No abstract

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn 2؉ than in the presence of Mg 2؉ . When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3 end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>50 M) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B viral hepatitis (23). Although acute HCV infection is often asymptomatic, nearly 80% of cases resolve to chronic hepatitis, which may lead to progressive liver disease, such as cirrhosis, and liver failure. Chronic HCV infection is also associated with the development of hepatocellular carcinoma (23). It is estimated that 170 million people worldwide and more than 4 million people in the United States are currently infected with HCV (2, 65). Obviously, HCV infection remains a major threat to the public health all over the world.HCV is an enveloped RNA virus containing a singlestranded positive-sense RNA genome approximately 9.5 kb in length (14,31,56). The RNA genome consists of a 5Ј-untranslated region (5Ј UTR) of 341 nucleotides (12, 13), a large open reading frame (ORF) encoding a single polypeptide of 3,010 to 3,040 amino acids (14, 31, 56), and a 3Ј-untranslated region (3Ј UTR) of variable length (33,57,66). HCV is similar in amino acid sequence and genome organization to flaviviruses and pestiviruses (41), and therefore HCV was classified ...

The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular, the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural, pharmacologic, and pharmacokinetic properties of RDEA119/BAY 869766, an allosteric MEK inhibitor, are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo, RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma, colon, and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data, we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice, RDEA119/BAY 869766 has the potential for use as a once-or twice-daily oral treatment for cancer. RDEA119/BAY 869766, an exquisitely selective, orally available MEK inhibitor, has been selected for clinical development because of its potency and favorable pharmacokinetic profile.

Hepatitis C virus (HCV) isHepatitis C virus (HCV) is a leading cause of chronic liver disease, which affects around 170 million people worldwide. Infections are initially acute, and in many cases the symptoms are mild. However, around 80% of patients eventually develop a persistent chronic infection which can result in steatosis, fibrosis, cirrhosis, liver failure, and, in some cases, hepatocellular carcinoma. The main treatments currently available for chronically infected patients use a combination of pegylated alpha interferon and ribavirin, but these still result in a sustained antiviral response in only about 50% of genotype 1 infections (4). Consequently, more effective antivirals that target either the virus proteins directly or the host cell proteins required during HCV replication are currently being developed. In order to ensure that successful antivirals are generated, it is important that all aspects of the HCV life cycle and HCV-associated pathology are well understood.One way in which the different host processes that are an essential part of the HCV replication cycle can be studied is to investigate the effect that HCV infection has on cellular gene expression. RNA microarray hybridization is routinely used to investigate host gene expression and allows the entire transcriptomic profile of the cell to be characterized. Microarray analysis of HCV-infected cells can provide an insight into the genes involved in host cell antiviral responses, genes that are essential for the HCV replication cycle, and genes that contribute to HCV-associated liver pathology. Microarray expression profiling has already been used to study host gene expression in cells transfected with RNA encoding either individual HCV genes, HCV subgenomic replicons, or the full-length HCV genome. These studies have demonstrated that the replication of the HCV genome results in the regulation of a small number of host genes involved in lipid metabolism, cellular immunity, proliferation, apoptosis, and molecular transport (2,5,15,32). These studies have provided interesting insights into the HCV replication cycle. However, the biological significance of gene expression patterns identified is less clear, since the full virus replication cycle, including the processes of viral entry, assembly, and exit, does not take place.The recent discovery of JFH-1, a genotype 2a HCV clone that can undergo a complete infection cycle in cell culture, provides the opportunity to characterize the true effect of HCV infection on host gene expression (51). A recent study investigated the effects that a J6/JFH-1 chimera had on the gene expression profile of Huh7.5 cells during a time course infection with time points of 24, 48, 72, 96, and 120 h (53). The number of host genes regulated during infection was much higher than that previously observed for cells permitting only genome replication, indicating that the full replication cycle has additional effects on host gene expression.In this study, we present the results from an investigation

The guanosine analogs BMS-200475 and lobucavir have previously been shown to effectively suppress propagation of the human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) in 2.2.15 liver cells and in the woodchuck animal model system, respectively. This repression was presumed to occur via inhibition of the viral polymerase (Pol) by the triphosphate (TP) forms of BMS-200475 and lobucavir which are both produced in mammalian cells. To determine the exact mode of action, BMS-200475–TP and lobucavir-TP, along with several other guanosine analog-TPs and lamivudine-TP were tested against the HBV, WHV, and duck hepatitis B virus (DHBV) polymerases in vitro. Estimates of the 50% inhibitory concentrations revealed that BMS-200475–TP and lobucavir-TP inhibited HBV, WHV, and DHBV Pol comparably and were superior to the other nucleoside-TPs tested. More importantly, both analogs blocked the three distinct phases of hepadnaviral replication: priming, reverse transcription, and DNA-dependent DNA synthesis. These data suggest that the modest potency of lobucavir in 2.2.15 cells may be the result of poor phosphorylation in vivo. Kinetic studies revealed that BMS-200475–TP and lobucavir-TP competitively inhibit HBV Pol and WHV Pol with respect to the natural dGTP substrate and that both drugs appear to bind to Pol with very high affinities. Endogenous sequencing reactions conducted in replicative HBV nucleocapsids suggested that BMS-200475–TP and lobucavir-TP are nonobligate chain terminators that stall Pol at sites that are distinct yet characteristically two to three residues downstream from dG incorporation sites.

Pro-C3 is a useful test to predict fibrogenesis and monitor disease progression. Moreover, it could differentiate mild from moderate disease. Pro-C3 may become a promising blood parameter be included in future studies for monitoring disease progression and eventually for evaluation of potential antifibrotic therapies.

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1)proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions. Adeno-associated virus (AAV) is a nonpathogenic human parvovirus with a biphasic life cycle (reviewed in reference 45). The linear, single-stranded DNA genome of AAV possesses inverted terminal repeats (ITRs) at either end that fold into hairpin structures and serve as the origin of replication. There are two open reading frames that encode the replication (Rep) and structural (Cap) proteins. The two large Rep proteins (Rep68 and Rep78) are required for replication and possess multiple activities, including specific DNA binding and sitespecific endonuclease nicking at the viral ITR (45). Productive AAV infection in cell culture requires helper functions that can be supplied by coinfection with a second virus. In the absence of helper virus, the AAV genome stably integrates into the host genome. Helper viruses that enable efficient AAV replication include adenovirus (Ad) and viruses of the herpesvirus group, such as herpes simplex virus types 1 and 2 (HSV-1 and -2) (12), human herpesvirus 6 (51), and human cytomegalovirus (HCMV) (43). Replication of AAV can also be achieved in several cell lines by the addition of a variety of genotoxic agents (67-69), and autonomous replication has been demonstrated in cultured differentiating keratinocytes (44). A basic question in AAV biology is the nature of the helper effect supplied by the different helper viruses as well as by other permissive conditions. Genetic analysis using mutant helper viruses and expression of individual helper virus genes has defined gene products that are required for efficient AAV replication. The helper functions of Ad are well characterized and are supplied by E1a, E1b, E2a, E4, and the VA RNA (36, 50). Specific functions have been assigned for these proteins and their predominant role appears to be in regulation of gene expression for AAV proteins. T...

DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7.