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Antiviral Research (v.87, #1)

Editorial Board (pp. ii).
Notes to Authors (pp. co3).

A small molecule inhibits HCV replication and alters NS4B's subcellular distribution by Paul D. Bryson; Nam-Joon Cho; Shirit Einav; Choongho Lee; Vincent Tai; Jill Bechtel; Mohan Sivaraja; Chris Roberts; Uli Schmitz; Jeffrey S. Glenn (pp. 1-8).
Hepatitis C Virus (HCV) is a leading cause of liver disease and represents a significant public health challenge. Treatments for this disease are inadequate and improved antiviral therapies are necessary. Several such antivirals are in development, most of which target the well-characterized NS3 protease or the NS5B polymerase. In contrast, the nonstructural 4B (NS4B) protein, though essential for HCV RNA replication, has been the subject of few pharmacological studies. One of the functions ascribed to this protein is the ability to form intracellular membrane-associated foci (MAF), which are believed to be related to the sites of viral replication. Here, we report the identification of a small molecule that inhibits HCV replication and disrupts the organization of these MAF. Genetic analysis links the compound's mode of action to the NS4B gene product, and transient transfections of NS4B-GFP demonstrate that treatment with this compound can lead to the formation of novel elongated assemblies of NS4B. Furthermore, an in vitro dynamic light scattering assay provides evidence that the second amphipathic helix of NS4B may be the target of the drug. Our results demonstrate that this molecule represents a new potential class of HCV inhibitors and also provides us with a useful tool for studying the HCV life cycle.

Keywords: HCV; Inhibitor; NS4B; Amphipathic helix


Hijacking hepatitis C viral replication with a non-coding replicative RNA by Juliette Bitard; Gaëlle Chognard; Estelle Dumas; Julie Rumi; Cyril Masante; Kathleen Mahias; Thérèse Astier-Gin; Michel Ventura (pp. 9-15).
The current treatments used against RNA viruses have a limited efficacy and are often hampered by the induction of side-effects. The specific delivery of antiviral proteins in infected cells should increase their efficiency and reduce their impact on healthy cells. Here, we describe the development of a new approach which takes advantage of the viral replication machinery to specifically target the antiviral protein expression to the infected cells. The strategy is based on the delivery of a non-coding (−)RNA carrying the structures required for the binding of the viral replication complex and the complementary sequence of an antiviral gene. The viral replication complex replicates the (−)RNA similarly to the viral genome to give a coding (+)RNA from which the antiviral protein will be expressed. As non-infected cells do not express the replication complex, this specific machinery can be used to target virus-infected cells without affecting healthy cells. We show that this approach can be successfully applied to the hepatitis C virus. In both replicon-harboring cells (genotype 1b) and JFH-1 infected cells (genotype 2a), nrRNAs induced a strong decrease in genomic RNA and viral protein NS5A. These effects were correlated with a strong activation of several interferon-stimulating genes.

Keywords: HCV replication; Antiviral strategyAbbreviations; HCV; hepatitis C virus; RdRp; RNA-dependent RNA polymerase; RC; replication complex; UTR; untranslated region


Detection of the rapid emergence of the H275Y mutation associated with oseltamivir resistance in severe pandemic influenza virus A/H1N1 09 infections by Bin Wang; Dominic E. Dwyer; Christopher C. Blyth; Maly Soedjono; Haijing Shi; Alison Kesson; Mala Ratnamohan; Ken McPhie; Anthony L. Cunningham; Nitin K. Saksena (pp. 16-21).
In 2009 a new swine-origin influenza virus A/H1N1 (A/H1N1 09) emerged, causing the century's first pandemic. Most isolates of the new A/H1N1 09 virus are susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance has been detected. Using rolling circle amplification (RCA) technology, 96 A/H1N1 09-specific RT-PCR positive clinical samples collected from 80 oseltamivir-treated and untreated patients were screened for the presence of the H275Y mutation. Samples positive for 275Y mutation by RCA were cloned and sequenced for confirmation. From 25 patients who had been treated with oseltamivir and remained A/H1N1 09 RT-PCR positive, we identified three (12%) individuals with the H275Y mutation: one immuno-suppressed adult, one immuno-competent adult and one child. Samples collected at multiple time points from the two adults showed a switch from wild-type oseltamivir-sensitive 275H to oseltamivir-resistant 275Y population after 9 days of treatment. The child had the 275Y mutation detected after being persistently A/H1N1 09 RT-PCR positive while receiving oseltamivir treatment. Resistance was not detected in 17 pre-treatment samples and 54 A/H1N1 09 RT-PCR positive outpatients. RCA demonstrates the rapid emergence of the H275Y resistance mutation in individuals with severe A/H1N1 09 infection receiving neuraminidase inhibitors. Rapid detection of oseltamivir resistance in severe infection is essential for patients to receive maximum therapeutic benefit. In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in newly emerging strains should be a priority.

