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Antiviral Research (v.76, #3)
Protease inhibitor resistance in HIV-infected patients: Molecular and clinical perspectives by Jorge L. Martinez-Cajas; Mark A. Wainberg (pp. 203-221).
The problem of HIV-1 drug resistance has established a need for new compounds that retain activity against mutated resistant viral isolates. Fortunately, a number of new compounds have recently been developed that possess excellent activity against HIV-1 strains that contain as many as eight relevant drug-resistance mutations in the viral protease (PR) gene. These newer protease inhibitors (PI) are characterized by higher genetic barrier for drug resistance, meaning that higher numbers of mutations are required for resistance to develop in comparison with older members of the PI family of drugs. Thus, different PIs can be used sequentially in HIV therapy in a manner that can overcome previous drug resistance and potentially forestall the development of additional resistance mutations in the viral PR. All currently used PIs, in general, require ritonavir to be used as a pharmalogical boosting agent. There is a need to develop novel PIs, that will not require such boosting, and that are also characterized by potent antiviral activity and a high genetic barrier for resistance.
Keywords: Protease inhibitors; HIV; Cross-resistance; Mutations; Genetic barrier; Salvage therapy
Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing by Chuan Young Ng; Feng Gu; Wai Yee Phong; Yen-Liang Chen; Siew Pheng Lim; Andrew Davidson; Subhash G. Vasudevan (pp. 222-231).
Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2′- C-methyladenosine (EC50=2.42±0.59μM), or ribavirin (EC50=6.77±1.33μM), mycophenolic acid (EC50=1.31±0.27μM) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery.
Keywords: Abbreviations; DEN; dengue; DEN-2; dengue virus type 2; NGC; New Guinea C; NS; non-structural protein; C; core; E; envelope; EGFP; enhanced green fluoresecent protein; prM; pre-membrane; FCS; fetal calf serum; HRP; horse radish peroxidase; PBS; phosphate-buffered saline; EC; 50; 50% effective concentration; CC; 50; 50% cytotoxic concentrationFlavivirus; Dengue; Replicon; NS5 inhibitor; High-throughput screening
Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells by Ju-Tao Guo; Tianlun Zhou; Haitao Guo; Timothy M. Block (pp. 232-240).
Although alpha interferon (IFN-α) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-α inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-α. We found that IFN-α efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin–Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-α did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-α treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-α-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.
Keywords: Interferon; Hepatitis B; Adenovirus; Vectors
Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells by Ya-Ching Lin; Hao-Chan Juan; Yi-Chen Cheng (pp. 241-251).
In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2ppm for 1h, the production of IL-1β, IL-6 and TNF-α was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48h. While infected cells were exposed to ozone for 2h at 1.5 or 2ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.
Keywords: Enterovirus 71; Ozone; Inhibition of virus replication; Cytokine production
Bovine lactoferrin prevents the entry and intercellular spread of herpes simplex virus type 1 in Green Monkey Kidney cells by Maria Grazia Ammendolia; Magda Marchetti; Fabiana Superti (pp. 252-262).
Lactoferrin (Lf) is a multifunctional glycoprotein that plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Bovine lactoferrin (bLf) has been recognized as a potent inhibitor of human herpetic viruses, such as cytomegalovirus and herpes simplex virus type 1 and 2. BLf has been found to prevent viral infection by binding to heparan sulphate containing proteoglycans that also act as cell receptors for herpetic viruses.In this study we further investigated the inhibiting activity of bLf against herpes simplex virus type 1 (HSV-1) in Green Monkey Kidney (GMK) cells and found that, in addition to the viral adsorption step, bLf also targets the HSV-1 entry process and cell-to-cell viral spread. Our study showed that the inhibition of HSV-1 infectivity by bLf is dependent on its interaction with specific structural viral proteins. Apart from the prevention of early phases of viral infection, cell-to-cell spread inhibition activity of HSV-1 by bLf confirmed that this protein is an outstanding candidate for the treatment of herpetic infections since it would offer the advantage to prevent also viral infections caused by cell-associated virus.
Keywords: Lactoferrin; HSV-1; GMK cells; Viral entry; Viral spreading
Neuraminidase inhibitor drug susceptibility differs between influenza N1 and N2 neuraminidase following mutagenesis of two conserved residues by Hui-Ting Ho; Aeron C. Hurt; Jenny Mosse; Ian Barr (pp. 263-266).
Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity.The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA.This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.
Keywords: Influenza; Neuraminidase inhibitors; Conserved residues; N1; Mutagenesis