Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus.

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBVWTCe) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBVETV-RL180M/S202G/M204V). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC50], ∼30 nM) and HBVWTCe plasmid-transfected Huh7 cells (IC50, 206 nM) and efficiently suppressed ETV-resistant HBVETV-RL180M/S202G/M204V (IC50, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 µM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBVWTCe-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBVETV-RL180M/S202G/M204V viremia by ∼1 log in HBVETV-RL180M/S202G/M204V-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBVWTCe reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBVETV-RL180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBVWTCe RT and HBVETV-RL180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.

Bi-allelic mutations of LONP1 encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Novel Hepatitis B Virus Capsid-Targeting Antiviral That Aggregates Core Particles and Inhibits Nuclear Entry of Viral Cores.

An estimated 240 million are chronically infected with hepatitis B virus (HBV), which can lead to liver disease, cirrhosis, and hepatocellular carcinoma. Currently, HBV treatment options include only nucleoside reverse transcriptase inhibitors and the immunomodulatory agent interferon alpha, and these treatments are generally not curative. New treatments with novel mechanisms of action, therefore, are highly desired for HBV therapy. The viral core protein (Cp) has gained attention as a possible therapeutic target because of its vital roles in the HBV life cycle. Several classes of capsid assembly effectors (CAEs) have been described in detail, and these compounds all increase capsid assembly rate but inhibit HBV replication by different mechanisms. In this study, we have developed a thermal shift-based screening method for CAE discovery and characterization, filling a much-needed gap in high-throughput screening methods for capsid-targeting molecules. Using this approach followed by cell-based screening, we identified the compound HF9C6 as a CAE with low micromolar potency against HBV replication. HF9C6 caused large multicapsid aggregates when capsids were assembled in vitro and analyzed by transmission electron microscopy. Interestingly, when HBV-expressing cells were treated with HF9C6, Cp was excluded from cell nuclei, suggesting that this compound may inhibit nuclear entry of Cp and capsids. Furthermore, mutational scanning of Cp suggested that HF9C6 binds the known CAE binding pocket, indicating that key Cp-compound interactions within this pocket have a role in determining the CAE mechanism of action.

Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions.

DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 3'-5' exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes. Here, we review the association of DNA polymerases in cancer from the A and B families, which participate in lesion bypass, and conduct gene replication. We also discuss possible therapeutic interventions that could be used to maneuver the role of these enzymes in tumorigenesis.

Small molecule inhibitors block Gas6-inducible TAM activation and tumorigenicity.

TAM receptors (Tyro-3, Axl, and Mertk) are a family of three homologous type I receptor tyrosine kinases that are implicated in several human malignancies. Overexpression of TAMs and their major ligand Growth arrest-specific factor 6 (Gas6) is associated with more aggressive staging of cancers, poorer predicted patient survival, acquired drug resistance and metastasis. Here we describe small molecule inhibitors (RU-301 and RU-302) that target the extracellular domain of Axl at the interface of the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. These inhibitors effectively block Gas6-inducible Axl receptor activation with low micromolar IC50s in cell-based reporter assays, inhibit Gas6-inducible motility in Axl-expressing cell lines, and suppress H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model. Furthermore, using homology models and biochemical verifications, we show that RU301 and 302 also inhibit Gas6 inducible activation of Mertk and Tyro3 suggesting they can act as pan-TAM inhibitors that block the interface between the TAM Ig1 ectodomain and the Gas6 Lg domain. Together, these observations establish that small molecules that bind to the interface between TAM Ig1 domain and Gas6 Lg1 domain can inhibit TAM activation, and support the further development of small molecule Gas6-TAM interaction inhibitors as a novel class of cancer therapeutics.

CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+ Lon protease.

CODAS syndrome is a multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. Using whole-exome and Sanger sequencing, we identified four LONP1 mutations inherited as homozygous or compound-heterozygous combinations among ten individuals with CODAS syndrome. The individuals come from three different ancestral backgrounds (Amish-Swiss from United States, n = 8; Mennonite-German from Canada, n = 1; mixed European from Canada, n = 1). LONP1 encodes Lon protease, a homohexameric enzyme that mediates protein quality control, respiratory-complex assembly, gene expression, and stress responses in mitochondria. All four pathogenic amino acid substitutions cluster within the AAA(+) domain at residues near the ATP-binding pocket. In biochemical assays, pathogenic Lon proteins show substrate-specific defects in ATP-dependent proteolysis. When expressed recombinantly in cells, all altered Lon proteins localize to mitochondria. The Old Order Amish Lon variant (LONP1 c.2161C>G[p.Arg721Gly]) homo-oligomerizes poorly in vitro. Lymphoblastoid cell lines generated from affected children have (1) swollen mitochondria with electron-dense inclusions and abnormal inner-membrane morphology; (2) aggregated MT-CO2, the mtDNA-encoded subunit II of cytochrome c oxidase; and (3) reduced spare respiratory capacity, leading to impaired mitochondrial proteostasis and function. CODAS syndrome is a distinct, autosomal-recessive, developmental disorder associated with dysfunction of the mitochondrial Lon protease.

