Peitang Huang

Highly pathogenic avian influenza A virus H5N1 NS1 protein induces caspase-dependent apoptosis in human alveolar basal epithelial cells.

BackgroundIt is widely considered that the multifunctional NS1 protein of influenza A viruses contributes significantly disease pathogenesis by modulating a number of virus and host-cell processes, but it is highly controversial whether this non-structural protein is a proapoptotic or antiapoptotic factor in infected cells.ResultsNS1 protein of influenza A/chicken/Jilin/2003 virus, a highly pathogenic H5N1 strain, could induce apoptosis in the carcinomic human alveolar basal epithelial cells (A549) by electron microscopic and flow cytometric analyses. NS1 protein-triggered apoptosis in A549 cells is via caspase-dependent pathway.ConclusionsInfluenza A virus NS1 protein serves as a strong inducer of apoptosis in infected human respiratory epithelial cells and plays a critical role in disease pathogenesis.

The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: Implication for virus-induced apoptosis

BackgroundOur previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown.ResultsTo characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3.ConclusionsThe present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

UBE2W interacts with FANCL and regulates the monoubiquitination of fanconi anemia protein FANCD2

Fanconi anemia (FA) is a rare cancer-predisposing genetic disease mostly caused by improper regulation of the monoubiquitination of Fanconi anemia complementation group D2 (FANCD2). Genetic studies have indicated that ubiquitin conjugating enzyme UBE2T and HHR6 could regulate FANCD2 monoubiquitination through distinct mechanisms. However, the exact regulation mechanisms of FANCD2 monoubiquitination in response to different DNA damages remain unclear. Here we report that UBE2W, a new ubiquitin conjugating enzyme, could regulate FANCD2 monoubiquitination by mechanisms different from UBE2T or HHR6. Indeed, UBE2W exhibits ubiquitin conjugating enzyme activity and catalyzes the monoubiquitination of PHD domain of Fanconi anemia complementation group L (FANCL) in vitro. UBE2W binds to FANCL, and the PHD domain is both necessary and sufficient for this interaction in mammalian cells. In addition, over-expression of UBE2W in cells promotes the monoubiquitination of FANCD2 and down-regulated UBE2W markedly reduces the UV irradiation-induced but not MMC-induced FANCD2 monoubiquitination. These results indicate that UBE2W regulates FANCD2 monoubiquitination by mechanisms different from UBE2T and HRR6. It may provide an additional regulatory step in the activation of the FA pathway.

The interaction between the PARP10 protein and the NS1 protein of H5N1 AIV and its effect on virus replication.

BackgroundDuring the process that AIV infect hosts, the NS1 protein can act on hosts, change corresponding signal pathways, promote the translation of virus proteins and result in virus replication.ResultsIn our study, we found that PARP domain and Glu-rich region of PARP10 interacted with NS1, and the presence of NS1 could induce PARP10 migrate from cytoplasm to nucleus. NS1 high expression could reduce the endogenous PARP10 expression. Cell cycle analysis showed that with inhibited PARP10 expression, NS1 could induce cell arrest in G2-M stage, and the percentage of cells in G2-M stage rise from the previous 10%-45%, consistent with the cell proliferation result. Plague forming unit measurement showed that inhibited PARP10 expression could help virus replication.ConclusionsIn a word, our results showed that NS1 acts on host cells and PARP10 plays a regulating role in virus replication.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells.

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the −486/−214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The −486/−214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Interaction of influenza virus NS1 protein with growth arrest-specific protein 8

NS1 protein is the only non-structural protein encoded by the influenza A virus, and it contributes significantly to disease pathogenesis by modulating many virus and host cell processes. A two-hybrid screen for proteins that interact with NS1 from influenza A yielded growth arrest-specific protein 8. Gas8 associated with NS1 in vitro and in vivo. Deletion analysis revealed that the N-terminal 260 amino acids of Gas8 were able to interact with NS1, and neither the RNA-binding domain nor the effector domain of NS1 was sufficient for the NS1 interaction. We also found that actin, myosin, and drebrin interact with Gas8. NS1 and β-actin proteins could be co-immunoprecipitated from extracts of transfected cells. Furthermore, actin and Gas8 co-localized at the plasma membrane. These results are discussed in relation to the possible functions of Gas8 protein and their relevance in influenza virus release.

Cloning and functional analysis of 5′‐upstream region of the Pokemon gene

Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5′‐upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG‐U and NEG‐D elements were involved in negative regulation of the Pokemon promoter, whereas the POS‐D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG‐U, NEG‐D and POS‐D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG‐U and NEG‐D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG‐U and NEG‐D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.

Fanconi anemia and ubiquitination.

Fanconi anemia (FA) is a rare recessive hereditary disease characterized clinically by congenital defects, progressive bone-marrow failure, and cancer predisposition. Cells from FA patients exhibit hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). To date, at least 12 FA genes have been found deleted or mutated in FA cells, and 10 FA gene products form a core complex involved in FA/BRCA2 DNA repair pathway?FA pathway. The ubiquitin E3 ligase FANCL, an important factor of FA core complex, co-functions with a new ubiquitin conjugating enzyme UBE2T to catalyze the monoubiquitination of FANCD2. FANCD2-Ub binds BRCA2 to form a new complex located in chromatin foci and then take part in DNA repair process. The deubiquitylating enzyme USP1 removes the mono-ubiquitin from FANCD2-Ub following completion of the repair process, then restores the blocked cell cycle to normal order by shutting off the FA pathway. In a word, the FANCD2 activity adjusted exquisitely by ubiquitination and/or deubiquitination in vivo may co-regulate the FA pathway involving in variant DNA repair pathway.

Creation of a Six-fingered Artificial Transcription Factor That Represses the Hepatitis B Virus HBx Gene Integrated into a Human Hepatocellular Carcinoma Cell Line

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection–related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection–related HCC.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.