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Antiviral Research (v.98, #1)

Inside Back Cover: Notes to Authors (pp. ibc).

Prelim II: Editorial Board (pp. ii).

Equal efficacy between the two types of peginterferon on HCV: A matter of relapse? by Etienne Brochot; Eric Nguyen-Khac; Gilles Duverlie (pp. 1-3).

► Equivalent results in terms of sustained virologic response between the two types of peginterferon alfa (2a and 2b). ► We analyzed studies comparing the two types of peginterferon with data concerning relapse (8538 patients). ► Peginterferon 2a is associated with a higher relapse rate. ► Pharmacokinetics characteristics can explain these data.Two types of peginterferon alfa (2a and 2b) are available for the treatment of chronic hepatitis C genotype 1 or non-1. Comparative studies of bitherapy suggest equivalent results in terms of sustained virologic response for the two types of interferon possibly with a slight advantage in favor of type 2a. However, these studies report only limited data concerning relapse.We analyzed studies comparing the two types of peginterferon with data concerning relapse. In the 6 studies examined (8538 patients), peginterferon 2a was clearly associated with a much higher rate of end of treatment response but also a higher relapse rate than type 2b (29.3% vs 21.1%; relative risk of relapse with peginterferon 2a:1.54 [1.35–1.76], p=0.0024). These data are highlighted in overweight patients. We tried to explain these differences between these two types of interferon by discussing in terms of pharmacokinetics.Peginterferon remains the cornerstone of HCV treatment and must be carefully chosen on the basis of the patient’s history and cofactors. These data are important and must be considered in the era of DAA.

Keywords: HCV therapy; Pegylated interferon; HCV relapse

Enhanced Influenza VLP vaccines comprising matrix-2 ectodomain and nucleoprotein epitopes protects mice from lethal challenge by Xiao Gao; WenJuan Wang; YuFeng Li; ShaoGeng Zhang; YueQiang Duan; Li Xing; ZhongPeng Zhao; PeiRui Zhang; ZhiWei Li; RuiSheng Li; Xiliang Wang; PengHui Yang (pp. 4-11).

► The 3M2e-NP-HBc VLP expressing the M2e and nucleoprotein (NP) epitopes were generated. ► The VLP induced robust M2e specific antibodies and cellular immune response in mice. ► The VLP protected mice from lethal pandemic 2009 H1N1 and HPAI H5N1 virus challenge.The matrix protein 2ectodomain (M2e) of the influenza A virus is a rational target antigen candidate for the development of a universal influenza virus-like particle (VLP) vaccine. In this study, a recombinant M2 protein with three tandem copies of M2e (3M2e), nucleoprotein (NP) epitopes and hepatitis B virus core (HBc), were expressed in Escherichia coli and purified by column chromatography. Mice immunized with 3M2e-NP-HBc in combination with an oil-in-water SP01 adjuvant produced robust M2e specific antibodies and cellular immune responses. Most importantly, the 3M2e-NP-HBc VLP vaccine provided enhanced protection against a lethal challenge with pandemic 2009 H1N1 and HPAI H5N1 virus through increased survival rates, a significant decrease in viral replication, and obvious alleviation of histopathological lung changes in challenged mice. Our results imply that a cellular immune response to NP is a plausible mechanism mediating this enhanced protection. These findings suggest that 3M2e-NP-HBc VLP has great potential as the basis development of a broadly protective influenza vaccine.

Keywords: Cross-protection; Influenza vaccine; M2e; Nucleoprotein

Computer-aided identification, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus by Marcella Bassetto; Tine De Burghgraeve; Leen Delang; Alberto Massarotti; Antonio Coluccia; Nicola Zonta; Valerio Gatti; Giampiero Colombano; Giovanni Sorba; Romano Silvestri; Gian Cesare Tron; Johan Neyts; Pieter Leyssen; Andrea Brancale (pp. 12-18).

► A homology model for the CHIKV nsP2 protease was constructed and validated. ► A new class of inhibitors of CHIKV replication was identified by virtual screening. ► SARs of the compound class were explored. ► Several analogues with low μM activity in the cell-based assay were identified.Chikungunya virus (CHIKV) is an Arbovirus that is transmitted to humans primarily by the mosquito species Aedes aegypti. Infection with this pathogen is often associated with fever, rash and arthralgia. Neither a vaccine nor an antiviral drug is available for the prevention or treatment of this disease. Albeit considered a tropical pathogen, adaptation of the virus to the mosquito species Aedes albopictus, which is also very common in temperate zones, has resulted in recent outbreaks in Europe and the US. In the present study, we report on the discovery of a novel series of compounds that inhibit CHIKV replication in the low μM range. In particular, we initially performed a virtual screening simulation of ∼5million compounds on the CHIKV nsP2, the viral protease, after which we investigated and explored the Structure–Activity Relationships of the hit identified in silico. Overall, a series of 26 compounds, including the original hit, was evaluated in a virus-cell-based CPE reduction assay. The study of such selective inhibitors will contribute to a better understanding of the CHIKV replication cycle and may represents a first step towards the development of a clinical candidate drug for the treatment of this disease.

Keywords: Antiviral; Chikungunya; CHIKV; Homology model; Molecular dynamics; Virtual screening

Screening and identification of compounds with antiviral activity against hepatitis B virus using a safe compound library and novel real-time immune-absorbance PCR-based high throughput system by Jason Lamontagne; Courtney Mills; Richeng Mao; Cally Goddard; Dawei Cai; Haitao Guo; Andy Cuconati; Timothy Block; Xuanyong Lu (pp. 19-26).

► A high throughput screening system utilizing a novel immune-absorbance qPCR assay was established. ► Using this system, 2000 compounds with known safety profiles were screened for activity against HBV. ► Eight compounds were shown to have anti-hepatitis B virus (HBV) activity. ► The anti-HBV mechanism of proparacaine and chlorophyllide has been further studied.There are now seven nucleoside/tide analogues, along with interferon-α, that are approved by the FDA for the management of chronic hepatitis B virus (HBV) infection, a disease affecting hundreds of millions of people worldwide. These medications, however, are limited in usefulness, and significant side effects and the emergence of viral escape mutants make the development of novel and updated therapeutics a pressing need in the treatment of HBV. With this in mind, a library containing 2000 compounds already known to be safe in both humans and mice with known mechanisms of action in mammalian cells were tested for the possibility of either antiviral activity against HBV or selective toxicity in HBV producing cell lines. A modified real-time immune-absorbance-polymerase chain reaction (IA-PCR) assay was developed for this screen, utilizing cells that produce and secrete intact HBV virions. In this procedure, viral particles are first captured by an anti-HBs antibody immobilized on a plate. The viral load is subsequently assessed by real-time PCR directly on captured particles. Using this assay, eight compounds were shown to consistently reduce the amount of secreted HBV viral particles in the culture medium under conditions that had no detectable impact on cell viability. Two compounds, proparacaine and chlorophyllide, were shown to reduce HBV levels 4- to 6-fold with an IC₅₀ of 1 and 1.5μM, respectively, and were selected for further study. The identification of these compounds as promising antiviral drug candidates against HBV, despite a lack of previous recognition of HBV antiviral activity, supports the validity and utility of testing known compounds for “off-pathogen target” activity against HBV, and also validates this IA-PCR assay as an important tool for the detection of anti-viral activity against enveloped viruses.

Keywords: Hepatitis B virus; Polymerase chain reaction; Immune-absorbance polymerase chain reaction; Proparacaine; Chlorophyllide; Antiviral drug

Chronic hepatitis B: What should be the goal for new therapies? by Timothy M. Block; Robert Gish; Haitao Guo; Anand Mehta; Andrea Cuconati; W. Thomas London; Ju-Tao Guo (pp. 27-34).

