Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-(3)H]uridine or [methyl-(3)H]thymidine or (32)PO(4) (3-). Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-(3)H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [(3)H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-(3)H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-(3)H]uridine-labeled DNA banded at the same position as the (32)P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures.     

Interleukin-18 Protects Mice against Acute Herpes Simplex Virus Type 1 Infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-γ), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-γ release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-γ production was found in uninfected mice administered IL-18. Although IFN-γ has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor N^G-monomethyl-l-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-γ produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.Interleukin-18 (IL-18) is a newly cloned murine and human cytokine (28, 36) previously called gamma interferon (IFN-γ)-inducing factor. It is synthesized by activated macrophages and has a structural relationship to the IL-1 family (5). Precursor IL-18 is processed by IL-1β-converting enzyme and is cleaved into mature IL-18 (11). IL-18 induces IFN-γ production by murine helper T cells and NK cells and stimulates T-cell proliferation and NK activation (18, 28). Moreover, IL-18 augments the Fas ligand-mediated cytotoxic activity of the Th1 clone and the NK cell clone (8, 35). Thus, IL-18 shares some biological activities with IL-12, although no significant homology between the two cytokines has been detected at the protein level (34). Furthermore, treatment with IL-12 and IL-18 has a synergistic effect on IFN-γ production (2, 14, 38, 40).According to a review by Nash (27), not only nonspecific or innate immunity, such as that from IFN, NK cells, or macrophages, but also specific or adaptive immunity is important in protection against herpesvirus infection. Herpes simplex virus is known to be an IFN inducer (13). IFN is produced at an early stage of virus infection. In addition to the direct inhibition of viral replication, it enhances the efficiency of the adaptive (specific) immune response by stimulating increased expression of major histocompatibility complex class I and II or by activating macrophages and NK cells. In protection from infection by herpesviruses, especially cytomegalovirus, NK cells have been major effector cells because of the correlation of increased susceptibility to cytomegalovirus infection with the absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome patients and beige mice (27). Upon target cell disruption, NK and cytotoxic T cells share not only the perforin but also the Fas ligand as an effector molecule (4, 20, 37). Recently, nitric oxide (NO) was reported to be involved in host defense against bacteria, fungi, parasites, and viruses (10, 16, 19, 39). NO produced by herpes simplex virus type 1 (HSV-1)-infected macrophages is reported to inhibit viral replication (7). CD4⁺ T cells, macrophages, IFN-γ, and tumor necrosis factor (TNF) are important in adaptive immunity against HSV-1 infection. The Th2 response exacerbates HSV-1-induced disease (25).Recently a protective role of IL-18 was reported in microbial infections (6, 17). Here, we demonstrate that IL-18 treatment protects mice from acute viral infection via both IFN-γ-dependent and -independent pathways. Although IFN-γ has the ability to induce NO production by a variety of cells, including macrophages (9), it is not likely to be important, at least in the inhibition of HSV-1 proliferation at the injection site of IL-18-treated mice. Furthermore, the protective effect of IL-18 on HSV-1 infection also does not seem to require complete NK cell activity in our experimental system, whereas our colleagues have already reported that deletion of NK cells by administration of anti-asialo GM1 antibody resulted in lowering of the improved survival rate of tumor-bearing mice treated with IL-18 (23).     

单纯疱疹病毒致病模型的研究

对单纯疱疹病毒（HSV）感染小白鼠致病特点进行了观察。小白鼠感染HSV第4天后开始发病,感染后2h血液内可分离出病毒,第48小时病毒血症水平和病毒检出率较高。不同组织病毒分布不同,脑、神经节在感染后第72小时病毒滴度较高,心、肝组织在第5天达到高峰。结果说明所建立的HSV致病模型可用于评价抗HSV药物。     

PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

宫颈疾患中人乳头瘤病毒和疱疹病毒Ⅱ型DNA的检测

本文应用HPV11,16,18型和HSV-2N/BglⅡ、HSV-2L/HindⅢDNA片段等五个分子探针,通过斑点杂交技术对79例宫颈疾患(包括50例宫颈癌和29例宫颈糜烂)组织DNA进行了检测,结果发现宫颈癌组织HPV16,18和11的阳性率分别为44％,12％和4％,而宫颈糜烂组织中HPV16,18和11的阳性率分别为14％,7％和14％;且3例标本HPV16和HPV18均呈弱杂交反应;在被检的所有宫颈癌组织中各有2例分别与HSV-2N/BglⅡHSV-2L/HindⅢ弱杂交,宫颈糜烂组织无一例阳性。结果提示,HPV在宫颈癌的发生过程中可能起主要作用,HSV-2的作用尚不确定,可能与HPV起协同作用。     

Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo⁻) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo⁻ HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo⁻ HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.Herpes simplex virus type 1 (HSV-1), the prototypic member of the family of Herpesviridae and that of the alphaherpesviridae subfamily, has served as the model for understanding the replication of herpesvirus genomes during lytic virus replication (29). The 152-kbp genome of herpes simplex virus type 1 (HSV-1) possesses approximately 85 genes, 7 of which have been shown to be necessary and sufficient for viral DNA replication within host cells (reviewed in references 5 and 38). These seven genes encode a DNA polymerase (pol) and its processivity factor (UL42), a heterotrimeric complex containing a DNA helicase (UL5), primase (UL52), and noncatalytic accessory protein (UL8), a single-stranded DNA binding protein (infected cell protein 8 [ICP-8]), and an origin binding protein with DNA helicase activity (UL9). There is strong evidence in support of the circularization of the linear virion DNA shortly after entry, and DNA replication then is thought to initiate at one or more of the three redundant origins of replication (29, 38). At least in the earliest stages of viral DNA replication, UL9 protein is required, presumably to bind to and unwind the DNA and to attract the other DNA replication proteins (29, 38). The electron microscopic examination of pulse-labeled replicating HSV-1 DNA indicates the presence of lariats, eye-forms, and D-forms (21), which is consistent with bidirectional theta-like replication from origins. To date, however, no biochemical assay has demonstrated origin-dependent DNA replication in vitro. However, in the absence of UL9, the other six HSV DNA replication proteins can support initiation and replication from a circular single-stranded DNA (ssDNA) template in an origin-independent fashion (15, 26), resembling the rolling-circle mode of replication thought to occur during the later stages of viral replication.Although nicks and small gaps have been observed in isolated replicating and virion DNA (38), the evidence for bidirectional duplex synthesis, the rapid rate of viral DNA replication, and the absence of long stretches of ssDNA in replicating and mature DNA isolated from HSV-1-infected cells suggest that leading- and lagging-strand synthesis are closely coordinated in vivo. Falkenberg et al. (15) used a minicircle DNA template with a strand bias and the six essential HSV-1 DNA replication proteins needed for rolling circle replication to demonstrate lagging-strand synthesis in vitro. However, replication from the parental strand template (leading-strand synthesis) was more efficient than synthesis from the complementary-strand template (lagging-strand synthesis). These results suggest the possibility that one or more host functions required for efficient lagging-strand synthesis or for its close coordination with leading-strand synthesis is missing in such in vitro systems.Although leading- and lagging-strand syntheses share many of the same requirements for bulk DNA synthesis, lagging-strand synthesis is a more complex process. Because the direction of polymerization of lagging-strand intermediates is opposite the direction of replication fork movement, lagging-strand synthesis requires that priming and extension occur many times to produce discontinuous segments called Okazaki fragments (reviewed in reference 25). Okazaki fragments need to be processed to remove the RNA primer, to fill in the area previously occupied by the RNA, and to seal the remaining nick between fragments, all of which must occur efficiently, accurately, and completely. Failure to do so would result in the accumulation of DNA breaks, multiple mutations, delayed DNA replication, and/or cell death (16, 61).In eukaryotes, what is currently known regarding the process of lagging-strand synthesis is based on genetic and biochemical studies with Saccharomyces cerevisiae and on in vitro reconstitution studies to define the mammalian enzymes required for simian virus 40 (SV40) T-antigen-dependent DNA replication (17, 37, 44, 57, 58). These studies have revealed that the extension of a newly synthesized Okazaki fragment DNA with pol δ causes the strand displacement of the preceding fragment to produce a 5′ flap (25). Results suggest that flap endonuclease 1 (Fen-1) is the activity responsible for the removal of the bulk of the 5′ flaps generated (1, 44, 48), although dna2 protein may facilitate the removal of longer flaps coated with the ssDNA binding protein complex (2, 44). In addition, the overexpression of exonuclease I can partially compensate for the loss of Fen-1 function in yeast (24, 51). For the proper processing of lagging-strand intermediates, the entire 5′ flap and all of the RNA primer need to be removed, and the gap must be filled to achieve a ligatable nick. DNA ligase I has been shown to be the enzyme involved in sealing Okazaki fragments in yeast and in humans (3, 31, 50, 56, 57). DNA ligase I requires a nick in which there is a 5′ phosphate on one end and a 3′ hydroxyl linked to a deoxyribose sugar entity on the other, and it works poorly in the presence of mismatches (54). The close coordination of Fen-1 and DNA ligase I activities for Okazaki fragment processing is facilitated by the interactions of these proteins with proliferating cell nuclear antigen (PCNA), the processivity factor for pol δ and ɛ (6, 30, 32, 46, 52, 53).HSV-1 does not appear to encode a protein with DNA ligase activity or one that can specifically cleave 5′ flaps, although it does encode a 5′-to-3′ exonuclease activity (UL12 [10, 20]) and a 3′-to-5′ exonuclease activity that is part of the HSV-1 pol catalytic subunit (27). As for most eukaryotes, RNA primers are essential for HSV-1 DNA synthesis, as demonstrated by the presence of oligoribonucleotides in replicating DNA in vivo (4), by the well-characterized ability of the UL52 protein in complex with the UL5 helicase activity to synthesize oligoribonucleotide primers on ssDNA in vitro (11, 13), and by the requirement of the conserved catalytic residues in the UL52 primase in vitro and in HSV-1-infected cells (14, 26). It is the strand displacement activity of pol δ that produces the 5′ flaps that are key to the removal of RNA primers during Okazaki fragment processing (6, 25). However, we previously demonstrated that wild-type HSV-1 DNA polymerase possesses poor strand displacement activity (62), in contrast to mammalian DNA pol δ (25). Thus, it is not apparent how RNA primers would be removed when encountered by HSV-1 pol during HSV-1 lagging-strand synthesis or how such intermediates would be processed.We wished to test the hypothesis that the nick translation activity of mammalian Fen-1 could function in collaboration with HSV-1 pol to facilitate the proper removal of RNA primers and/or short flaps to produce the ligatable products required for Okazaki fragment processing. In this report, we have examined the ability of wild-type and exonuclease-deficient (exo⁻) HSV-1 pol, which differ in their respective strand displacement activities, to extend model lagging-strand substrates in the presence or absence of mammalian Fen-1. Our results demonstrate that both wild-type and exo⁻ HSV-1 pol can cooperate with and enhance Fen-1 activity to achieve a ligatable nick in vitro. Moreover, colocalization and coimmunoprecipitation studies reveal a physical association of Fen-1 with HSV-1 DNA replication proteins, supporting a model for the involvement of Fen-1 in HSV-1 DNA replication.     