Keywords: Influenza; A/H1N1 09; Oseltamivir resistance; 275Y; Immuno-suppressed; Rolling Circle; Amplification


Drug-resistant mutation patterns in CRF01_AE cases that failed d4T+3TC+nevirapine fixed-dosed, combination treatment: Follow-up study from the Lampang cohort by Siriphan Saeng-aroon; Naho Tsuchiya; Wattana Auwanit; Panasda Isarangura Na Ayuthaya; Panita Pathipvanich; Pathom Sawanpanyalert; Archawin Rojanawiwat; Mari Kannagi; Koya Ariyoshi; Wataru Sugiura (pp. 22-29).
HIV/AIDS patients are treated in Thailand's national antiretroviral treatment (ART) program with a generic combination tablet of stavudine, lamivudine, and nevirapine (GPOvir). To determine GPOvir-resistant mutations, HIV-1 sequences of 59 GPOvir-failure cases from the Lampang cohort were compared with sequences from 76 randomly selected ART-naïve cases. The GPOvir-failure cases had not only known stavudine-, lamivudine- and nevirapine-resistant mutations, but also V118I, G196E, and H221Y. Among the 59 GPOvir-failure cases, 29 were ART-naïve prior to GPOvir (naïve group), and 30 had previous ART (exposed group). To clarify the effect of previous ART in drug-resistant acquisition pathways, naïve and exposed groups were compared. The exposed group had predominantly thymidine analogue-related mutations, whereas the naïve group had a higher prevalence of Q151M and K103N mutations. M184V lamivudine resistance was most frequent in both naïve and exposed groups. To identify which mutations in CRF01_AE pol were polymorphisms, the connection and RNase domains were also analyzed. CRF01_AE-specific polymorphisms were found in 19 residues, and GPOvir-failure cases had significantly higher frequency of N348I, E399D, P537S, and I542M. Our results expand identification of mutations in CRF01_AE pol that are polymorphisms by also analyzing the connection and RNase H domains.

Keywords: CRF01_AE; Polymorphism; Drug resistance; Connection domain; RNase H; GPOvir


BSA conjugates bearing multiple copies of the basic domain of HIV-1 Tat: Prototype for the development of multitarget inhibitors of extracellular Tat by Antonella Bugatti; Paola Chiodelli; Joseph Rosenbluh; Abraham Loyter; Marco Rusnati (pp. 30-39).
The transactivating factor (Tat) of HIV-1 is involved in AIDS progression and associated pathologies. Tat possesses a basic amino acid sequence implicated in heparan sulfate proteoglycan (HSPG)-mediated internalization, nuclear localization and transactivation by Tat and in the interaction of Tat with integrins and with the vascular endothelial growth factor receptor 2 (KDR) (kinase insert domain receptor). A BSA conjugate bearing an average of four copies of a peptide representing the basic domain/nuclear localization signal of Tat (BSA-Tat-NLS) inhibits transactivation by Tat exogenously added to cells but not by Tat endogenously produced after cell transfection with a tat cDNA, indicating that BSA-Tat-NLS does not interfere with Tat at an intracellular level. Surface plasmon resonance (SPR) experiments revealed that BSA-Tat-NLS binds to the HSPG analogue heparin. Accordingly, BSA-Tat-NLS binds to HSPGs of HL3T1 cell surface and inhibits HSPG-dependent Tat internalization. BSA-Tat-NLS retains its inhibitory potential when pre-incubated with HL3T1 cells before Tat administration, possibly by masking cell-surface HSPGs thus preventing Tat binding and internalization. SPR experiments revealed that BSA-Tat-NLS binds also to integrin αvβ3 and KDR. Accordingly, it inhibits pro-angiogenic endothelial cell adhesion to Tat and motogenesis. In conclusion, BSA-Tat-NLS binds/masks three different cell-surface receptors of Tat inhibiting different biological activities. These data point to BSA-Tat-NLS as a prototype for the development of Tat-antagonists endowed with a multitargeted mechanism of action.