► Clinical end points are proposed to define a “functional” cure of hepatitis B. ► Virological end points are proposed to guide therapy and define a virological cure. ► New drugs should be sought that rapidly reduce cccDNA, antigenemia and DNA load. ► Combinations of novel antiviral and current drugs may achieve a “functional cure”.Chronic hepatitis B can currently be medically managed with either pegylated interferon-alpha (pegIFN-α) or one of the five nucleos(t)ide analog Direct Acting Antivirals (DAAs) that inhibit the hepatitis B virus (HBV) DNA polymerase. While pegIFN-α is effective in approximately one-third of the treated patients, the polymerase inhibitors significantly reduce viral load in the vast majority of those treated. However, neither pegIFN-α nor nucleosi(t)de analogs are capable of reliably eliminating the virus and achieving a cure. Moreover, the interferons and polymerase inhibitors are recommended by US, European and Asian professional society practice guidelines for use in only a subset of those infected with HBV. This subset is the population with the greatest levels of circulating viral DNA and abnormal liver function. Although this is the population at the highest risk for cirrhosis and liver cancer, those who fall outside the treatment guidelines, with low levels of viral replication and normal serum ALTs, may also benefit from antiviral therapy. The questions are thus: are new classes of drugs needed to manage chronic hepatitis B? Is a cure possible? Is a cure even necessary? It is therefore important to define the meaning of a cure and determine what the goals of new therapies should be. In this article, we address those questions and propose two operational definitions of medically attainable cures. The first is a “functional cure” based on the clinical outcome, in which the patient’s life expectancy becomes the same as that of an individual who has resolved his HBV infection without therapy. Because such an outcome cannot be measured over the short term, we also define an “apparent virological cure,” based on the stable off-drug suppression of HBV viremia and antigenemia and the normalization of ALTs and other laboratory tests. We suggest that such a virological cure should be the goal of future therapeutics in all patients with chronic hepatitis B. The extent to which a virological cure predicts a functional cure will only be determined by long-term follow-up.

Keywords: Hepatitis B virus; Chronic hepatitis B; Direct-acting antivirals

An iminosugar with potent inhibition of dengue virus infection in vivo by Stuart T. Perry; Michael D. Buck; Emily M. Plummer; Raju A. Penmasta; Hitesh Batra; Eric J. Stavale; Kelly L. Warfield; Raymond A. Dwek; Terry D. Butters; Dominic S. Alonzi; Steven M. Lada; Kevin King; Brennan Klose; Urban Ramstedt; Sujan Shresta (pp. 35-43).

► UV-4 provides effective inhibition of dengue virus infection in vivo. ► Treatment with UV-4 in vivo reduces viral titers in key tissues. ► Cytokine levels are reduced in UV-4 treated mice. ► First study to thoroughly characterize the effect of an iminosugar on DENV.The aim of the present study was to evaluate the ability of the iminosugar drug UV-4 to provide in vivo protection from lethal dengue virus (DENV) challenge. This study utilized a well-described model of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)-like lethal disease in AG129 mice lacking the type I and II interferon receptors. Herein, we present UV-4 as a potent iminosugar for controlling DENV infection and disease in this mouse model. Specifically, administration of UV-4 reduced mortality, as well as viremia and viral RNA in key tissues, and cytokine storm. In addition, UV-4 treatment can be delayed, and it does not alter the anti-DENV antibody response. These results have set the foundation for development of UV-4 as a DENV-specific antiviral in phase I human clinical trials.

Keywords: Abbreviations; DENV; dengue virus; DF; dengue fever; DHF/DSS; dengue hemorrhagic fever/dengue shock syndrome; YFV; yellow fever virus; JEV; Japanese encephalitis virus; ADE; antibody dependent enhancement; ER; endoplasmic reticulum; DNJ; deoxynojirimycin; NGC; New Guinea C; GE; genomic equivalents; PFU; plaque forming units; PrM; pre-membrane; E; envelope; LD; ₉₀; 90% lethal doseDengue virus; Iminosugar; α-Glucosidase; Host-targeted antiviral; Mouse model of DHF/DSS

Oleanolic acid and ursolic acid: Novel hepatitis C virus antivirals that inhibit NS5B activity by Lingbao Kong; Shanshan Li; Qingjiao Liao; Yanni Zhang; Ruina Sun; Xiangdong Zhu; Qinghua Zhang; Jun Wang; Xiaoyu Wu; Xiaonan Fang; Ying Zhu (pp. 44-53).

