Oncogenic Transformation of Hamster Cells after Exposure to Herpes Simplex Virus Type 2

THE possibility of a relationship between herpes simplex viruses (HSV) and human cancer has been suggested^1–4 chiefly on the basis of studies of the epidemiology of cervical cancer, but so far it has not been possible to demonstrate that human herpes viruses can induce primary transformation of normal cells. Injection of herpes simplex virus type 1 (ref. 5) or type 2 (ref. 6) into Syrian hamsters rarely leads to the production of a tumour and it has been difficult to demonstrate herpes viral antigens in tumour cells. Human herpes simplex viruses grown in vitro are characterized by the rapidity with which the infected cell is destroyed, so that cell transformation is impossible, but this effect can be mitigated by inactivation of the herpes virus by ultraviolet irradiation. Indeed, this procedure may have the additional advantage that viral infectivity is removed more quickly than the viral transforming potential⁷.     

Properties of Hamster Embryo Fibroblasts Transformed In Vitro After Exposure to Ultraviolet-Irradiated Herpes Simplex Virus Type 2

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

Oncogenic Transformation of Hamster Embryo Cells After Exposure to Inactivated Herpes Simplex Virus Type 1

The in vitro transformation of hamster embryo fibroblasts by herpes simplex virus type 1 (HSV-1) after exposure of the virus to UV irradiation is described. Cell transformation was induced by 2 out of 12 strains of HSV-1 that were tested for transforming potential. Cells transformed by the KOS strain of HSV-1 were not oncogenic when injected into newborn Syrian hamsters. However, cells transformed by HSV-1 strain 14-012 induced tumors in 47% of the newborn hamsters injected. HSV-specific antigens were found in the cytoplasm of cells transformed by both virus strains. Sera from tumor-bearing hamsters contained HSV-1- and HSV-2-neutralizing antibodies as well as antibodies which reacted specifically with HSV antigens by the indirect immunofluorescence technique. Hamster oncornavirus antigens were not detected by immunofluorescence methods. These observations represent the first evidence of the oncogenic potential of HSV-1.     

Oxidative Stress Enhances Neurodegeneration Markers Induced by Herpes Simplex Virus Type 1 Infection in Human Neuroblastoma Cells

Mounting evidence suggests that Herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer’s disease (AD). Previous work from our laboratory has shown HSV-1 infection to induce the most important pathological hallmarks of AD brains. Oxidative damage is one of the earliest events of AD and is thought to play a crucial role in the onset and development of the disease. Indeed, many studies show the biomarkers of oxidative stress to be elevated in AD brains. In the present work the combined effects of HSV-1 infection and oxidative stress on Aβ levels and autophagy (neurodegeneration markers characteristic of AD) were investigated. Oxidative stress significantly potentiated the accumulation of intracellular Aβ mediated by HSV-1 infection, and further inhibited its secretion to the extracellular medium. It also triggered the accumulation of autophagic compartments without increasing the degradation of long-lived proteins, and enhanced the inhibition of the autophagic flux induced by HSV-1. These effects of oxidative stress were not due to enhanced virus replication. Together, these results suggest that HSV-1 infection and oxidative damage interact to promote the neurodegeneration events seen in AD.     

Clinical Reactivations of Herpes Simplex Virus Type 2 Infection and Human Immunodeficiency Virus Disease Progression Markers

Background

The natural history of HSV-2 infection and role of HSV-2 reactivations in HIV disease progression are unclear.

Methods

Clinical symptoms of active HSV-2 infection were used to classify 1,938 HIV/HSV-2 co-infected participants of the Women''s Interagency HIV Study (WIHS) into groups of varying degree of HSV-2 clinical activity. Differences in plasma HIV RNA and CD4+ T cell counts between groups were explored longitudinally across three study visits and cross-sectionally at the last study visit.

Results

A dose dependent association between markers of HIV disease progression and degree of HSV-2 clinical activity was observed. In multivariate analyses after adjusting for baseline CD4+ T cell levels, active HSV-2 infection with frequent symptomatic reactivations was associated with 21% to 32% increase in the probability of detectable plasma HIV RNA (trend p = 0.004), an average of 0.27 to 0.29 log10 copies/ml higher plasma HIV RNA on a continuous scale (trend p<0.001) and 51 to 101 reduced CD4+ T cells/mm³ over time compared to asymptomatic HSV-2 infection (trend p<0.001).

