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Antiviral Research (v.77, #3)
A screening method for identifying disruptions in interferon signaling reveals HCV NS3/4a disrupts Stat-1 phosphorylation by Karla J. Helbig; Evelyn Yip; Erin M. McCartney; Nicholas S. Eyre; Michael R. Beard (pp. 169-176).
Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak–Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000U/ml IFN and 30mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. Conclusion: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak–Stat signaling pathway. We show that HCV NS3/4a is able to block this pathway at the stage of Stat-1 serine 727 phosphorylation.
Keywords: Interferon; Jak–Stat; Hepatitis C virus
Characterization of resistance mutations against HCV ketoamide protease inhibitors by Xiao Tong; Stephane Bogen; Robert Chase; V. Girijavallabhan; Zhuyan Guo; F. George Njoroge; Andrew Prongay; Anil Saksena; Angela Skelton; Ellen Xia; Robert Ralston (pp. 177-185).
An issue of clinical importance in the development of new antivirals for HCV is emergence of resistance. Several resistance loci to ketoamide inhibitors of the NS3/4A protease have been identified (residues V36, T54, R155, A156, and V170) by replicon and clinical studies. Using SCH 567312, a more potent protease inhibitor derived from SCH 503034 (boceprevir) series, we identified two new positions (Q41 and F43) that confer resistance to the ketoamide class. The catalytic efficiency of protease enzymes was not affected by most resistance mutations, whereas replicon fitness varied with specific mutations. SCH 503034 and another ketoamide inhibitor, VX-950 (telaprevir), showed moderate losses of activity against most resistance mutations (≤10-fold); the highest resistance level was conferred by mutations at A156 locus. Although SCH 503034 and VX-950 bind similarly to the active site, differences in resistance level were observed with specific mutations. Changes at V36 and R155 had more severe impact on VX-950, whereas mutations at Q41, F43 and V170 conferred higher resistance to SCH 503034. Structural analysis of resistance mutations on inhibitor binding is discussed.
Keywords: Resistance mutation; Boceprevir; SCH 503034; HCV replicon; Protease
Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo by Zhixin Feng; Ping Jiang; Xianwei Wang; Yufeng Li; Wenming Jiang (pp. 186-194).
Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS), which is responsible for the heavy economic losses in stockbreeding. There are no specific antiviral drugs for treatment of the virus infection. We have now constructed two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against either ORF1 (rAdS1) or ORF2 (rAdS2) of PCV2 and measured the inhibition of PCV2 replication. The results showed that delivery of these shRNAs by recombinant adenovirus into PK15 cells could induce a significant inhibition of viral RNA and DNA replication and protein synthesis level in cells subsequently infected with PCV2. The antiviral effect was dose-dependent and could sustain at least for 120h and the inhibition of virus replication could be significantly strengthened by combination of rAdS1 with rAdS2. Mice injected with shRNA before PCV2 infection showed substantial and low level of PCV2 DNA replication in the spleen during the period of 21–28 days post-PCV2 infection. These results indicated that shRNAs generated by adenovirus could sufficiently and continuously inhibit PCV2 infection in vitro as well as in vivo. The adenovirus based shRNA targeting ORF1 and ORF2 of PCV2 might be a new potential alternative strategy for controlling PCV2 infection.
Keywords: PCV2; RNAi; shRNA; Recombinant adenovirus
Inhibition of hepatitis C virus RNA replicons by peptide aptamers by Alla Trahtenherts; Meital Gal-Tanamy; Romy Zemel; Larisa Bachmatov; Shelly Loewenstein; Ran Tur-Kaspa; Itai Benhar (pp. 195-205).
Hepatitis C virus infection is a major worldwide health problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be important. With the aim of inhibiting NS3 protease activity as a means to inhibit HCV replication we used a novel bacterial genetic screen to isolate NS3-inhibiting peptide aptamers.We have isolated and characterized seven NS3-inhibiting peptide aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic RNA replicons undergo upon intracellular expression of these peptide aptamers, assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected replicon cells.The peptide aptamers inhibited NS3 protease activity in vitro with an IC50 in the low micromolar range. Upon transfection, aptamers inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA replicons. Aptamer-based intracellular immunization may emerge as a promising antiviral approach to interfere with the life cycle and pathogenicity of HCV.
