Electron Microscopy of Herpes Simplex Virus IV. Studies with Ferritin-conjugated Antibodies

Small aggregates of viral antigen were encountered in the nuclear matrix. The capsids did not tag with antibodies specific for the virus or for the host cell. This observation remains unexplained. Nuclear and cytoplasmic membranes, as well as the envelope of the virus, reacted with both types of antibodies and appear, therefore, to contain host cell and viral protein. Large amounts of viral antigen are synthesized within the cytoplasm. This antigen was either diffusely spread or localized at the surface of membranes. The surface of infected cells contains viral antigen, which accumulates as infection progresses. At circumscribed sites, the cell wall becomes altered antigenically and structurally so as to resemble the envelope of the virus. Hypotheses are presented regarding the manner in which cell fusion occurs.     

Specific Deposition of Passively Transferred Monoclonal Antibodies against Herpes Simplex Virus Type 1 in Rat Brain Infected with the Virus

The kinetics of human monoclonal antibody (anti-gB) to herpes simplex virus type 1 (HSV-1) were investigated after intravenous injection of anti-gB into an HSV-1 encephalitis animal model. Immunohistochemical study revealed specific deposition of passively tansferred anti-gB in the hippocampus and thalamus of the infected rat brain, and it bound to the same neurons in which HSV-1 antigen was positively stained. To examine the macroscopic distribution of anti-gB in the infected brain, we undertook an ¹²⁵I-labeled anti-gB injection study, and the same distribution of ¹²⁵I-labeled anti-gB deposition was observed by brain semimicroautoradiography as in the immunohistochemical study. These results suggest that anti-gB easily permeates the capillary wall and is deposited in the inflammatory site where HSV-1-specific antigen is detectable. The use of radioisotope-labeled anti-gB injection and external brain imaging could lead to a noninvasive diagnostic tool for the early detection of HSV-1 antigen in cases of suspected HSV-1 encephalitis.     

Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives.     

Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

Electron Microscopy of Herpes Simplex Virus III. Effect of Hydroxyurea

The effect of hydroxyurea on the development of herpes virus is mediated through its inhibitory action on deoxyribonucleic acid (DNA) synthesis. Concentrations of the drug that suppress the production of infectious virus cause typical developmental anomalies: failure in formation of the normally dense cores or "complete" viral particles, and either faulty or no envelopment of viral capsids by membranes. The synthesis of viral capsids and virus-stimulated nuclear and cytoplasmic membranes, however, is not interrupted. Combining these results with those of time sequence experiments, the following hypotheses can be presented regarding viral development. Protein synthesis, which is characterized by capsids enclosing cores of low density, precedes DNA synthesis, which is characterized by the appearance of dense cores. Capsids with dense cores are selectively transported to the cytoplasm. Envelopment generally takes place as capsids pass from the nucleus to the cytoplasm. The process of envelopment is also selective, with the result that the majority of particles that have an envelope contain a full quota of DNA.     

Chromosomal and Autoradiographic Studies of Cells Infected with Herpes Simplex Virus

The induction of chromosomal aberrations by herpes simplex virus (HSV) and the interaction between viral deoxyribonucleic acid (DNA) and chromosomes have been studied (i) by infection of the BHK-21 line of hamster kidney cells at multiplicities ranging from 0.5 to 100 followed by 1-hr pulses of (3)H-thymidine preceding preparation at varying intervals of metaphases and autoradiography, and (ii) by use of (3)H-thymidine-labeled HSV for infection, chromosomal, and autoradiographic analyses at intervals thereafter. The results revealed that (i) chromosomal lesions develop prior to, and thus are independent of, viral DNA synthesis; (ii) HSV is capable of replicating in cells during the late G-2 period or in metaphase; (iii) most of the viral DNA remains unassociated with chromosomes and is not detectable at sites of chromosomal lesions; (iv) the capacity of the virus to cause chromosomal aberrations is four times less sensitive to inactivation by ultraviolet (UV) irradiation than its infectious property; and (v) after large doses of UV, invasion of the nuclei by the irradiated virus is reduced. These observations indicate that the chromosomal lesions induced by HSV result most likely from action of an early enzyme under control of the viral genome. This explanation is proposed also for the effects of adenovirus type 12 on chromosomes.     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

