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Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional–translational feedback loop of clock genes. Brain and muscle ARNT-like 1 (BMAL1) is one of the core clock genes and transcription factors that represents a positive arm of this autoregulatory circadian clock system. Despite the indispensable role of BMAL1 in the circadian rhythm, the molecular mechanisms underlying translational control of BMAL1 are largely unknown. Here, using murine NIH-3T3 cells, gene constructs, and a variety of biochemical approaches, including RNAi- and luciferase reporter gene–based assays, along with immunoblotting, in vitro transcription, quantitative real-time PCR, and real-time bioluminescence experiments, we show that translation of Bmal1 is negatively regulated by an RNA-binding protein, heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Interestingly, we found that hnRNP Q rhythmically binds to a specific region of the Bmal1 mRNA 5′ UTR and controls its time-dependent expression. Moreover, we demonstrate that knockdown of hnRNP Q modulates BMAL1 protein oscillation amplitude without affecting mRNA rhythmic patterns. Furthermore, hnRNP Q depletion increases the mRNA oscillation amplitudes of BMAL1-regulated target genes. Together, our results suggest that hnRNP Q plays a pivotal role in both Bmal1 translation and BMAL1-regulated gene expression.

Approximately 71 million people suffer from hepatitis C virus (HCV) infection worldwide. Persistent HCV infection causes liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, resulting in approximately 400,000 deaths annually. Effective direct-acting antiviral agents (DAAs) have been developed and are currently used for HCV treatment targeting the following three proteins: NS3/4A proteinase that cleaves the HCV polyprotein into various functional proteins, RNA-dependent RNA polymerase (designated as NS5B), and NS5A, which is required for the formation of double membrane vesicles serving as RNA replication organelles. At least one compound inhibiting NS5A is included in current HCV treatment regimens due to the high efficacy and low toxicity of drugs targeting NS5A. Here we report fluorene compounds showing strong inhibitory effects on GT 1b and 3a of HCV. Moreover, some compounds were effective against resistance-associated variants to DAAs. The structure-activity relationships of the compounds were analyzed. Furthermore, we investigated the molecular bases of the inhibitory activities of some compounds by the molecular docking method.

Hepatitis C virus (HCV) causes liver diseases, such as hepatitis, liver cirrhosis, steatosis, and hepatocellular carcinoma. To understand the life cycle and pathogenesis of HCV, the one-step growth of HCV in a cell culture system was analyzed using a highly infectious variant of the JFH1 clone. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 h (h) after infection, followed by the active synthesis of positive-strand RNAs. Our measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.

Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway.

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265-269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.

Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either “conditioned” Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naive Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.

The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.

The subcellular location of phospholipase D1 (PLD1) and its activation by protein kinase C alpha (PKC alpha) were examined by subcellular fractionation and by microscopic observation of green fluorescent protein-fused PLD1 (GFP-PLD1) or PKC alpha (GFP-PKC alpha) in fibroblastic 3Y1 cells. Major PLD1 immunoreactivity and PKC alpha-stimulated PLD activity segregated with a plasma membrane marker, even though a significant amount was co-fractionated with markers for endoplasmic reticulum (ER) and Golgi. Upon treatment with phorbol myristate acetate (PMA), PKC alpha translocated from the cytosolic fraction to the membrane fraction to which PLD1 also localized. GFP-PLD1 was found in the plasma membrane as well as a in a perinuclear compartment consistent with ER and Golgi and in other dispersed vesicular structures in the cytoplasm. However, most of GFP-PKC alpha was translocated from the cytosol to the plasma membrane after treatment with PMA. From these results, we concluded that the plasma membrane is the major site of PLD1 activation by PKC alpha in 3Y1 cells.

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no β-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A “descending molecular see-saw” model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

A dicistronic poliovirus W1-P1/E/P2,3-1 with the genotype [PV]5'NTR-P1-[EMCV]IRES-[PV]P2,3-3'NTR (Molla, Jang, Paul, Reuer, and Wimmer, 1992, Nature 356, 255) was used to investigate whether the viral proteinase 2Apro, whose primary function in proteolytic processing was erased through the insertion of an internal ribosomal entry site (IRES) element into the ORF of the polyprotein, had other function(s) in viral replication. Deletion of 2Apro from W1-P1/E/P2,3-1 rendered the corresponding transcripts unable to replicate whereas partial deletion of 2Apro or an exchange of Cys109 (an amino acid of the catalytic triad of the proteinase) to Ala reduced RNA replication. No cytopathic effects were observed after transfection with any of the three dicistronic constructs containing mutant 2A, and no virus was recovered after attempts to expand a possibly low yield of mutant virus. In contrast, insertion of the IRES of encephalomyocarditis virus (EMCV) into the ORF of the poliovirus polyprotein at the cleavage site between 2Apro and 2B yielded the novel dicistronic virus W1-P1,2A/E/2BC,P3-1 with the genotype [PV]5'NTR-P1-2A-[EMCV]IRES-[PV]2BC-P3-3'NTR, expressing a small plaque phenotype. These results indicate that neither the intact P2 polypeptide nor the cleavage fragment 2AB of P2 is required for viral proliferation. On the other hand, 2Apro appears to be an essential component in RNA replication as no viral RNA synthesis can be observed by reverse transcription/PCR in cells transfected with dicistronic RNA lacking this viral polypeptide.