (Ⅶ),caffeic acid (Ⅷ),4-hydroxylcinnamic acid (Ⅸ),?-sitosterol (Ⅹ),daucosterol (Ⅺ),and 5-hydroxyl-1,4-naphthoquinone (ⅩⅡ).Conclusion The compounds Ⅰ,Ⅳ,and Ⅵ—Ⅸ are isolated from J.mandshurica for the first time.