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单纯疱疹病毒1型起源结合蛋白UL9与DNA的复合物作为新型抗病毒药物设计的平台。

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.

作者信息

Bazhulina N P, Surovaya A N, Gursky Y G, Andronova V L, Moiseeva E D, Nikitin Capital A Cyrillic M, Golovkin M V, Galegov G А, Grokhovsky S L, Gursky G V

机构信息

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

出版信息

J Biomol Struct Dyn. 2014;32(9):1456-73. doi: 10.1080/07391102.2013.820110. Epub 2013 Jul 24.

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.

摘要

单纯疱疹病毒1型起始结合蛋白(OBP)是一种由UL9基因编码的DNA解旋酶。该蛋白以序列特异性方式结合到病毒复制起点,即两个OriS位点和一个OriL位点。为了寻找OBP活性的有效抑制剂,我们获得了在大肠杆菌细胞中表达的重组起始结合蛋白。通过PCR扩增UL9基因,并将其插入修饰后的质粒pET14的NdeI和KpnI位点之间。重组蛋白与Box I和Box II序列结合,并具有解旋酶和ATP酶活性。在ATP和病毒蛋白ICP8(单链DNA结合蛋白)存在的情况下,起始蛋白诱导最小OriS双链体(约80 bp)解旋。该蛋白还结合到一条单链DNA(OriS*)上,该单链DNA在3'端含有一个稳定的Box I-Box III发夹和一个不稳定的富含AT的发夹。在本研究中,合成了新的小沟结合配体,它们能够抑制培养的Vero细胞中病毒诱导的细胞病变效应的发展。通过荧光方法、凝胶迁移率变动分析和足迹分析对这些化合物与DNA和合成寡核苷酸的结合进行了研究。足迹研究表明,铂-双嗜中性菌素及相关分子对结合到OriS中的AT间隔区表现出偏好。这些药物稳定了富含AT区域的结构,并抑制了AT碱基对的波动打开,而这是OBP解开DNA的前提条件。通过测量最小OriS双链体中连接到寡核苷酸5'端和3'端的荧光团之间的福斯特共振能量转移(FRET)效率,研究了在有无嗜中性菌素衍生物存在的情况下OriS的ATP依赖性解旋动力学。结果与OBP是DNA Holiday连接(HJ)结合解旋酶的推测一致。该蛋白在没有ATP的情况下诱导OriS双链体的构象变化(弯曲和部分解链)并刺激HJ形成。双嗜中性菌素的抗病毒活性与其抑制A+T簇中AT碱基对的波动打开的能力以及稳定对应于最小双链体OriS上链的单链寡核苷酸中富含AT发夹结构的能力相关。研究了双嗜中性菌素在细胞培养中的抗病毒活性及其对HSV1感染实验动物的治疗效果。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2709/4066892/231c816aa6b1/tbsd32_1456_f1.jpg

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