RESUMEN
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.
Asunto(s)
Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Multimerización de Proteína , Ubiquitina-Proteína Ligasas , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Cristalografía por Rayos X , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Modelos Moleculares , Humanos , Dominios Proteicos , Pliegue de Proteína , Secuencia de Aminoácidos , Conformación Proteica en Lámina betaRESUMEN
Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Humanos , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Células Receptoras Sensoriales/metabolismoRESUMEN
We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39, resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0-, ICP8-, and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8- virus, but not the ICP0- virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases.IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0-) virus and a replication-incompetent (ICP8-) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development.
Asunto(s)
Herpesvirus Humano 1/genética , Vacunas contra Herpesvirus/inmunología , Queratitis Herpética/prevención & control , Proteínas Virales/genética , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Queratitis Herpética/inmunología , Queratitis Herpética/virología , Ratones , Mutación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Células Vero , Proteínas Virales/inmunología , Latencia del Virus , Replicación ViralRESUMEN
In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCE HSV-1 is a major human pathogen that infects â¼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.
Asunto(s)
Proteínas de la Cápside , Herpes Simple , Herpesvirus Humano 1/fisiología , Mutación Puntual , Activación Viral/genética , Latencia del Virus/genética , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Herpes Simple/genética , Herpes Simple/metabolismo , Herpes Simple/patología , Vacunas contra el Virus del Herpes Simple/genética , Vacunas contra el Virus del Herpes Simple/metabolismo , Ratones , Células Vero , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
The cyclin-dependent kinase 5 (CDK-5) activating protein, p35, is important for acute herpes simplex virus 1 (HSV-1) replication in mice. This report shows that HSV-1 increases p35 levels, changes the primary localization of CDK-5 from the nucleus to the cytoplasm, and enhances CDK-5 activity during lytic or acute infection. Infected neurons also stained positive for the DNA damage response (DDR) marker γH2AX. We propose that CDK-5 is activated by the DDR to protect infected neurons from apoptosis.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Neuronas/virología , Fosfotransferasas/biosíntesis , Replicación Viral , Animales , Apoptosis , Daño del ADN , Histonas/análisis , Ratones NoqueadosRESUMEN
The ubiquitin-proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.
Asunto(s)
Western Blotting/métodos , Herpesvirus Humano 1/enzimología , Proteínas Inmediatas-Precoces/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Herpesvirus Humano 1/patogenicidad , UbiquitinaciónRESUMEN
The herpes simplex virus 1 (HSV-1) immediate-early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cells, for virus-induced chaperone-enriched (VICE) domain formation, and for normal expression of a subset of viral late proteins. Additionally, ICP22 is important for optimal acute viral replication in vivo. Previous studies have shown that the US1 gene that encodes ICP22, produces an in-frame, N-terminally truncated form of ICP22, known as US1.5. To date, studies conducted to characterize the functions of ICP22 have not separated its functions from those of US1.5. To determine the individual roles of ICP22 and US1.5, we made viral mutants that express either ICP22 with an M90A mutation in the US1.5 initiation codon (M90A) or US1.5 with three stop codons introduced upstream of the US1.5 start codon (3×stop). Our studies showed that, in contrast to M90A, 3×stop was unable to replicate efficiently in the eyes and trigeminal ganglia of mice during acute infection, to efficiently establish a latent infection, or to induce VICE domain formation and was only mildly reduced in its replication in restrictive HEL-299 cells and murine embryonic fibroblasts (MEFs). Both mutants enhanced the expression of the late viral proteins virion host shutoff (vhs) and glycoprotein C (gC) and inhibited viral gene expression mediated by HSV-1 infected cell protein 0 (ICP0). When we tested our mutants' sensitivity to type I interferon (beta interferon [IFN-ß]) in restrictive cells, we noticed that the plating of the ICP22 null (d22) and 3×stop mutants was reduced by the addition of IFN-ß. Overall, our data suggest that US1.5 partially complements the functions of ICP22.
