Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis

AbstractDuring HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This “HSV-1-dependent” apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSΔ27)}. Unlike HEp-2/HeLa cells, vBSΔ27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis

The Virion Host Shut-Off (vhs) Protein Blocks a TLR-Independent Pathway of Herpes Simplex Virus Type 1 Recognition in Human and Mouse Dendritic Cells

Molecular pathways underlying the activation of dendritic cells (DCs) in response to Herpes Simplex Virus type 1 (HSV-1) are poorly understood. Removal of the HSV virion host shut-off (vhs) protein relieves a block to DC activation observed during wild-type infection. In this study, we utilized a potent DC stimulatory HSV-1 recombinant virus lacking vhs as a tool to investigate the mechanisms involved in the activation of DCs by HSV-1. We report that the release of pro-inflammatory cytokines by conventional DC (cDC) during HSV-1 infection is triggered by both virus replication-dependent and replication-independent pathways. Interestingly, while vhs is capable of inhibiting the release of cytokines during infection of human and mouse cDCs, the secretion of cytokines by plasmacytoid DC (pDC) is not affected by vhs. These data prompted us to postulate that infection of cDCs by HSV triggers a TLR independent pathway for cDC activation that is susceptible to blockage by the vhs protein. Using cDCs isolated from mice deficient in both the TLR adaptor protein MyD88 and TLR3, we show that HSV-1 and the vhs-deleted virus can activate cDCs independently of TLR signaling. In addition, virion-associated vhs fails to block cDC activation in response to treatment with TLR agonists, but it efficiently blocked cDC activation triggered by the paramyxoviruses Sendai Virus (SeV) and Newcastle Disease Virus (NDV). This block to SeV- and NDV-induced activation of cDC resulted in elevated SeV and NDV viral gene expression indicating that infection with HSV-1 enhances the cell's susceptibility to other pathogens through the action of vhs. Our results demonstrate for the first time that a viral protein contained in the tegument of HSV-1 can block the induction of DC activation by TLR-independent pathways of viral recognition

Comparison of HEp-2 and Vero Cell Responses Reveal Unique Proapoptotic Activities of the Herpes Simplex Virus Type 1 α0 Gene Transcript and Product

Previous studies have provided evidence suggesting a role for apoptosis in the control of Herpes Simplex Virus 1 (HSV-1) latency. HSV-1 induces and then later blocks apoptosis in infected cells. The immediate early viral gene α0, which synthesizes the ICP0 protein, is necessary and sufficient for HSV-1-induced apoptosis in human epithelial (HEp-2) cells. While previous research showed that ICP0 protein synthesis is not necessary for HSV-1-induced apoptosis in infected HEp-2 cells, circumstantial evidence suggested that it might be needed in infected African green monkey kidney (Vero) cells. In this study, we determined the specific aspects of α0 needed to trigger apoptosis in these two cell types. HEp-2 cells transfected with α0 expressing plasmids that generated either full-length, truncated, or no detectable (multiple stop codons) ICP0 protein died through apoptosis. This indicates that ICP0 protein is not necessary for α0-induced apoptosis and that α0 mRNA alone has apoptotic induction properties in HEp-2 cells. We next investigated the primary structure of α0’s mRNA to better define its proapoptotic ability. Since α0 is one of the few HSV-1 genes that are spliced, we transfected cells with a plasmid expressing ICP0 from cDNA copy, pcDNAICP0. The cells transfected with pcDNAICP0 underwent apoptosis at a level equivalent to those transfected with the genomic copy of α0, which indicates that neither splicing events nor introns are required for the apoptotic function of α0 in HEp-2 cells. Next, we studied the ability of α0 to cause apoptosis in Vero cells. Since HSV-1-induced apoptosis in Vero cells requires protein synthesis early in infection, proteins synthesized with immediate early kinetics may facilitate apoptosis. Vero cells were transfected with plasmids producing either full-length ICP0 or ICP0 truncated at codon 212. Full-length ICP0, but not truncated ICP0, induced apoptosis in Vero cells. Together, these results suggest that α0 gene expression triggers apoptosis, but ICP0 protein is needed to facilitate apoptosis in Vero cells. In addition, ICP0’s facilitation activity may lie in its carboxyl-terminated domain. Thus, our results demonstrate that α0’s mRNA and protein possess proapoptotic properties. The requirement for ICP0 protein during HSV-dependent apoptosis appears to be cell type specific

APP Processing Induced by Herpes Simplex Virus Type 1 (HSV-1) Yields Several APP Fragments in Human and Rat Neuronal Cells

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Overview.

The Concise Guide to PHARMACOLOGY 2017/18 is the third in this series of biennial publications. This version provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13882/full. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are eight areas of focus: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

ICP0 Gene Expression Is a Herpes Simplex Virus Type 1 Apoptotic Trigger

Apoptosis is a highly regulated programmed cell death process which is activated during normal development and by various stimuli, such as viral infection, which disturb cellular metabolism and physiology. That herpes simplex virus type 1 (HSV-1) induces apoptosis but then prevents its killing of infected cells is well-established. However, little is known about the viral factor/event which triggers the apoptotic process. We previously reported that infections with either (i) a temperature-sensitive virus at its nonpermissive temperature which does not inject viral DNA into nuclei or (ii) various UV-inactivated wild-type viruses do not result in the induction of apoptosis (C. M. Sanfilippo, F. N. W. Chirimuuta, and J. A. Blaho, J. Virol. 78:224-239, 2004). This indicates that virus receptor binding/attachment to cells, membrane fusion, virion disassembly/tegument dispersal, virion RNAs, and capsid translocation to nuclei are not responsible for induction and implicates viral immediate-early (IE) gene expression in the process. Here, we systematically evaluated the contribution of each IE gene to the stimulation of apoptosis. Using a series of viruses individually deleted for α27, α4, and α22, we determined that these genes are not required for apoptosis induction but rather that their products play roles in its prevention, likely through regulatory effects. Sole expression of α0 acted as an “apoptoxin” that was necessary and sufficient to trigger the cell death cascade. Importantly, results using a recombinant virus which contains a stop codon in α0 showed that it was not the ICP0 protein which acted as the apoptotic inducer. Based on these findings, we propose that α0 gene expression acts as an initial inducer of apoptosis during HSV-1 infection. This represents the first description of apoptosis induction in infected cells triggered as a result of expression of a single viral gene. Expression of apoptotic viral genes is a unique mechanism through which human pathogens may modulate interactions with their host cells