Novel inhibition mechanism and potent antiviral activity of translocation-deficient reverse transcriptase inhibitors [abstract]

Abstract only availableNucleoside RT inhibitors (NRTIs) are among the most potent anti-HIV agents and act as chain terminators because they lack a 3'OH. However, this feature can reduce affinity for RT compared to the analogous dNTP substrate, as well as reduced intracellular conversion to the active dNTP. To overcome this, it was shown that certain nucleosides that retain the 3'OH and have substitutions at the 4' ribose and 2 position of the base have exceptional antiviral properties. One of these compounds, 4'-ethynyl, 2-fluoro deoxy-adenosine (4'E-2FdA) is the most potent NRTI inhibitor against wild-type and multi-drug resistant HIV viruses described to date. We have recently reported that 4'E-2FdA acts as a chain terminator despite the presence of an accessible 3'OH. We show that after 4'E-2FdA-MP incorporation, RT does not bind the next incoming dNTP. We analyzed RT translocation on different sequences terminated with 4'E-2FdA-MP, and found that even at sequences when RT is naturally found post-translocated, the inhibitor prevents translocation. This decrease in translocation efficiency explains the reduced binding of the next incoming dNTP and the termination of elongation. While the inhibitor stabilizes the pre-translocated 4'E-2FdA-MP-terminated primer, the pyrophosphate-dependent excision rate of 4'E-2FdA-MP was not very high compared to ddAMP. In conclusion, this highly potent chain termination activity arises from difficulty of the primer 3'-terminus to translocate following incorporation of the compound, and not from simple steric hindrance due to the 4' substitution. Therefore, we propose that 4'E-2FdA is a Translocation-Deficient Reverse Transcriptase Inhibitor (TDRTI) that acts by a novel mechanism.NIH grant to S. Sarafiano

CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.United States. National Institutes of Health (DK085713)National Cancer Institute (U.S.) (P30-CA14051)National Institute of Environmental Health Sciences (P30-ES002109)United States. National Institutes of Health (1K08DK101754

Visualization of positive and negative sense viral RNA for probing the mechanism of direct-acting antivirals against hepatitis C virus

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action

Characterization of the mechanism of action of the ultra-potent HIV inhibitor 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine [abstract]

Abstract only availableRetroviruses rely on the enzyme reverse transcriptase (RT) to perform the reverse transcription of its genome from single-stranded RNA into double-stranded DNA, which can then be integrated into the host's genome by the action of the viral integrase enzyme. There are currently sixteen antiretroviral agents used for the treatment of HIV infections. Highly active antiviral therapy (HAART) is based on a combination of at least 3 anti-HIV drugs. It has slowed down the progression of AIDS and decreased mortality. RT is one of the main targets for these antiretroviral drugs. One class of drugs targeting the reverse transcriptase is the nucleoside analogue RT inhibitors (NRTIs). NRTIs compete with natural nucleotides for incorporation in the elongating DNA chain by HIV-1 RT. Once incorporated, they act as chain-terminators because they lackthe 3'OH group which is required for further nucleotide incorporation. Prolonged use of these drugs leads to drug-resistant HIV strains. To overcome drug resistance, novel inhibitors that are active against NRTI-resistant viruses are being developed. NRTIs containing a modification at the 4' position of their sugar moiety have been synthesized by Hiroaki Mitsuya and his colleagues. One of these analogues, 4'ethynyl-2-fluoro-2'-deoxyadenosine (4'-E-2-F dA) was shown to be ultra-potent against wild-type and drug resistant HIV-1. Unlike other nucleoside analogues, 4'-E-2-F dA has a hydroxyl group at the 3' position. The purpose of this project is to understand the mechanism of RT inhibiton by 4'-E-2-F dA. In order to determine its mechanism of action, in vitro primer extension assays as well as gel mobility shift assays were used. Using primer extension assays, we determined that the active form of 4'-E-2-F dA, 4'-E-2-F dA-triphosphate (TP), acts as a chain terminator at physiological concentrations of nucleotides, despite the presence of a 3'OH. We first hypothesized that the presence of 4'-E-2-F dA-monophosphate (MP) at the 3' end of the primer destabilized the RT/DNA complex. The RT/DNA complex is not affected by the presence of 4'-E-2-F dAMP as observed in gel mobility shift assays. We next hypothesised that RT was not able to bind to the next incoming nucleotide to form a ternary complex. Indeed, we found that the presence of 4'-E-2-F dAMP at the 3' end of the primer severly impair the formation of a stable ternary complex. In conclusion, we found that 4'-E-2-F dA inhibits DNA elongation by RT by acting as chain-terminator despite the presence of the 3'OH. In order to do so, 4'-E-2-F dAMP blocks the binding of the next incoming nucleotide. This is a novel mechanism of inhibition that results in the most efficient blocking of HIV activity reported to date for any NRTI. Our findings have generated interest from two pharmaceutical companies that wish to develop it as a next-generation therapeutic for the treatment of HIV infection

Discovery and Mechanistic Characterization of Novel SARS Coronavirus Inhibitors that Block Viral Entry [abstract]

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionSevere acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS-associated coronavirus (SARS-CoV). Viral entry is a key target step for therapies because it can prevent the propagation of virus at early stages of the disease. We used a cell-based assay to identify inhibitors of SARS-CoV entry. We prepared a pseudotyped virus in which the core is from HIV and envelop is from the SARS-CoV (HIV-luc/SARS env). This pseudotyped virus was used to infect, 293T cells expressing the receptor for SARS-CoV, Aangiotensin-converting enzyme-2 (ACE2). Using this assay we screened a chemical library of more than 2000 compounds and identified three compounds that specifically inhibit entry of the HIV-luc/SARS env. These compounds did not inhibit another pseudotyped virus which had same core from HIV but envelop was from Vesicular Stomatitis Virus. The compounds had strong potencies (EC50s were 2.9, 4.8 and 5.8 µM) and low cytotoxicities (high CC50s) resulting in promising Selectivity Indices (CC50/EC50 were >175, >65, and >86, respectively). Importantly, the compounds were found to have excellent antiviral activities, blocking SARS-CoV replication at low nM concentrations. Only one of the compounds was a moderate inhibitor of cathepsin L, a cellular protease whose activity is required to process the SARS-CoV env glycoprotein (Spike) and allow viral entry. Moreover, none of the compounds affects the cleavage activity of furin, another host protease, which may also be involved in SARS-CoV entry. Using a flow cytometry binding assay, we found that all three compounds decrease binding of the SARS-CoV Spike receptor binding domain to ACE2 receptor expressed on the surface of 293Tcells. Hence, we have discovered three promising compounds as the first small molecule inhibitors that can block receptor-dependent entry of SARS-CoV

Experimental variables that affect human hepatocyte MV transduction in liver chimeric mice

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah(-/-)) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah(-/-) chimera model, attention to which may improve reproducibility between findings from different laboratories

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 μM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 μM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. © 2013 Tavis et al

Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC(50)s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC(50) = 12 µM and EC(90) = 20 µM).We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD