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Antiviral Research (v.72, #3)
Resveratrol inhibition of varicella-zoster virus replication in vitro by John J. Docherty; Thomas J. Sweet; Erin Bailey; Seth A. Faith; Tristan Booth (pp. 171-177).
Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.
Keywords: Resveratrol; Varicella-zoster virus; Cell infection
DNA immunization with 2C FMDV non-structural protein reveals the presence of an immunodominant CD8+, CTL epitope for Balb/c mice by Annette Malene Barfoed; Fernando Rodriguez; Dominic Therrien; Belen Borrego; Francisco Sobrino; Søren Kamstrup (pp. 178-189).
Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-γ) induction in CD8+, CD44hi splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-γ production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses.
Keywords: Foot and mouth disease virus; Non-structural proteins; CTL epitope; DNA vaccination; Intracellular cytokine staining; Flow cytometry
Inhibition of hepatitis B virus X gene expression by 10-23 DNAzymes by Wei Hou; Qin Ni; Jianer Wo; Minwei Li; Kezhou Liu; Liwei Chen; Zhongrong Hu; Ronghua Liu; Minjun Hu (pp. 190-196).
The X protein (HBx) of human hepatitis B virus (HBV) is a transcriptional activator protein. The HBx protein plays an important role in viral replication in HBV infected cells and the liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Therefore, the repression of HBx gene expression by 10-23 DNAzymes might be a good way to inhibit HBV replication and counteract HBV-related liver diseases. We designed three 10-23 DNAzymes with different substrate-recognition domains. When each of the 10-23 DNAzymes were cotransfected into human AD293 cells with HBx-EGFP expression plasmid, they could all reduce the level of HBx mRNA as well as the HBx-EGFP protein. These results suggest that the 10-23 DNAzymes might be used for gene therapy of liver diseases caused by HBV.
Keywords: 10-23 DNAzyme; HBV; HBx; Inhibition; Gene expression
Antiviral activity of Arthrospira-derived spirulan-like substances by Sabine Rechter; Tanja König; Sabrina Auerochs; Stefanie Thulke; Hauke Walter; Heike Dörnenburg; Christian Walter; Manfred Marschall (pp. 197-206).
Natural substances offer interesting pharmacological perspectives for antiviral drug development in regard to broad-spectrum antiviral properties and novel modes of action. In this study we analyzed polysaccharide fractions isolated from Arthrospira platensis. Fractions containing intracellular or extracellular spirulan-like molecules showed a pronounced antiviral activity in the absence of cytotoxic effects. Using specific assays for the quantification of viral replication in vitro, these substances exhibited strong inhibition of human cytomegalovirus, herpes simplex virus type 1, human herpesvirus type 6 and human immunodeficiency virus type 1, while only weak or no inhibition was noted for Epstein-Barr virus and influenza A virus. Considering herpesviruses, antiviral effects were most pronounced when the cells were preincubated with the substances prior to the addition of virus, indicating that antiviral action may be primarily targeted to virus entry. However, an inspection of the inhibition of human cytomegalovirus protein synthesis clearly demonstrated that intracellular steps also contributed to the antiviral effect. In the case of human immunodeficiency virus, inhibition occurred at a stage later than viral entry. Thus, spirulan-like substances possess a marked antiherpesviral and anti-HIVactivity based on different modes of action. Further development of these substances might yield novel candidates of broad-spectrum antiviral drugs.
Keywords: Human pathogenic viruses; Drug development; Spirulan-like substances; Mode of action; Broad-spectrum antiviral activity
Solution conformation of an immunodominant epitope in the hepatitis B virus preS2 surface antigen by Seung-Wook Chi; Do-Hyoung Kim; Jae-Sung Kim; Myung Kyu Lee; Kyou-Hoon Han (pp. 207-215).