Conclusions

HIV induced CD4+ T cell loss was associated with frequent symptomatic HSV-2 reactivations. However, effect of HSV-2 reactivations on HIV disease progression markers in this population was modest and appears to be dependent on the frequency and severity of reactivations. Further studies will be necessary to determine whether HSV-2 reactivations contribute to acceleration of HIV disease progression.     

Virus-Specific Antigens in Hamster Cells Transformed by Rous Sarcoma Virus

Virus-specific antigens were studied in hamster cells transformed by Rous sarcoma virus (RSV). Antigens were localized in the cytoplasm, as demonstrated by fluorescent antibody staining of fixed cells as well as by complement fixation (CF) following subcellular fractionation. Cytoplasmic extracts were analyzed by velocity and isopycnic centrifugation. CF antigens were found in a soluble form and in association with membranes and polyribosomes. Isolated plasma membranes had no CF antigen. Both soluble and particulate fractions with CF activity contained the same antigenic determinants by Ouchterlony analysis. These antigenic determinants were identical to those released by ether treatment of RSV.     

Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.     

The Genome Sequence of Herpes Simplex Virus Type 2

The genomic DNA sequence of herpes simplex virus type 2 (HSV-2) strain HG52 was determined as 154,746 bp with a G+C content of 70.4%. A total of 74 genes encoding distinct proteins was identified; three of these were each present in two copies, within major repeat elements of the genome. The HSV-2 gene set corresponds closely with that of HSV-1, and the HSV-2 sequence prompted several local revisions to the published HSV-1 sequence (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531–1574, 1988). No compelling evidence for the existence of any additional protein-coding genes in HSV-2 was identified.The complete 152-kbp genomic DNA sequence of herpes simplex virus type 1 (HSV-1) was published in 1988 (56) and since then has been very widely employed in a great range of research on HSV-1. Additionally, results from this most studied member of the family Herpesviridae have fed powerfully into research on other herpesviruses. In contrast, although a substantial number of individual gene sequences have been determined for the other HSV serotype, HSV-2, the complete genome sequence for this virus has not been available hitherto. In this paper we report the sequence of the genome of HSV-2, strain HG52.At a gross level the 155-kbp genome of HSV-2 is viewed as consisting of two extended regions of unique sequence (U_L and U_S), each of which is bounded by a pair of inverted repeat elements (TR_L-IR_L and IR_S-TR_S) (17, 66) (Fig. ​(Fig.1).1). There is a directly repeated sequence of some 254 bp at the genome termini (the a sequence), with one or more copies in the opposing orientation (the a′ sequence) at the internal joint between IR_L and IR_S (21). U_L plus its flanking repeats is termed the long (L) region, and U_S with its flanking repeats is termed the short (S) region. In individual molecules of HSV-2 DNA, the L and S components may be linked with each in either orientation, so that DNA preparations contain four sequence-orientation isomers, one of which is defined as the prototype (66). The sequences of the terminal and internal copies of R_L and of R_S are considered to be indistinguishable. Open in a separate windowFIG. 1Overall organization of the genome of HSV-2. The linear double-stranded DNA is represented, with the scale at the top. The unique portions of the genome (U_L and U_S) are shown as heavy solid lines, and the major repeat elements (TR_L, IR_L, IR_S, and TR_S) are shown as open boxes. For each pair of repeats the two copies are in opposing orientations. As indicated, TR_L, U_L, and IR_L are regarded as comprising the L region, and IR_S, U_S, and TR_S are regarded as comprising the S region. Plasmid-cloned fragments used for sequence determination are indicated at the bottom: BamHI and HindIII fragments are indicated by B and H, respectively, followed by individual fragment designations in lowercase; KH and HK indicate KpnI/HindIII fragments as described in the text.This paper presents properties of the HSV-2 DNA sequence and our present understanding of its content of protein-coding genes and other elements. We are also interested in comparative analysis of the HSV-1 and HSV-2 genomes to examine processes of molecular evolution which have occurred since the two species diverged, and we intend to pursue this topic in a separate paper.     

Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by CD4 T Cells Infiltrating Human Genital Herpes Lesions

The local cellular immune response to herpes simplex virus (HSV) is important in the control of recurrent HSV infection. The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity, inhibition of viral growth, lymphokine secretion, and support of humoral and CD8 responses. The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions. Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase, encoded by genes U_L49 and U_L50, respectively, are T-cell antigens. Separate epitopes in VP22 were defined for T-cell clones from each of three patients. Reactivity with the tegument protein encoded by U_L21 was identified for an additional patient. Three new epitopes were identified in VP16, a tegument protein associated with VP22. Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells. These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines.     

疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

目的：探讨疱疹病毒Ⅱ型（HSV-2）感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法：病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果：相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异（P〈0.05）;分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异（P〉0.05）。结论：初步在人神经母细胞瘤细胞株SH—SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。     

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.     

Caspase 9 Is Essential for Herpes Simplex Virus Type 2-Induced Apoptosis in T Cells

Herpes simplex virus type 2 (HSV-2) induces apoptosis in T cells by a caspase-dependent mechanism. Apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways. Here, we show that the initiator caspase for the intrinsic pathway is activated in T cells following HSV-2 exposure. To determine the respective contributions of intrinsic and extrinsic pathways, we assessed apoptosis in Jurkat cells that are deficient in caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. Our results indicate HSV-2-induced apoptosis in T cells occurs via the intrinsic pathway.Herpes simplex virus (HSV) reactivation in human results in accumulation and persistence of virus-specific CD4⁺ and CD8⁺ cells at the site of reactivation (15, 16). Despite such immune responses, virus reactivation can occur on >75% of days for some individuals (14). Reactivation of the virus in the midst of continued cell-mediated immunity demonstrates the ability of the virus to circumvent the host''s immune system. HSV antigens were readily detected from T cells isolated from human HSV lesions, indicating that T cells are infected with HSV in vivo (1). In vitro, HSV-infected T cells undergo apoptosis (5-7, 10), and we have additionally demonstrated that apoptosis occurs in Jurkat cells, a T-cell leukemia line, and primary CD4⁺ T lymphocytes isolated from human peripheral blood mononuclear cells following exposure to HSV type 2 (HSV-2)-infected human foreskin fibroblasts (5). These results suggest that induction of T-cell death is a mechanism by which the virus limits the effectiveness of local cell-mediated immunity during reactivation.Since T cells are most likely exposed to HSV-2 via infected epithelial cells in vivo, we examined T-cell apoptosis following exposure to infected fibroblasts in vitro. To evaluate whether HSV-2-exposed primary T cells undergo apoptosis by a caspase-dependent mechanism, we examined activation of caspase 3 in human CD4⁺ cells after exposure to HSV-2-infected fibroblasts. Human foreskin fibroblasts (American Type Culture Collection, Manassas, VA) were mock infected or infected with the HSV-2 HG52 strain at a multiplicity of infection of 5. At 6 h postinfection, CD4⁺ cells were exposed to mock-infected or HSV-2-infected fibroblasts at a ratio of approximately 3:1 (lymphocytes to fibroblasts). CD4⁺ cells were isolated to >95% purity using MACS CD4 microbeads (Miltenyi Biotec, Inc., Auburn, CA) immediately prior to experiments with human peripheral mononuclear cells (Memorial Blood Centers, St. Paul, MN) that were stimulated with phytohemagglutinin (Sigma Aldrich, St. Louis, MO) for 48 h and then maintained with interleukin-2 (Invitrogen, Carlsbad, CA) as previously described (5). After 4 h of incubation, CD4⁺ cells were harvested and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 0.3 ng/ml interleukin-2. At 24 h and 48 h postexposure, CD4⁺ cells were probed with a fluorescence-labeled antibody against activated caspase 3 (BD Biosciences, San Jose, CA). Data were collected on a FACSCanto (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). Side scatter and fluorescence were used as dual parameters to clearly gate the cell population with activated caspase 3, and an identical gate was applied to all samples within each experiment. As shown in Fig. ​Fig.1,1, the percentage of cells with activated caspase 3 was 23% in HSV-2-exposed CD4⁺ cells compared with 5% in mock-exposed cells at 24 h postexposure. At 48 h, the percentage of cells with activated caspase 3 increased to 39% in HSV-2-exposed cells compared to 8% in mock-exposed cells. These percentages are comparable to those for Jurkat cells in our previous report (19% for virus-exposed cells versus 5% for mock-exposed cells at 24 h postexposure) (5).Open in a separate windowFIG. 1.Activation of caspase 3 in CD4⁺ cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ cells were isolated from human peripheral mononuclear cells and exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with an antibody for activated caspase 3 and analyzed by flow cytometry. (A) A representative flow cytometry analysis from three independent experiments is shown. Side scatter was used as a second parameter to identify the cell populations. An identical gate was applied to all samples within each experiment. Increased percentages of cells with caspase 3 activation are seen following exposure to HSV-2-infected fibroblasts compared to those following exposure to mock-infected fibroblasts. (B) Mean percentages of CD4⁺ cells with caspase 3 activation from three independent experiments at 24 h and 48 h postexposure are shown with standard errors bars.Efficient infection of T cells by HSV upon exposure to HSV-infected human fibroblasts via cell-to-cell spread has been recently demonstrated (1). To delineate the relationship between virus infection and apoptosis, Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then coprobed with antibodies for activated caspase 3 and HSV-2 ICP10. As shown in Fig. ​Fig.2,2, two antibodies demonstrated largely mutually exclusive populations in cells exposed to HSV-2-infected fibroblasts. We obtained similar results with antibodies against other HSV-2 antigens, including glycoprotein B, ICP5, and ICP8 (data not shown). The results suggest that infected cells may be inducing apoptosis in uninfected cells via a bystander effect, similar to what has been proposed for HSV-1-infected activated cytotoxic T cells (11). Alternatively, the activation of apoptosis may result in degradation of viral antigens in infected cells.Open in a separate windowFIG. 2.Expression of HSV-2 antigen and caspase 3 activation in Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Jurkat cells were first exposed to mock- or HSV-2-infected fibroblasts and then coprobed with antibodies for HSV-2 ICP10 and activated caspase 3 at 24 h postexposure. A representative flow cytometry analysis from three independent experiments is shown. Each antibody detected largely mutually exclusive cell populations following exposure to HSV-2-infected fibroblasts.Our results demonstrate an activation of caspase 3 in HSV-2-exposed T cells, suggesting a caspase-mediated apoptosis. Previous studies have supported such a mechanism, as caspase inhibitors block virus-induced cell death (5, 10). Caspase-dependent apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways (3). Here, we sought to determine the respective contribution of each pathway in HSV-2-induced T-cell death.To investigate the contribution of individual apoptosis pathways in HSV-2-exposed T cells, we first evaluated mitochondrial membrane potential in CD4⁺ and Jurkat cells following virus exposure. Mitochondria play a major role in the intrinsic apoptosis pathway (2, 3). T cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then probed with chloromethyl-X-rosamine (CMXRos; Invitrogen) at 24 h and 48 h postexposure. CMXRos is a lipophilic cationic fluorescent dye whose staining of cells is dependent on negative mitochondrial membrane potential, and a loss of staining indicates a loss of mitochondrial potential (2, 9). As shown in Fig. ​Fig.3,3, a loss of mitochondrial membrane potential was observed in increased percentages of HSV-2-exposed CD4⁺ and Jurkat cells compared to those for mock-exposed cells. The percentage of cells with a loss of mitochondrial membrane potential reached 49% for CD4⁺ cells and 92% for Jurkat cells at 48 h postexposure. The changes in mitochondrial membrane potential in HSV-2-exposed T cells suggest that the intrinsic apoptosis pathway is activated in these cells. We previously reported that Jurkat and CD4⁺ cells were equally susceptible to apoptosis induced by anti-Fas monoclonal antibody (5). A recent report suggests that activated primary T cells are less susceptible to HSV infection than are Jurkat cells (1). Thus, we believe that a lower percentage of CD4⁺ cells with a loss of mitochondrial membrane potential than Jurkat cells is due to lower susceptibility to HSV infection.Open in a separate windowFIG. 3.A loss of mitochondrial membrane potential in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ and Jurkat cells were exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with chloromethyl-X-rosamine (CMXRos). (A) Increased percentages of CD4⁺ cells with a loss of mitochondrial membrane potential are seen following exposure to HSV-2-infected fibroblasts compared to those seen after mock exposure. Shown are mean percentages of cells with a loss of mitochondrial membrane potential at 24 h and 48 h postexposure, with standard error bars, from three independent experiments. (B) Shown are mean percentages of cells with a loss of mitochondrial membrane potential for Jurkat cells at 24 h and 48 h postexposure, with standard error bars, from three independent experiments.To further define the involvement of intrinsic and extrinsic apoptosis pathways in HSV-2-induced T-cell apoptosis, we investigated whether caspase 8, an initiator protease for the extrinsic pathway, and caspase 9, an initiator protease for the intrinsic pathway, are activated in HSV-2-exposed T cells. CD4⁺ and Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. Cell lysates were made at 24 h postexposure, and immunoblots were performed with 30 to 50 μg of protein per well, as described previously (5), with anti-caspase 8 and anti-caspase 9 antibodies (Cell Signaling, Danvers, MA). A 24-h time point was chosen for the immunoblots in order to minimize the detection of baseline apoptotic activity that can be seen with primary CD4⁺ cells in cultures with a longer incubation time.As shown in Fig. ​Fig.4,4, CD4⁺ cells that were exposed to HSV-2 had an increase in cleaved, activated fragments of caspase 9. No clear differences between mock-infected and HSV-2-exposed CD4⁺ cells were seen for caspase 8 cleavage. In contrast, increases in both cleaved caspase 8 and cleaved caspase 9 were evident in Jurkat cells following virus exposure. Together, these results indicate that caspase 9 is activated in HSV-2-exposed T cells and suggest activation of the intrinsic apoptosis pathway. Although activation of caspase 8 was seen with Jurkat cells, the contribution of the extrinsic pathway remained in doubt for virus-induced apoptosis in CD4⁺ cells.Open in a separate windowFIG. 4.Detection of cleaved caspase 8 and caspase 9 in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were assayed for uncleaved and cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from more than three independent experiments are shown. Blots demonstrate increases in cleaved caspase 9 for CD4⁺ and Jurkat cells. An increase in cleaved caspase 8 is also seen for Jurkat cells.To determine the respective contributions of each pathway, we assessed apoptosis in Jurkat cells that are deficient in either caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. We chose caspase 3 activation to assess apoptosis in HSV-2-exposed cells, because convergence of extrinsic and intrinsic apoptosis pathways occurs at caspase 3.Jurkat cells deficient in caspase 8 or FADD (American Type Culture Collection) have been previously described (8), and we confirmed the absence of caspase 8 and FADD in respective cell lines (Fig. ​(Fig.5A).5A). We also investigated the parental Jurkat cell A3 subclone and verified that both caspase 8 and FADD were expressed as expected (Fig. ​(Fig.5A).5A). Parental and deficient Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. At 24 h postexposure, cells were probed with fluorescence-labeled antibody against activated caspase 3. As shown in Fig. ​Fig.5B,5B, cells deficient in caspase 8 or FADD underwent apoptosis at levels similar to those of parental cells as determined by caspase 3 activation. The results indicate that neither caspase 8 nor FADD is required for HSV-2-induced apoptosis in Jurkat cells. We additionally evaluated for cleavage products of caspase 8 and caspase 9 in these cells. As shown in Fig. ​Fig.5C,5C, cleaved fragments of caspase 9 were seen in all three cell types following HSV-2 exposure. Interestingly, cleaved fragments of caspase 8 were detected in both parental and FADD-deficient cells, suggesting that FADD is not required for cleavage of caspase 8 following HSV-2 exposure.Open in a separate windowFIG. 5.Loss of caspase 8 or FADD expression does not impede HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of I2.1, I9.2, and A3 cell lysates were probed with antibodies for procaspase 8, FADD, and β-actin. FADD is not expressed in I2.1 cells, while caspase 8 is not expressed in I9.2 cells. The parental cell line A3 is a subclone of Jurkat cells that expresses both caspase 8 and FADD. β-Actin expression is shown as a control. (B) Percentages of cells with caspase 3 activation are comparable in cells with caspase 8 or FADD deficiency and in parental A3 cells following exposure to HSV-2. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate increases in cleaved caspase 8 for I2.1 and A3 cells that were exposed to HSV-2. Increases in cleaved caspase 9 are seen for all three cell types.Jurkat cells deficient in caspase 9 expression and the corresponding caspase 9-reconstituted clones (12, 13) were a gift of Ingo Schmitz (University of Dusseldorf, Dusseldorf, Germany). Immunoblot analysis confirmed the presence or absence of caspase 9 in both cell lines (Fig. ​(Fig.6A).6A). Cells were exposed to mock- or HSV-2-infected fibroblasts and analyzed for activation of caspase 3 as described above. Following exposure to HSV-2, cells deficient in caspase 9 were protected from apoptosis as determined by caspase 3 activation (Fig. ​(Fig.6B).6B). In contrast, cells with reconstituted caspase 9 showed an increased percentage of caspase 3 activation. An evaluation of caspase 8 and caspase 