Keywords: HCV; NS3 serine protease; Liver diseases; Peptide aptamers; RNA replicons
The role of helioxanthin in inhibiting human hepatitis B viral replication and gene expression by interfering with the host transcriptional machinery of viral promoters by Ya Ping Tseng; Yueh Hsiung Kuo; Cheng-Po Hu; King-Song Jeng; Damodar Janmanchi; Chih Hsiu Lin; Chen Kung Chou; Sheau Farn Yeh (pp. 206-214).
A non-nucleosidic compound, Helioxanthin (HE-145), was found to suppress HBV gene expression and replication in HCC cells. To understand the molecular mode of action of HE-145 on HBV gene expression, the effects of HE-145 on four viral promoter activities using luciferase as a reporter were examined. It was found that HE-145 selectively suppresses surface antigen promoter II (SPII) and core promoter (CP) but has no effect on surface antigen promoter I (SPI) or promoter for X gene (Xp). The suppressive effects of HE-145 on either SPII or CP activity is liver-specific, since no suppressive activity of HE-145 was observed when CP or SPII promoter activity was assayed in non-liver cells such as HeLa or 293T. To examine the mode of action of HE-145, EMSA analysis revealed that HE-145 decreased the DNA-binding activity of nuclear extract of HepA2 cells to specific cis element of HBV promoter for core antigen, including peroxisome proliferator-activated receptors (PPARs), PPARs binding site (PPRE), α-fetoprotein transcription factor (FTF), and Sp1. Ectopic expression of PPARγ or HNF4α partially reversed the HE-145-mediated suppression of HBV RNA. Therefore, HE-145 may represent a novel class of anti-HBV agents which selectively modulate transcriptional machinery of human liver cells to suppress HBV gene expression and replication.
Keywords: Abbreviations; HE-145; helioxanthin; HBV; hepatitis B virus; HBsAg; hepatitis B surface antigen; HBeAg; hepatitis B e antigen; HCC; hepatocellular carcinoma; CP; core promoter; SPI; surface promoter I; SPII; surface promoter II; XP; X promoter; 3TC; Lamivudine, (−)β-; l; -2′,3′-dideoxy-3′-thiacytidine; EIA; enzyme immunoassay; ELISA; enzyme-linked immunosorbent assay; EMSA; electrophoretic mobility shift assayHelioxanthin; Hepatitis B virus; Viral promoters; Hepatic nuclear factors; Human hepatocellular carcinoma cells
Prophylactic and therapeutic intervention of Punta Toro virus ( Phlebovirus, Bunyaviridae) infection in hamsters with interferon alfacon-1 by Brian B. Gowen; Min-Hui Wong; Kie-Hoon Jung; Lawrence M. Blatt; Robert W. Sidwell (pp. 215-224).
Punta Toro virus (PTV) is a member of the Bunyaviridae family, genus Phlebovirus, related to the highly pathogenic Rift Valley fever virus (RVFV). It produces a disease in hamsters that models severe Rift Valley fever (RVF) in humans. The recent outbreak of RVF in Kenya stresses the need to identify prophylactic and therapeutic measures for preventing and treating severe forms of disease. To this end, interferon (IFN) alfacon-1 (consensus IFN-α) was evaluated in cell culture against RVFV and PTV, and in the hamster PTV infection model. Survival outcome following treatment initiated pre- and post-virus challenge and the suppression of viral burden and liver disease in infected hamsters was determined. Pre-treatment of cell cultures with IFN alfacon-1 induced marked antiviral activity against both viruses. Intraperitoneal treatment of hamsters initiated 4h prior to infection with PTV was highly protective and greatly limited liver disease and systemic and liver viral burden. Complete protection from a highly lethal challenge dose was afforded by treatment initiated 36h following viral inoculation. Although efficacy was much reduced, IFN alfacon-1 therapy was still beneficial when started as late as 3–5 days post-virus exposure. These studies suggest that IFN alfacon-1 may be an effective treatment for early intervention following infection with RVFV.