We have determined the solution conformation of the major B cell epitope (residues 123–145, adrl23 hereafter) in the preS2 region of hepatitis B virus known to be associated with infection neutralization. The adrl23 shows an “L� shaped helix-turn-helix topology with two β-turns formed by residues Ala130-Asp133 and Asp133-Val136 intervening the N- and C-terminal helices. The N-terminal α-helix consists of residues Ser124-Gln129 whereas the C-terminal 310 helix is formed by residues Val136-Tyr140. The β-turns overlap partially with the putative “conformational� epitope. The overall topology of adrl23 is primarily maintained by hydrophobic interactions involving Phe127, Leu131, Leu132, Val136, and Tyr140 that are clustered on one side of the molecule. An additional hydrophobic stabilization comes from Phe141 that is buried inside the concave side of the molecule. A network of hydrogen bonds formed among Thr125, His128, and Arg137 further contribute to the “boomerang-shaped� architecture of adrl23. The N-terminus of adrl23 is immobile due to a hydrogen bond between the N-terminal amide proton of Asn123 and the hydroxyl oxygen of Thr126. The side chains of Asp133, Arg135, Val136, Leu139, and Tyr140 that were shown to be important for binding to a monoclonal antibody H8 mAb are surface exposed.
Keywords: Abbreviations; HBV; hepatitis B virus; mAb; monoclonal antibody; NMR; nuclear magnetic resonance; RMSD; root mean square deviation; COSY; shift correlation spectroscopy; TOCSY; total COSY; NOESY; nuclear Overhauser enhancement spectroscopy; CSI; chemical shift indexHepatitis B virus; Surface antigen; PreS2; Monoclonal antibody; NMR
Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication by S.P. Ong; B.G.H. Choo; J.J.H. Chu; M.L. Ng (pp. 216-223).
RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.
Keywords: Flavivirus; Envelope protein; Non-structural NS5 protein; siRNA
Antiviral effects of mifepristone on human immunodeficiency virus type-1 (HIV-1): Targeting Vpr and its cellular partner, the glucocorticoid receptor (GR) by Elizabeth A. Schafer; Narasimhan J. Venkatachari; Velpandi Ayyavoo (pp. 224-232).
The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the optimal infection of primary monocytes/macrophages and other non-dividing cells. Vpr interacts with the cellular glucocorticoid receptor (GR) and transactivates the HIV-1 LTR through glucocorticoid response element (GRE), an event that can be blocked by the GR antagonist, mifepristone. Results demonstrated that Vpr-induced transactivation of the HIV-1 LTR was inhibited by mifepristone in a dose-dependent manner by >60% at a 10μM concentration. Infectivity assays using X4 and R5 viruses demonstrated antiviral effects on a dose-dependent regimen of mifepristone. The effects of mifepristone were also tested in latently infected cells that could be activated with extracellular Vpr protein and results indicated specific inhibition of virus reactivation in the presence of this antagonist.
Keywords: HIV-1; Antiviral; Vpr; Mifepristone; Glucocorticoid receptor
StpC-based gene therapy targeting latent reservoirs of HIV-1 by Lorianne Stehouwer Turner; Alexander Y. Tsygankov; Earl E. Henderson (pp. 233-241).
The ability of HIV-1 to form latent reservoirs presents a major obstacle to eradication. One approach to elimination of the latent reservoir is induction therapy, whereby cells harboring latent virus are activated and therefore initiate virus replication. We have constructed a lentiviral vector encoding Herpesvirus saimiri subgroup C saimiri transformation-associated protein (StpC), which has been shown to modulate HIV-1 replication, under the control of a cytomegalovirus promoter in order to determine the ability of StpC to upregulate latent HIV-1. We have included a suicide gene, herpes simplex virus thymidine kinase (TK), under the control of the HIV-1 long terminal repeat (LTR) promoter. We hypothesized that upon StpC expression in latently infected cells induction of virus replication and subsequent production of viral transactivators of the LTR will activate expression of the tk gene, sensitizing the cells to the nucleoside analogue ganciclovir (GCV). Transduction of the latently infected cell line J1.1 resulted in increased virus replication. In the presence of GCV transduced cells exhibited decreased HIV-1 replication, inhibition of cell proliferation, and increased apoptosis. This prototype vector serves as a proof of concept of the utility of gene-based induction agents and suicide genes as a new method for targeting reservoirs of latent HIV-1.
Keywords: HIV-1; Latency; Herpesvirus saimiri; StpC; Suicide gene; Lentiviral vector
Resveratrol suppresses nuclear factor-κB in herpes simplex virus infected cells by Seth A. Faith; Thomas J. Sweet; Erin Bailey; Tristan Booth; John J. Docherty (pp. 242-251).