Asunto(s)
Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Chlorocebus aethiops , Femenino , Herpes Simple/genética , Herpesvirus Humano 1/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Ratones , Chaperonas Moleculares/genética , Células Vero , Proteínas Reguladoras y Accesorias Virales/genética , Replicación ViralRESUMEN
Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in sensory neurons and can reactivate from latency under stress conditions. To promote lytic infection, the virus must interact with specific cellular factors to evade the host's antiviral defenses. The HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), activates transcription of viral genes, in part, by mediating the degradation of certain cellular proteins that play a role in host antiviral mechanisms. One component of the cellular defenses that ICP0 disrupts is the suborganelle, nuclear domain 10 (ND10), by inducing the degradation and dissociation of the major organizer of ND10, a promyelocytic leukemia (PML) and ND10 constituent, Sp100. Because previously identified domains in ICP0 explain only partially how it directs the degradation and dissociation of PML and Sp100, we hypothesized that additional regions within ICP0 may contribute to these activities, which in turn facilitate efficient viral replication. To test this hypothesis, we used a series of ICP0 truncation mutants and examined PML protein levels and PML and Sp100 immunofluorescence staining in human embryonic lung cells. Our results demonstrate that two overlapping regions within the central N-terminal portion of ICP0 (residues 212 to 311) promoted the dissociation and degradation of PML and dissociation of Sp100 (residues 212 to 427). In conclusion, we have identified two additional regions in ICP0 involved in altering ND10 antiviral defenses in a cell culture model of HSV-1 infection.
Asunto(s)
Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Herpes Simple/metabolismo , Herpesvirus Humano 1/enzimología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Antígenos Nucleares/genética , Autoantígenos/genética , Regulación Viral de la Expresión Génica , Herpes Simple/genética , Herpes Simple/virología , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Unión Proteica , Proteolisis , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication.
Asunto(s)
Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/metabolismo , Replicación Viral , Sustitución de Aminoácidos , Animales , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Línea Celular , Humanos , Proteínas Inmediatas-Precoces/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Proteína de la Leucemia Promielocítica , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.
RESUMEN
PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses.
RESUMEN
Herpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.
Asunto(s)
ADN Viral/genética , Genoma Viral , Herpesvirus Humano 1/genética , Análisis de Secuencia de ADN , ADN Viral/química , Humanos , Mutación INDEL , Datos de Secuencia Molecular , Polimorfismo de Nucleótido SimpleRESUMEN
The herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent compared to other wild-type strains of HSV-1. To help us better understand the genetic determinants that lead to differences in the pathogenicity of McKrae and other HSV-1 strains, we sequenced its genome. Comparing the sequence of McKrae's genome to that of strain 17 revealed that the genomes differ by at least 752 single nucleotide polymorphisms (SNPs) and 86 insertion/deletion events (indels). Although the majority of these polymorphisms reside in noncoding regions, 241 SNPs and 10 indels alter the protein-coding sequences of 58 open reading frames. Some of these variations are expected to contribute to the pathogenic phenotype of McKrae.
Asunto(s)
Genoma Viral , Herpesvirus Humano 1/genética , Animales , Secuencia de Bases , Chlorocebus aethiops , Herpesvirus Humano 1/clasificación , Herpesvirus Humano 1/aislamiento & purificación , Datos de Secuencia Molecular , Células Vero/virología , Proteínas Virales/genéticaRESUMEN
We utilized a high-throughput cell-based assay to screen several chemical libraries for inhibitors of herpes simplex virus 1 (HSV-1) gene expression. From this screen, four aurora kinase inhibitors were identified that potently reduced gene expression during HSV-1 lytic infection. HSV-1 is known to interact with cellular kinases to regulate gene expression by modulating the phosphorylation and/or activities of viral and cellular proteins. To date, the role of aurora kinases in HSV-1 lytic infection has not been reported. We demonstrated that three aurora kinase inhibitors strongly reduced the transcript levels of immediate-early (IE) genes ICP0, ICP4, and ICP27 and impaired HSV-1 protein expression from all classes of HSV-1, including ICP0, ICP4, ICP8, and gC. These restrictions caused by the aurora kinase inhibitors led to potent reductions in HSV-1 viral replication. The compounds TAK 901, JNJ 7706621, and PF 03814735 decreased HSV-1 titers by 4,500-, 13,200-, and 8,400-fold, respectively, when present in a low micromolar range. The antiviral activity of these compounds correlated with an apparent decrease in histone H3 phosphorylation at serine 10 (H3S10ph) during viral infection, suggesting that the phosphorylation status of H3 influences HSV-1 gene expression. Furthermore, we demonstrated that the aurora kinase inhibitors also impaired the replication of other RNA and DNA viruses. These inhibitors significantly reduced yields of vaccinia virus (a poxvirus, double-stranded DNA, cytoplasmic replication) and mouse hepatitis virus (a coronavirus, positive-sense single-strand RNA [ssRNA]), whereas vesicular stomatitis virus (rhabdovirus, negative-sense ssRNA) yields were unaffected. These results indicated that the activities of aurora kinases play pivotal roles in the life cycles of diverse viruses. IMPORTANCE We have demonstrated that aurora kinases play a role during HSV-1 lytic infection. Three aurora kinase inhibitors significantly impaired HSV-1 immediate-early gene expression. This led to a potent reduction in HSV-1 protein expression and viral replication. Together, our results illustrate a novel role for aurora kinases in the HSV-1 lytic cycle and demonstrate that aurora kinase inhibitors can restrict HSV-1 replication. Furthermore, these aurora kinase inhibitors also reduced the replication of murine coronavirus and vaccinia virus, suggesting that multiple viral families utilize the aurora kinases for their own replication.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Virus ARN , Animales , Ratones , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Línea Celular , Herpes Simple/genética , ADN/metabolismo , ARN/metabolismo , Estadios del Ciclo de VidaRESUMEN
In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.