Keywords: Punta Toro virus; Phlebovirus; Interferon; Interferon alfacon-1; Antiviral; Hamster
Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV) by John DeVincenzo; Jeffrey E. Cehelsky; Rene Alvarez; Sayda Elbashir; Jens Harborth; Iva Toudjarska; Lubomir Nechev; Veeravagu Murugaiah; Andre Van Vliet; Akshay K. Vaishnaw; Rachel Meyers (pp. 225-231).
Small interfering RNAs (siRNAs) work through RNA interference (RNAi), the natural RNA inhibitory pathway, to down-regulate protein production by inhibiting targeted mRNA in a sequence-specific manner. ALN-RSV01 is an siRNA directed against the mRNA encoding the N-protein of respiratory syncytial virus (RSV) that exhibits specific in vitro and in vivo anti-RSV activity. The results of two safety and tolerability studies with ALN-RSV01 involving 101 healthy adults (65 active, 36 placebo, single- and multiple dose, observer-blind, randomized dose-escalation) are described. Intranasal administration of ALN-RSV01 was well tolerated over a dose range up through 150mg as a single dose and for five daily doses. Adverse events were similar in frequency and severity to placebo (normal saline) and were transient, mild to moderate, with no dose-dependent trend. The frequency or severity of adverse events did not increase with increasing ALN-RSV01 exposure. All subjects completed all treatments and assessments with no early withdrawals or serious adverse events. Physical examinations, vital signs, ECGs and laboratory tests were normal. Systemic bioavailability of ALN-RSV01 was minimal. ALN-RSV01 appears safe and well tolerated when delivered intranasally and is a promising therapeutic candidate for further clinical development.
Keywords: Respiratory syncytial virus; RNA interference; Small interfering RNA; RNA-induced silencing complex; ALN-RSV01; Antiviral
Rapid identification of inhibitors that interfere with poliovirus replication using a cell-based assay by Yu-Chen Hwang; Justin Jang-Hann Chu; Priscilla L. Yang; Wilfred Chen; Marylynn V. Yates (pp. 232-236).
A small molecule library containing 480 known bioactive compounds was screened for antiviral activity against poliovirus (PV) using a cellular fluorescence resonance energy transfer (FRET) assay for viral protease activity. The infected reporter cells treated with the viral replication-suppressing compounds were examined via fluorescence microscope 7.5h postinfection. Twelve molecules showed moderate to potent antiviral activity at concentrations less than 32μM during the primary screening. Three compounds, anisomycin, linoleic acid, and lycorine, were chosen for validation. A dose-dependent cytotoxicity assay and a secondary screening using conventional plaque assay were conducted to confirm the results. The developed method can be used for rapid screening for molecules with antiviral activity.
Keywords: Abbreviations; 2A; pro; 2A protease; FRET; fluorescence resonance energy transferCellular sensor; Protease; Viral replication; FRET
Characterization of a cidofovir-resistant HHV-6 mutant obtained by in vitro selection by Pascale Bonnafous; David Boutolleau; Lieve Naesens; Claire Deback; Agnès Gautheret-Dejean; Henri Agut (pp. 237-240).
Cidofovir (CDV) was used for in vitro selection of a human herpesvirus 6 (HHV-6) mutant with decreased susceptibility to this drug. The resulting mutant was highly resistant to CDV as compared to its sensitive counterpart (inhibitory concentration 50% (IC50): 213μM versus 1.8μM). Its replication fitness was not impaired. Genotypic characterization of the resistant virus revealed a mutation in the U38 gene encoding the viral DNA polymerase. The resulting R798I amino acid change was located in the conserved domain VII close to the highly conserved motif KKRY interacting with the DNA primer–template duplex, and is likely responsible for the high-level resistance to CDV, even though a definite virological and/or biochemical confirmation is required. The possible emergence of such changes in HHV-6 DNA polymerase in patients receiving CDV therapy should be taken into account in the treatment of HHV-6 infections.
Keywords: HHV-6; Cidofovir; Resistance; DNA polymerase