Effects of substitutions at the 4' and 2 positions on the bioactivity of 4'-ethynyl-2-fluoro-2'-deoxyadenosine.

Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4'-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2'-exo/C-3'-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4'-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4'-ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.

Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase.

RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Biochemical, inhibition and inhibitor resistance studies of xenotropic murine leukemia virus-related virus reverse transcriptase.

We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (k(off)) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.

K70Q adds high-level tenofovir resistance to "Q151M complex" HIV reverse transcriptase through the enhanced discrimination mechanism.

HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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). SIGNIFICANCE: 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.

Combination of synthetic and natural products as pesticides (CSYNAP): a new class of antifungal agents.

In the present communication some dehydrated dialdol products such as 1, 5 - Diphenyl pent - 1, 4 - diene - 3 - one (A1); 1, 9 - Diphenylnon - 1, 3, 6, 8 - tetraene - 5 - one (A2); 1, 5 - di (2 - hydroxyphenyl) pent - 1, 4 - diene - 3 - one (A3); 1, 5 - difuran pent - 1, 4 - diene - 3 - one (A4); 1, 5 - di [4 - bis (N, Ndimethyl) phenyl] pent - 1, 4 - diene - 3 - one (A5) were screened for their antifungal activity. To reduce their adverse effect on the environment, for the first time, we have attempted to screen the antifungal activity of these synthetic compounds in conjunction with selected natural products. The natural products that were used in our study include Nicotine tobaccum and Neem oil (Azadirachta indica). A set of 15 samples was tested against highly pathogenic and of extensive host range fungi Sclerotium rolfsii, Rhizactonia bataticola, Fusarium udum. The filter paper disc assay to monitor antifungal effect revealed significant and interesting results. We found that the use of the combination of natural and synthetic pesticides is more effective and environmentally healthy compared to just synthetic chemicals and/or less available natural products. These results obtained from the combined use of natural and synthetic chemicals lead us to suggest to a new class of less toxic but more effective pesticides. We call it group as CSYNAP, i. e. Combination of SYnthetic and NAtural products as Pesticides.

Transactivation of Abl by the Crk II adapter protein requires a PNAY sequence in the Crk C-terminal SH3 domain.

To gain a better understanding of how Crk II regulates the function of the Abl tyrosine kinase, we explored the function of the C-terminal linker and SH3 domain, a region of Crk II that is still poorly understood. Molecular modeling, tryptophan fluorescence, and covariation sequence alignment indicate that the Crk-SH3-C has a unique binding groove and RT loop not observed in typical SH3 domains. Based on these models, we made a series of mutations in the linker and in residues predicted to destabilize the putative binding pocket and RT loop. In Abl transactivation assays, Y222F and P225A mutations in the linker resulted in strong transactivation of Abl by Crk II. However, mutations predicted to be at the surface of the Crk SH3-C were not activators of Abl. Interestingly, combinations of activating mutations of Crk II with mutations in the highly conserved PNAY sequence in the SH3-C inactivated the activating mutations, suggesting that the SH3-C is necessary for activation. Our data provide insight into the role of highly conserved residues in the Crk-SH3-C, suggesting a mechanism for how the linker and the Crk-SH3-C function in the transactivation of the Abl tyrosine kinase.

Cleavage site selection within a folded substrate by the ATP-dependent lon protease.

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.

Lysine 152 of MuLV reverse transcriptase is required for the integrity of the active site.

Comparison of the three-dimensional structure of the active sites of MuLV and HIV-1 reverse transcriptases shows the presence of a lysine residue (K152) in the substrate-binding region in MuLV RT, while its equivalent position in HIV-1 RT is occupied by a glycine (G112). To investigate the role of K152 in the mechanism of the polymerase reaction catalyzed by MuLV RT, four mutant RTs, namely, K152A, K152R, K152E, and K152G, were generated and biochemically characterized. All muteins exhibited reduced polymerase activity on both RNA and DNA template-primers with K152E being the most defective. The template-primer binding affinity and the processivity of DNA synthesis, however, remained unchanged. The steady-state kinetic characterization showed little change in K(m.dNTP) (except for that of K152E) and an approximately 3-10-fold decrease in k(cat) depending upon the template-primer and mutational substitutions. The ddNTP resistance patterns were unchanged for all muteins, suggesting no participation of K152 in ddNTP recognition. The ability of individual muteins to add dNTP on the covalently cross-linked enzyme-template-primer complex was significantly decreased. These results together with the analysis of the ion pairs in the catalytic apparatus of MuLV RT suggest that K152 participates in maintaining the integrity of the active site of MuLV RT. Examination of the prepolymerase ternary complex formation showed that neither the wild type nor any of the K152 muteins of MuLV RT are capable of forming stable ternary complexes. This property is in contrast to that of HIV-1 RT, which readily forms stable ternary complexes under similar conditions. These results further indicate that the catalytic mechanism of MuLV RT is significantly different from that of HIV-1 RT, despite the presence of a number of conserved motifs and amino acid residues.