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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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EMSA studies in the nondenaturing 10% polyacrylamide gel of complexes formed by OBP with single-stranded oligonucleotide S8 (panel a), partial duplex (S3* + S7) and oligomer (S3* + S7 + S8*) (panel b). Lane 0, oligonucleotide S8* alone (panel a), ternary complex (S3* + S7* + S8*) and partial duplex (S3* + S7) in the absence of OBP and ATP (panel b). In panels a, b, and c, lane 1 corresponds to a complex of OBP with single-stranded oligonucleotide S8* (panel a), duplex (S3* + S7*) (panel b) and oligomer (S3* + S7* + S8*) (panel c) in the absence of ATP. Lane 2 corresponds to the same complex incubated for 30 min at 37°C in 50 mM Tris-HCl buffer (pH 7.8) in the presence of 5 mM dithiothreitol, 15 mM MgCl2, 10% (v/v) glycerol; .01% (v/v) Triton X-100 and 20 mM ATP. The reaction mixtures were cooled to room temperature and loaded on a 10% nondenaturing polyacrylamide gel (19:1) with a thickness of .4 mm. Electrophoresis was carried out for 120 min at 800 V and temperature 20 °C.
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Figure 11: EMSA studies in the nondenaturing 10% polyacrylamide gel of complexes formed by OBP with single-stranded oligonucleotide S8 (panel a), partial duplex (S3* + S7) and oligomer (S3* + S7 + S8*) (panel b). Lane 0, oligonucleotide S8* alone (panel a), ternary complex (S3* + S7* + S8*) and partial duplex (S3* + S7) in the absence of OBP and ATP (panel b). In panels a, b, and c, lane 1 corresponds to a complex of OBP with single-stranded oligonucleotide S8* (panel a), duplex (S3* + S7*) (panel b) and oligomer (S3* + S7* + S8*) (panel c) in the absence of ATP. Lane 2 corresponds to the same complex incubated for 30 min at 37°C in 50 mM Tris-HCl buffer (pH 7.8) in the presence of 5 mM dithiothreitol, 15 mM MgCl2, 10% (v/v) glycerol; .01% (v/v) Triton X-100 and 20 mM ATP. The reaction mixtures were cooled to room temperature and loaded on a 10% nondenaturing polyacrylamide gel (19:1) with a thickness of .4 mm. Electrophoresis was carried out for 120 min at 800 V and temperature 20 °C.

Mentions: Figure 11 shows the EMSA analysis of the products present in the reaction mixture after incubation of the oligomer (S3* + S7* + S8*) with OBP and ATP for 30 min at 37 °C and subsequent cooling of the reaction mixture to room temperature. EMSA studies show that the amount of free radioactively labeled oligomers (S3* + S7 + S8*) in the solution decreased after addition of OBP. Slowly migrating species are formed that may represent a 4-ways HJ built by two oligomers (S3* + S7* + S8*) each forming a 1:1 complex with OBP. After incubation of the reaction mixture at 37 °C for 30 min in the presence of ATP and Mg+2 ions, the presumed complex of HJ with OBP (designated as X+UL9) partially dissociates, and a slowly migrating complex of OBP with a folded form of oligonucleotide S3* is observed. In addition, free (S3* + S7* + S8*) oligomers and oligonucleotide S8* are also present in the reaction mixture (Figure 11, panel C). The dissociation of HJ in the presence of ATP and Mg+2 ions is reminiscent of complexes of UvrD and RecG helicases with synthetic HJs (Carter et al., 2012; Whitby & Lloyd, 1998).


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)

EMSA studies in the nondenaturing 10% polyacrylamide gel of complexes formed by OBP with single-stranded oligonucleotide S8 (panel a), partial duplex (S3* + S7) and oligomer (S3* + S7 + S8*) (panel b). Lane 0, oligonucleotide S8* alone (panel a), ternary complex (S3* + S7* + S8*) and partial duplex (S3* + S7) in the absence of OBP and ATP (panel b). In panels a, b, and c, lane 1 corresponds to a complex of OBP with single-stranded oligonucleotide S8* (panel a), duplex (S3* + S7*) (panel b) and oligomer (S3* + S7* + S8*) (panel c) in the absence of ATP. Lane 2 corresponds to the same complex incubated for 30 min at 37°C in 50 mM Tris-HCl buffer (pH 7.8) in the presence of 5 mM dithiothreitol, 15 mM MgCl2, 10% (v/v) glycerol; .01% (v/v) Triton X-100 and 20 mM ATP. The reaction mixtures were cooled to room temperature and loaded on a 10% nondenaturing polyacrylamide gel (19:1) with a thickness of .4 mm. Electrophoresis was carried out for 120 min at 800 V and temperature 20 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: EMSA studies in the nondenaturing 10% polyacrylamide gel of complexes formed by OBP with single-stranded oligonucleotide S8 (panel a), partial duplex (S3* + S7) and oligomer (S3* + S7 + S8*) (panel b). Lane 0, oligonucleotide S8* alone (panel a), ternary complex (S3* + S7* + S8*) and partial duplex (S3* + S7) in the absence of OBP and ATP (panel b). In panels a, b, and c, lane 1 corresponds to a complex of OBP with single-stranded oligonucleotide S8* (panel a), duplex (S3* + S7*) (panel b) and oligomer (S3* + S7* + S8*) (panel c) in the absence of ATP. Lane 2 corresponds to the same complex incubated for 30 min at 37°C in 50 mM Tris-HCl buffer (pH 7.8) in the presence of 5 mM dithiothreitol, 15 mM MgCl2, 10% (v/v) glycerol; .01% (v/v) Triton X-100 and 20 mM ATP. The reaction mixtures were cooled to room temperature and loaded on a 10% nondenaturing polyacrylamide gel (19:1) with a thickness of .4 mm. Electrophoresis was carried out for 120 min at 800 V and temperature 20 °C.
Mentions: Figure 11 shows the EMSA analysis of the products present in the reaction mixture after incubation of the oligomer (S3* + S7* + S8*) with OBP and ATP for 30 min at 37 °C and subsequent cooling of the reaction mixture to room temperature. EMSA studies show that the amount of free radioactively labeled oligomers (S3* + S7 + S8*) in the solution decreased after addition of OBP. Slowly migrating species are formed that may represent a 4-ways HJ built by two oligomers (S3* + S7* + S8*) each forming a 1:1 complex with OBP. After incubation of the reaction mixture at 37 °C for 30 min in the presence of ATP and Mg+2 ions, the presumed complex of HJ with OBP (designated as X+UL9) partially dissociates, and a slowly migrating complex of OBP with a folded form of oligonucleotide S3* is observed. In addition, free (S3* + S7* + S8*) oligomers and oligonucleotide S8* are also present in the reaction mixture (Figure 11, panel C). The dissociation of HJ in the presence of ATP and Mg+2 ions is reminiscent of complexes of UvrD and RecG helicases with synthetic HJs (Carter et al., 2012; Whitby & Lloyd, 1998).

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