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Antiviral Research (v.80, #1)

Notes to Authors (pp. co3).

Editorial Board (pp. ii).

Flavivirus methyltransferase: A novel antiviral target by Hongping Dong; Bo Zhang; Pei-Yong Shi (pp. 1-10).

Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2′-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of nonstructural protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5′ end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m⁷GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA→m⁷GpppA-RNA→m⁷GpppAm-RNA, using S-adenosyl-l-methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2′-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2′-O methylation. In the context of virus, West Nile viruses (WNVs) defective in N-7 methylation are non-replicative; however, WNVs defective in 2′-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.

Keywords: Flavivirus NS5; Methyltransferase; Flavivirus replication; Antiviral therapy; West Nile virus; Dengue virus; Yellow fever virus; Japanese encephalitis virus; Tick-borne encephalitis virus

Closing the door on flaviviruses: Entry as a target for antiviral drug design by Rushika Perera; Mansoora Khaliq; Richard J. Kuhn (pp. 11-22).

With the emergence and rapid spread of West Nile virus in the United States since 1999, and the 50–100 million infections per year caused by dengue virus globally, the threat of flaviviruses as re-emerging human pathogens has become a reality. To support the efforts that are currently being pursued to develop effective vaccines against these viruses, researchers are also actively pursuing the development of small molecule compounds that target various aspects of the virus life cycle. Recent advances in the structural characterization of the flaviviruses have provided a strong foundation towards these efforts. These studies have provided the pseudo-atomic structures of virions from several members of the genus as well as atomic resolution structures of several viral proteins. Most importantly, these studies have highlighted specific structural rearrangements that occur within the virion that are necessary for the virus to complete its life cycle. These rearrangements occur when the virus must transition from immature, to mature, to fusion-active states and rely heavily on the conformational flexibility of the envelope (E) protein that forms the outer glycoprotein shell of the virus. Analysis of these conformational changes can suggest promising targets for structure-based antiviral design. For instance, by targeting the flexibility of the E protein, it might be possible to inhibit required rearrangements of this protein and trap the virus in a specific state. This would interfere with a productive flaviviral infection. This review presents a structural perspective of the flavivirus life cycle and focuses on the role of the E protein as an opportune target for structure-based antiviral drug design.

Keywords: Flaviviruses; Dengue virus; West Nile virus; Envelope glycoprotein; Antiviral therapy

The flavivirus polymerase as a target for drug discovery by Hélène Malet; Nicolas Massé; Barbara Selisko; Jean-Louis Romette; Karine Alvarez; Jean Claude Guillemot; Hughes Tolou; Thai Leong Yap; Subash G. Vasudevan; Julien Lescar; Bruno Canard (pp. 23-35).

Flaviviruses are emerging pathogens of increasingly important public health concern in the world. For most flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) neither vaccine nor antiviral treatment is available. The viral RNA-dependent RNA polymerase (RdRp) non-structural protein 5 (NS5) has no equivalent in the host cell and is essential for viral replication. Here, we give an overview of the current knowledge regarding Flavivirus RdRp function and structure as it represents an attractive target for drug design. Flavivirus RdRp exhibits primer-independent activity, thus initiating RNA synthesis de novo. Following initiation, a conformational change must occur to allow the elongation process. Structure-function studies of Flavivirus RdRp are now facilitated by the crystal structures of DENV (serotype 3) and WNV RdRp domains. Both adopt a classic viral RdRp fold and present a closed pre-initiation conformation. The so-called priming loop is thought to provide the initiation platform stabilizing the de novo initiation complex. A zinc-ion binding site at the hinge between two subdomains might be involved in opening up the RdRp structure towards a conformation for elongation. Using two different programs we predicted common potential allosteric inhibitor binding sites on both structures. We also review ongoing approaches of in vitro and cell-based screening programs aiming at the discovery of nucleosidic and non-nucleosidic inhibitors targeting Flavivirus RdRps.

Keywords: Abbreviations; BVDV; bovine viral diarrhea virus; CS; cyclization sequences; DENV; dengue virus; ds; double strand; HCV; hepatitis C virus; HTS; high-throughput screening; MTase; methyltransferase; NI; nucleoside analogue inhibitors; NLS; nuclear localization sequence; NNI; non-nucleoside analogue inhibitors; RdRp; RNA-dependent RNA polymerase; RF; replicative form; RI; replicative intermediate; ss; single strand; tP; triphosphate; TR; terminal regions; WNV; West Nile virus; YFV; yellow fever virusDengue virus; West Nile virus; Flavivirus; RNA-dependent RNA polymerase; De novo; Drug design; Crystal structure

Vector design for liver-specific expression of multiple interfering RNAs that target hepatitis B virus transcripts by Lindsey L. Snyder; Jonathan M. Esser; Catherine J. Pachuk; Laura F. Steel (pp. 36-44).

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence-specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue-specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes.

Keywords: RNAi; Hepatitis B virus; Anti-viral therapy; MicroRNA; RNA pol II promoter; Expression vector

Retinoids inhibit measles virus in vitro via nuclear retinoid receptor signaling pathways by Claire Trottier; Sophie Chabot; Koren K. Mann; Myrian Colombo; Avijit Chatterjee; Wilson H. Miller Jr; Brian J. Ward (pp. 45-53).

Measles virus (MV) infects 30 million children every year, resulting in more than half a million deaths. Vitamin A (retinol) treatment of acute measles can reduce measles-associated mortality by 50–80%. We sought to determine whether or not retinoids can act directly to limit MV output from infected cells. Physiologic concentrations of retinol were found to inhibit MV output in PBMC and a range of cell lines of epithelial and endothelial origin (40–50%). Near complete inhibition of viral output was achieved in some cells/lines treated with all- trans retinoic acid (ATRA) and 9- cis RA (9cRA). Important attenuation of the anti-MV effect of retinoids in R4 cells, a subclone of a retinoid-responsive cell line (NB4) deficient in RAR signaling, demonstrates that this effect is mediated at least in part by nuclear retinoid receptor signaling pathways. Inhibition of MV replication could not be fully explained as a result of retinoid effects on cell differentiation, proliferation or viability, particularly at low retinoid concentrations (1–10nM). These data provide the first evidence that retinoids can directly inhibit MV in vitro, and raise the possibility that retinoids may have similar actions in vivo.

Keywords: Vitamin A; Retinoic acid receptor; Paramyxovirus; Measles virus

Glycoside analogs of β-galactosylceramide, a novel class of small molecule antiviral agents that inhibit HIV-1 entry by Himanshu Garg; Nicholas Francella; Kurissery A. Tony; Line A. Augustine; Joseph J. Barchi Jr.; Jacques Fantini; Anu Puri; David R. Mootoo; Robert Blumenthal (pp. 54-61).

The interaction between HIV gp120 and galactose-containing cell surface glycolipids such as GalCer or Gb₃ is known to facilitate HIV binding to both CD4⁺ as well as CD4⁻ cells. In an effort to develop small molecule HIV-1 entry inhibitors with improved solubility and efficacy, we have synthesized a series of C-glycoside analogs of GalCer and tested their anti HIV-1 activity. The analogs were tested for gp120 binding using a HIV-1 (IIIB) V3-loop specific peptide. Two of the six analogs that interfered with gp120 binding also inhibited HIV Env-mediated cell-to-cell fusion and viral entry in the absence of any significant cytotoxicity. Analogs with two side chains did not show inhibition of fusion and/or infection under identical conditions. The inhibition of virus infection seen by these compounds was not coreceptor dependent, as they inhibited CXCR4, CCR5 as well as dual tropic viruses. These compounds showed inhibition of HIV entry at early steps in viral infection since the compounds were inactive if added post viral entry. Temperature-arrested state experiments showed that the compounds act at the level of virus attachment to the cells likely at a pre-CD4 engagement step. These compounds also showed inhibition of VSV glycoprotein-pseudotyped virus. The results presented here show that the glycoside derivatives of GalCer with simple side chains may serve as a novel class of small molecule HIV-1 entry inhibitors that would be active against a number of HIV isolates as well as other enveloped viruses.

Keywords: HIV-1; Galactosyl Ceramide; Glycosides; Anti HIV; Fusion; Envelope glycoprotein

Anti-herpes simplex virus (HSV-1) activity of oxyresveratrol derived from Thai medicinal plant: Mechanism of action and therapeutic efficacy on cutaneous HSV-1 infection in mice by Taksina Chuanasa; Jurairatana Phromjai; Vimolmas Lipipun; Kittisak Likhitwitayawuid; Mikiko Suzuki; Pornpen Pramyothin; Masao Hattori; Kimiyasu Shiraki (pp. 62-70).

Oxyresveratrol, a major compound purified from Artocarpus lakoocha, a Thai traditional medicinal plant, was evaluated for its mechanism of action and therapeutic efficacy on cutaneous herpes simplex virus (HSV) infection in mice. The inhibitory concentrations for 50% HSV-1 plaque formation of oxyresveratrol, three clinical isolates, thymidine kinase (TK)-deficient and phosphonoacetic acid (PAA)-resistant HSV-1 were 19.8, 23.3, 23.5, 24.8, 25.5 and 21.7μg/ml, respectively. Oxyresveratrol exhibited the inhibitory activity at the early and late phase of viral replication and inhibited the viral replication with pretreatment in one-step growth assay of HSV-1 and HSV-2. Oxyresveratrol inhibited late protein synthesis at 30μg/ml. The combination of oxyresveratrol and acyclovir (ACV) produced synergistic anti-HSV-1 effect, as characterized by the isobologram of plaque inhibition. Mice orally treated with oxyresveratrol (500mg/kg/dose) dose at 8h before and three times daily had significant delay in herpetic skin lesion development ( P<0.05). Topical application of 30% oxyresveratrol ointment five times daily significantly delayed the development of skin lesions and protected mice from death ( P<0.0001).

Keywords: Oxyresveratrol; Acyclovir; Herpes simplex virus (HSV); Synergism; Therapeutic efficacy; Medicinal herb

Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41 by Hiroki Nishikawa; Eiichi Kodama; Ayako Sakakibara; Ayako Fukudome; Kazuki Izumi; Shinya Oishi; Nobutaka Fujii; Masao Matsuoka (pp. 71-76).

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.

Keywords: HIV-1; Gp41; Fusion; ELISA; Screening; Alkaline phosphatase

In vitro susceptibility of sea lion poxvirus to cidofovir by Hendrik H. Nollens; Frances M.D. Gulland; Elliott R. Jacobson; Jorge A. Hernandez; Paul A. Klein; Michael T. Walsh; Richard C. Condit (pp. 77-80).

Parapoxviruses of seals and sea lions are commonly encountered pathogens with zoonotic potential. The antiviral activity of the antiviral compounds isatin-beta-thiosemicarbazone, rifampicin, acyclovir, cidofovir and phosphonoacetic acid against a parapoxvirus (SLPV-1) isolated from a Californian sea lions ( Zalophus californianus) was evaluated. Cidofovir was able to reduce virus-induced cytopathic effect of SLPV-1 in confluent monolayers when used in concentrations greater than 2μg/ml. A decreasing virus yield was observed in the presence of increasing concentrations of cidofovir, which confirmed the ability of cidofovir to inhibit SLPV-1 replication. The in vitro efficacy of cidofovir against SLPV-1 indicates the therapeutic potential of cidofovir for the treatment of infections of humans and pinnipeds with parapoxviruses of seals and sea lions. This study confirms the previously proposed therapeutic potential of cidofovir for the treatment of parapoxvirus infections.

Keywords: Isatin-beta-thiosemicarbazone; Rifampicin; Acyclovir; Cidofovir; Phosphonoacetic acid; Sea lion; Seal; Parapoxvirus; Poxvirus; Treatment

Mutations close to functional motif IV in HSV-1 UL5 helicase that confer resistance to HSV helicase–primase inhibitors, variously affect virus growth rate and pathogenicity by Subhajit Biswas; Laurence S. Tiley; Holger Zimmermann; Alexander Birkmann; Hugh J. Field (pp. 81-85).

Herpes simplex virus (HSV) helicase–primase (HP) is the target for a novel class of antiviral compounds, the helicase–primase inhibitors (HPIs), e.g. BAY 57-1293. Although mutations in herpesviruses conferring resistance to nucleoside analogues are commonly associated with attenuation in vivo, to date, this is not necessarily true for HPIs. HPI-resistant HSV mutants selected in tissue culture are reported to be equally pathogenic compared to parental virus in animal models. Here we demonstrate that a slow-growing HSV-1 mutant, with the BAY 57-1293-resistance mutation Gly352Arg in UL5 helicase, is clearly less virulent than its wild-type parent in a murine zosteriform infection model. This contrasts with published results obtained for a mutant containing a different HPI-resistance substitution (Gly352Val) at the same location, since this mutant was reported to be fully pathogenic. We believe our report to be the first to describe an HPI-resistant HSV-1 mutant, that is markedly less virulent in vivo and slowly growing in tissue culture compared to the parental strain. Another BAY 57-1293-resistant UL5 mutant (Lys356Gln), which showed faster growth characteristics in cell culture, however, was at least equally virulent compared to the parent strain.

Keywords: Herpes simplex virus; BAY 57-1293; Antiviral; Resistance; Mutant

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Antiviral Research (v.80, #1)