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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.

Bazhulina NP, Surovaya AN, Gursky YG, Andronova VL, Moiseeva ED, Nikitin CA, Golovkin MV, Galegov GА, Grokhovsky SL, Gursky GV - J. Biomol. Struct. Dyn. (2013)

Bottom Line: 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.The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase.The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.

View Article: PubMed Central - PubMed

Affiliation: a V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , ul. Vavilova 32, 119991 , Moscow , Russia .

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.

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The nucleotide sequence at the HSV1 origin of replication OriS (A). The positions of DNA segments carrying palindromic sequences are indicated by arrows. These segments contain binding sites I and III for helicase (Boxes I and III), the A + T cluster and Box II. D and A – donor and acceptor molecules covalently linked to the 5′- and 3′-ends of the oligonucleotide in the minimal duplex OriS. Indicated are the proposed stem and loop structures for a fragment (63 nt) of the upper chain in the minimal OriS duplex (panels B and C).
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Figure 1: The nucleotide sequence at the HSV1 origin of replication OriS (A). The positions of DNA segments carrying palindromic sequences are indicated by arrows. These segments contain binding sites I and III for helicase (Boxes I and III), the A + T cluster and Box II. D and A – donor and acceptor molecules covalently linked to the 5′- and 3′-ends of the oligonucleotide in the minimal duplex OriS. Indicated are the proposed stem and loop structures for a fragment (63 nt) of the upper chain in the minimal OriS duplex (panels B and C).

Mentions: Figure 1 shows the nucleotide sequence of the OriS and the hypothetical hairpin structures formed in the upper strand of the minimal duplex OriS (63 base pairs) after separation of DNA strands (Aslani, Macao, Simonsson, & Elias, 2001; Aslani, Olsson, & Elias, 2002; Bazhulina et al., 2012; Surovaya et al., 2010). In the present study, kinetics of DNA unwinding by OBP was monitored by measuring the efficiency of the resonance energy transfer between the fluorophore and quencher covalently linked to the 5′- and 3′-ends of the oligonucleotide. The data obtained suggest that Pt-bis-netropsin inhibits the unwinding of the OriS duplex by OBP. We investigated antiviral activities of bis-netropsins and their therapeutic effects on the HSV1-infected laboratory animals.


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.

Bazhulina NP, Surovaya AN, Gursky YG, Andronova VL, Moiseeva ED, Nikitin CA, Golovkin MV, Galegov GА, Grokhovsky SL, Gursky GV - J. Biomol. Struct. Dyn. (2013)

The nucleotide sequence at the HSV1 origin of replication OriS (A). The positions of DNA segments carrying palindromic sequences are indicated by arrows. These segments contain binding sites I and III for helicase (Boxes I and III), the A + T cluster and Box II. D and A – donor and acceptor molecules covalently linked to the 5′- and 3′-ends of the oligonucleotide in the minimal duplex OriS. Indicated are the proposed stem and loop structures for a fragment (63 nt) of the upper chain in the minimal OriS duplex (panels B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066892&req=5

Figure 1: The nucleotide sequence at the HSV1 origin of replication OriS (A). The positions of DNA segments carrying palindromic sequences are indicated by arrows. These segments contain binding sites I and III for helicase (Boxes I and III), the A + T cluster and Box II. D and A – donor and acceptor molecules covalently linked to the 5′- and 3′-ends of the oligonucleotide in the minimal duplex OriS. Indicated are the proposed stem and loop structures for a fragment (63 nt) of the upper chain in the minimal OriS duplex (panels B and C).
Mentions: Figure 1 shows the nucleotide sequence of the OriS and the hypothetical hairpin structures formed in the upper strand of the minimal duplex OriS (63 base pairs) after separation of DNA strands (Aslani, Macao, Simonsson, & Elias, 2001; Aslani, Olsson, & Elias, 2002; Bazhulina et al., 2012; Surovaya et al., 2010). In the present study, kinetics of DNA unwinding by OBP was monitored by measuring the efficiency of the resonance energy transfer between the fluorophore and quencher covalently linked to the 5′- and 3′-ends of the oligonucleotide. The data obtained suggest that Pt-bis-netropsin inhibits the unwinding of the OriS duplex by OBP. We investigated antiviral activities of bis-netropsins and their therapeutic effects on the HSV1-infected laboratory animals.

Bottom Line: 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.The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase.The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.

View Article: PubMed Central - PubMed

Affiliation: a V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , ul. Vavilova 32, 119991 , Moscow , Russia .

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.

Show MeSH
Related in: MedlinePlus