ABSTRACT
Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies bis-netropsins get bound selectively to an A+T-cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA bis-netropsins stabilize a structure of the A+T-cluster and inhibit thermal fluctuation-induced opening of AT- base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3?- and 5?-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT- rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.
Subject(s)
Antiviral Agents/pharmacology , DNA Replication/drug effects , DNA, Viral/metabolism , DNA-Binding Proteins , Herpes Simplex , Herpesvirus 1, Human/enzymology , Netropsin/pharmacology , Viral Proteins , Animals , Chlorocebus aethiops , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Herpes Simplex/drug therapy , Herpes Simplex/enzymology , Mice , Mice, Inbred BALB C , Netropsin/analogs & derivatives , Vero Cells , Viral Proteins/antagonists & inhibitors , Viral Proteins/metabolismABSTRACT
The protein binding to the origin of replication of the herpes simplex virus type 1 (HSV-1) is DNA helicase encoded by the UL9 gene of the herpes virus. The protein specifically binds to two binding sites in the viral DNA replication origins OriS or OriL. In order to determine the role of the UL9 protein in the initiation of replication and find efficient inhibitors of the UL9 activity, we have synthesized a recombinant UL9 protein expressed in E. coli cells. It was found that the recombinant UL9 protein binds to Boxes I and II in the OriS and possesses the DNA helicase and ATPase activities. In a complex with a fluorescent analog of ATP, two molecules of the ATP analog bind to one protein dimer molecule. It was also found that the UL9 protein in the dimer form can bind simultaneously to two DNA fragments, each containing specific binding sites for the protein. The interaction of the recombinant UL9 protein with the 63-mer double and single-stranded oligonucleotides OriS and OriS* has been investigated, which correspond to the origin of replication of herpes simplex virus. From the titrations of OriS and OriS* by ethidium bromide in the presence and absence of the UL9 protein, the equilibrium affinity constants of the protein binding to OriS and OriS* have been determined. A DNase I footprinting study showed that bis-linked netropsin derivatives exhibit preferences for binding to the AT-cluster in the origin of replication OriS and inhibit the fluctuation opening of AT-base pairs in the AT-cluster. The drugs also prevent the formation of an intermediate conformation of OriS* that involves a disordered tail at the 3'-end and stable Box I-Box III hairpin to which the UL9 helicase selectively binds. The stabilization by bis-netropsins of the AT-rich hairpin at its 3' end can inhibit the helicase activity. It was concluded that the antiviral activity of bis-netropsins may be associated with the inhibitory effects of bis-netropsins on these two stages of the reaction catalyzed by helicase UL9.
Subject(s)
Antiviral Agents/chemistry , DNA Helicases/chemistry , DNA, Viral/chemistry , DNA-Binding Proteins/chemistry , Herpesvirus 1, Human/enzymology , Netropsin/analogs & derivatives , Viral Proteins/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Antiviral Agents/therapeutic use , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , Herpes Simplex/drug therapy , Herpes Simplex/enzymology , Herpesvirus 1, Human/genetics , Netropsin/chemistry , Netropsin/therapeutic use , Protein Binding/drug effects , Protein Binding/physiology , Protein Multimerization/drug effects , Protein Multimerization/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Replication Origin/physiology , Viral Proteins/geneticsSubject(s)
Cell Survival/drug effects , Herpesvirus 1, Human/drug effects , Herpesvirus 1, Human/physiology , Netropsin/administration & dosage , Vaccinia virus/drug effects , Vaccinia virus/physiology , Animals , Antiviral Agents/administration & dosage , Chlorocebus aethiops , Cytotoxins/administration & dosage , Dose-Response Relationship, Drug , Vero CellsABSTRACT
Optimal conditions of protein scanning by atomic force microscopy were developed. Proteins of different molecular masses (950-11.5 kDa) and different three-dimensional organization were used to investigate the structural features of proteins. The most distinct images of proteins were obtained using a tip with the free amplitude in the range of 5-15 nm and with the set-point amplitude in the regime of repulsion from the sample. The method allowed one to clearly recognize the structural details of large molecules such as immunoglobulins IgM and IgG1 and Ricinus agglutinin. The revealing of the structural properties of proteins with molecular masses of 60 kDa and less was limited by the sharpness of probe tips used in the present study. It was shown that, by a quantitative analysis of the geometric parameters of molecules, it is possible to distinguish IgG1, Ricinus agglutinin, and ricin.