Gene delivery to neurons: Is herpes simplex virus the right tool for the job?

Herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into neurons. HSV vectors can facilitate a range of molecular studies on postmitotic neurons and may ultimately be used for somatic cell gene therapy for certain neurologic diseases. In this article, the salient features of the pathogenesis and molecular biology of HSV relevant to its use as a vector are described, along with an overview of the methods used to derive these vectors. The accomplishments which have been made to date using the HSV vector system are discussed, with emphasis on the issues of this technology which remain to be addressed. HSV has the potential to be a most useful tool for neuronal cell transgenesis and it is likely that important neurobiological questions will be answered using this vector system.     

细胞周期蛋白依赖性蛋白激酶2在单纯疱疹病毒复制中的作用

细胞周期蛋白依赖性蛋白激酶(CDK)与单纯疱疹病毒(HSV)等多种重要人类疾病病毒的复制密切相关.但具体哪种CDK是病毒复制所必需的还不清楚.本文用不同剂量的HSV-1-KOS株(以下简称HSV)感染CDK2功能缺陷型宿主细胞,结果发现HSV在CDK2功能缺陷型宿主细胞中的复制具有感染剂量依赖性;一步生长曲线分析结果表明其在CDK2功能缺陷型宿主细胞中的复制较在正常细胞延迟3h;感染6h时CDK2活性被诱导,9h时活性最大;CDK2活性增加后HSV-1即进入快速的裂解性复制.提示CDK2可能在HSV复制的启动中起着某种重要作用.     

Utilization of herpes simplex PCR assays for cerebrospinal fluid in a pediatric health care setting

An assessment was made of the utilization and impact of a diagnostic polymerase chain reaction (PCR) assay for the diagnosis of herpes simplex viruses (HSV) 1 and 2 in cerebrospinal fluid of children who attended a Canadian pediatric referral centre. One hundred and three assays were performed on specimens from 103 patients during the period August 1997 to September 1998. Patient ages ranged from newborn to 16 years. Indications for HSV PCR included seizures with or without fever (56.3%), aseptic meningitis (16.5%), and encephalopathy with or without fever (10.7%). Only 2 of 103 (1.9%) assays were positive, including one each for HSV1 and HSV2. Control specimens that were seeded with virus indicated inhibition for 24.3, 8.8, and 6.8% of assays for HSV1, HSV2, and both HSV1 and HSV2, respectively. The mean turn-around time for HSV PCR was 2.5 days, and 90.3% were completed in less than 5 days. Acyclovir was administered to 78.6% of the patients overall; the results of the HSV PCR impacted on the treatment courses for 36 individuals. Nevertheless, 16.5% of patients continued to receive extended courses of antiviral therapy despite negative HSV PCR assays. Although it is desirable to decrease the frequency of PCR inhibitions and to further decrease the interval to assay completion, HSV PCR does have a significant impact on antiviral use in this setting.     

Herpes simplex virus type 1 infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b

We recently demonstrated that herpes simplex virus type 1 (HSV 1) induces a receptor on human umbilical vein endothelial cells for complement component C3b (C3bR). We assigned this receptor function to HSV 1 viral glycoprotein C (gC) based on several observations: tunicamycin, which prevents glycosylation and expression of N-linked glycoproteins on the surface of infected cells, markedly reduced expression of the C3bR; monoclonal antibodies to HSV 1 gC blocked detection of the C3bR, whereas monoclonal antibodies to other HSV 1 glycoproteins (gB, gD, gE) had no effect; and the MP mutant of HSV 1, which fails to express gC, did not induce C3bR. We now report that HSV 1 induces C3bR on a wide variety of cell types including bovine thoracic aorta and pulmonary artery endothelial cells, human embryonic lung and embryonic foreskin fibroblasts, and human embryonic kidney cells. To date, all cells studied that are permissive to HSV 1 express C3bR, although the pattern of rosetting of C3b-coated erythrocytes varies among the cell strains examined. We also demonstrate that C3bR expression is not a general response of human umbilical vein endothelial cells to injury, because three other viruses (adenovirus 7, measles, and mumps) do not induce C3bR after infection of these cells. Previously we had shown that among herpes simplex viruses, a variety of HSV 1 strains induce C3bR, whereas HSV 2 strains do not. We now demonstrate that other herpes family viruses (CMV and VZV) do not express C3bR. Therefore, C3bR expression appears to be unique for HSV 1 and occurs on a wide variety of cells permissive to this virus.     

