Human immunodeficiency virus coinfection with hepatitis B virus leads to a decrease in extracellular and intracellular hepatitis B antigen.

Chronic hepatitis B virus (HBV) infection could cause severe liver disease including cirrhosis, hepatocellular carcinoma, and end-stage liver failure in HIV-positive individuals. The available data from clinical studies suggest that HIV infection modulates the HBV-specific T cell response. However, the virological and molecular aspects of HIV-HBV coinfection are currently poorly understood due to the lack of appropriate model systems. In this study, the effect of HIV infection on the life cycle of HBV was explored using an in vitro model system. The present data show that the extracellular and intracellular hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) decrease significantly in HepG2 cells cotransfected with HIV NL4-3 and pHBV1.3 as compared to those cells transfected only with pHBV1.3. Moreover, a significant decrease in HBV DNA and mRNA expression was also observed in the cotransfected cells. HIV Rev protein, an RNA-bound regulatory protein, could significantly decrease the expression levels of extracellular and intracellular HBsAg and HBeAg by mediating the expression of HBV mRNA in cells cotransfected with plasmids containing HIV-1 Rev and pHBV1.3. Further experiments demonstrate that HIV Rev manipulated neither the promoters of HBV nor the nuclear export of HBV mRNA. These results from the in vitro model system might provide clues to further understand the rapid progression of liver disease in HIV-HBV-coinfected patients.

MiR373 and miR542-5p regulate the replication of enterovirus 71 in rhabdomyosarcoma cells / 生物工程学报

MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we examined miRNAs effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of miRNAs screening system. 3'untranslated region (UTR) dual luciferase reporter analysis was used to identify putative miRNA targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then we screened the miRNAs that may target to 5'-UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. The study showed that miR373 and miR542-5p could suppress the replication of EV71 virus through binding to the 5'-UTR gene. Cellular miRNAs could regulate the replication of EV71 virus in host cells, and our paper should report the role of miR373 and miR542-5p in this regulation for the first time. Our findings supported the notion that the cellular miRNAs might be essential in the host-pathogen interactions.

Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

Enterovirus 71 (EV71) is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II) which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

High level expression of 5-helix protein in HIV gp41 heptad repeat regions and its virus fusion-inhibiting activity / 生物工程学报

The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines / 中国病毒学

The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA,and cloned into pMD 18-T vector respectively.The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced.32 BALB/c mice (6-week-old) were divided into four groups at random.Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 μg of plasmid pHM6-m,30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) respectively.A additional group of mice were injected with 100 μ1 PBS as controls.Two weeks later,all mice were challenged with homologous H5N1 avian influenza virus,and observed in the following 12 days.The survival rates of mice in the pHM6-m group,the pHM6-np group and mixed plasmids group were 62.5% ,25.0% and 50.0%,respectively.Results showed that effective protection could be provided by either pHM6-m or pHM6-np,but pHM6-m provided a better protective effect than pHM6-np.

An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain.

Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD). Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content beta-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

Cloning, expression and purification of the C-terminal section of murine heat shock protein gp96 / 生物工程学报

Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.

Expression and purification of heptad repeat region of the mumps virus F protein and analysis of characteristics / 生物工程学报

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.