单纯疱疹病毒1型单链结合蛋白ICP8研究进展

在单纯疱疹病毒1型（Herpes simplex virus 1,HSV-1）中，感染细胞蛋白8(Infected cell protein 8,ICP8)是由UL29基因编码的一种单链DNA结合蛋白（Single strand DNA-binding protein,SSBP），该蛋白在病毒复制中必不可缺，具有维持单链DNA的稳定性，并与其他病毒蛋白相互结合，促进病毒复制室的形成，介导晚期基因的表达。除此之外，ICP8还具有促进UL9编码的起源结合蛋白（Origin binding protein,OBP）和UL5/UL8/UL52蛋白的酶活性，与UL12蛋白共同介导链交换等功能。本文就上述目前国内外对HSV-1 ICP8的研究进展作一综述，以期为后续研究ICP8的作用机制提供参考。     

螺旋藻多糖抗单纯疱疹病毒作用的实验研究

在体外进行了钝顶螺旋藻多糖(polysaccharides fromSpirulina platensis,PSP)抗单纯疱疹病毒活性的研究。以不同剂量的PSP分别作用于HSV-1及HSV-2病毒复制周期的各个环节,以病毒半数感染量(TCID50),细胞病变效应(CPE),蚀斑形成单位(PFU),MTT染色细胞保护率(MTT法)作为评价指标,判断PSP的抗病毒效果;FQ-PCR检测PSP抗病毒作用的时效关系。结果表明PSP对Vero细胞毒性极低(TC50为1750μg/mL),对HSV-1及HSV-2均无直接灭活作用,可阻滞HSV-1及HSV-2病毒吸附和抑制感染细胞内病毒的复制,但不影响病毒的释放;FQ-PCR结果显示随着PSP浓度及作用时间的增加,PSP对HSV-1病毒DNA的抑制作用明显增强,具有良好的剂量和时效关系。提示PSP抗HSV-1及HSV-2病毒作用的机制与抑制病毒吸附和感染细胞内病毒的生物合成有关。     

核内不均一核糖核蛋白A2B1功能及其在病毒感染中的作用研究进展

核内不均一核糖核蛋白A2B1(hnRNPA2B1)是核不均一核糖核蛋白家族（hnRNPs）成员，在各种组织中广泛表达，具有识别和结合RNA和DNA模体的功能，其在细胞生命活动，如新生转录物的包装、选择性剪接和翻译调控中起重要作用。研究发现，hnRNPA2B1在病毒感染和抗病毒免疫方面扮演重要的角色，具有识别胞内病毒核酸，激发抗病毒免疫通路，调控病毒感染、复制及病毒释放等功能。本文就hnRNPA2B1的结构、功能，尤其是在DNA和RNA病毒感染中所涉及的作用机制作一综述。     

HSV-1间层蛋白VP22转录调控功能的分析

HSV-1间层蛋白是一类重要的功能蛋白,在病毒复制的多个环节中具有重要意义．为了解主要间层蛋白VP22在病毒复制过程中的生物学特性,本实验利用氯霉素乙酰转移酶系统,分析了VP22对病毒启动子的转录调控功能,结果表明VP22对HSV-1觯,TK和gC基因启动子明显具有剂量效应关系的转录抑制作用;VP22也能抑制病毒不同转录调控因子（VP16和ICP0）对启动子的转录激活作用,尤其明显抑制ICP0对TK和gC基因启动子的转录激活作用;VP22能明显抑制组蛋白乙酰转移酶PCAF对ICP0转录激活的促进作用,此抑制作用较VP22抑制ICP0的转录激活作用更为显著．VP22对其他病毒启动子的转录抑制效应,在内对照实验β-gal活性分析中也得到了证明．     