► The Chinese herb Fructus Ligustri Lucidi (FLL) aqueous extract significantly inhibits HCV replication. ► Ursolic acid and oleanolic acid are anti-HCV components within FLL aqueous extract. ► Ursolic acid and oleanolic acid directly inhibits HCV NS5B activity as noncompetitive inhibitors. ► Ursolic acid and oleanolic acid were demonstrated to be potential broad-spectrum antivirals.Hepatitis C virus (HCV) infects up to 170 million people worldwide and causes significant morbidity and mortality. Unfortunately, current therapy is only curative in approximately 50% of HCV patients and has adverse side effects, which warrants the need to develop novel and effective antivirals against HCV. We have previously reported that the Chinese herb Fructus Ligustri Lucidi (FLL) directly inhibited HCV NS5B RNA-dependent RNA polymerase (RdRp) activity (Kong et al., 2007). In this study, we found that the FLL aqueous extract strongly suppressed HCV replication. Further high-performance liquid chromatography (HPLC) analysis combined with inhibitory assays indicates that oleanolic acid and ursolic acid are two antiviral components within FLL aqueous extract that significantly suppressed the replication of HCV genotype 1b replicon and HCV genotype 2a JFH1 virus. Moreover, oleanolic acid and ursolic acid exhibited anti-HCV activity at least partly through suppressing HCV NS5B RdRp activity as noncompetitive inhibitors. Therefore, our results for the first time demonstrated that natural products oleanolic acid and ursolic acid could be used as potential HCV antivirals that can be applied to clinic trials either as monotherapy or in combination with other HCV antivirals.

Keywords: HCV; NS5B; Oleanolic acid; Ursolic acid; Antivirals

BST2/Tetherin inhibits hepatitis C virus production in human hepatoma cells by Xiao-Ben Pan; Xiao-Wang Qu; Dong Jiang; Xing-Liang Zhao; Jin-Chao Han; Lai Wei (pp. 54-60).

► HCV production in Huh7.5 cells is inhibited by overexpression of BST2 in a dose dependent manner. ► HCV core protein co-localizes with highly expressed.BST2 in Huh7.5 cells. ► BST2 is not likely to be a major contributor to the antiviral effect of IFN-α.Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-α)-based therapies. IFN-induced cell membrane protein BST2 (also known as CD317, HM1.24 or tetherin) has been reported to tether a broad range of lipid-enveloped viruses on cell surfaces. However, whether HCV is sensitive to BST2 remains controversial. Here we established a Huh7.5-BST2-TO cell line, in which BST2 expression is regulated by tetracycline. Our results showed that the effect of BST2 on inhibiting HCV production was dependent on its expression level. Highly expressed BST2 reduced the yield of cell-free HCV virions but did not affect the efficiency of HCV infection and genome replication. Co-localization of HCV core protein and BST2 was detected by immunofluorescence in certain cells with high expression, but not in cells with low BST2 expression. Furthermore, inhibition of IFN-α induced BST2 expression in Huh7.5 cells by siRNA technology slightly reduced the antiviral response of the cytokine against HCV, but only at low IFN-α concentration. While overexpression of BST2 inhibited HCV replication in this system, BST2 is therefore not likely to be a major contributor to the antiviral effect of IFN-α.

Keywords: HCV; BST2; Interferon

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus by Jordi M. Argilaguet; Eva Pérez-Martín; Sergio López; Martin Goethe; J.M. Escribano; Katrin Giesow; Günther M. Keil; Fernando Rodríguez (pp. 61-65).