Resveratrol inhibits herpes simplex virus (HSV) replication by an unknown mechanism. Previously it was suggested that this inhibition may be mediated through a cellular factor essential for HSV replication [Docherty, J.J., Fu, M.M., Stiffler, B.S., Limperos, R.J., Pokabla, C.M., DeLucia, A.L., 1999. Resveratrol inhibition of herpes simplex virus replication. Antivir. Res. 43, 145–155]. After examining numerous cellular factors, we report that resveratrol suppresses NF-kappaB (NF-κB) activation in HSV infected cells. Reports have indicated that HSV activates NF-κB during productive infection and this may be an essential aspect of its replication scheme [Patel, A., Hanson, J., McLean, T.I., Olgiate, J., Hilton, M., Miller, W.E., Bachenheimer, S.L., 1998. Herpes simplex type 1 induction of persistent NF-kappa B nuclear translocation increases the efficiency of virus replication. Virology 247, 212–222; Gregory, D., Hargett, D., Holmes, D., Money, E., Bachenheimer, S.L., 2004. Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-RelA/p65 pathway. J. Virol. 78, 13582–13590]. Electromobility shift assays determined that resveratrol, in a dose dependent and reversible manner, suppressed activation of NF-κB in Vero cells infected with HSV-1, HSV-2 and acyclovir resistant HSV-1. Furthermore, resveratrol did not protect IκBα, a cytoplasmic NF-κB inhibitor, from degradation in HSV-1 infected cells. Immunohistochemical studies demonstrated that RelA/p65, a component of the dimeric NF-κB complex, translocated to the nucleus of HSV-1 infected cells in the presence of resveratrol. Finally, direct effects on viral transcription and DNA synthesis were evaluated. Real-time RT-PCR analysis showed that resveratrol treatment of infected cells resulted in reductions of mRNA for ICP0, ICP4, ICP8 and HSV-1 DNA polymerase by 2.1-, 3.3-, 3.8- and 3.1-fold, respectively. Plus, mRNA for glycoprotein C, an HSV late gene, was completely absent in the presence of resveratrol. Lastly, quantitative PCR showed that resveratrol significantly blocked HSV DNA synthesis. Cumulatively, these data indicate that resveratrol (i) suppresses HSV induced activation of NF-κB within the nucleus and (ii) impairs expression of essential immediate-early, early and late HSV genes and synthesis of viral DNA.
Keywords: Resveratrol; Herpes; NF-κB; Suppression
Antiviral activity of substituted homoisoflavonoids on enteroviruses by Sabrina Tait; Anna Laura Salvati; Nicoletta Desideri; Lucia Fiore (pp. 252-255).
The antiviral activity of homoisoflavonoids, a class of flavonoids, was determined in vitro against a large panel of enteroviruses. The inhibition of viral replication was monitored on BGM (Buffalo Green Monkey) cells, and the concentration required for 50% inhibition (IC50), as well as the selectivity index (SI) were determined. None of the substances were effective against Sabin type 1 poliovirus (PV1), but most of them showed a low cytotoxicity and a marked antiviral activity against Coxsackie virus B1, B3, B4, A9 and echovirus 30.
Keywords: Antivirals; Homoisoflavonoids; Enteroviruses
Erratum to “Pharmacology of current and promising nucleosides for the treatment of human immunodeficiency viruses� [Antiviral Res. 71 (2006) 322–334] by Raymond F. Schinazi; Brenda I. Hernandez-Santiago; Selwyn J. Hurwitz (pp. 256-256).
Nucleoside antiretroviral agents are chiral small molecules that have distinct advantages compared to other classes including long intracellular half-lives, low protein binding, sustained antiviral response when a dose is missed, and ease of chemical manufacture. They mimic natural nucleosides and target a unique but complex viral polymerase that is essential for viral replication. They remain the cornerstone of highly active antiretroviral therapy (HAART) and are usually combined with non-nucleoside reverse transcriptase and protease inhibitors to provide powerful antiviral responses to prevent or delay the emergence of drug-resistant human immunodeficiency virus (HIV). The pharmacological and virological properties of a selected group of nucleoside analogs are described. Some of the newer nucleoside analogs have a high genetic barrier to resistance development. The lessons learned are that each nucleoside analog should be treated as a unique molecule since any structural modification, including a change in the enantiomeric form, can affect metabolism, pharmacokinetics, efficacy, toxicity and resistance profile.
Keywords: Antiviral agents; Pharmacology; Nucleoside analogs; HIV