Keywords: AIDS; HIV; Transactivation; Heparan sulfate proteoglycans; Integrins; Vascular endothelial growth factor receptor 2; Endothelial cells; Angiogenesis


Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine by Kazuto Tajiri; Tatsuhiko Ozawa; Aishun Jin; Yoshiharu Tokimitsu; Masami Minemura; Hiroyuki Kishi; Toshiro Sugiyama; Atsushi Muraguchi (pp. 40-49).
Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.

Keywords: Hepatitis B virus (HBV); Cell-microarray system; Hepatitis B surface antigen (HBsAg); Monoclonal antibody; Neutralization; Vaccination


Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals by Licui Wang; Qiuxia Fu; Yafeng Dong; Yong Zhou; Shuaizheng Jia; Juan Du; Fang Zhao; Yingli Wang; Xiaohui Wang; Jianchun Peng; Shuhua Yang; Linsheng Zhan (pp. 50-56).
The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANluc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NS5A/B. We proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANluc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-α. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals.

Keywords: NS3/4A protease; Split firefly luciferase complementation strategy; In vivo imaging


Monitoring hepatitis C virus (HCV) RNA-dependent RNA polymerase oligomerization by a FRET-based in vitro system by Itxaso Bellón-Echeverría; Alberto José López-Jiménez; Pilar Clemente-Casares; Antonio Mas (pp. 57-66).
Hepatitis C virus (HCV) is a positive-strand RNA virus ((+)RNA) that replicates its genome in replication complexes (RC) associated to endoplasmic reticulum (ER)-derived micro-vesicles. One key protein in these complexes is NS5B, a viral enzyme that shows the RNA binding and RNA-dependent RNA polymerase (RdRp) activities. For this reason, NS5B protein has become one of the most important targets for designing new antiviral therapy compounds. Recently, it has been demonstrated that NS5B interacts itself forming oligomers, and mutations that disrupt these interactions are lethal for polymerase function. Therefore, NS5B oligomerization could be a new target for the design of anti-HCV compounds. In this study we describe a new accurate method to analyze NS5B–NS5B interactions by using Förster-resonance-energy transfer (FRET). This method allows analyses of the conditions, mainly ionic strength, driving the interactions between NS5B-cyan and NS5B-citrine constructs. Experiments using different combinations of point mutants rendered FRET values from zero to around 100%, suggesting the geometry of the interaction. Finally, oligomerization experiments in the presence of non-nucleoside inhibitor (NNI) PF-254027 gave a statistically significant reduction in the FRET signal, suggesting a new connection between NS5B oligomerization and NNI binding.

Keywords: RNA polymerase; HCV; Oligomerization; Inhibitors; FRET


Effects of therapy using a helicase–primase inhibitor (HPI) in mice infected with deliberate mixtures of wild-type HSV-1 and an HPI-resistant UL5 mutant by Soumi Sukla; Subhajit Biswas; Alexander Birkmann; Peter Lischka; Helga Ruebsamen-Schaeff; Holger Zimmermann; Hugh J. Field (pp. 67-73).
Point mutations in the HSV-1 UL5 (helicase) gene confer resistance to helicase–primase inhibitors (HPIs), e.g. BAY 57-1293. Such mutations normally occur at a frequency of ≤10−6PFU. However, individual HSV-1 laboratory strains and some clinical isolates contained resistance mutations (e.g. UL5: Lys356Asn) at 10−4PFU. To address the possibility that pre-existing mutants at high frequency might have an impact on therapy using HPIs, deliberate mixtures were prepared to contain the SC16 UL5: Lys356Asn mutant in SC16 wild-type in the proportion of 1/500 or 1/50PFU. Mice were infected in the neck-skin with 5×104PFU/mouse of wt alone, mutant alone, or the respective mixture. The mutant could not be detected in infectious virus yields from mice inoculated with the 1/500 mixture. However, resistant mutant was recovered from some treated mice inoculated with the 1/50 mixture. All mice inoculated with mixtures remained responsive to BAY 57-1293-therapy with no increase in clinical signs compared to treatment of wt-infected mice.