► A BacMam vector expressing three ASFV antigens confers partial protection against ASFV infection. ► Four out of six immunized pigs remained viremia-free after sub-lethal ASFV infection. ► The protection was obtained in the absence of antibodies prior to ASFV challenge. ► The protection correlated with the detection of specific T-cells at 17days post-infection.Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

Keywords: BacMam; ASFV; IFNγ; Vaccine

Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155 by Aixia Zhai; Jun Qian; Wenping Kao; Aimei Li; Yujun Li; Junming He; Qingmeng Zhang; Wuqi Song; Yingmei Fu; Jing Wu; Xiaobei Chen; Hui Li; Zhaohua Zhong; Hong Ling; Fengmin Zhang (pp. 66-75).

► Borna disease virus (BDV)-encoded P protein inhibited miR-155 expression. ► miR-155 positively regulated the production of type I interferons (IFNs). ► miR-155 inhibited the expression of SOCS1 and SOCS3. ► miR-155 suppressed the expression of BDV P protein.It has been reported that the Borna disease virus (BDV) encoded phosphoprotein (P protein) can inhibit the activity of Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK-1), thus preventing the induction of type I interferon (IFN). However, the effects of microRNA on the regulation of BDV infection and the host’s immune response have not been characterized. miR-155 was predicted to be complementary to the BDV P mRNA by RNAhybrid software. Here, we showed that miR-155 was down-regulated in BDV persistently infected human oligodendroglial (OL/BDV) cells and that the BDV P protein, but not the X protein, directly inhibited miR-155 expression in cells. When miR-155 was over-expressed, the inhibition of type I IFNs by BDV in cells was reversed, and the expression of type I IFNs was increased. When miR-155 expression was specifically blocked, cellular IFN expression and the induction of IFN by poly I:C treatment were suppressed. Furthermore, miR-155 promoted type I IFN production by targeting suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Mutations in the nt1138–nt1158 region of SOCS3 abandoned the impact of miR-155 on the expression of SOCS3-enhanced green fluorescent protein (EGFP). The levels of BDV P mRNA and protein were significantly decreased in OL/BDV cells when miR-155 was over-expressed; however, miR-155-mutation did not affect the expression of BDV P-EGFP. Thus, BDV persistent infection inhibited the expression of type I IFNs through the suppression of miR-155, and miR-155 played an important immune regulatory role in BDV persistent infection.

Keywords: miR-155; SOCS; IFN; BDV; Persistent infection

Comparison of vRNA and cRNA based reporters for detection of influenza replication by Yue Wang; Brian Landman; Changqing Wu; Jack Gelb; Serguei Golovan (pp. 76-84).

► vRNA and cRNA based replicon reporters were designed for detection of influenza. ► Reporters show strong correlation between influenza replication and luciferase signal. ► vRNA based reporter was more sensitive in influenza detection. ► Application of reporters to study kinetics of antiviral siRNA is demonstrated. ► Possibility of secondary replication of progeny vRNA is suggested.In this study, RNA polymerase I expressed replicons containing EGFP and luciferase reporter genes controlled by influenza vRNA or cRNA promoters were compared side-by-side in the ability to detect influenza RNA-dependent RNA polymerase activity as an indicator of influenza replication. Results showed the vRNA based Luc reporter was more sensitive to early detection of influenza virus at 6h post infection ( p<0.05), and at 10-fold lower titer (MOI=0.001). Lower sensitivity of cRNA based Luc reporter constructs was due to its background expression, 2-fold lower expression, and around 4h delay in expression of luciferase. Despite these differences, both cRNA- and vRNA-based reporters demonstrated strong correlation between MOI and luciferase signal, and can be used for effective and early detection of influenza infection in vitro. Further, we demonstrated that these reporters can be used successfully to study the kinetics of antiviral drugs including siRNA. Our results also suggest that progeny vRNAs might participate not only in secondary transcription but also in secondary replication. The developed cRNA and vRNA reporters may help with further elucidation of the replication model of influenza A virus.

Keywords: Abbreviations; cRNA; complementary RNA; hpi; hours post infection; Pol; polymerase; RdRp; RNA-dependent RNA polymerase; RNAi; RNA interference; siRNA; short interfering RNA; vRNA; virion genomic RNAInfluenza; vRNA; cRNA; RNA interference; Replication

Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors by Katie Chan; Margaret Robinson; Huiling Yang; Sophia Cornew; William E. Delaney IV (pp. 85-92).

