Hepatitis C virus core protein recruits nucleolar phosphoprotein B23 and coactivator p300 to relieve the repression effect of transcriptional factor YY1 on B23 gene expression.

Hepatitis C virus (HCV) core has a pleiotropic effect on various promoters. In this study, we found that the expression of nucleolar phosphoprotein B23 was enhanced in HCV core-expressing cells and, moreover, HCV core interacts directly with the C-terminal end of B23. Using sucrose gradient centrifugation analysis and immunoprecipitation assays, HCV core was found in a large complex containing B23 and its interacting partner transcription factor YY1. Both B23 and HCV core associated with YY1 in the central GA/GK-rich and C-terminal zinc finger domain. These physical interactions between core, B23, and YY1 led to ternary complex formation that was bound to the YY1 response element. In a transient cotransfection experiment, relief of the trans-suppression activity of YY1 on the YY1-response element-driven reporter by core and B23 was found. This is also true when examining the effects of these three constructs on the B23 promoter-driven reporter. Additionally, chromatin immunoprecipitation assays indicated that a transcriptional activation complex consisting of core, together with B23, p300, and YY1, was recruited to the YY1 response element of B23 promoter, and this probably occurred through complex formation between core and these three cellular transcription regulators. This is different from the situation in the absence of core, where YY1 and histone deacetylase 1, but not B23 and p300, were associated on the YY1 element as the transcription repression complex. Together, our results indicate that HCV core can recruit B23 and p300 to relieve the repression effect of YY1 on B23 promoter activity, a property that requires the intrinsic histone acetyltransferase activity of p300. Thus, because these three core-associated cellular transcription regulators have a multitude of cellular interacting proteins and are involved in a versatility of cellular processes, the complex formation described here may partially account for the pleiotropic effects of core protein on gene expression and cellular function in HCV-infected cells.

DDX3, a DEAD box RNA helicase, is deregulated in hepatitis virus-associated hepatocellular carcinoma and is involved in cell growth control.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide and is highly correlated with hepatitis virus infection. Our previous report shows that a DEAD box RNA helicase, DDX3, is targeted and regulated by hepatitis C virus (HCV) core protein, which implicates the involvement of DDX3 in HCV-related HCC development. In this study, the potential role of DDX3 in hepatocarcinogenesis is investigated by examining its expression in surgically excised human HCC specimens. Here we report the differential deregulation of DDX3 expression in hepatitis virus-associated HCC. A significant downregulation of DDX3 expression is found in HCCs from hepatitis B virus (HBV)-positive patients, but not from HCV-positive ones, compared to the corresponding nontumor tissues. The expression of DDX3 is differentially regulated by the gender and, moreover, there is a tendency that the downregulation of DDX3 expression in HCCs is more frequent in males than in females. Genetic knockdown of DDX3 with small interfering RNAs (siRNA) in a nontransformed mouse fibroblast cell line, NIH-3T3, results in a premature entry to S phase and an enhancement of cell growth. This enhanced cell cycle progression is linked to the upregulation of cyclin D1 and the downregulation of p21(WAF1) in the DDX3 knockdown cells. In addition, constitutive reduction of DDX3 expression increases the resistance of NIH-3T3 cells to serum depletion-induced apoptosis and enhances the ras-induced anchorage-independent growth, indicating the involvement of DDX3 in cell growth control. These findings together with the previous study suggest that the deregulation of DDX3, a DEAD box RNA helicase with cell growth-regulatory functions, is involved in HBV- and HCV-associated pathogenesis.

YB-1 disrupts mismatch repair complex formation, interferes with MutSα recruitment on mismatch and inhibits mismatch repair through interacting with PCNA.

Y-box binding protein-1 (YB-1) is highly expressed in tumors and it participates in various cellular processes. Previous studies indicated that YB-1 binds to mispaired DNA and interacts with several mismatch repair (MMR)-related factors. However, its role in the MMR system remains undefined. Here, we found that YB-1 represses mutS homolog 6 (MSH6)-containing MMR complex formation and reduces MutSα mismatch binding activity by disrupting interactions among MMR-related factors. In an effort to elucidate how YB-1 exerts this inhibitory effect, we have identified two functional proliferating cell nuclear antigen (PCNA)-interacting protein (PIP)-boxes that mediate YB-1/PCNA interaction and locate within the C-terminal region of YB-1. This interaction is critical for the regulatory role of YB-1 in repressing MutSα mismatch binding activity, disrupting MutSα/PCNA/G/T heteroduplex ternary complex formation and inhibiting in vitro MMR activity. The differential regulation of 3' and 5' nick-directed MMR activity by YB-1 was also observed. Moreover, YB-1 overexpression is associated with the alteration of microsatellite pattern and the enhancement of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced and spontaneous mutations. Furthermore, upregulation of other PIP-box-containing proteins, such as myeloid cell leukemia-1 (Mcl-1) and inhibitor of growth protein 1b (ING1b), has no impact on MMR complex formation and mutation accumulation, thus revealing the significant effect of YB-1 on regulating the MMR system. In conclusion, our study suggests that YB-1 functions as a PCNA-interacting factor to exert its regulatory role on the MMR process and involves in the induction of genome instability, which may partially account for the oncogenic potential of YB-1.

Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting as an eIF4E inhibitory protein.

DDX3 is a human RNA helicase with plethoric functions. Our previous studies have indicated that DDX3 is a transcriptional regulator and functions as a tumor suppressor. In this study, we use a bicistronic reporter to demonstrate that DDX3 specifically represses cap-dependent translation but enhances hepatitis C virus internal ribosome entry site-mediated translation in vivo in a helicase activity-independent manner. To elucidate how DDX3 modulates translation, we identified translation initiation factor eukaryotic initiation factor 4E (eIF4E) as a DDX3-binding partner. Interestingly, DDX3 utilizes a consensus eIF4E-binding sequence YIPPHLR to interact with the functionally important dorsal surface of eIF4E in a similar manner to other eIF4E-binding proteins. Furthermore, cap affinity chromatography analysis suggests that DDX3 traps eIF4E in a translationally inactive complex by blocking interaction with eIF4G. Point mutations within the consensus eIF4E-binding motif in DDX3 impair its ability to bind eIF4E and result in a loss of DDX3's regulatory effects on translation. All these features together indicate that DDX3 is a new member of the eIF4E inhibitory proteins involved in translation initiation regulation. Most importantly, this DDX3-mediated translation regulation also confers the tumor suppressor function on DDX3. Altogether, this study demonstrates regulatory roles and action mechanisms for DDX3 in translation, cell growth and likely viral replication.

Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF.

Several members of the tumour necrosis factor receptor (TNF-R) superfamily can induce cell death. For TNF-R1, Fas/APO-1, DR3, DR6, TRAIL-R1 and TRAIL-R2, a conserved 'death domain' in the intracellular region couples these receptors to activation of caspases. However, it is not yet known how TNF receptor family members lacking a death domain, such as TNF-R2, CD40, LT-betaR, CD27 or CD30, execute their death-inducing capability. Here we demonstrate in different cellular systems that cytotoxic effects induced by TNF-R2, CD40 and CD30 are mediated by endogenous production of TNF and autotropic or paratropic activation of TNF-R1. In addition, stimulation of TNF-R2 and CD40 synergistically enhances TNF-R1-induced cytotoxicity. These findings describe a novel pro-apoptotic mechanism induced by some members of the TNF-R family.