A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B.

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event.

Mechanistic insights into the suppression of drug resistance by human immunodeficiency virus type 1 reverse transcriptase using alpha-boranophosphate nucleoside analogs.

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively.

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core.

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different.

Defect turbulence in a spiral wave pattern in the torsional Couette flow.

Our experimental study is devoted to the transition to defect turbulence of a periodic spiral wave pattern occurring in the flow between a rotating and a stationary disk. As the rotation rate Omega of the disk is increased, the radial phase velocity of the waves changes its sign: The waves that propagate first outward on average, then become stationary and finally propagate inward. As they become stationary, the nucleation of topological defects breaks the periodicity of the pattern.

A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate.

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV).

A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions.

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not.

The molecular mechanism of multidrug resistance by the Q151M human immunodeficiency virus type 1 reverse transcriptase and its suppression using alpha-boranophosphate nucleotide analogues.

Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer.

Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue.

The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2',3'-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2',3'-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo.

Crystal structure of a monocotyledon (maize ZMGlu1) beta-glucosidase and a model of its complex with p-nitrophenyl beta-D-thioglucoside.

The maize beta-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.