Core Promoter Activity Research Articles

Solamargine acts as an antiviral by interacting to MZF1 and targeting the core promoter of the hepatitis B virus gene.

Hepatitis B virus (HBV) infection is still a serious threat to global health and can lead to a variety of liver diseases, including acute and chronic hepatitis, liver cirrhosis, liver failure, hepatocellular carcinoma (HCC), and so on. At present, there are mainly two kinds of drugs for the treatment of hepatitis B at home and abroad: interferon (IFN) and nucleoside/nucleotide analogs (NAs). In recent years, natural compounds have been considered an important source for the development of new anti-HBV drugs due to their complex structure, diverse components, high efficiency, and low toxicity. Many studies have demonstrated that Solamargine has significant anticancer activity, but the antiviral effect is rarely studied. This study aimed to verify the anti-HBV effect of Solamargine and to explore the specific mechanism. The relative expression of HBV pregenomic RNA (pgRNA) was detected by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR). Northern blot and western blot were used to detect the relative expression of HBV pgRNA and target protein. PCR was used in the construction of HBV pg-promoter, ENII/BCP, and a series of gene deletion mutant fluorescent reporter vectors. The fluorescence relative expression of each mutant was detected by Renilla luciferase assay. By binding to MZF1 (Myeloid zinc finger protein 1, MZF1), Solamargine inhibits HBV core promoter activity, reduces pregenomic RNA level, and inhibits HBV, achieving antiviral effects.

Paclitaxel-induced Immune Dysfunction and Activation of Transcription Factor AP-1 Facilitate Hepatitis B Virus Replication.

Hepatitis B virus (HBV) reactivation is commonly observed in individuals with chronic HBV infection undergoing antineoplastic drug therapy. Paclitaxel (PTX) treatment has been identified as a potential trigger for HBV reactivation. This study aimed to uncover the mechanisms of PTX-induced HBV reactivation in vitro and in vivo, which may inform new strategies for HBV antiviral treatment. The impact of PTX on HBV replication was assessed through various methods including enzyme-linked immunosorbent assay, dual-luciferase reporter assay, quantitative real-time PCR, chromatin immunoprecipitation, and immunohistochemical staining. Transcriptome sequencing and 16S rRNA sequencing were employed to assess alterations in the transcriptome and microbial diversity in PTX-treated HBV transgenic mice. PTX enhanced the levels of HBV 3.5-kb mRNA, HBV DNA, HBeAg, and HBsAg both in vitro and in vivo. PTX also promoted the activity of the HBV core promoter and transcription factor AP-1. Inhibition of AP-1 gene expression markedly suppressed PTX-induced HBV reactivation. Transcriptome sequencing revealed that PTX activated the immune-related signaling networks such as IL-17, NF-κB, and MAPK signaling pathways, with the pivotal common key molecule being AP-1. The 16S rRNA sequencing revealed that PTX induced dysbiosis of gut microbiota. PTX-induced HBV reactivation was likely a synergistic outcome of immune suppression and direct stimulation of HBV replication through the enhancement of HBV core promoter activity mediated by the transcription factor AP-1. These findings propose a novel molecular mechanism, underscoring the critical role of AP-1 in PTX-induced HBV reactivation.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

Oxidative stress sensor Keap1 recognizes HBx protein to activate the Nrf2/ARE signaling pathway, thereby inhibiting hepatitis B virus replication.

The Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is one of the most important defense mechanisms against oxidative stress. We previously reported that a cellular hydrogen peroxide scavenger protein, peroxiredoxin 1, a target gene of transcription factor Nrf2, acts as a novel HBV X protein (HBx)-interacting protein and negatively regulates hepatitis B virus (HBV) propagation through degradation of HBV RNA. This study further demonstrates that the Nrf2/ARE signaling pathway is activated during HBV infection, eventually leading to the suppression of HBV replication. We provide evidence suggesting that Keap1 interacts with HBx, leading to Nrf2 activation and inhibition of HBV replication via suppression of HBV core promoter activity. This study raises the possibility that activation of the Nrf2/ARE signaling pathway is a potential therapeutic strategy against HBV. Our findings may contribute to an improved understanding of the negative regulation of HBV replication by the antioxidant response.