9 cleavage in these cells showed that cleaved fragments were seen only in the cells reconstituted with caspase 9 (Fig. ​(Fig.6C).6C). To confirm that the findings were applicable to the parental Jurkat cell line, we evaluated the effect of a caspase 9 inhibitor, Z-LEHD-fmk (R&D Biosystems, Minneapolis, MN) on Jurkat cells that were exposed to mock- or HSV-2-infected fibroblasts. Jurkat cells were incubated with 100 μM Z-LEHD-fmk or a dimethyl sulfoxide (DMSO) solvent control for 1 h before exposure to fibroblasts, throughout the exposure, and postexposure. At 24 h postexposure, the percentage of virus-exposed cells with activated caspase 3 was inhibited by Z-LEHD-fmk to 3%, identical to the baseline percentage seen with mock-exposed cells with DMSO solvent control (Fig. ​(Fig.6D).6D). Together, these findings indicate that caspase 9 is required for HSV-2-induced apoptosis and cleavage of both caspase 8 and caspase 9 in Jurkat cells.Open in a separate windowFIG. 6.Caspase 9 is required for HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of JMR and F9 cell lysates probed with antibodies for procaspase 9 and β-actin. Caspase 9 is not expressed in JMR cells, a subclone of Jurkat cells. Caspase 9 expression is reconstituted in F9 cells, which are JMR cells stably transfected with a plasmid expressing caspase 9. (B) JMR cells with caspase 9 show protection from HSV-2-induced apoptosis, while F9 cells with reconstituted caspase 9 are susceptible to apoptosis induced by the virus. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from JMR and F9 cells were exposed to mock- or HSV-2-infected fibroblasts and then probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate an increase in cleaved caspase 8 and caspase 9 for F9 cells but not JMR cells. (D) Jurkat cells that were incubated with Z-LEHD-fmk show protection from HSV-2-induced apoptosis compared to results with the DMSO solvent control at 24 h postexposure. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments.Our results provide evidence that exposure to HSV-2-infected fibroblasts leads to apoptosis in T cells. It is important to note that our method of activation and maintenance of human peripheral blood mononuclear cells enriches for CD4⁺ cells. Phytohemagglutinin and interleukin-2 nonspecifically activate these cells, and no specific immune response to HSV-2 is expected. Further studies are necessary to determine if our observations with human CD4⁺ cells are applicable to other primary lymphocyte cell types and to virus-specific immune cells. Despite these limitations, the similarity in the pattern of change in apoptotic markers in Jurkat and primary human CD4⁺ cells suggests that apoptosis occurs by a similar mechanism in the two cell types.It is our prediction that the ability of HSV-2 to kill T cells plays a role in mitigating the cell-mediated immune response in vivo. In the present study, we have begun to elucidate the mechanism behind apoptosis in T cells following exposure to HSV-2-infected fibroblasts. Viral antigens could be detected in virus-exposed Jurkat cells, suggesting infection that occurs by cell-to-cell spread. We previously reported that expression of HSV-1 ICP0 and HSV-2 ICP10 results in apoptosis of transfected Jurkat cells (4). Therefore, we predicted that infected cells would be positive for apoptotic markers in the present study. However, viral antigens and activated caspase 3 were largely mutually exclusive in virus-exposed cells. We are currently undertaking studies to further decipher the relationship between viral infection and apoptosis to determine whether apoptosis occurs by a bystander effect in T cells.Our results revealed the requirement of caspase 9 in HSV-2-induced apoptosis of Jurkat cells. The finding that neither caspase 8 nor FADD was required suggests that the extrinsic pathway is not involved in HSV-2-induced apoptosis in Jurkat cells. Consistent with this finding, cleavage of caspase 9 was clearly demonstrated in virus-exposed CD4⁺ cells compared to mock-exposed cells, while cleavage of caspase 8 was not evident. Although similarities between HSV-2-induced apoptosis in CD4⁺ and Jurkat cells predict that similar mechanisms are likely to be responsible in both cell types, the applicability of the finding to other types of T cells remains to be elucidated.In conclusion, the present study demonstrated activation of intrinsic apoptosis pathways in Jurkat and primary human CD4⁺ cells following exposure to HSV-2. Further understanding of virus-induced apoptosis in T cells, which is predicted to be an immune-evasion mechanism during reactivation, is expected to provide means to prevent HSV-2 reactivation in infected individuals.     

Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.