单纯疱疹病毒1型立即早期蛋白ICP22研究进展

单纯疱疹病毒1型(Herpes simplex virus 1,HSV-1)感染细胞蛋白22(Infected Cell Protein 22,ICP22)是Us1基因编码的一种翻译后修饰多功能蛋白,为HSV-1的五种立即早期蛋白之一。HSV-1 ICP22能与不同的细胞和病毒成分相互作用来执行不同的功能,包括改变RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ/PolⅡ)的磷酸化状态、参与抵抗细胞对病毒复制的消极作用、引起细胞周期蛋白A和B水平降低、介导修饰拓扑异构酶Ⅱα、参与胞核内病毒诱导的分子伴侣富集(Virus-induced chaperone-enriched,VICE)区域的形成及促进病毒新生核衣壳的初次包装。这些作用大多与调节病毒在胞核中有效复制相关。此外,HSV-1 ICP22还在限制细胞中的病毒复制、病毒致病力和潜伏感染建立过程中发挥重要作用,但ICP22发挥这些作用的机制尚未知。本文就上述目前国内外对HSV ICP22的研究进展作一综述,以期为后续研究提供参考。     

TRIM家族蛋白在病毒感染中作用的研究进展

三基序蛋白家族(tripartite motif,TRIM)是参与不同细胞功能的一大类具有E3泛素连接酶活性的蛋白质，在宿主抗病毒免疫应答中发挥着重要的作用。TRIM家族蛋白可通过提高宿主固有免疫应答或直接降解病毒蛋白发挥抗病毒活性；部分病毒有时也可利用TRIM家族蛋白调控细胞因子表达促进自身感染。本文综述了TRIM家族蛋白在病毒复制中的作用及相关机制的研究进展，为研究病毒感染机制提供参考。     

HSV-1 UL41基因编码蛋白的RNase活性及其调控

单纯疱疹病毒1型(herpes simplex virus type-1,HSV-1)UL41基因编码一种皮层蛋白,该蛋白质具有核糖核酸酶(ribonuclease,RNase)活性,能特异性地降解一些宿主和病毒信使RNA(messenger RNA,m RNA),参与宿主免疫逃逸,与其他蛋白质相互作用调控其RNase活性。该文概述了HSV-1UL41基因编码蛋白的RNase活性及其调控机制,以期为该基因的深入研究提供参考。     

靶向ICP4的小干扰RNA对HSV-1病毒复制能力的影响

HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。     

动物宿主microRNAs抗病毒作用机制研究进展

microRNAs(miRNAs)是一种长度为22nt的非编码RNA分子,能够结合mRNA,影响翻译效率,调控蛋白的表达。近来多项研究表明,动物宿主miRNAs通过多种机制抑制病毒的复制,如直接靶向病毒基因、干扰病毒复制所需因子、调节先天性免疫、调节细胞凋亡、调节细胞免疫,发挥抗病毒效应。本文归纳总结宿主miRNAs对病毒复制的调控机制,讨论miRNAs类抗病毒药物的应用情况,并展望该类药物的发展趋势。     

Sequences at the C-terminus of the herpes simplex virus type 1 UL30 protein are dispensable for DNA polymerase activity but not for viral origin-dependent DNA replication.

The UL30 protein of herpes simplex virus type 1 (HSV-1) is a catalytically active DNA polymerase which is present in virus infected cells in a heterodimeric complex with an accessory subunit, the UL42 polypeptide. Both proteins are essential for viral DNA synthesis but because the UL42 protein is much more abundant it has been difficult to determine whether its role is related to, or independent of, its interaction with the UL30 protein in vivo. Since the C-terminal region of UL30 has been shown to be important for interaction with the UL42 protein but dispensable for DNA polymerase activity, a recombinant baculovirus which overexpresses a UL30 protein truncated by 27 amino acids at its C-terminus was constructed and used to assess the significance of the protein-protein interaction. The mutated protein was as active as wildtype (wt) UL30 in a DNA polymerase assay in which activated calf thymus DNA was used as template. However, in contrast to the wt protein, the activity of the truncated polymerase on this template was not stimulated by addition of purified UL42. A monoclonal antibody against the UL42 protein co-precipitated the full length but not truncated polymerase from extracts of cells which had been co-infected with a UL42-expressing recombinant baculovirus. Finally, the truncated protein was not active in a transient assay for HSV-1 origin-dependent DNA replication performed in insect cells in tissue culture. These results indicate that sequences at the C-terminus of the UL30 protein which are dispensable for DNA polymerase activity play essential roles both in viral DNA replication and interaction with the UL42 protein, and strongly suggest that the interaction between the proteins is important in vivo.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.     