Asunto(s)
Oftalmopatías/virología , Herpes Simple/virología , Herpesvirus Humano 1/patogenicidad , Proteínas Inmediatas-Precoces/genética , Mutación/genética , Ganglio del Trigémino/virología , Ubiquitina-Proteína Ligasas/genética , Activación Viral , Replicación Viral , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Oftalmopatías/metabolismo , Femenino , Genoma Viral , Herpes Simple/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de Aminoácido , Transcripción Genética , Ganglio del Trigémino/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células Vero , Latencia del VirusRESUMEN
We previously isolated an HSV-1 mutant, KOS-NA, that contains two non-synonymous mutations in UL39. One of the mutations, resulting in an R950H amino acid substitution in ICP6, renders KOS-NA severely neuro-attenuated and significantly reduces HSV-1 latency. Vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated eye diseases even at a very low immunizing dose, indicating its utility as a vaccine scaffold. Because KOS-NA contains a neuro-attenuating mutation in a single gene, we sought to improve its safety by deleting a portion of the UL29 gene whose protein product, ICP8, is essential for viral DNA replication. Whereas KOS-NA reduced replication of HSV-1 challenge virus in the corneal epithelium and protected mice against blepharitis and keratitis induced by the challenge virus, KOS-NA/8- and an ICP8- virus were significantly less efficacious except at higher doses. Our results suggest that the capacity to replicate, even at significantly reduced levels compared with wild-type HSV-1, may be an important feature of an effective vaccine. Means to improve safety of attenuated viruses as vaccines without compromising efficacy should be sought.
Asunto(s)
Herpesvirus Humano 1 , Animales , Chlorocebus aethiops , Replicación del ADN , ADN Viral , Herpesvirus Humano 1/genética , Ratones , Vacunas Atenuadas , Células Vero , Proteínas Virales/genética , Replicación ViralRESUMEN
Herpes simplex virus 1 (HSV-1) is a human pathogen capable of establishing lifelong latent infections that can reactivate under stress conditions. A viral immediate early protein that plays important roles in the HSV-1 lytic and latent infections is the viral E3 ubiquitin ligase, ICP0. ICP0 transactivates all temporal classes of HSV-1 genes and facilitates viral gene expression. ICP0 also impairs the antiviral effects of interferon (IFN)-ß, a component of host innate defenses known to limit viral replication. To begin to understand how ICP0 allows HSV-1 to disarm the IFN-ß response, we performed genetic analyses using a series of ICP0 truncation mutants in the absence and presence of IFN-ß in cell culture. We observed that IFN-ß pretreatment of cells significantly impaired the replication of the ICP0 truncation mutants, n212 and n312, which code for the first 211 and 311 amino acids of ICP0, respectively; this effect of IFN-ß correlated with decreased HSV-1 early and late gene expression. This increased sensitivity to IFN-ß was not as apparent with the ICP0 mutant, n389. Our mapping studies indicate that loss of 77 amino acids from residues 312 to 388 in the N-terminal half of ICP0 resulted in a virus that was significantly more sensitive to cells pre-exposed to IFN-ß. This 77 amino acid region contains a phospho-SUMO-interacting motif or -SIM, which we propose participates in ICP0's ability to counteract the antiviral response established by IFN-ß. IMPORTANCE Interferons (IFNs) are secreted cellular factors that are induced by viral infection and limit replication. HSV-1 is largely refractory to the antiviral effects of type 1 IFNs, which are synthesized shortly after viral infection, in part through the activities of the viral regulatory protein, ICP0. To understand how ICP0 impedes the antiviral effects of type 1 IFNs, we used a series of HSV-1 ICP0 mutants and examined their viral replication and gene expression levels in cells stimulated with IFN-ß (a type 1 IFN). Our mapping data identifies a discrete 77 amino acid region in the N-terminal half of ICP0 that facilitates HSV-1 resistance to IFN-ß. This region of ICP0 is modified by phosphorylation and binds to the posttranslational modification SUMO, suggesting that HSV, and potentially other viruses, may counteract type 1 IFN signaling by altering SUMO and/or SUMO modified cellular proteins.