单纯疱疹病毒1型DNA聚合酶辅助亚基UL42的研究进展

单纯疱疹病毒1型(Herpes simplex virus type 1, HSV-1) UL42作为病毒编码的DNA聚合酶辅助亚基之一,是一种多功能蛋白,其在催化和调节病毒在细胞核内的有效复制发挥了重要的作用。已知UL42能提高DNA聚合酶催化亚基UL30的持续合成能力,激活病毒DNA聚合酶活性;介导DNA聚合酶的入核;与DNA模板链结合,提高病毒复制的保真度,以及含有抑制DNA聚合酶活性的肽段,提示其在病毒复制过程中也可能具有负调控作用。近期亦有报道显示,UL42能够阻断肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)激活的核转录因子(nuclear factor kappa-B,NF-κB)信号通路以及干扰素调控因子3(interferon regulatory factor 3, IRF-3)的功能,提示其在病毒逃逸宿主天然免疫反应中发挥了一定的功能,但具体的作用机制尚不明确。本文对目前国内外HSV-1 UL42的结构特点、主要功能、作用机制及其在抗病毒药物研发中的研究进展进行综述,为后续揭示病毒致病机制和抗病毒药物的研发提供参考。     

Mutational Analysis of the Herpes Simplex Virus Type 1 DNA Packaging Protein UL33

