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Supplementary Figure 1 from Activation of Ribosomal RNA Transcription by Hepatitis C Virus Involves Upstream Binding Factor Phosphorylation via Induction of Cyclin D1

Supplementary Figure 2 from 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 NH2-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. [Cancer Res 2009;69(5):2057–64]

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys(382)-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G(1)-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

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.

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. Non-structural 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.Weconfirmed anNS5A/HSPinteraction 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.Atransient 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.

We have previously identified HSC70 in complex with viral NS5A along with other heat shock proteins. HSC70 has been reported to modulate HCV infectivity. Here, we further analyze the NS5A/HSC70 interaction biochemically and investigate the role of HSC70 in viral life cycle. We demonstrate that HSC70 knockdown significantly affects virus production with no cytotoxicity. We observed that HSC70 knockdown did not affect viral RNA replication and protein production. In fact, we found a slight, but statistically significant increase in viral RNA and protein levels likely caused by compensatory upregulation of HSP70, which we have previously shown to be required for viral protein production. NS5A-augmented IRES-mediated translation was also not inhibited by HSC70 knockdown and, rather, slightly increased. In contrast, we discovered that intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains direct...

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)

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 NH2-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. [Cancer Res 2009;69(5):2057–64]

Glucose transporter isoform-3 (GLUT3) is the trophoblastic facilitative glucose transporter. To investigate the role of this isoform in embryonic development, we created a novel GLUT3-null mouse and observed arrested early embryonic development and loss at neurulation stage when both alleles were mutated. This loss occurred despite the presence of other related isoforms, particularly GLUT1. In contrast, when a single allele was mutated, despite increased embryonic cell apoptosis, adaptive changes in the subcellular localization of GLUT3 and GLUT1 in the preimplantation embryo led to postimplantation survival. This survival was compromised by decreased GLUT3-mediated transplacental glucose transport, causing late-gestation fetal growth restriction. This yielded young male and female adults demonstrating catch-up growth, with normal basal glucose, insulin, insulin-like growth factor-I and IGF-binding protein-3 concentrations, fat and lean mass, and glucose and insulin tolerance. We conclude that GLUT3 mutations cause a gene dose-dependent early pregnancy loss or late-gestation fetal growth restriction despite the presence of embryonic and placental GLUT1 and a compensatory increase in system A amino acid placental transport. This critical life-sustaining functional role for GLUT3 in embryonic development provides the basis for investigating the existence of human GLUT3 mutations with similar consequences during early pregnancy.

A small inhibitor RNA (IRNA) isolated from yeast has previously been shown to efficiently block poliovirus and hepatitis C virus IRES-mediated translation by sequestering mammalian RNA-binding (transacting) factors that play important roles in cap-independent translation. Here we have investigated the IRNA-binding proteins that might be involved in cap-independent translation in the yeast Saccharomyces cerevisiae . We have identified Zuotin, a DnaJ chaperone protein similar to mammalian HSP-40 chaperone, which interacts strongly with IRNA. Using ZUO1 -deleted S. cerevisiae , we demonstrate a preferential requirement of Zuo1p for cap-independent translation mediated by the 5′ untranslated region of the yeast TFIID mRNA. Further studies using zuo1 Δ S. cerevisiae complemented with various Zuo1p mutants indicate that the DnaJ domain of Zuo1p, known to influence its interaction with HSP-70, significantly affects cap-independent translation. These results demonstrate for the first time a role for an established chaperone protein in cap-independent translation of a cellular mRNA.