Electron Microscopy of Herpes Simplex Virus: II. Sequence of Development

Examination of infected cells at sequential intervals after infection revealed that the first viral forms to appear were capsids enclosing cores of low density. Not until the 6th hr were dense cores encountered, and at approximately the same time enveloped virus was seen. Envelopment occurred most frequently in close proximity to the nuclear surface, although the process was also encountered within the nuclear matrix and in the cytoplasm. There was often extensive proliferation of the nuclear membrane. Envelopment of the virus by budding from the cell surface was not observed. It was concluded that enveloped virus consitutes the infectious particle and that the unenveloped capsid is unstable outside the cell. Nevertheless, it is likely that capsids enclosing infectious nucleic acid can pass directly from one cell to another after fusion has taken place.     

Ribonucleotide Reductase Activity of Synchronized KB Cells Infected with Herpes Simplex Virus

The replication of herpes simplex virus (HSV) is unimpeded in KB cells which have been blocked in their capacity to synthesize deoxyribonucleic acid (DNA) by high levels of thymidine (TdR). Studies showed that the presence of excess TdR did not prevent host or viral DNA replication in HSV-infected cells. In fact, more cellular DNA was synthesized in infected TdR-blocked cells than in uninfected TdR-blocked cells. This implies that the event which relieved the TdR block was not specific for viral DNA synthesis but allowed some cellular DNA synthesis to occur. These results suggested that HSV has a means to insure a pool of deoxycytidylate derivatives for DNA replication in the presence of excess TdR. We postulated that a viral-induced ribonucleotide reductase was present in the cell after infection which was not inhibited by thymidine triphosphate (TTP). Accordingly, comparable studies of the ribonucleotide reductase found in infected and uninfected KB cells were made. We established conditions that would permit the study of viral-induced enzymes in logarithmically growing KB cells. A twofold stimulation in reductase activity was observed by 3 hr after HSV-infection. Reductase activity in extracts taken from infected cells was less sensitive to inhibition by exogenous (TTP) than the enzyme activity present in uninfected cells. In fact, the enzyme extracted from infected cells functioned at 60% capacity even in the presence of 2 mm TTP. These results support the idea that a viral-induced ribonucleotide reductase is present after HSV infection of KB cells and that this enzyme is relatively insensitive to inhibition by exogenous TTP.     

Alterations of Neutral Glycolipids in Cells Infected with Syncytium-Producing Mutants of Herpes Simplex Virus Type 1

The isolation of syncytium-producing mutants of herpes simplex virus type 1 (KOS strain), which cause extensive cell fusion during otherwise normal infections, has been reported previously (S. Person, R. W. Knowles, G. S. Read, S. C. Warner, and V. C. Bond, J. Virol. 17:183-190, 1976). Seven of these mutants, plus two syncytial strains obtained elsewhere, were used to compare the incorporation of labeled galactose into neutral glycolipids of mock-infected, wild-type-infected, and syncytially infected human embryonic lung cells. Five predominant cellular glycolipid species were observed, denoted GL-1 through GL-5 in order of increasing oligosaccharide chain length; for example, GL-1 and GL-2 correspond to glycolipids that contain mono- and disaccharide units, respectively. Wild-type virus infection caused an increase in galactose incorporation into GL-1 and GL-2 relative to GL-3 through GL-5. For a single labeling interval from 4 to 10 h after adsorption, syncytial infections generally resulted in a relatively greater incorporation into more complex glycolipids than did wild-type infections. One mutant, syn 20, was compared with wild-type virus throughout infection by using a series of shorter labeling pulses and appeared to delay by at least 2 h the alterations observed during wild-type infections. These alterations are apparently due to defects in synthesis, since prelabeled cellular glycolipids were not differentially degraded during mock or virus infection.     

Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

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。     

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.     

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.