Luciano Cellai

Diminished RNA primer usage associated with the L74V and M184V mutations in the reverse transcriptase of human immunodeficiency virus type 1 provides a possible mechanism for diminished viral replication capacity.

ABSTRACT The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA3Lys and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.

Pyrophosphorolytic excision of nonobligate chain terminators by hepatitis C virus NS5B polymerase.

ABSTRACT Nonobligate chain terminators, such as 2′-C-methylated nucleotides, block RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3′-hydroxyl group can be excised in the presence of pyrophosphate (PPi), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2′-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2′-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2′-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2′-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.

NMR structure of the chimeric hybrid duplex r(gcaguggc)⋅r(gcca)d(CTGC) comprising the tRNA-DNA junction formed during initiation of HIV-1 reverse transcription

A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc)⋅r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA–DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5xa0Å in the RNA/RNA stem to about 4.5xa0Å in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of ∼11xa0Å and ∼8.5xa0Å, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in induced-fit-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.

Pyrazinoporphyrazines with Externally Appended Pyridine Rings. 13. Structure, UV–Visible Spectral Features, and Noncovalent Interaction with DNA of a Positively Charged Binuclear (ZnII/PtII) Macrocycle with Multimodal Anticancer Potentialities

We investigated with spectroscopic techniques the noncovalent interaction of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation, [(PtCl(2))(CH(3))(6)LZn](6+), and its octacationic analogue [(CH(3))(8)LZn](8+), lacking the cis-platin-like functionality, with a 21-mer double strand (ds) 5-d[GGG(TTAGGG)(3)]-3/3-d[CCC(AATCCC)(3)]-5, as model for B-DNA. Both hexacation and octacation tend to aggregate in water. The structure as well as the ground and excited-state electronic properties of the Zn(II)/Pt(II) hexacation [(PtCl(2))(CH(3))(6)LZn](6+) in water solution were investigated using density functional theory (DFT) and time-dependent DFT (TDDFT) methods. TDDFT calculations of the lowest excited states of [(PtCl(2))(CH(3))(6)LZn](6+) in water provided an accurate description of the Q-band spectral region. In particular, the calculated optical spectra were in agreement with the experimental ones, obtained in the presence of micelles favoring complete disruption of the aggregates. The model for dsDNA binding that emerges from the analysis of UV-vis absorption and time-resolved fluorescence data shows the presence of complexes of 1 dsDNA molecule with 1, 2, and 4 macrocycles. Comparing the results for the hexacation [(PtCl(2))(CH(3))(6)LZn](6+) with those for the [(CH(3))(8)LZn](8+)octacation, we observed a higher degree of monomerization for the [(PtCl(2))(CH(3))(6)LZn](6+) derivative.

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).

Tetra-2,3-pyrazinoporphyrazines with externally appended pyridine rings. 10. A water-soluble bimetallic (Zn(II)/Pt(II)) porphyrazine hexacation as potential plurimodal agent for cancer therapy: exploring the behavior as ligand of telomeric DNA G-quadruplex structures.

The behavior of a bimetallic water-soluble (Zn(II)/Pt(II)) porphyrazine hexacation as ligand of G-quadruplex (G4) structures adopted by a human telomeric DNA sequence has been examined with different spectroscopic techniques. In K(+) rich solution the hexacationic Zn(II) porphyrazine ligand bearing a peripheral cis-platin-like functionality changes the G-quadruplex conformational equilibrium of the human telomeric sequence 5-d[AGGG(TTAGGG)(3)]-3 and drives it exclusively toward a very stable parallel G4 form in the complex with 2:1 stoichiometry. An increase of the melting temperature of more than 20 °C is observed in this complex compared to the G4 alone. On the contrary ligand binding to G-quadruplex of the same telomeric sequence in Na(+) rich solution neither markedly influences the predominant basket conformation nor confers increased thermal stability to the G4 structure.

Control of Template Positioning during de Novo Initiation of RNA Synthesis by the Bovine Viral Diarrhea Virus NS5B Polymerase

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5′-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3′-end of the template can remain opposite to the 5′-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3′-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.

Synthesis and activity studies of N-[ω-N′-(adamant-1′-yl)aminoalkyl]-2-(4′-dimethylaminophenyl)acetamides: In the search of selective inhibitors for the different molecular forms of acetylcholinesterase

A series of N-[omega-N-(adamant-1-yl)aminoalkyl]-2-(4- dimethylaminophenyl)acetamides were synthesized and tested as acetylcholinesterase inhibitors. A significant selectivity toward acetylcholinesterases from various natural sources, mainly differing in their quaternary structure and solubility, was pointed out. The interest of this kind of molecules as potential therapeutic agents for Alzheimers disease is discussed.