Poliovirus (PV) is the prototype virus of a large group of medically important viruses (picornaviruses) that include those inducing poliomyelitis (polioviruses), infectious hepatitis (hepatitis A virus), the common cold (rhinoviruses), and encephalitis and myocarditis (coxsackie viruses) (31). The single-stranded, plus-polarity RNA genome (∼7,500 nucleotides) of PV (18, 30) is translated into one large polyprotein, which is cotranslationally processed by virally encoded proteases 2Apro, 3Cpro, and 3CDpro into mature viral structural and nonstructural proteins (22). The viral proteases have been studied extensively and found to be very specific in polyprotein cleavage; 3Cpro and 3CDpro cleave the polyprotein at glutamine-glycine (Q-G) bonds while the 2Apro cleaves only at tyrosine-glycine (Y-G) bonds (20). The proteases do not cleave every potential cleavage site within the polyprotein; other determinants such as accessibility and context of the cleavage site are also important. Accurate initiation of transcription by RNA polymerase (Pol) II requires the assembly of a multiprotein complex on the core promoter around the mRNA start site (13). The multiprotein complex, consisting of at least seven to eight general transcription factors (GTFs) and RNA Pol II form a preinitiation complex at specific cis-acting elements (promoters) on the DNA template. The most common cis-acting element, the TATA box, is situated approximately 25 nucleotides upstream of the transcription start site. Among the GTFs in the complex, the TATA-binding protein (TBP) has been studied extensively (29). The TBP was first identified through its role in Pol II transcription, where it associates with 13 or 14 TBP-associated factors to form the TFIID complex (34). The unique aspect of TBP is its additional presence in complexes required for RNA Pol I (SL1) and RNA Pol III (TFIIIB) transcription. In both SL1 and TFIIIB complexes, TBP associates with a distinct set of TBP-associated factors (7, 33). Thus, TBP appears to be involved in all three RNA polymerase-mediated transcriptions. TBP has a bipartite structure with a highly conserved C-terminal core domain (amino acids 159 to 339), which folds as a molecular saddle, and is responsible for DNA binding via the concave underside. Additionally, the core also interacts with GTFs and other regulatory proteins via the solvent-exposed convex side. In contrast, the N-terminal region (amino acids 1 to 141) is variable in both length and sequence but is well conserved among vertebrates. It modulates DNA binding by interacting with the core domain and is characterized by a glutamine repeat region (Fig. ​(Fig.1).1). Recent studies have underscored the importance of the TBP N-terminal domain in transcriptional regulation both in yeast and mammalian systems (15, 21). Moreover, deletion of 55 and 96 amino acids from the N-terminal domain of TBP leads to inactivation of TATA-mediated transcription from the U6 small nuclear RNA promoter (24). FIG. 1. Shutoff of RNA polymerase II-mediated transcription does not correlate with 3Cpro-induced cleavage at the 18th glutamine-glycine site in TBP. (A) The domain structure of TBP consisting of the core, N-terminal with the glutamine stretch (Q), and acidic ... Infection of HeLa cells with PV causes a severe decrease in cellular transcription catalyzed by all three cellular RNA polymerases (10). Transcription mediated by RNA polymerase I (Pol I) is inhibited first, at 1 to 2 h postinfection, followed by inhibition of Pol II and Pol III transcription at approximately 3 and 4 h postinfection, respectively. Crawford et al. first showed that PV-induced inhibition of transcription observed in vivo could be recapitulated in vitro (8). Further studies showed that the viral protease 3Cpro alone was responsible for the shutoff of transcription by all three cellular RNA polymerases (36). Recent studies have shown that 3Cpro enters the nucleus in the form of its precursor 3CD (32), which presumably undergoes autocatalysis to generate 3Cpro in the nucleus of infected cells. A primary target of 3Cpro in PV-infected cells was previously identified to be the TATA-binding protein (6). Consistent with this observation, the TFIID complex isolated from PV-infected HeLa cells was transcriptionally inactive in an in vitro reconstituted transcription assay compared to the TFIID isolated from uninfected cells (19). Moreover, purified TBP could be directly cleaved by the purified 3Cpro in vitro, and the addition of purified TBP could completely restore both basal and activated transcription from the TATA and initiator promoters in HeLa cell extracts from PV-infected cells (37). Examination of the human TBP sequence revealed three Q-G sites at positions 12, 18, and 112 (Fig. ​(Fig.1).1). Subsequent in vitro studies using purified components showed that only the 18th Q-G site in TBP could be efficiently cleaved by 3Cpro both in vitro and in vivo (9). To determine if cleavage of TBP at the 18th Q-G bond is responsible for the shutoff of transcription in PV-infected cells, we expressed and purified a recombinant TBP that lacks the N-terminal 18 amino acids (called ΔN18 TBP). If cleavage at the 18th Q-G bond is the primary cause of transcriptional inactivation of TBP, then ΔN18 TBP should not be able to restore transcription in PV-infected cell extracts. We found that ΔN18 TBP was as active as the wild-type (wt) TBP in fully restoring Pol II transcription in PV-infected cell extracts from the adenovirus major late promoter (Ad MLP). We also found that the transcriptional activity of ΔN18 TBP was comparable to that of wt TBP in an in vitro transcription reconstitution assay. Since both genetic and biochemical evidence suggested that 3Cpro was involved in the shutoff of transcription and that TBP was the primary target of 3Cpro, we set out to determine if TBP was being cleaved by 3Cpro at sites other than the 18th Q-G bond. We report here identification of a Q-S site at position 104 to 105 of TBP, which appears to be cleaved by the viral protease 3Cpro. We also demonstrate that an alanine residue at P4 is critical for 3C-mediated cleavage of the 104th Q-S bond. A truncated form of TBP lacking the first 100 amino acids is significantly less active transcriptionally than the wt TBP in vitro. Finally, we demonstrate that a stable HeLa cell line expressing a recombinant TBP (rTBP) resistant to cleavage by the viral proteases is significantly refractive to the shutoff of transcription by poliovirus compared to the control cell line expressing wt TBP. Infection with poliovirus of the HeLa cell line expressing the noncleavable form of TBP shows small plaques and significantly reduced viral RNA synthesis compared to the control TBP cell line, suggesting that a defect in the shutoff of transcription can lead to inefficient replication of the virus.

Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.

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.

The origin recognition complex (ORC), first identified in Saccharomyces cerevisiae (sc), is a six-subunit protein complex that binds to DNA origins. Here, we report the identification and cloning of cDNAs encoding the six subunits of the ORC of Schizosaccharomyces pombe (sp). Sequence analyses revealed that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from S. cerevisiae , Xenopus , Drosophila , and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Chuang and Kelly [(1999) Proc. Natl. Acad. Sci. USA 96, 2656–2661], the C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction of S. pombe chromatin with 1 M NaCl, or after DNase I treatment, yielded the six-subunit ORC, whereas extraction with 0.3 M resulted in five-subunit ORC lacking spOrc4p. The spORC can be reconstituted in vitro with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits required the removal of DNA from reaction mixture by DNase I. This suggests that a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may contribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication in S. pombe .

It was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.

Yeast replication origins have a modular arrangement of essential DNA sequences containing the ARS consensus sequence (ACS) flanked by auxiliary DNA elements which stimulate origin function. One of the auxiliary elements identified at several origins is a DNA replication enhancer that binds the Abf1p protein. We have isolated an ARS sequence from Saccharomyces cerevisiae based on its ability to bind Abf1p. Here we present a detailed molecular dissection of this ARS, designated ARS 1501, and we demonstrate that it functions as a genomic replication origin on chromosome XV . Mutagenesis of the Abf1p DNA-binding sites revealed that these sequences did not contribute significantly to ARS function. Instead, a new DNA element important for replication, designated REN1501, has been located 5' to the T-rich strand of the ACS. We show that REN1501 functions in either orientation and at variable distances from the ACS, defining this element as a DNA replication enhancer. Most significantly, point mutations within this element decreased the stability of plasmids bearing ARS 1501, suggesting that REN1501 binds a protein important for replication initiation. Only three elements found at origins are known to specifically bind proteins. These include the ARS essential sequences and the Abf1p and Rap1p DNA-binding sites. We show that the function of REN1501 at the origin cannot be replaced by a Rap1p DNA-binding site or a site that binds the transcriptional factor Gal4p and can only be partially substituted for by an Abf1p recognition sequence. This implies that the role of the REN1501 element at the ARS 1501 origin is specific, and suggest that the frequency of origin firing in eukaryotic cells may be regulated by origin-specific enhancers.