► A genotype 1b/2a chimeric HCV reporter virus system in cell culture was developed. ► Key adaptive mutations enable robust replication and high viral titers. ► The virus system described facilitates routine antiviral screening. ► Robust reporter signals facilitate early virus life-cycle characterization assays. ► Potential host editing of viral RNA was observed during viral adaptation.The development of JFH1 based intergenotypic recombinants that exploit the unique replication characteristics of JFH1 has made it possible to study infectious hepatitis C virus (HCV) encoding the structural genes of additional HCV genotypes. To facilitate the study of 1b structural proteins, we aimed to develop a robust 1b/2a chimera encoding a humanized Renilla luciferase reporter gene (1b/2a hRluc). The unadapted genome replicated efficiently but produced very low titers of infectious virus. Adaptation by continuous passage over a novel Huh-7 Lunet clone improved viral titers approximately 100-fold but caused an unexpected decline in luciferase activity, limiting the utility of the reporter-containing virus. Genotypic analysis revealed 17 adenosine to guanosine (A to G) nucleotide mutations in the luciferase gene and two potential adaptive mutations. To overcome the problems of low viral titers and editing of the luciferase gene during viral adaptation, six adaptive mutations previously identified in a non-reporter 1b/2a HCV genome were introduced into the 1b/2a hRluc genome. This resulted in the immediate production of high-titer viral stocks (approximately 1000-fold greater than the parental virus) that could efficiently infect naïve cells and generate robust luciferase signals. The improved sensitivity of the luciferase reporter also facilitated time of addition studies validating the utility of this system for characterizing the early steps of HCV infection. Thus, the development of the 1b/2a hRluc reporter virus described here provides a versatile tool for discovery of inhibitors targeting the early steps of the viral life cycle and genotype 1b structural genes.

Keywords: Abbreviations; ADAR1; adenosine deaminases acting on RNA; DMEM; Dulbecco’s modified Eagle medium; EC; ₅₀; 50% effective concentration; FMDV; foot and mouth disease virus; GAPDH; glutaraldehyde-3-phosphate dehydrogenase; HCV; hepatitis C virus; HCVcc; hepatitis C virus in cell culture; IFN-α; interferon-α; MAb; monoclonal antibody; MOI; multiplicity of infection; PVDF; polyvinylidene fluoride; S:B; signal:background; TCID; ₅₀; 50% tissue culture infective doseHCVcc; Adaptive mutations; Con1; JFH1; Virus production; Luciferase reporter

Molecular basis of human immunodeficiency virus type 1 drug resistance: Overview and recent developments by Luis Menéndez-Arias (pp. 93-120).

► Current antiretroviral drugs target HIV entry, genome replication, integration and virion maturation. ► Reverse transcriptase inhibitors are the backbone of current antiretroviral therapies. ► HIV-1 drug resistance constitutes a major hurdle towards treatment efficacy. ► This review provides an overview of current therapies and how HIV acquires resistance to them.The introduction of potent combination therapies in the mid-90s had a tremendous effect on AIDS mortality. However, drug resistance has been a major factor contributing to antiretroviral therapy failure. Currently, there are 26 drugs approved for treating human immunodeficiency virus (HIV) infections, although some of them are no longer prescribed. Most of the available antiretroviral drugs target HIV genome replication (i.e. reverse transcriptase inhibitors) and viral maturation (i.e. viral protease inhibitors). Other drugs in clinical use include a viral coreceptor antagonist (maraviroc), a fusion inhibitor (enfuvirtide) and two viral integrase inhibitors (raltegravir and elvitegravir). Elvitegravir and the nonnucleoside reverse transcriptase inhibitor rilpivirine have been the most recent additions to the antiretroviral drug armamentarium. An overview of the molecular mechanisms involved in antiretroviral drug resistance and the role of drug resistance-associated mutations was previously presented (Menéndez-Arias, L., 2010. Molecular basis of human immunodeficiency virus drug resistance: an update. Antiviral Res. 85, 210–231). This article provides now an updated review that covers currently approved drugs, new experimental agents (e.g. neutralizing antibodies) and selected drugs in preclinical or early clinical development (e.g. experimental integrase inhibitors). Special attention is dedicated to recent research on resistance to reverse transcriptase and integrase inhibitors. In addition, recently discovered interactions between HIV and host proteins and novel strategies to block HIV assembly or viral entry emerge as promising alternatives for the development of effective antiretroviral treatments.