TRIM56 impairs HBV infection and replication by inhibiting HBV core promoter activity

Members of the tripartite motif (TRIM) protein family strongly induced by interferons (IFNs) are parts of the innate immune system with antiviral activity. However, it is still unclear which TRIMs could play important roles in hepatitis B virus (HBV) inhibition. Here, we identified that TRIM56 expression responded in IFN-treated HepG2-NTCP cells and HBV-infected liver tissues, which was a potent IFN-inducible inhibitor of HBV replication. Mechanistically, TRIM56 suppressed HBV replication via its Ring and C-terminal domain. C-terminal domain was essential for TRIM56 translocating from cytoplasm to nucleus during HBV infection. Further analysis revealed that TRIM56's Ring domain targeted IκBα for ubiquitination. This modification induced phosphorylation of p65, which subsequently inhibited HBV core promoter activity, resulting in the inhibition of HBV replication. The p65 was found to be necessary for NF-κB signal pathway to inhibit HBV replication. We verified our findings using HepG2-NTCP and primary human hepatocytes. Our findings reveal that TRIM56 is a critical antiviral immune effector and exerts an anti-HBV activity via NF-κB signal pathway, which is essential for inhibiting transcription of HBV covalently closed circular DNA.

A 314-bp SINE insertion in the ZNF2 promoter region may act as a repressor related to regulation of fat deposition in pigs

Retrotransposons, a type of DNA fragment that can mobilize itself on genome, can generate genetic variations and develop for molecular markers based on the insertion polymorphism. Zinc finger proteins (ZNFs) are among the most abundant proteins in eukaryotic animals, and their functions are extraordinarily diverse and particularly important in gene regulation. In the current study, bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms (RIPs) in six ZNF genes (ZNF2, ZNF3, ZNF7, ZNF8, ZNF10 and ZNF12). Six RIPs in these ZNFs, including one short interspersed nuclear element (SINE) RIP in intron 1 and one long interspersed nuclear element 1 (L1) RIP in intron 3 of ZNF2, one SINE RIP in 5′ flanking region and one SINE RIP in intron 2 of ZNF3, one SINE RIP in 3′ UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12, were discovered and their presence was confirmed by PCR. The impact of the SINE RIP in the first intron of ZNF2, which is close to the core promoter of ZNF2, on the gene activity was investigated by dual-luciferase assay in three cell lines. Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly (P<0.01) in cervical cancer cells and porcine primary embryonic fibroblasts (HeLa and PEF), thus SINE may act as a repressor. This SINE RIP also significantly (P<0.05) affected the corrected back fat thickness in Yorkshire pigs. The corrected back fat thickness of individuals with SINE insertion in the first intron of ZNF2 was significantly (P<0.05) higher than that of individuals without SINE insertion. In summary, our data suggested that RIPs play important roles in the genetic variations of these ZNF genes and SINE RIP in the intron 1 of ZNF2 may provide a useful molecular marker for the screening of fat deposition in the pig breeding.

Forkhead O Transcription Factor 4 Restricts HBV Covalently Closed Circular DNA Transcription and HBV Replication through Genetic Downregulation of Hepatocyte Nuclear Factor 4 Alpha and Epigenetic Suppression of Covalently Closed Circular DNA via Interacting with Promyelocytic Leukemia Protein.