Chromatin control of herpes simplex virus lytic and latent infection

Herpes simplex viruses (HSV) can undergo a lytic infection in epithelial cells and a latent infection in sensory neurons. During latency the virus persists until reactivation, which leads to recurrent productive infection and transmission to a new host. How does HSV undergo such different types of infection in different cell types? Recent research indicates that regulation of the assembly of chromatin on HSV DNA underlies the lytic versus latent decision of HSV. We propose a model for the decision to undergo a lytic or a latent infection in which HSV encodes gene products that modulate chromatin structure towards either euchromatin or heterochromatin, and we discuss the implications of this model for the development of therapeutics for HSV infections.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.     

Herpes simplex viruses and induction of interferon responses

Herpes simplex viruses (HSV) are human pathogens responsible for a variety of diseases,including localized mucocutaneous lesions,encephalitis,and disseminated diseases.HSV infection leads to rapid induction of innate immune responses.A critical part of this host response is the type I IFN system including the induction of type I IFNs,IFN-mediated signaling and amplification of IFN response.This provides the host with immediate countermeasure during acute infection to limit initial viral replication and to facilitate an appropriate adaptive immune response.However,HSV has devised multiple strategies to evade and interfere with innate immunity.This review will focus on the induction of type I IFN response by HSV during acute infection and current knowledge of mechanisms by which HSV interferes with this induction process.     

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.     

Herpes simplex virus in postradiation cervical smears. A morphologic and immunocytochemical study.

From January 1987 to August 1988, cytomorphologic criteria of both herpes simplex virus (HSV) and radiation effects were observed in Papanicolaou smears from 3 of 1,340 patients who had received radiotherapy for squamous cell carcinoma of the cervix. Avidin-biotin immunoperoxidase staining, using a rabbit IgG polyclonal HSV antibody, confirmed the presence of HSV antigen in those three postradiation smears. Both multinucleated molded cells and epithelial cells that lacked cytopathic effects were positive for HSV. Three other postradiation smears from these cases were similarly positive for HSV antigen; the one preradiation smear was negative. In situ hybridization and immunoperoxidase studies on sections from the preradiation biopsies were negative: severely altered neoplastic cells showed no reactivity. The absence of HSV markers in the preradiation specimens suggests that the HSV infections were secondary to the radiotherapy; further studies are needed to prove this association and to assess the possible mechanisms. These cases clearly indicate that the overlapping features of radiation and viral effects (such as multinucleation) may be present simultaneously.     

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。     

Anti-CeHV1 antibodies of two cynomolgus macaques cross-react with HSV2 but not HSV1 antigens in ELISA

BACKGROUND: The aim of the study was to compare the cross-reactivity of macaque anti-CeHV1 antibodies with type 1 and type 2 human herpes simplex viruses (HSV1 and HSV2). METHODS: We studied the serum of 344 animals which had been tested either positive (n = 39) or negative (n = 305) for the presence of CeHV1 antibodies by expert laboratories. Macaque serums were studied by means of two ELISA: one based on HSV1 antigen-coated wells, the other on polystyrene beads coated with HSV1 and HSV2 antigens in approximately equal proportions. RESULTS: In the serum of two animals originating from Vietnam, we found anti-CeHV1 antibodies cross-reacting with HSV2 but not with HSV1 antigens. For the serum with the highest titer, inhibition by soluble antigens confirmed the high affinity of the antibodies for HSV2 antigens. CONCLUSIONS: Tests using HSV1 and HSV2 in a combined way are better suited to macaque screening than tests using only HSV1 antigens.     

Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.

When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.     

