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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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Changes in the (S3 + S7 + S8) oligomer structure and aggregation state which are induced by OBP in the absence (A) and presence of Pt-bis-netropsin (B) and ATP. I0 is the fluorescence intensity of the free (S3 + S7 + S8) oligomer at 559 nm. Excitation wavelength – 510 nm, emission and excitation slit widths are 5 and 10 nm, respectively. Additions of OBP, Pt-bis-Nt and ATP to the (S3 + S7 + S8) oligomer solutions are indicated by arrows. Concentrations: ATP – 1.8 × 10−2 M, (S3 + S7 + S8) – 1.1 × 10−7 M, UL9 helicase – 3.95 × 10−7 M, Pt-bis-Nt – 8.9 × 10−7 M. The solid lines corresponding to the dependencies on time I559(t) (panels: A and B) and I670(t) (panel C) show the best fits to the experimental data points. Conditions are indicated in the legend to Figure 8.
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Figure 10: Changes in the (S3 + S7 + S8) oligomer structure and aggregation state which are induced by OBP in the absence (A) and presence of Pt-bis-netropsin (B) and ATP. I0 is the fluorescence intensity of the free (S3 + S7 + S8) oligomer at 559 nm. Excitation wavelength – 510 nm, emission and excitation slit widths are 5 and 10 nm, respectively. Additions of OBP, Pt-bis-Nt and ATP to the (S3 + S7 + S8) oligomer solutions are indicated by arrows. Concentrations: ATP – 1.8 × 10−2 M, (S3 + S7 + S8) – 1.1 × 10−7 M, UL9 helicase – 3.95 × 10−7 M, Pt-bis-Nt – 8.9 × 10−7 M. The solid lines corresponding to the dependencies on time I559(t) (panels: A and B) and I670(t) (panel C) show the best fits to the experimental data points. Conditions are indicated in the legend to Figure 8.

Mentions: Consistent with these results are the observations obtained for the (S3 + S7 + S8) oligomer which differs from the (S3 + S6) duplex in that the oligonucleotides S7 and S8 replace oligonucleotide S6 in a complex with the oligonucleotide S3 (Figure 10). After annealing of the oligonucleotides S3, S7, and S8, the upper strand of the oligomer contained a 3′-terminal single-stranded tail of 18 nucleotides long. The analogue of the cyanine dye Cy5 is tethered to the 3′-end of the oligonucleotide S8, whereas the donor (R6G) is attached to the 5′-end of the oligonucleotide S7. The observed quenching effect reflects a change in the efficiency of energy transfer from the excited donor to the acceptor attached to the 5′- and 3′- ends of the oligonucleotides S7 and S8. Consistent with this are the observations that a decrease of the fluorescence intensity at 559 nm is accompanied by an increase of the fluorescence intensity at 670 nm (Figure 10, panels A and C). This can be attributed to the energy transfer from the excited donor (R6G) to the acceptor (Cy5) conjugated with oligonucleotides S7 and S8, respectively.


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)

Changes in the (S3 + S7 + S8) oligomer structure and aggregation state which are induced by OBP in the absence (A) and presence of Pt-bis-netropsin (B) and ATP. I0 is the fluorescence intensity of the free (S3 + S7 + S8) oligomer at 559 nm. Excitation wavelength – 510 nm, emission and excitation slit widths are 5 and 10 nm, respectively. Additions of OBP, Pt-bis-Nt and ATP to the (S3 + S7 + S8) oligomer solutions are indicated by arrows. Concentrations: ATP – 1.8 × 10−2 M, (S3 + S7 + S8) – 1.1 × 10−7 M, UL9 helicase – 3.95 × 10−7 M, Pt-bis-Nt – 8.9 × 10−7 M. The solid lines corresponding to the dependencies on time I559(t) (panels: A and B) and I670(t) (panel C) show the best fits to the experimental data points. Conditions are indicated in the legend to Figure 8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Changes in the (S3 + S7 + S8) oligomer structure and aggregation state which are induced by OBP in the absence (A) and presence of Pt-bis-netropsin (B) and ATP. I0 is the fluorescence intensity of the free (S3 + S7 + S8) oligomer at 559 nm. Excitation wavelength – 510 nm, emission and excitation slit widths are 5 and 10 nm, respectively. Additions of OBP, Pt-bis-Nt and ATP to the (S3 + S7 + S8) oligomer solutions are indicated by arrows. Concentrations: ATP – 1.8 × 10−2 M, (S3 + S7 + S8) – 1.1 × 10−7 M, UL9 helicase – 3.95 × 10−7 M, Pt-bis-Nt – 8.9 × 10−7 M. The solid lines corresponding to the dependencies on time I559(t) (panels: A and B) and I670(t) (panel C) show the best fits to the experimental data points. Conditions are indicated in the legend to Figure 8.
Mentions: Consistent with these results are the observations obtained for the (S3 + S7 + S8) oligomer which differs from the (S3 + S6) duplex in that the oligonucleotides S7 and S8 replace oligonucleotide S6 in a complex with the oligonucleotide S3 (Figure 10). After annealing of the oligonucleotides S3, S7, and S8, the upper strand of the oligomer contained a 3′-terminal single-stranded tail of 18 nucleotides long. The analogue of the cyanine dye Cy5 is tethered to the 3′-end of the oligonucleotide S8, whereas the donor (R6G) is attached to the 5′-end of the oligonucleotide S7. The observed quenching effect reflects a change in the efficiency of energy transfer from the excited donor to the acceptor attached to the 5′- and 3′- ends of the oligonucleotides S7 and S8. Consistent with this are the observations that a decrease of the fluorescence intensity at 559 nm is accompanied by an increase of the fluorescence intensity at 670 nm (Figure 10, panels A and C). This can be attributed to the energy transfer from the excited donor (R6G) to the acceptor (Cy5) conjugated with oligonucleotides S7 and S8, respectively.

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