Cervical cancer is one of the leading causes of female death world wide with approximately 50,000 deaths per year. We identified a 300 kb deletion of chromosome 11q13 in cervical cancer and mapped eleven genes to this region. Of these, phosphofurin acidic cluster sorting −1 (PACS-1) gene encodes a multi-functional sorting protein involved in secretory pathway membrane traffic. Expression studies showed the presence of an abnormal PACS-1 transcript in tumorigenic cell lines. Protein expression studies in paired normal and cervical tumors showed over-expression of PACS-1 protein in tumor specimens. RT-PCR indicated that protein over-expression was related to over-expression at the RNA level. These findings together with the absence of detectable mutations in PACS-1 exons in HeLa or SiHa cervical carcinoma cell lines raised the possibility that dysregulation of a microRNA (miRNA) may alter PACS-1 expression in cervical cancer. Consistent with this possibility, a UCSC genome database search showed the presence of binding sites for miRNA449a in the 3′ UTR (untranslated region) of the PACS-1 RNA transcript. Co-transfection of PACS-1 3′UTR luciferase reporter with miRNA449a resulted in reduced expression of luciferase. Expression of miR449a in HeLa and SiHa cells led to reduced expression of PACS-1 and increased expression of p53 and p21 proteins. FACS analysis of HeLa cells transfected with miR449a showed a reduction in S phase indicating the growth suppressive effect of miR449a. These results indicate that miR449a regulates PACS-1 expression and loss of miR449a expression leads to over-expression of PACS-1. This in turn seems to result in the destabilization of p53 in cervical cancer. Our findings suggest PACS-1 plays a role in cervical cancer development and its expression is regulated post-transcriptionally by miRNA449a. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4048.

Host cell transcription mediated by all three RNA polymerases is rapidly inhibited after infection of mammalian cells with poliovirus (PV). Both genetic and biochemical studies have shown that the virus-encoded protease 3C cleaves the TATA-binding protein and other transcription factors at glutamine–glycine sites and is directly responsible for host cell transcription shut-off. PV replicates in the cytoplasm of infected cells. To shut-off host cell transcription, 3C or a precursor of 3C must enter the nucleus of infected cells. Although the 3C protease itself lacks a nuclear localization signal (NLS), amino acid sequence examination of 3D identified a potential single basic type NLS, KKKRD, spanning amino acids 125–129 within this polypeptide. Thus, a plausible scenario is that 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis ultimately leading to cleavage of transcription factors in the nucleus. Using transient transfection of enhanced green fluorescent protein (EGFP) fusion polypeptides, we demonstrate here that both 3CD and 3D are capable of entering the nucleus in PV-infected cells. However, both polypeptides remain in the cytoplasm in uninfected HeLa cells. Mutagenesis of the NLS sequence in 3D prevents nuclear entry of 3D and 3CD in PV-infected cells. We also demonstrate that 3CD can be detected in the nuclear fraction from PV-infected HeLa cells as early as 2 h postinfection. Significant amount of 3CD is found associated with the nuclear fraction by 3–4 h of infection. Taken together, these results suggest that both the 3D NLS and PV infection are required for the entry of 3CD into the nucleus and that this may constitute a means by which viral protease 3C is delivered into the nucleus leading to host cell transcription shut-off.

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.

We previously identified HSP70 and HSC70 in complex with NS5A in a proteomic screen. Here, coimmunoprecipitation studies confirmed NS5A/HSC70 complex formation during infection, and immunofluorescence studies showed NS5A and HSC70 to colocalize. Unlike HSP70, HSC70 knockdown did not decrease viral protein levels. Rather, intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A whereas only the HSP70 nucleotide binding domain does. Knockdown of both HSC70 and HSP70 demonstrated an additive reduction in virus production. This data suggests that HSC70 and HSP70 play discrete roles in the viral life cycle. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection.