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Antiviral Research (v.86, #3)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Marine compounds and their antiviral activities by Jarred Yasuhara-Bell; Yuanan Lu (pp. 231-240).

Available treatments for many infectious diseases are limited. In particular, diseases caused by viral pathogens have demonstrated the need for new medicines, due to the increasing appearance of resistance to these available treatments. Thousands of novel compounds have been isolated from various marine organisms and tested for pharmacological properties, many of which are commercially available. The screening of natural products derived from marine species for antiviral activity has yielded a considerable number of active crude aqueous and organic solvent extracts. Today, over 40 compounds are commercially available in pharmacological markets, including alternative antiviral medicines or those being tested as potential antiviral drugs. Many more are being tested as potential antiviral drugs at the preclinical and clinical stages. The growing interest in marine-derived antiviral compounds, along with the development of new technology in marine cultures and extraction, will significantly expedite the current exploration of the marine environment for compounds with significant pharmacological applications, which will continue to be a promising strategy and new trend for modern medicine.

Keywords: Marine microorganisms; Marine compounds; Antiviral activities

Simvastatin potentiates the anti-hepatitis B virus activity of FDA-approved nucleoside analogue inhibitors in vitro by Ted Bader; Brent Korba (pp. 241-245).

Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. We report that a particular statin, simvastatin (SIM), exhibits strong in vitro anti-HBV activity. Moreover, a combination of SIM with each of the individual nucleos(t)ide analogues lamivudine (LMV), adefovir (ADV), tenofovir (TEN) and entecavir (ETV), showed synergistic antiviral activity. Combination drug treatments were performed in the HepG2.2.15 cell line. Compound combinations were centered on a mixture designed to deliver approximately equipotent (not necessarily equimolar) concentrations of each agent, based on the ninety percent viral inhibition monotherapy values. SIM interacted favorably with all four licensed anti-HBV nucleos(t)ide analogues, especially at molar ratios that approximate combinations likely to be used clinically. As the relative concentration of SIM was raised to an excess, the overall favorability of the interactions progressively increased.SIM displayed about equal degrees of synergy with ADV and TDF. The highest degree of synergy was observed at the 300:1 combination of SIM with ETV. Interactions with LMV were the least favorable. The in vitro potential shown here may greatly augment anti-HBV therapy clinically.

Keywords: Abbreviations; HBV; hepatitis B virus; HCC; hepatocellular carcinoma; IFN; pegylated alfa-interferons; NA; nucleoside analogue; HBeAg; hepatitis B “e” antigen; ALT; alanine aminotransferase; HBV DNA; hepatitis B virus deoxyribonucleic acid; DNA; deoxyribonucleic acid; FDA; Food and Drug Administration (USA); HMG CoA; 3-hydroxy-3-methylglutaryl-coenzyme A; RNA; ribonucleic acid; HBsAg; hepatitis B surface antigen; SIM; simvastatin; LMV; lamivudine; ADV; adefovir; TDF; tenofovir; ETV; entecavir; NIAID; National Institute of Allergy and Infectious Diseases (USA); HBcAg; hepatitis B core antigen; EC; ₅₀; fifty percent viral inhibition; EC; ₉₀; ninety percent viral inhibition; CC50; 50% cell cytotoxicity; SI; selectivity index; AST; aspartate aminotransferase; HBx; “x” protein for hepatitis B virusHepatitis B virus; Simvastatin; Mevalonate; Entecavir; Tenofovir

Phenotypic and genotypic characterization of acyclovir-resistant clinical isolates of herpes simplex virus by A. Sauerbrei; S. Deinhardt; R. Zell; P. Wutzler (pp. 246-252).

Sixteen herpes simplex virus type 1 (HSV-1) and four type 2 (HSV-2) isolates resistant to acyclovir (ACV) were characterized retrospectively for drug resistance. Phenotypic testing was performed by means of tetrazolium reduction assay and genotypic analysis was carried out by sequencing of thymidine kinase (TK) and DNA-polymerase (pol) genes. All strains were characterized as cross-resistant to penciclovir, brivudin and susceptible to cidofovir. In addition, three strains were resistant to foscarnet. Genotypic analysis revealed two to seven non-synonymous mutations in the TK gene of HSV-1 and one to seven non-synonymous mutations in the DNA pol gene of HSV-1 and 2 associated with the gene polymorphism. Seventeen strains contained at least one non-synonymous resistant-related mutation in the TK gene and three strains, which were additionally foscarnet-resistant, revealed one resistance-associated mutation in the DNA pol gene. In most strains, resistant-related mutations in TK gene represented frameshift mutations and single non-synonymous nucleotide substitutions of conserved gene regions. However, numerous amino acid changes could not be interpreted clearly as accounting for resistance. In conclusion, further studies, e.g. site-directed mutagenesis experiments are required to characterize mutations of the TK and DNA pol genes in ACV-resistant viral strains as part of viral gene polymorphism or as cause of drug resistance.

Keywords: Herpes simplex virus; Phenotypic resistance; Genotypic resistance; Thymidine kinase; DNA polymerase

The rare HIV-1 gp41 mutations 43T and 50V elevate enfuvirtide resistance levels of common enfuvirtide resistance mutations that did not impact susceptibility to sifuvirtide by Kris Covens; Sarah Megens; Nathalie Dekeersmaeker; Kabamba Kabeya; Jan Balzarini; Stéphane De Wit; Anne-Mieke Vandamme; Kristel Van Laethem (pp. 253-260).

Mutations that are selected at low frequency and/or reside outside the enfuvirtide target region, amino acid 36–45 of gp41, might still be important determinants for drug resistance. This study aimed to investigate the phenotypic impact against enfuvirtide and sifuvirtide of uncharacterized gp41 mutations 42G, 43T and 50V, selected in patients failing enfuvirtide-containing regimens. As single mutations, neither 42G, 43T nor 50V conferred resistance to enfuvirtide. However, 50V increased slightly resistance levels for 36D, 38M, 43D or 43T as did 43T for 38M. All mutants displayed a reduced replication capacity, except 42S, 50V and 36D±50V. None of the mutants displayed resistance to the next-generation fusion inhibitor sifuvirtide. This study highlights the necessity to confirm the in vitro effect of infrequently selected mutations as 42G was not associated with enfuvirtide resistance whereas 43T and 50V should be considered as secondary enfuvirtide resistance mutations.

Keywords: Phenotypic effect; Infrequent mutations; Enfuvirtide; Sifuvirtide

Efficacy of 2′-C-methylcytidine against yellow fever virus in cell culture and in a hamster model by Justin G. Julander; Ashok K. Jha; Jung-Ae Choi; Kie-Hoon Jung; Donald F. Smee; John D. Morrey; Chung K. Chu (pp. 261-267).

Yellow fever virus (YFV) continues to cause outbreaks of disease in endemic areas where vaccine is underutilized. Due to the effectiveness of the vaccine, antiviral development solely for the treatment of YFV is not feasible, but antivirals that are effective in the treatment of related viral diseases may be characterized for potential use against YFV as a secondary indication disease. 2′-C-methylcytidine (2′-C-MeC), a compound active against hepatitis C virus, was found to have activity against the 17D vaccine strain of YFV in cell culture (EC₉₀=0.32μg/ml, SI=141). This compound was effective when added as late as 16h after virus challenge of Vero cells. When administered to YFV-infected hamsters 4h prior to virus challenge at a dose as low as 80mg/kg/d, 2′-C-MeC was effective in significantly improving survival and other disease parameters (weight change, serum ALT, and liver virus titers). Disease was improved when compound was administered beginning as late as 3 d post-virus infection. Broadly active antiviral compounds, such as 2′-C-MeC, represent potential for the development of compounds active against related viruses for the treatment of YFV.

Keywords: Yellow fever virus; Hamster; 2′-C-methylcytidine; Treatment; Antiviral

Slow binding–tight binding interaction between benzimidazol-2-one inhibitors and HIV-1 reverse transcriptase containing the lysine 103 to asparagine mutation by Alberta Samuele; Emmanuele Crespan; Samanta Vitellaro; Anna-Maria Monforte; Patrizia Logoteta; Alba Chimirri; Giovanni Maga (pp. 268-275).

Novel benzimidazol-2-one non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been recently identified, through rational structure-based molecular modeling and docking approaches, as highly effective inhibitors of the wild type and drug-resistant HIV-1 reverse transcriptase (RT). These compounds also showed potent anti-HIV activities against viral strains, superior to the clinically approved NNRTI efavirenz. However, they were still of limited efficacy towards the K103N mutant. Here we report a detailed enzymatic analysis elucidating the molecular mechanism of interaction between benzimidazol-2-one derivatives and the K103N mutant RT. The loss of potency of these molecules towards the K103N RT was specifically due to a reduction of their association rate to the enzyme. Unexpectedly, these compounds showed a strongly reduced dissociation rate from the K103N mutant, as compared to the wild type enzyme, suggesting that, once occupied by the drug, the mutated binding site could achieve a more stable interaction with these molecules. The characterization of this slow binding–tight binding mutant-specific mechanism of interaction may pave the way to the design of more effective new generation benzimidazol-2-one NNRTIs with promising drug resistant profile and minimal toxicity.

Keywords: Antiretroviral therapy; Non-nucleoside HIV-1 reverse transcriptase inhibitors; Drug resistant HIV-1 mutant; Benzimidazolones

Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice by Jenny Rowe; Rebecca J. Greenblatt; Dongmei Liu; Jennifer F. Moffat (pp. 276-285).

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400μM) and phosphonoacetic acid (PAA, 1mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase),l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC₉₉ doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7mg/kg/day) was as effective as PAA (36mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV.

Keywords: Varicella-zoster virus; Antiviral; Cyclin dependent kinase; Roscovitine; Kinase inhibitor; Skin organ culture; SCID-Hu mouse

Lipophile-conjugated sulfated oligosaccharides as novel microbicides against HIV-1 by Joanna Said; Edward Trybala; Elin Andersson; Ken Johnstone; Ligong Liu; Norbert Wimmer; Vito Ferro; Tomas Bergström (pp. 286-295).

With the aim of providing compounds suitable for further development as microbicides active against human immunodeficiency virus 1 (HIV-1) a library containing 37 lipophile-conjugated sulfated oligosaccharides was screened for antiviral and virucidal activity against this virus. Four highly active compounds had low drug inhibition concentrations (IC₅₀) for HIV-1 and inactivated viral particles, suggestive of virucidal properties. Two of these compounds comprising a sulfated tetrasaccharide linked to a cholestanol group by a glycosidic bond, showed low toxicity and high selectivity indices. The two compounds were active both against CCR5 and dual-tropic CCR5/CXCR4 clinical HIV-1 isolates. Since herpes simplex virus type 2 (HSV-2) may be a cofactor for HIV-1 infection, the virucidal effect of the compounds was demonstrated against both viruses when mixed and incubated together on permissive cells. Incubation of compounds with serum, and to a lesser degree, cervical secretions, reduced the HIV-1 inactivating capacity, which suggests the need for molecular modification to reduce host protein binding. Considering the virucidal effect and low toxicity, these sulfated oligosaccharides with lipophilic tails may offer new possibilities of microbicide development.

Keywords: HIV-1; HSV-2; Microbicides; Virucidal activity

Dengue virus replicons: Production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity by Nicolas Massé; Andrew Davidson; François Ferron; Karine Alvarez; Mike Jacobs; Jean-Louis Romette; Bruno Canard; Jean-Claude Guillemot (pp. 296-305).

The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood. In this study we produced a novel dengue virus type 2 (DV2) subgenomic replicon that expresses a fusion protein comprised of Enhanced Green Fluorescent Protein (EGFP) and Puromycin N-Acetyltransferase (PAC). We successfully established BHK, COS and Huh7 cell lines that stably expressed the DV2 replicon. Using EGFP as a reporter of DV replication complex activity, we set up a new HTS assay. The assay was validated using the inhibitor ribavirin, confirmed by flow cytometry analysis and the analysis of NS5 expression by Western-blot analysis. In order to develop a system to test antivirals against the NS5 proteins of all four DV serotypes in a similar cellular environment, the replicon was further modified, to allow easy exchange of the NS5 gene between DV serotypes. As proof of principle, a chimeric replicon in which the DV2 NS5 gene was substituted with that of DV type 3 was stably expressed in BHK cells and used in ribavirin inhibition studies. The assays described in this study will greatly facilitate DV drug discovery by serving as primary or complementary screening. The approach should be applicable to the development of fluorescent cell-based HTS assays for other flaviviruses, and useful for the study of many aspects of DV, including viral replication and pathogenesis.

