Xuefei Cai

Enhancement of canonical Wnt/β-catenin signaling activity by HCV core protein promotes cell growth of hepatocellular carcinoma cells.

Background The Hepatitis C virus (HCV) core protein has been implicated as a potential oncogene or a cofactor in HCV-related hepatocellular carcinoma (HCC), but the underlying mechanisms are unknown. Overactivation of the Wnt/β-catenin signaling is a major factor in oncogenesis of HCC. However, the pathogenesis of HCV core-associated Wnt/β-catenin activation remains to be further characterized. Therefore, we attempted to determine whether HCV core protein plays an important role in regulating Wnt/β-catenin signaling in HCC cells. Methodology Wnt/β-catenin signaling activity was investigated in core-expressing hepatoma cells. Protein and gene expression were examined by Western blot, immunofluorescence staining, RT-qPCR, and reporter assay. Principal Findings HCV core protein significantly enhances Tcf-dependent transcriptional activity induced by Wnt3A in HCC cell lines. Additionally, core protein increases and stabilizes β-catenin levels in hepatoma cell line Huh7 through inactivation of GSK-3β, which contributes to the up-regulation of downstream target genes, such as c-Myc, cyclin D1, WISP2 and CTGF. Also, core protein increases cell proliferation rate and promotes Wnt3A-induced tumor growth in the xenograft tumor model of human HCC. Conclusions/Significance HCV core protein enhances Wnt/β-catenin signaling activity, hence playing an important role in HCV-associated carcinogenesis.

Sirtuin 1 Regulates Hepatitis B Virus Transcription and Replication by Targeting Transcription Factor AP-1

ABSTRACT Chronic hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma. Nevertheless, the molecular mechanism of HBV replication remains elusive. SIRT1 is a class III histone deacetylase that is a structure component of the HBV cccDNA minichromosome. In this study, we found by using microarray-based gene expression profiling analysis that SIRT1 was upregulated in HBV-expressing cells. Gene silencing of SIRT1 significantly inhibited HBV DNA replicative intermediates, 3.5-kb mRNA, and core protein levels. In contrast, the overexpression of SIRT1 augmented HBV replication. Furthermore, SIRT1 enhanced the activity of HBV core promoter by targeting transcription factor AP-1. The c-Jun subunit of AP-1 was bound to the HBV core promoter region, as demonstrated by using a chromatin immunoprecipitation assay. Mutation of AP-1 binding site or knockdown of AP-1 abolished the effect of SIRT1 on HBV replication. Finally, SIRT1 inhibitor sirtinol also suppressed the HBV DNA replicative intermediate, as well as 3.5-kb mRNA. Our study identified a novel host factor, SIRT1, which may facilitate HBV replication in hepatocytes. These data suggest a rationale for the use of SIRT1 inhibitor in the treatment of HBV infection.

HCV core protein interacts with Dicer to antagonize RNA silencing.

RNA silencing is a form of nucleic acid-based immunity against viruses in plants and invertebrate animals. Successful viral infection requires evasion or suppression of gene silencing. Here, we report that the core protein of Hepatitis C virus (HCV) acts as a potent suppressor of RNA silencing (SRS). We have found that the HCV core protein inhibits RNA silencing induced by short hairpin RNAs (shRNAs) but not by synthetic small interfering RNAs (siRNAs) in various mammalian cells. We have further demonstrated that HCV core protein directly interacts with Dicer, an RNase enzyme that generates siRNA in host cells. The HCV core protein has been shown to inhibit the function of Dicer to process double-stranded RNAs (dsRNAs) into siRNAs. Through deletion analysis, we have found that the N-terminal domain is required for core protein to antagonize RNA silencing activity of Dicer enzyme. Thus, our results suggest that HCV core protein may abrogate host cell RNA silencing defense by suppressing the ability of Dicer to process precursor dsRNAs into siRNAs. This anti-Dicer ability of core protein may contribute to the persistent viral infection and pathogenesis of HCV.

Hepatitis B virus protein up-regulated HLJ1 expression via the transcription factor YY1 in human hepatocarcinoma cells.

The hepatitis B virus (HBV) protein plays a major role in hepatocellular carcinoma (HCC) development. However, its contribution to tumor invasion and metastasis has not been established so far. HLJ1 was recently cloned and classified as a member of the heat shock protein 40 family (Hsp40/DnaJ) which is abundantly expressed in HBV-related tumors, might be involved in tumor progression. In this study, the role of HBV in activation of HLJ1 was investigated. In HepG2 cells with transit or stable expression of HBV, HLJ1 expression was activated by HBV. The activity assay of HLJ1 promoter revealed that HBV up-regulated HLJ1 expression through the transcription factor YY1 sites within the HLJ1 promoter. YY1 expression was significantly up-regulated by HBV in a concentration-dependent manner. Knockdown of YY1 expression could partially reduce the HBV-induced HLJ1 activation which indicated that YY1 would be involved in HBV-induced HLJ1 expression. In conclusion, our data showed that HBV could promote HLJ1 expression by up-regulating the transcription factor YY1, and this provided a new insight of the mechanism of HBV induction in tumor metastasis.

