title: Complex of the herpes simplex virus type 1 origin binding protein UL9 with DNA as a platform for the design of a new type of antiviral drugs.
abstract: The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80 bp). The protein also binds to a single-stranded DNA (OriS*) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3'-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5'- and 3'- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А + Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.
authors.affiliation: a V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , ul. Vavilova 32, 119991 , Moscow , Russia .
authors.forename: N P
authors.initials: NP
authors.lastname: Bazhulina
authors.forename: A N
authors.initials: AN
authors.lastname: Surovaya
authors.forename: Y G
authors.initials: YG
authors.lastname: Gursky
authors.forename: V L
authors.initials: VL
authors.lastname: Andronova
authors.forename: E D
authors.initials: ED
authors.lastname: Moiseeva
authors.forename: Capital A Cyrillic M
authors.initials: CA
authors.lastname: Nikitin
authors.forename: M V
authors.initials: MV
authors.lastname: Golovkin
authors.forename: G А
authors.initials: GА
authors.lastname: Galegov
authors.forename: S L
authors.initials: SL
authors.lastname: Grokhovsky
authors.forename: G V
authors.initials: GV
authors.lastname: Gursky
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D009429:Netropsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D009944:Organoplatinum Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009944:Organoplatinum Compounds; D011994:Recombinant Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; C068200:Pt-bis-netropsin; D009429:Netropsin; D004265:DNA Helicases
keywords: DNA helicase; FRET method; antiviral activity; bis-linked netropsin derivatives; herpes simplex virus type 1; replication origin binding protein