The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity.

Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.     

Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template.

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.     

A mutation in the human herpes simplex virus type 1 UL52 zinc finger motif results in defective primase activity but can recruit viral polymerase and support viral replication efficiently

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase/primase complex consisting of UL5, UL8, and UL52. UL5 contains conserved helicase motifs, while UL52 contains conserved primase motifs, including a zinc finger motif. Although HSV-1 and HSV-2 UL52s contain a leucine residue at position 986, most other herpesvirus primase homologues contain a phenylalanine at this position. We constructed an HSV-1 UL52 L986F mutation and found that it can complement a UL52 null virus more efficiently than the wild type (WT). We thus predicted that the UL5/8/52 complex containing the L986F mutation might possess increased primase activity; however, it exhibited only 25% of the WT level of primase activity. Interestingly, the mutant complex displayed elevated levels of DNA binding and single-stranded DNA-dependent ATPase and helicase activities. This result confirms a complex interdependence between the helicase and primase subunits. We previously showed that primase-defective mutants failed to recruit the polymerase catalytic subunit UL30 to prereplicative sites, suggesting that an active primase, or primer synthesis, is required for polymerase recruitment. Although L986F exhibits decreased primase activity, it can support efficient replication and recruit UL30 efficiently to replication compartments, indicating that a partially active primase is capable of recruiting polymerase. Extraction with detergents prior to fixation can extract nucleosolic proteins but not proteins bound to chromatin or the nuclear matrix. We showed that UL30 was extracted from replication compartments while UL42 remained bound, suggesting that UL30 may be tethered to the replication fork by protein-protein interactions.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication.

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.     

An Intertypic Herpes Simplex Virus Helicase-Primase Complex Associated with a Defect in Neurovirulence Has Reduced Primase Activity

R13-1 is an intertypic recombinant virus in which the left-hand 18% of the herpes simplex virus type 1 (HSV-1) genome is replaced by homologous sequences from HSV-2. R13-1 is nonneurovirulent and defective in DNA replication in neurons. The defect was localized to the UL5 open reading frame by using marker rescue analysis (D. C. Bloom and J. G. Stevens, J. Virol. 68:3761–3772, 1994). To provide conclusive evidence that UL5 is the only HSV-2 gene involved in the restricted replication phenotype of R13-1, we have characterized the phenotype of a recombinant virus (IB1) in which only the UL5 gene of HSV-1 was replaced by HSV-2 UL5. Data from 50% lethal dose determinations and the in vivo yields of virus suggested that IB1 has the same phenotypic characteristics as R13-1. UL5 is the helicase component of a complex with helicase and primase activities. All three subunits of this complex (UL5, UL8, and UL52) are required for viral DNA replication in all cell types. The intertypic complex HSV-2 UL5–HSV-1 UL8–HSV-1 UL52 was purified and biochemically characterized. The primase activity of the intertypic complex was 10-fold lower than that of HSV-1 UL5–HSV-1 UL8–HSV-1 UL52. The ATPase activity was comparable to that of the HSV-1 enzyme complex, and although the helicase activity was threefold lower, this did not interfere with the synthesis of leading strands by the HSV polymerase. One explanation for these findings is that the interactions between the subunits of the helicase-primase intertypic complex that are important for the full function of each subunit are inappropriate or weak.     

The three-component helicase/primase complex of herpes simplex virus-1

Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase–primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase–primase complex for development of alternative anti-viral treatments.