ABSTRACTNuclear located hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) remains the key obstacle to cure chronic hepatitis B (CHB). In our previous investigation, it was found that FoxO4 could inhibit HBV core promoter activity through downregulating the expression of HNF4α. However, the exact mechanisms whereby FoxO4 inhibits HBV replication, especially its effect on cccDNA, remain unclear. Here, our data further revealed that FoxO4 could effectively inhibit cccDNA mediated transcription and HBV replication without affecting cccDNA level. Mechanistic study showed that FoxO4 could cause epigenetic suppression of cccDNA. Although FoxO4-mediated downregulation of HNF4α contributed to inhibiting HBV core promoter activity, it had little effect on cccDNA epigenetic regulation. Further, it was found that FoxO4 could colocalize within promyelocytic leukemia protein (PML) nuclear bodies and interact with PML. Of note, PML was revealed to be critical for FoxO4-mediated inhibition of cccDNA epigenetic modification and of the following cccDNA transcription and HBV replication. Furthermore, FoxO4 was found to be downregulated in HBV-infected hepatocytes and human liver tissues, and it was negatively correlated with cccDNA transcriptional activity in CHB patients. Together, these findings highlight the role of FoxO4 in suppressing cccDNA transcription and HBV replication via genetic downregulation of HNF4α and epigenetic suppression of cccDNA through interacting with PML. Targeting FoxO4 may present as a new therapeutic strategy against chronic HBV infection.IMPORTANCE HBV cccDNA is a determining factor for viral persistence and the main obstacle for a cure of chronic hepatitis B. Strategies that target cccDNA directly are therefore of great importance in controlling persistent HBV infection. In present investigation, we found that FoxO4 could efficiently suppress cccDNA transcription and HBV replication without affecting the level of cccDNA itself. Further, our data revealed that FoxO4 might inhibit cccDNA function via a two-part mechanism: one is to epigenetically suppress cccDNA transcription via interacting with PML, and the other is to inhibit HBV core promoter activity via the genetic downregulation of HNF4α. Of note, HBV might dampen the expression of FoxO4 for its own persistent infection. We propose that manipulation of FoxO4 may present as a potential therapeutic strategy against chronic HBV infection.

Large‐scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it “computes” expression remains poorly understood. To dissect its function, we carried out a comprehensive structure–function analysis in Drosophila. First, we performed a genome‐wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters’ activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.

From the Editor’s desk…

From the Editor’s desk…

Vitamin D signaling inhibits HBV activity by directly targeting the HBV core promoter

Clinical and epidemiological studies support a role for vitamin D in suppressing hepatitis B virus (HBV). This antiviral role of vitamin D is widely attributed to vitamin D receptor (VDR)/retinoid X receptor–mediated regulation of host immunomodulatory genes through vitamin D response elements (VDREs) in their promoters. Here, we investigated the ability of calcitriol (1α,25-dihydroxyvitamin D3, metabolically activated vitamin D) to directly regulate HBV activity through this signaling pathway. We observed that calcitriol selectively inhibited only the HBV core promoter without affecting the HBV-PreS1, HBV-PreS2/S, or HBx promoters. We then identified a VDRE cluster in the HBV core promoter that is highly conserved across most HBV genotypes. Disruption of this VDRE cluster abrogated calcitriol-mediated suppression of the HBV core promoter. Furthermore, we showed that VDR interacts directly with the VDRE cluster in the HBV core promoter independent of retinoid X receptor. This demonstrates that calcitriol inhibits HBV core promoter activity through a noncanonical calcitriol-activated VDR pathway. Finally, we observed that calcitriol suppressed expression of the canonical HBV core promoter transcripts, pregenomic RNA, and precore RNA in multiple HBV cell culture models. In addition, calcitriol inhibited the secretion of hepatitis B “e” antigen and hepatitis B surface antigen (HBV-encoded proteins linked to poor disease prognosis), without affecting virion secretion. Our findings identify VDR as a novel regulator of HBV core promoter activity and also explain at least in part the correlation of vitamin D levels to HBV activity observed in clinical studies. Furthermore, this study has implications on the potential use of vitamin D along with anti-HBV therapies, and lays the groundwork for studies on vitamin D-mediated regulation of viruses through VDREs in virus promoters.

Core promoter activity contributes to chromatin-based regulation of internal cryptic promoters.

During RNA polymerase II (RNA Pol II) transcription, the chromatin structure undergoes dynamic changes, including opening and closing of the nucleosome to enhance transcription elongation and fidelity. These changes are mediated by transcription elongation factors, including Spt6, the FACT complex, and the Set2-Rpd3S HDAC pathway. These factors not only contribute to RNA Pol II elongation, reset the repressive chromatin structures after RNA Pol II has passed, thereby inhibiting aberrant transcription initiation from the internal cryptic promoters within gene bodies. Notably, the internal cryptic promoters of infrequently transcribed genes are sensitive to such chromatin-based regulation but those of hyperactive genes are not. To determine why, the weak core promoters of genes that generate cryptic transcripts in cells lacking transcription elongation factors (e.g. STE11) were replaced with those from more active genes. Interestingly, as core promoter activity increased, activation of internal cryptic promoter dropped. This associated with loss of active histone modifications at the internal cryptic promoter. Moreover, environmental changes and transcription elongation factor mutations that downregulated the core promoters of highly active genes concomitantly increased their cryptic transcription. We therefore propose that the chromatin-based regulation of internal cryptic promoters is mediated by core promoter strength as well as transcription elongation factors.

Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection

Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and invivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.

HBV Core Promoter Inhibition by Tubulin Polymerization Inhibitor (SRI-32007).

Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.

Nuclear receptor E75 is a transcription suppressor of the Litopenaeus vannamei small subunit hemocyanin gene

Hemocyanin is a respiratory protein that possesses multiple physiological and immunological functions in shrimp. However, the transcriptional regulation of the hemocyanin gene is still poorly understood. Here, the nuclear receptor E75 of Litopenaeus vannamei (LvE75) was identified as one of the transcriptional regulators that modulates the transcription of the small molecular weight hemocyanin gene of L. vannamei (LvHMCs) by inhibiting its core promoter activity in a Dual-luciferase assay. In silico analysis revealed that the core promoter (designated HsP3), which is located at +1517/+1849 bp of LvHMCs contained a putative E75 binding motif (“ACGGAAT”, spanning +1812/+1818 bp). Further, LvE75 was shown to inhibit the core promoter activity by direct binding. Importantly, in vivo silencing of LvE75 resulted in a significant upregulation in the mRNA and protein expression of LvHMCs gene. Taken together, our present results provide direct evidence that LvE75 is a transcriptional suppressor of the LvHMCs gene expression.

Steroid receptor coactivator 3 inhibits hepatitis B virus gene expression through activating Akt signaling to prevent HNF4\u03b1 nuclear translocation

BackgroundChronic hepatitis B virus (HBV) infection is one of the most serious global public health problems. The role of steroid receptor coactivator 3 (SRC-3) in HBV biosynthesis is unknown. The aim of this study is to investigate the function of SRC-3 in regulating HBV biosynthesis both in vitro and in vivo and to identify the underlying mechanism.ResultsIn this study, we found that knockdown of SRC-3 could increase the levels of HBV mRNA and HBV proteins HBsAg and HBeAg in human liver cancer cell line HepG2 transfected with pHBV1.3 plasmids. In contrast, enforced expression of SRC-3 in SRC-3-knockdown HepG2 cells reduced the levels of HBV mRNA and HBV proteins HBsAg and HBeAg. Knockdown of SRC-3 dampened the Akt signaling, which has been shown to play a negative role in HBV transcription. Ectopic expression of constitutively activated Akt impaired the enhancement of HBV transcription by SRC-3 knockdown, indicating that SRC-3 inhibits HBV transcription by enhancing Akt signaling. Both SRC-3 and constitutively activated Akt could inhibit hepatocyte nuclear factor 4α (HNF4α)-mediated upregulation of HBV core promoter activity by preventing HNF4α nuclear translocation. Consistent with the in vitro results, in an in vivo chronic HBV replication mouse model developed by hydrodynamic injection of pHBV1.3 plasmids into mouse tail vein, enforced expression of SRC-3 in mouse liver reduced the levels of HBV mRNA in the liver and HBV antigens in serum, whereas knockout of SRC-3 in mouse increased the levels of HBV mRNA in the liver and HBV antigens in the serum.ConclusionOur study suggests that SRC-3 inhibits HBV gene expression by activating Akt signaling to prevent HNF4α nuclear translocation.

FoxO4 inhibits HBV core promoter activity through ERK-mediated downregulation of HNF4α

Hepatitis B virus (HBV) infection remains a global health problem, causing nearly one million deaths annually. Forkhead box O (FoxO) transcription factors play important roles in modulating diverse physiological processes. Recent studies show that FoxOs are involved in antiviral responses. In present investigation, we found that HBV induced significant down-regulation of FoxO4 protein, while had little effect on the expression of FoxO1 and FoxO3. Further study showed that FoxO4 displayed inhibitory effect on HBV transcription and replication both in vitro and in vivo. Mechanistically, it was found that FoxO4 exerted its anti-HBV activity by targeting HBV core promoter. Further, FoxO4 was revealed to inhibit HBV core promoter activity via downregulating hepatocyte nuclear factor-4α (HNF4α), and ERK signaling was required for FoxO4-mediated suppression of HNF4α and HBV core promoter activity. Together, these data indicated that FoxO4 displayed anti-HBV activity by suppressing HNF4α expression via activation of ERK pathway, and targeting FoxO4 might present as a novel therapeutic strategy against HBV infection.