Keywords: Abbreviations; DV; dengue virus; DV2; DV serotype 2; DV3; DV serotype 3; EGFP; Enhanced Green Fluorescent Protein; HTS; high throughput screening; IRES; internal ribosome entry site; MTase; methyltransferase; NS5; non-structural protein 5; PAC; Puromycin N-Acetyltransferase; RdRp; RNA-dependent RNA polymeraseDengue virus; Replicon; HTS; NS5; Antiviral

Antiviral activity of selected antimicrobial peptides against vaccinia virus by Ketha V.K. Mohan; Shilpakala Sainath Rao; Chintamani D. Atreya (pp. 306-311).

Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1–PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10–30-fold reduction. The EC₅₀ values of PD3, PD4 and RW3 were found to be 40μg/ml, 50μg/ml and 6.5μM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90–100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents.

Keywords: AMPs; RW peptides; Platelet-derived peptides; Microbicidal; pfu

The antiretroviral potency of emtricitabine is approximately 3-fold higher compared to lamivudine in dual human immunodeficiency virus type 1 infection/competition experiments in vitro by Daniel Drogan; Pia Rauch; Daniel Hoffmann; Hauke Walter; Karin J. Metzner (pp. 312-315).

The increasing number of antiretroviral drugs leads to mounting possibilities of combinations for the antiretroviral therapy (ART) of HIV-1 infected patients. Thus, it is of interest to determine the most potent combination of antiretroviral drugs for the first ART to delay the development of drug resistance. We have investigated the differences in the inhibitory potencies of the nucleoside reverse transcriptase inhibitors (NRTI) lamivudine (3TC) and emtricitabine (FTC) using an in vitro model based on simultaneous infection of T cells with drug-sensitive and drug-resistant viruses. Changes of frequencies in these virus populations have been measured by allele-specific real-time PCR allowing simultaneous quantification of different HIV-1 variants in the same sample. We show that the suppression of drug-sensitive viruses is significantly enhanced by FTC compared to 3TC. Mathematical modeling of the distinct rates of suppression of drug-sensitive viruses revealed an approximately 3-fold higher antiretroviral potency for FTC compared to 3TC.

Keywords: HIV-1; Drug resistance; M184V; 3TC; FTC; Dual HIV-1 infection/competition model

Conservation of HHV-6 DNA polymerase processivity factor sequence and predicted structure suggests it as a target for antiviral development by Pascale Bonnafous; Moira Verbelen; Stéphanie Petrella; Claire Deback; Agnès Gautheret-Dejean; David Boutolleau; Lieve Naesens; Henri Agut (pp. 316-319).

The replication of human herpesvirus-6 (HHV-6) DNA is catalyzed by the viral DNA polymerase pU38 and the processivity factor pU27 which stabilizes the enzyme on the DNA template. The genetic polymorphism of pU27 among 46 clinical strains of HHV-6 variant A or B and four strains resistant to antivirals was investigated. Overall, 28 amino acid changes (7.6%) and a two-amino acid deletion were identified among the 368 residues of pU27, when using the U1102 (variant A) sequence as the reference. Eleven amino acid changes (3.0%) specifically differentiated both variants. The median intravariant amino acid variability was 1.2% and 0.3% for A and B, respectively. Except for a single change, the pU27 sequence of multi-drug resistant HHV-6 strains was also conserved. Structural models of pU27 for variants A and B were derived from that of the human cytomegalovirus homologue pUL44, and showed either identical or very similar residues in the regions interacting with viral DNA polymerase and viral DNA molecule. As pU27 is both highly conserved and essential for viral replication, it might constitute an interesting target for antiviral chemotherapy.

Keywords: HHV-6; U27; DNA polymerase; Antiviral

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Antiviral Research (v.86, #3)