T Cell Receptor Signaling Pathways： New Targets for Herpes Simplex Virus

Herpes simplex viruses (HSV-1 and HSV-2) cause global morbidity and synergistically correlate with HIV infection.HSV exists life-long in a latent form in sensory neurons with intermittent reactivation,in despite of host immune surveillance.While abundant evidence for HSV interfering with innate immune responses so as to favor the replication and propagation of the virus,several lines of evidence declare that HSV attenuates adaptive immunity by various mechanisms,including but not limited to the ablation of antigen presentation,induction of apoptosis,and interruption of cellular signaling.In this review,we will focus on the perturbative role of HSV in Tcells signaling.     

Different forms of membrane-associated herpes simplex virus glycoproteins induce functionally distinct subsets of herpes simplex virus-specific suppressor T cells.

Previous studies have shown that two types of virus-specific suppressor T cells (Ts) are induced in mice made tolerant with herpes simplex virus (HSV)-infected spleen cells (SC). One type of Ts blocks the afferent phase of the delayed hypersensitivity response to HSV (Ts-aff), and the other blocks the efferent or effector phase (Ts-eff). In this report we show that the induction requirements for these suppressor populations differ. Injection of SC infected for 6 h with HSV at a multiplicity of infection of 5 or less or treated with heat-inactivated virus induced only Ts-aff. Similar results were seen with SC incubated for 90 min in virus-free preparations containing only viral proteins. In contrast, the Ts-eff population was induced only by SC treated for 6 h with infectious HSV at a multiplicity of infection of 10. Collectively, these data indicate that Ts-aff are induced by adsorbed HSV antigens on SC, whereas Ts-eff are induced by nascent HSV antigens expressed on infected SC. In addition to their induction requirements, the two types of regulatory cells differ in their expression of effector function. Ts-eff but not Ts-aff require a cyclophosphamide-sensitive target cell in the immune recipient for suppressor function. The possible identity of this target cell and the significance of the different induction requirements between the two types of Ts are discussed.     

Modified-self-induced modulation of the immune response to herpes simplex virus: effect on antibody formation, cytotoxic T lymphocyte induction, and survival

C3H/HeJ mice were injected i.v. with soluble herpes simplex virus (HSV) envelope antigens coupled to syngeneic splenocytes 7 days before challenge i.p. with either soluble HSV antigens in complete Freund's adjuvant or infectious HSV. Fourteen days post challenge, and anti-HSV antibody hyporesponsiveness was observed in the HSV modified-self pretreated animals that was 50% or less than the control response. Both forms of HSV challenge were equally effective. This unresponsiveness was specific for HSV when compared with a concomitant response to chicken red blood cell (CRBC) challenge. Challenge with infectious HSV afforded us the opportunity to investigate the effect of HSV modified-self pretreatment on the induction of HSV-specific cytotoxic T lymphocytes (CTL). In vitro restimulation of in vivo-primed splenocytes with infectious HSV generated HSV-specific CTL. The degree of HSV-specific CTL response was found to be variable in animals that received the HSV modified-self pretreatment with respect to control animals. Survival was significantly greater in HSV modified-self pretreated mice after infectious HSV challenge than in control animals.     

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.     

Herpes simplex virus-enhanced cationic lipid/DNA-mediated transfection

Liposome plasmid DNA complexes (lipoplexes) are often inefficient in mediating gene transfer and expression because of DNA degradation in lysosomal vesicles. Because herpes simplex virus (HSV) enters cells by fusion of the virus envelope with the plasma membranes, thereby overriding the endosomal pathway, HSV/lipoplex mixtures could be useful for improving gene transfer particularly when the mixture uses highly defective HSV particles that fail to express cytotoxic viral gene products. To evaluate this possibility, lipoplexes composed of cationic liposomes and lacZ reporter plasmids were compared for their ability to transduce cells in culture in the presence and absence of infectious HSV particles. The results showed that HSV increased the efficiency of cell transduction by approximately 4-100-fold compared with lipoplex vector alone, depending on the cell type targeted for gene delivery. The increased efficiency of transduction was virus dose dependent and required virus entry.     

溶瘤单纯疱疹病毒治疗技术及其进展

溶瘤病毒治疗是当前肿瘤治疗技术的研究热点,溶瘤单纯疱疹病毒是目前研究最多的,也是抗肿瘤作用最好的溶瘤病毒之一,本文就主要介绍溶瘤单纯疱疹病毒的优势、遗传操作、发展策略、研究进展及靶向性策略等。