Santanu Raychaudhuri

Histone Code Modifications Repress Glucose Transporter 4 Expression in the Intrauterine Growth-restricted Offspring

We examined transcriptional and epigenetic mechanism(s) behind diminished skeletal muscle GLUT4 mRNA in intrauterine growth-restricted (IUGR) female rat offspring. An increase in MEF2D (inhibitor) with a decline in MEF2A (activator) and MyoD (co-activator) binding to the glut4 promoter in IUGR versus control was observed. The functional role of MEF2/MyoD-binding sites and neighboring three CpG clusters in glut4 gene transcription was confirmed in C2C12 muscle cells. No differential methylation of these three and other CpG clusters in the glut4 promoter occurred. DNA methyltransferase 1 (DNMT1) in postnatal, DNMT3a, and DNMT3b in adult was differentially recruited with increased MeCP2 (methyl CpG-binding protein) concentrations to bind the IUGR glut4 gene. Covalent modifications of the histone (H) code consisted of H3.K14 de-acetylation by recruitment of histone deacetylase (HDAC) 1 and enhanced association of HDAC4 enzymes. This set the stage for Suv39H1 methylase-mediated di-methylation of H3.K9 and increased recruitment of heterochromatin protein 1α, which partially inactivates postnatal and adult IUGR glut4 gene transcription. Further increased interactions in the adult IUGR between DNMT3a/DNMT3b and HDAC1 and MEF2D and HDAC1/HDAC4 and decreased association between MyoD and MEF2A existed. We conclude that epigenetic mechanisms consisting of histone code modifications repress skeletal muscle glut4 transcription in the postnatal period and persist in the adult female IUGR offspring.

The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production.

The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase‐based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES‐mediated translation, and small interfering RNA (siRNA)‐mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion. Conclusion: Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity. (HEPATOLOGY 2009.)

The interaction of cytoplasmic RNA viruses with the nucleus

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3C(pro), is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICalpha, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2A(pro), also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.

Epigenetics – A Science of Heritable Biological Adaptation

This annual review issue is dedicated to “Epigenetics” an emerging but fast growing field in the postgenomic era involving nonMendelian heritable changes in gene expression that are not mediated by alterations in Watson-Crick basepairing of the DNA sequence (1,2). Conrad Waddington (1905–1975) coined the term “epigenetics” and defined it as “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” (1,2). Both Darwin and Lamarck, the founders of evolutionary theory, predicted that “evolution may favor the development of self-guiding mechanisms, maximizing variability where and when it is most likely to yield positive changes while minimizing phenotypic variability when and where it is not needed” (1,2), supporting the general idea of nonrandom evolution. Epigenetic regulation mediates adaptation to the environment by the genome lending plasticity that translates into the presenting phenotype, particularly under “mismatched” environmental conditions (3). Most investigations focus on demonstrating chemical modifications of the DNA sequence and/or the nuclear histone proteins that in turn alter the configuration of chromatin affecting its packaging and accessibility within the nucleus. The state of chromatin is critically important for the transcription of genes. Thus, heterochromatin signifies tightly wound chromatin impeding access of specific DNA sequences (ciselements) by nuclear trans-activating proteins, a process that inhibits gene transcription. On the other hand, euchromatin is an active state, in which the loosely wound chromatin allows binding of the promoter region of genes by activating proteins resulting in active transcription. This simple concept, while serving as the ultimate process, is complicated by nuclear transport via the nuclear membrane pore or retention of critically important proteins, the complex nuclear arrangement of chromatin and proteins in a network creating loops and higher order structures, and recruitment of various enzymes and proteins to transcription initiation complexes, that are vital for either activating or repressing transcription (4,5), along with the newly evolving role for noncoding RNAs (6,7). The organization of chromatin in the nucleus allows the close proximity of the 5=and 3=ends of chromatin setting the stage for factors that mediate mRNA processing at the 3=-end to be involved in transcription at the 5=-end. Further, the approximation of chromosomes lends to the process of nonrandom translocation that determines a particular cellular phenotype (4,5). This self-organization of the nucleus is thought to be responsible for the formation of nucleoli either in the presence or absence of a scaffolding network consisting of lamin and/or actin filaments (4,5). Cytosine methylation of CpG dinucleotide clusters at the carbon 5 position found in close proximity to critically important cis-elements within the promoter can silence a gene. De novo and maintenance DNA methyl transferase enzyme isoforms (Dnmt1, 3a, 3b) responsible for methylating DNA and repressor methyl-binding domain (MBD 1, 2, 3) proteins or methyl CpG binding proteins (MeCP 1, 2) are necessary for mediating and/or modifying this reaction (8). Methylated DNA in turn attracts chromodomain binding proteins (heterochromatin protein, HP1) that maintain the heterochromatin state. What lends to further complexity is the long range chromatin interactions that occur which may not be evident at first pass when examining the promoter region of a gene where a concentration of unmethylated CpG islands tend to cluster (9). Usually CpGs in a GC rich region with excluded Alu repeats within a promoter are unmethylated and conserved across species while CpGs within or in close proximity to transposans or repeats are methylated (9). The mammalian one carbon metabolism provides the methyl groups for all biologic methylation reactions that in turn is dependent on methyl donors (methionine and choline) and co-factors (folic acid, vitamin B12, pyridoxal phosphate). Examples of the genome that carry methylated CpG regions as seen in the centromere, in the imprinting control regions (ICRs) which are characterized by differentially methylated regions (DMRs) are called “methylation marks” (4,8). These imprints resulting in gene silencing are seen with transposans, X-chromosome inactivation and parenterally imprinted genes causing mono-allelic gene expression (IGF 2, IGF2 receptor genes, Peg) (4). The genome of the immature primordial germ cells of the developing embryo (zygote and blastomeres) undergoes extensive demethylation allowing for totipotency while erasing methylation marks thereby repressing the somatic program (10). Subsequent re-establishment of appropriate sexdependent patterns of cytosine methylation during gametogenesis sets the stage for pluripotency that is maintained through embryonic development during many rounds of rapid cellular proliferation (mitosis) until final maturation of cell differentiation. The re-establishment of methylation imprints occurs Correspondence: Sherin U. Devaskar, M.D., David Geffen School of Medicine at UCLA, Department of Pediatrics, Division of Neonatology and Developmental Biology, 10833 Le Conte Avenue, Room B2-375 MDCC, Los Angeles, CA 90095, Phone: (310) 825-9436, Fax: (310) 267-0154; e-mail: SDevaskar@mednet.ucla.edu Statement of Financial Support: The authors are supported by grants from NICHD HD-25024, -33997, -41230 [S.U.D.] and -46979 [S.U.D., S.R.].