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

HBx mutations promote hepatoma cell migration through the Wnt/β-catenin signaling pathway.

HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β‐catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β‐catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV‐associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/β‐catenin signaling in HBx‐transfected HCC cells and HBV‐related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP‐luc activity and increased nuclear translocation of β‐catenin. Moreover, the HBx Combo mutant increased and stabilized β‐catenin levels through inactivation of glycogen synthase kinase‐3β, resulting in upregulation of downstream target genes such as c‐Myc, CTGF, and WISP2. Enhanced activation of Wnt/β‐catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β‐catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV‐associated hepatocarcinogenesis.

Structural Basis of the Novel S. pneumoniae Virulence Factor, GHIP, a Glycosyl Hydrolase 25 Participating in Host-Cell Invasion

Pathogenic bacteria produce a wide variety of virulence factors that are considered to be potential antibiotic targets. In this study, we report the crystal structure of a novel S. pneumoniae virulence factor, GHIP, which is a streptococcus-specific glycosyl hydrolase. This novel structure exhibits an α/β-barrel fold that slightly differs from other characterized hydrolases. The GHIP active site, located at the negatively charged groove in the barrel, is very similar to the active site in known peptidoglycan hydrolases. Functionally, GHIP exhibited weak enzymatic activity to hydrolyze the PNP-(GlcNAc)5 peptidoglycan by the general acid/base catalytic mechanism. Animal experiments demonstrated a marked attenuation of S. pneumoniae-mediated virulence in mice infected by ΔGHIP-deficient strains, suggesting that GHIP functions as a novel S. pneumoniae virulence factor. Furthermore, GHIP participates in allowing S. pneumoniae to colonize the nasopharynx and invade host epithelial cells. Taken together, these findings suggest that GHIP can potentially serve as an antibiotic target to effectively treat streptococcus-mediated infection.

Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy

Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long‐term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication‐competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion‐and‐ligation techniques to generate replication‐competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the “fragment substitution reaction” (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro. J. Med. Virol. 84:34–43, 2011.

Crystal structure of a novel dimer form of FlgD from P. aeruginosa PAO1

Crystal structure of a novel dimer form of FlgD from P. aeruginosa PAO1 Hua Zhou,1,2y Miao Luo,1,2y Xuefei Cai, Jian Tang, Siqiang Niu, Wenlu Zhang, Yuan Hu, Yibing Yin, Ailong Huang, Dacheng Wang, and Deqiang Wang* 1 Key Laboratory of Molecular Biology on Infectious Disease, Chongqing Medical University, YiXueYuanlu-1, Chongqing 400016, China 2 Key Laboratory of Diagnostic Medicine, Department of Laboratory Medicine, Chongqing Medical University, YiXueYuanlu-1, Chongqing 400016, China 3National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China

APOBEC3B edits HBV DNA and inhibits HBV replication during reverse transcription.

ABSTRACT Hepatitis B virus is a partially double‐stranded DNA virus that replicates by reverse transcription, which occurs within viral core particles in the cytoplasm. The cytidine deaminase APOBEC3B is a cellular restriction factor for HBV. Recently, it was reported that APOBEC3B can edit HBV cccDNA in the nucleus, causing its degradation. However, whether and how it can edit HBV core‐associated DNAs during reverse transcription is unclear. Our studies to address this question revealed the following: First, silencing endogenous APOBEC3B in an HBV infection system lead to upregulation of HBV replication. Second, APOBEC3B can inhibit replication of HBV isolates from genotypes (gt) A, B, C, and D as determined by employing transfection of plasmids expressing isolates from four different HBV genotypes. For HBV inhibition, APOBEC3B‐mediated inhibition of replication primarily depends on the C‐terminal active site of APOBEC3B. In addition, employing the HBV RNaseH‐deficient D702A mutant and a polymerase‐deficient YMHA mutant, we demonstrated that APOBEC3B can edit both the HBV minus‐ and plus‐strand DNAs, but not the pregenomic RNA in core particles. Furthermore, we found by co‐immunoprecipitation assays that APOBEC3B can interact with HBV core protein in an RNA‐dependent manner. Our results provide evidence that APOBEC3B can interact with HBV core protein and edit HBV DNAs during reverse transcription. These data suggest that APOBEC3B exerts multifaceted antiviral effects against HBV. HighlightsThe C‐terminal domain of APOBEC3B plays an important role in its deaminase‐dependent antiviral function.APOBEC3B can edit both the HBV minus‐ and plus‐ strand DNAs, but not the pregenomic RNA during reverse transcription.The cytoplasmic fraction of APOBEC3B can reduce HBV DNA levels dependent on its deamination.APOBEC3B can interact with HBV core protein.