Keywords: HSV; Antivirals; Helicase–primase inhibitor; Antiviral resistance; Murine infection model; BAY 57-1293


Naturally occurring hepatitis B virus (HBV) variants with primary resistance to antiviral therapy and S-mutants with potential primary resistance to adefovir in Argentina by María L. Cuestas; Cintia W. Rivero; María L. Minassian; Amalia I. Castillo; Emiliano A. Gentile; Julieta Trinks; Liliana León; Graciela Daleoso; Bernardo Frider; Carol Lezama; Marcela Galoppo; Gisela Giacove; Verónica L. Mathet; José R. Oubiña (pp. 74-77).
Hepatitis B virus (HBV) variants may either emerge in patients with chronic hepatitis B (CHB) as a result of positive selection pressure exerted by their own immune response, or during therapy with nucleos(t)ide analogues (NAs). Naturally occurring HBV variants with primary antiviral resistance are rarely observed. The aim of this study was to retrospectively analyze the (eventual) circulation of HBV variants with natural resistance to NAs currently used as therapy for CHB in Argentina. This study reports 13 cases of CHB-infected patients with natural antiviral resistance to at least one NA. Five of them were also carriers of S-variants that might escape the humoral immune system recognition with potential resistance to adefovir. In addition to the already reported A2 HBV subgenotype association to NAs natural resistance, E and F genotypes association to such resistance is described for the first time. These findings suggest that sequence analysis of the HBV reverse transcriptase might be an essential tool before starting antiviral therapy, in order to choose the proper NAs for optimizing the therapeutic management of chronically infected patients. Moreover, the circulation and transmission of S-mutants with resistance to such antiviral drugs should be of public health concern as they may represent an additional risk for the community.

Keywords: HBV; Antivirals; Naturally occurring resistance; S-mutants; Argentina


A novel nucleoside analog, 1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR), exhibits efficacy against a broad range of flaviviruses in vitro by Michael McDowell; Sarah R. Gonzales; Sidath C. Kumarapperuma; Marjan Jeselnik; Jeffrey B. Arterburn; Kathryn A. Hanley (pp. 78-80).
Antiviral therapies are urgently needed to control emerging flaviviruses such as dengue, West Nile, and yellow fever. Ribavirin (RBV) has shown activity against flaviviruses in cultured cells, but efficacy in animal models has generally been poor. In a preliminary screen of novel, synthetic 1-β-d-ribofuranosyl-azole analogs, two compounds, 1-β-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR) and 1-β-d-ribofuranosyl-4-ethynyl-[1,3]imidazole (IM18), significantly reduced the replication of dengue virus serotype 2 (DENV-2) in cultured Vero cells. In the current study we demonstrated that the effective concentration 50 (EC50) of ETAR for DENV-2 is substantially lower than both IM18 and RBV. Moreover, ETAR reduced the replication of five additional flaviviruses, including DENV serotypes 1, 3 and 4, Langat virus and Modoc virus, ≥1000-fold relative to untreated controls. Addition of exogenous guanosine to DENV-2 infected cells negated the antiviral effects of both RBV and ETAR, indicating that GTP depletion is a major mechanism of action for both drugs. ETAR represents a promising drug candidate for the treatment of flavivirus infections.

Keywords: Abbreviations; ETAR; 1-β-; d; -ribofuranosyl-3-ethynyl-[1,2,4]triazole; IM18; 1-β-; d; -ribofuranosyl-4-ethynyl-[1,3]imidazole; DENV; dengue virus; LGTV; Langat virus; MODV; Modoc virus; WNV; West Nile virus; RBV; ribavirinDengue virus; Ribavirin; Antiviral; Nucleoside analog; ETAR; Flavivirus


Humanized antibodies with broad-spectrum neutralization to avian influenza virus H5N1 by Yingwei Chen; Wenxin Luo; Wai Lan Wu; Zhong Fang; Lin Xia; Xun Gui; Yixin Chen; Honglin Chen; J. Wai-Kuo Shih; Ningshao Xia (pp. 81-84).
Hemagglutinin (HA), the major antigen on the surface of influenza viruses, is the primary target for neutralizing antibodies and vaccine design. However, frequent mutations in this gene allow the virus to evade host immune responses and conventional prophylaxis and treatment. In this report, we humanized 4D1 and 10F7 mouse monoclonal antibodies (mAbs) that, we had previously shown to display broad-spectrum neutralization to avian H5N1 virus. The genes of variable (V) regions of 4D1 and 10F7 mAbs were combined with constant region of human antibody to construct the chimeric antibodies (cAbs). The results of hemagglutinin inhibition (HI) and neutralization assays showed that 4D1 and 10F7 cAbs were functional and retained broad-spectrum reactivity. Antibody competitive ELISA and affinity tests indicated that the cAbs recognized the same epitope as the parent mAbs with similar affinity. In animal experiments, the 10F7 cAb showed full protection against lethal challenge of highly virulent avian H5N1 virus, A/BH Goose/QH/15C/2005, in all infected mice. These humanized broad-spectrum antibodies may be potentially important for the control of both current and future antigenic variants of H5N1 virus.

Keywords: H5N1; Humanized antibody; Broad-spectrum neutralization
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