Asunto(s)
Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Interferón Tipo I , Ubiquitina-Proteína Ligasas , Aminoácidos , Antivirales/farmacología , Herpesvirus Humano 1/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Interferón Tipo I/inmunología , Infección Latente/virología , Ubiquitina-Proteína Ligasas/genética , Proteínas Virales/genéticaRESUMEN
Herpesviruses are characterized by the ability to establish lifelong latent infections and to reactivate periodically, leading to recurrent disease. The herpes simplex virus type 1 (HSV-1) genome is maintained in a quiescent state in sensory neurons during latency, which is characterized by the absence of detectable viral protein synthesis. Cellular factors induced by stress may act directly on promoters within the latent viral genome to induce the transcription of viral genes and trigger reactivation. In order to identify which viral promoters are induced by stress and elucidate the cellular mechanism responsible for the induction, we generated a panel of HSV-1 promoter-luciferase constructs and measured their response to heat shock. Of the promoters tested, those of ICP0 and ICP22 were the most strongly upregulated after heat shock. Microarray analysis of lytically infected cells supported the upregulation of ICP0 and ICP22 promoters by heat shock. Mutagenic analysis of the ICP0 promoter identified two regions necessary for efficient heat-induced promoter activity, both containing predicted nuclear factor Y (NF-Y) sites, at bases -708 and -75 upstream of the transcriptional start site. While gel shift analysis confirmed NF-Y binding to both sites, only the site at -708 was important for efficient heat-induced activity. Reverse transcription-PCR analysis of selected viral transcripts in the presence of dominant-negative NF-Y confirmed the requirement for NF-Y in the induction of the ICP0 but not the ICP22 promoter by heat shock in lytically infected cells. These findings suggest that the immediate-early ICP0 gene may be among the first genes to be induced during the early events in HSV-1 reactivation, that NF-Y is important for this induction, and that other factors induce the ICP22 promoter.
Asunto(s)
Factor de Unión a CCAAT/fisiología , Regulación Viral de la Expresión Génica , Respuesta al Choque Térmico/genética , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular , Chlorocebus aethiops , Perfilación de la Expresión Génica , Genes Inmediatos-Precoces , Genes Virales , Humanos , Células Vero , Activación ViralRESUMEN
Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6ß (wt) and dlx3.1-6ß (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter:lacz expression cassette. A ≥10-fold difference in ß-galactosidase activity was observed in cells infected with KOS6ß compared to dlx3.1-6ß, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z'-factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.
Asunto(s)
Herpesvirus Humano 1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/genética , Activación Transcripcional/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Recolección de Datos , Expresión Génica , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Transcripcional/genéticaRESUMEN
Of the five herpes simplex virus type 1 immediate early (IE) proteins, the least is known about the function of ICP22 during productive infection and latency. Research characterizing the physical and functional properties of the protein has been limited because ICP22 has proven to be difficult to express in transient assays. In addition, genetic analysis of ICP22 has been complicated by the fact that the C terminus of ICP22 is expressed as a discrete protein product. In order to characterize properties of mutant and wild-type ICP22, we developed a transient expression system. We found that ICP22 can be expressed at detectable levels when placed under the control of the cytomegalovirus IE promoter, confirming recent observations by K. A. Fraser and S. A. Rice (J. Virol. 81:5091-5101, 2007). We extended this analysis to show that ICP22 can also be expressed from its own promoter in the presence of other viral factors, either by coexpression with ICP0 or by infection with an ICP22 null virus. Notably, infection of cells transfected with an ICP22 expression vector yielded ICP22 protein that was modified in a manner similar to that of ICP22 protein detected in wild-type-infected cells. We go on to demonstrate that the failure of ICP22 protein to be expressed in transiently transfected cells was not due to inactivity of the ICP22 promoter, but rather to the ability of ICP22 to inhibit expression of reporter gene activity, including its own, in transient assays. Of special note was the observation that expression of ICP22 was sufficient to prevent transactivation of reporter genes by ICP0. Finally, transient expression of ICP22 was sufficient to complement replication of an ICP22 null virus, demonstrating that this system can be used to study functional properties of ICP22. Collectively, this transient expression system facilitates tests of the physical and functional properties of ICP22 and ICP22 mutants prior to introduction of mutant genes into the viral genome.