A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation.

ABSTRACT La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal “La motif” which is required for efficient interaction of La with IRNA and viral 5′ untranslated region (5′-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5′-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5′-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.

Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle.

We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.

Shutoff of RNA Polymerase II Transcription by Poliovirus Involves 3C Protease-Mediated Cleavage of the TATA-Binding Protein at an Alternative Site: Incomplete Shutoff of Transcription Interferes with Efficient Viral Replication

ABSTRACT The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3Cpro to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3Cpro involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3Cpro involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3Cpro is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3Cpro-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

A cell-permeable peptide inhibits hepatitis C virus replication by sequestering IRES transacting factors.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. There is no effective therapy or vaccine available to HCV-infected patients other than interferon-ribavarin combination, which is effective in a relatively small percentage of infected patients. Our previous results have shown that a synthetic peptide (LAP) corresponding to the N-terminal 18 amino acids of the Lupus autoantigen (La) was a potent inhibitor of HCV IRES-mediated translation. We demonstrate here that LAP efficiently blocks HCV replication of infectious JFH1 virus in cell culture. Our data suggest that LAP forms complexes with IRES-transacting factors (ITAFs) PTB and PCBP2. LAP-mediated inhibition of HCV IRES-mediated translation in vitro could be fully rescued by recombinant PCB and PCBP2. Also transient expression of PTB / PCBP2 combination significantly restores HCV replication in LAP-inhibited cultures. These results suggest that ITAFs could be potential targets to block HCV replication.

Activation of ribosomal RNA transcription by hepatitis C virus involves upstream binding factor phosphorylation via induction of cyclin D1.

Hepatitis C virus (HCV) causes chronic infection in humans leading to liver cirrhosis and hepatocellular carcinoma. rRNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis, and changes in Pol I transcription rate are associated with profound alterations in the growth rate of the cell. Because rRNA synthesis is intimately linked to cell growth and frequently up-regulated in many cancers, we hypothesized that HCV might have the ability to activate rRNA synthesis in infected cells. We show here that rRNA promoter-mediated transcription is significantly (10- to 12-fold) activated in human liver-derived cells following infection with type 2 JFH-1 HCV or transfection with the subgenomic type 1 HCV replicon. Further analysis revealed that HCV nonstructural protein 5A (NS5A) was responsible for activation of rRNA transcription. Both the NH(2)-terminal amphipathic helix and the polyproline motifs of NS5A seem to be essential for rRNA transcription activation. The NS5A-dependent activation of rRNA transcription seems to be due to hyperphosphorylation and consequent activation of upstream binding factor (UBF), a Pol I DNA binding transcription factor. We further show that hyperphosphorylation of UBF occurs as a result of up-regulation of both cyclin D1 and cyclin-dependent kinase 4 by the HCV NS5A polypeptide. These results suggest that the endoplasmic reticulum-associated NS5A is able to transduce signals into the nucleoplasm via UBF hyperphosphorylation leading to rRNA transcription activation. These results could, at least in part, explain a mechanism by which HCV contributes to transformation of liver cells.

A cell‐permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A–mediated translation and virus production

NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)‐mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A‐driven augmentation of IRES‐mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES‐mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C‐terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same‐size control peptides from other NS5A domains. C34 also competitively inhibited NS5A‐augmented IRES‐mediated translation, whereas controls did not. Triple‐alanine scan mutagenesis determined that an exposed beta‐sheet hairpin in C34 was primarily responsible for NS5A‐augmented IRES‐mediated translation. Moreover, treatment with a 10–amino acid peptide derivative of C34 suppressed NS5A‐augmented IRES‐mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES‐mediated translation, identify a sequence‐specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin–mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A‐mediated IRES activity, sequence‐specific HSP recognition, and rational drug design. (HEPATOLOGY 2012;55:1662–1672)