The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging.During the packaging of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1), the cleavage of replicated concatemeric viral DNA into single-genome lengths is tightly coupled to its insertion into preassembled spherical procapsids. Upon genome insertion, the internal scaffold protein of the procapsid is lost, and the capsid shell angularizes. Genetic analysis has revealed that successful packaging requires a cis-acting DNA sequence (the a sequence) together with seven proteins, encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes (6, 10). By analogy with double-stranded bacteriophage, the encapsidation of HSV-1 DNA is thought to be mediated by a heteromultimeric terminase enzyme. It is envisaged that the terminase is involved in the recognition of packaging signals present in the concatemers and the association with procapsids via an interaction with the capsid portal protein. Terminase initiates packaging by cleaving at an a sequence present between adjacent genomes within concatemers and subsequently provides energy for genome insertion through the hydrolysis of ATP. Packaging is terminated by a second cleavage event at the next similarly orientated a sequence, resulting in the encapsidation of a unit-length genome.An accumulating body of evidence suggests that the HSV-1 terminase is comprised of the UL15, UL28, and UL33 gene products. Viruses lacking a functional version of any of these three proteins are unable to initiate DNA packaging, and uncleaved concatemers and abortive B-capsids (angularized forms containing scaffold but no DNA) accumulate in the nuclei of infected cells (2, 4, 5, 11, 25, 27, 30, 36, 38). Protein sequence comparisons revealed a distant relationship between UL15 and the large subunit of bacteriophage T4 terminase, gp17, including the presence of Walker A and B box motifs characteristic of ATP binding proteins (13). Subsequent experiments demonstrated that point mutations affecting several of the most highly conserved residues abolished the ability of the resulting mutant viruses to cleave and package viral DNA (26, 39). The UL28 component has been reported to interact with the viral DNA packaging signal (3), a property shared with the homologous protein of human cytomegalovirus (CMV), UL56 (9). Furthermore, both UL15 and UL28 are able to interact with UL6 (33, 37), which form a dodecameric portal complex through which DNA is inserted into the capsid (22, 23, 31). Within the terminase complex, strong interactions have previously been reported between UL15 and UL28 and between UL28 and UL33 (1, 7, 17, 19, 34). Evidence also suggests that UL15 and UL33 may be able to interact directly, albeit more weakly than UL28 and UL33 (7, 15). Temperature-sensitive (ts) lesions in UL33 or UL15 reduced both the interaction of the thermolabile protein with the other members of the terminase complex and viral growth at the nonpermissive temperature (36). Recent evidence suggests that the terminase complex assembles in the cytoplasm and is imported into the nucleus via a mechanism involving a nuclear localization signal within UL15 (35). UL15 is also necessary for the localization of the terminase to nuclear sites of DNA replication and packaging (15). At present, the enzymatic activities necessary for DNA packaging have not been demonstrated for either the complex or individual subunits of the HSV-1 terminase.This study concerns the UL33 protein, which, at 130 residues, is the smallest subunit of the presumptive terminase (7, 27). No specific role in terminase activity has yet been ascribed to UL33, but several possibilities have been proposed including (i) ensuring correct folding or assembly of the complex, (ii) regulating the functions of the other subunits, (iii) performing an essential enzymatic role per se, and (iv) ensuring correct localization of the terminase to sites of DNA packaging (7). However, recent immunofluorescence studies using mutants with defects in the individual terminase subunits suggest that UL33 is unlikely to be involved in this last function (15).In order to further investigate the role of UL33 in the cleavage-packaging process, we utilized transposon-mediated mutagenesis to introduce insertions of five codons throughout the UL33 ORF. We report the generation and characterization of 15 mutants in terms of their ability to support viral growth and DNA packaging and to interact with the terminase component UL28.     

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction wit...     

Differentiation Between Herpes Simplex Virus Type 1 and Type 2 Strains by Immunoelectroosmophoresis

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.     

Effect of Heparin on Hemagglutinin of Herpes Simplex Virus Type 1

Heparin inhibited the hemagglutinin activity of herpes simplex virus (HSV) type 1. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of HSV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by HSV. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis.     

The Influence of Stress Factors on the Reactivation of Latent Herpes Simplex Virus Type 1 in Infected Mice

This study shows that the influence of different stress factors impacts the reactivation of latent herpes simplex virus type 1 (HSV-1) specifically in the trigeminal ganglion of infected mice. Different stress factors including hyperthermia, hypothermia, fatigue, and immunosuppression were exerted on mice infected with HSV-1. These viral antigens were then detected in the trigeminal ganglion region of infected mice under the influence of each stress factor, with hyperthermia having the most influence on reactivation. Interestingly, an increase in IL-6 was also detected in mice subjected to hyperthermia. These studies therefore suggest that stress can induce the reactivation of latent HSV-1, possibly through the induction of IL-6, in the trigeminal ganglion region of infected mice. This reveals a new insight on the pathogenesis of relapse infection of HSV-1.     

单纯疱疹病毒1型VP16蛋白和VHS蛋白研究进展

单纯疱疹病毒1型(HSV-1)为有包膜的DNA病毒,能引起皮肤性疱疹、角膜炎、脑炎等症状。HSV-1感染细胞后,要么进入裂解性感染阶段,要么进入潜伏感染阶段。受感染的细胞常会启动免疫系统抵抗病毒,而病毒却通过某种机制巧妙地逃避宿主的免疫反应并进入潜伏。进入潜伏感染阶段的病毒又会因机体受某种刺激而被激活进入裂解感染期。在这期间,有两个关键的病毒蛋白一间层蛋白(Viral protein16,VP16)和内膜蛋白(Virion host shutoff protein,VHS)倍受关注,它们既是HSV-1的结构蛋白,在病毒复制晚期参与病毒颗粒的组装,同时又作为重要的功能蛋白…