Keywords: HIV; Reverse transcriptase; Drug resistance; Protease; Integrase; Entry inhibitors

Antiviral strategies combining antiretroviral drugs with RNAi-mediated attack on HIV-1 and cellular co-factors by Fatima Boutimah; Julia J.M. Eekels; Ying Poi Liu; Ben Berkhout (pp. 121-129).

► We analyse the anti-HIV potential of shRNA combinations targeting the viral replication cycle. ► The stably-expressed shRNAs target highly conserved viral RNA sequences or host-factor mRNAs. ► Some combinations provide enhanced and extended suppression of viral replication. ► We test the influence of some potent shRNAs on the activity of conventional antiretroviral drugs. ► High levels of additivity and synergy are recorded and need in vivo evaluation.To improve the care of HIV-1/AIDS patients there is a critical need to develop tools capable of blocking viral evolution and circumventing therapy-associated problems. An emerging solution is gene therapy either as a stand-alone approach or as an adjuvant to pharmacological drug regimens. Combinatorial RNAi by multiplexing antiviral RNAi inhibitors through vector-mediated delivery has recently shown significant superiority over conventional mono-therapies. Viral as well as cellular co-factor targets have been identified, but they are generally attacked separately. Here, we hypothesized that a mixture of shRNAs directed against highly conserved viral RNA sequences and the mRNAs of cellular components that are involved in HIV replication could restrict mutational escape by enhanced synergistic inhibition. We screened for potent silencer cocktails blending inhibitors acting scattered along the viral replication cycle. The results show enhanced and extended suppression of viral replication for some combinations. To further explore the power of combinatorial approaches, we tested the influence of RNAi-mediated knockdown on the activity of conventional antiretroviral drugs (fusion, RT, integrase and protease inhibitors). We compared the fold-change in IC₅₀ (FCIC₅₀) of these drugs in cell lines stably expressing anti-HIV and anti-host shRNAs and measured increased values that are up by several logs for some combinations. We show that high levels of additivity and synergy can be obtained by combining gene therapy with conventional drugs. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models.

Keywords: HIV-1; Gene therapy; shRNAs; Antiretroviral drugs; Drug RNAi combination therapy; Cellular co-factors

Novel DNA polymerase mutations conferring cytomegalovirus resistance: Input of BAC-recombinant phenotyping and 3D model by Sébastien Hantz; Sébastien Cotin; Eva Borst; Anthony Couvreux; Arielle Salmier; Isabelle Garrigue; Pierre Merville; Catherine Mengelle; Michel Attal; Martin Messerle; Sophie Alain (pp. 130-134).

► CMV clinical isolates with new polymerase mutations in two transplant patients. ► Mutations conferring low-level resistance to ganciclovir and high level resistance to cidofovir. ► Analyzed using BAC recombinant technology. ► Input of 3D model in resistance mechanism analysis.Long-term exposure to antiviral therapy in immunocompromised patients favors emergence of human cytomegalovirus (HCMV) resistance mutations. Two new UL54 DNA polymerase mutations (deletion of codon 524 and N408S substitution) identified in a kidney recipient and a bone marrow recipient respectively were characterized. Marker transfer experiment through recombination into a HCMV AD169 BAC demonstrated del524 and mutation N408S confer GCV and CDV resistance. These results suggest continued mutation of UL54 under selective antiviral pressure. Characterization of each new mutation is thus required to inform genotypic assays and to better understand the functional regions of UL54 for the development of novel antivirals.

Keywords: Cytomegalovirus; Ganciclovir; Cidofovir; Resistance; Transplant recipient; BAC; 3D-model

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Antiviral Research (v.98, #1)