A 14-bp functional deletion within the CMTM2 gene is significantly associated with litter size in goat

CKLF-like MARVEL transmembrane domain-containing 2 (CMTM2) plays an important role in animal reproduction, and abnormal CMTM2 expression leads to spermatogenesis disorders. This study was conducted to assess the role of CMTM2 in goat reproduction by investigating an insertion/deletion (indel) variation and its effect on litter size, and to reveal its functional mechanism. A 14-base pair (bp) indel was found at position −861 to −848 nt of the CMTM2 promoter in 1131 female Shaanbei white cashmere goats. Association analyses revealed that the 14-bp indel in dams was significantly correlated with the size of the first litter (P = 0.013), with the 14-bp deletion significantly decreasing litter size. Moreover, litter size at first kidding was significantly correlated with genotype frequencies (P = 0.019). Expression of the goat CMTM2 gene was detected in testes and ovaries. In male, CMTM2 was increased after the initiation of meiosis in the developing testis. In female, CMTM2 expression was higher in ovary from multiple prolific goats at first kidding compared to non-prolific goats. Moreover, both in testis after initiation of meiosis and in ovary, the homozygous 14-bp inserted genotype II was associated with increased CMTM2 expression compared to the heterozygous genotype ID (P < 0.05). To further explore whether the 14-bp indel was located in the core promoter activity region of the CMTM2 promoter, the five recombinant plasmids pGL3-pro1 (−1756 to +83), pGL3-pro2 (−1200 to +83), pGL3-pro3 (−763 to +83), pGL3-pro4 (−496 to +83), and pGL3-pro5 (−205 to +83) were co-transfected with pRL-TK into HEK293T and GC-1 cells. A luciferase reporter assay showed that the pGL3-pro2 and pGL3-pro5 vectors produced significantly stronger luminescence than the other vectors. Interestingly, the 14-bp indel locus was included in the pGL3-pro2 plasmid, suggesting that the indel was functional. Subsequently, the two recombinant plasmids pGL3-pro2-inserted-allele and pGL3-pro2-deleted-allele were co-transfected with pRL-TK into HEK293T and GC-1 cells, respectively. The luciferase reporter assay demonstrated that the deleted allele of CMTM2 showed significantly lower luminescence activity than the inserted allele (P < 0.05), which corresponded to a decrease in litter size in the deletion-deletion genotype. Therefore, our results suggest that a 14-bp deletion in the promoter region of CMTM2 is significantly correlated with litter size in goats; this marker seems promising for breeding selection for improving economically important traits in goats.

Interleukin-35 stimulates hepatitis B virus transcription and replication by targeting transcription factor HNF4α.

Hepatitis B virus (HBV) infection is a major health problem worldwide. Interleukin-35 (IL-35) is a definite immunosuppressive cytokine belonging to the IL-12 family. Nevertheless, the role of IL-35 in HBV replication remains elusive. In this study, we found that the level of HBV DNA replicative intermediates detected by qPCR and Southern blotting analysis was significantly increased by rhIL-35 in a dose-dependent manner. Moreover, HBV 3.5 kb mRNA levels were up-regulated by rhIL-35. The HBV core protein level as well as the HBsAg and HBeAg secretion levels were also increased by rhIL-35. Moreover, a mechanistic study demonstrated that IL-35 promoted HBV replication by enhancing the HBV core promoter activity. Importantly, hepatocyte nuclear factor 4α (HNF4α) was probably the target of IL-35. Mutation of the HNF4α-binding site on HBV core promoter or silencing HNF4α abolished the enhancement of HBV replication induced by IL-35. Finally, rhIL-35 was able to increase HBV replication in HBV transgenic mice. Taken together, our findings demonstrated that IL-35 has a novel role in HBV replication.

Tumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level.

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.