No abstract

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (>30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.Several studies have suggested that the antiviral activity of phosphorothioated oligonucleotides (PS-ONs) with different sequences or different intended mechanisms of action (i.e., antisense or sequence-specific aptamers) all work as inhibitors of human immunodeficiency virus (HIV) viral entry by binding to the V3 loop of gp120 and preventing CD4 interaction and attachment to the host cell (13, 43). The general conservation of this mechanism of action with different oligonucleotides having different sequences suggested that the antiviral activity of oligonucleotides against HIV was sequence independent.The sequence-independent nature of the antiviral activity of oligonucleotides (ONs) was more clearly demonstrated in several studies using degenerate or homopolymeric ONs (24,30,37). In these studies, the anti-HIV activities of 28-mer phosphorothioated polycytidylic, polyadenylic, and polythimidylic or fully degenerate ONs were studied in vitro, and in the case of polycytidylic ONs, it was also discovered that for ONs up to 28 bases in length increasingly larger ONs displayed more potent antiviral activity. The binding of 28-mer polycytidylic ONs to the V3 loop of gp120 was also described (38).PS-ONs are also polyanionic, and the antiviral activities of a variety of other classes of polyanions, such as sulfated saccharides, polysulfonates, polyhydroxycarboxylates, and polyoxometalates, have also been well established (11,28,31,34,35).There is good evidence to suggest that these compounds also interfere with viral attachment by binding to the V3 loop of gp120 (2,12,33,40).To further explore the sequence-independent mechanisms of t...

BACKGROUND & AIMS-Hepatitis C virus (HCV) gains entry into susceptible cells by interacting with cell surface receptor(s). Viral entry is an attractive target for antiviral development because of the highly conserved mechanism.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) represents a powerful experimental model for the study of the basic virology and pathogenesis of arenaviruses. In the present study, we used the LCMV model to evaluate the anti-viral potential of phosphorothioate oligonucleotides against arenaviruses. Our findings indicate that amphipathic DNA polymers (APs) are potent inhibitors of infection with a series of LCMV isolates with IC(50) in the low nanomolar range. APs target the surface glycoprotein (GP) of LCMV and block viral entry and cell-cell propagation of the virus, without affecting later steps in replication or release of progeny virus from infected cells. The anti-viral action of APs is sequence-independent but is critically dependent on their size and hydrophobicity. Mechanistically, we provide evidence that APs disrupt the interaction between LCMVGP and its cellular receptor, alpha-dystroglycan. Exposure of LCMV to APs does not affect the stability of the GP virion spike and has no effect on the conformation of a neutralizing antibody epitope, suggesting rather subtle changes in the conformation and/or conformational dynamics of the viral GP.