RESUMO
The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.
Assuntos
Herpesvirus Humano 2/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Chlorocebus aethiops , Herpesvirus Humano 2/patogenicidade , Fosforilação , Isoformas de Proteínas , Proteínas Quinases/fisiologia , Transfecção , Células Vero , Proteínas Estruturais Virais/análise , Proteínas Estruturais Virais/imunologiaRESUMO
Sequences from -420 to -70 from the ICPO transcriptional start site of herpes simplex virus type 1 are dispensable for reactivation from latency. A putative cAMP-response element (CRE) outside of this region was non-functional in both murine neuroblastoma (NB41A3) and rat pheochromocytoma (PC12) cells. Also, poor binding of cAMP-response element binding protein (CREB) was observed. Sequences from -95 to -37 are important for constitutive activity in NB41A3, PC12 and baby hamster kidney (BHK) cells. The TATA box and Sp1 site were also shown to be major contributors to constitutive activity. Finally, high constitutive activity of a deleted construct (-420 to -1) in NB41A3 and BHK cells suggests transcription initiates upstream of -420 in the absence of VP16. The implications of these observations regarding ICPO expression during the virus life-cycle are discussed.
Assuntos
Genes Virais , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , Cricetinae , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Genes Precoces , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Células PC12 , Ratos , Deleção de Sequência , Fator de Transcrição Sp1/metabolismo , TATA Box , Ubiquitina-Proteína Ligases , Ativação Viral/genéticaRESUMO
The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Virais/genética , Latência Viral , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Mutagênese , Células PC12 , Regiões Promotoras Genéticas , Ratos , Recombinação Genética , Células Vero , Proteínas Virais/fisiologia , Ativação Viral , Replicação ViralRESUMO
A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.
Assuntos
Infecções Oculares/prevenção & controle , Herpes Simples/prevenção & controle , Simplexvirus/imunologia , Vacinas Virais/uso terapêutico , Animais , Olho/efeitos da radiação , Olho/virologia , Camundongos , Camundongos Endogâmicos , Ribonucleases , Simplexvirus/genética , Raios Ultravioleta , Vacinas Atenuadas/uso terapêutico , Proteínas Virais/genética , Eliminação de Partículas ViraisRESUMO
Herpes simplex virus type 1 gene UL4 is predicted to encode a 199-amino-acid protein with a molecular mass of 21 5 kDa. We report here identification of this protein and its localization in the nuclei of infected cells. Antisera raised against oligopeptides corresponding to the C terminus of the predicted UL4 protein were used for identification of a 25 kDa protein as the product of the UL4 gene. This protein was not detected in cells infected with a UL4 defective mutant virus, but was synthesized by coupled in vitro transcription-translation of the UL4 gene. Synthesis of the 25 kDa protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis, indicating that the UL4 gene is expressed with gamma kinetics. Subcellular fractionation showed the protein to be localized in the nucleus. It was not detected in virions or light particles.
Assuntos
AMP Cíclico/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , AMP Cíclico/genética , Herpesvirus Humano 1/genética , Humanos , Dados de Sequência Molecular , Proteínas Virais/genéticaRESUMO
A quantitative ribonuclease protection assay (RPA) was developed in order to rapidly and accurately measure the levels and timing of latency-associated transcript (LAT) expression in ganglia latently infected with wild-type and mutant herpes simplex virus (HSV). Use of this assay in parallel with measurement of viral titers in murine trigeminal ganglia demonstrated that the peak of viral replication precedes the peak and subsequent plateau of LAT expression. This plateau of LAT expression was unaltered from Day 7 through the end of the experimental period on Day 28, suggesting that LAT does not further accumulate during latency of wild-type virus. RPA analyses of trigeminal ganglia latently infected with HSV-1 mutants containing specific alterations in the LAT TATA box, cyclic AMP-response element (CRE), and both TATA and CRE were performed. Mutation of the upstream TATA box reduced LAT expression to 25% of wild-type or marker-rescued virus levels, whereas mutation of the CRE did not significantly affect LAT expression in vivo whether in the presence or absence of the TATA box. These experiments demonstrate a specific requirement for the upstream promoter TATA box for wild-type LAT expression. Further examination of the role of the CRE and the TATA box by transient expression assays suggests that the CRE is important for inducible activity and that its interaction with the TATA box requires stereospecific alignment.
Assuntos
Regulação Viral da Expressão Gênica , Genes Reguladores , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Regiões Promotoras Genéticas , TATA Box , Transcrição Gênica , Latência Viral , Neoplasias das Glândulas Suprarrenais , Animais , Sequência de Bases , Chlorocebus aethiops , AMP Cíclico , Primers do DNA , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células PC12 , Feocromocitoma , Ratos , Gânglio Trigeminal/virologia , Células VeroAssuntos
Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Citomegalovirus/genética , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Células PC12 , Ratos , Tetraciclina/farmacologia , Células VeroRESUMO
The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.
Assuntos
Herpesvirus Humano 1/genética , Ceratite Herpética/microbiologia , Regiões Promotoras Genéticas , Transcrição Gênica , Replicação Viral , Animais , Sequência de Bases , Bucladesina/farmacologia , Cloranfenicol O-Acetiltransferase/biossíntese , Colforsina/farmacologia , Sequência Conservada , Biblioteca Genômica , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Camundongos , Dados de Sequência Molecular , Células PC12 , Recombinação Genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Herpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (< 1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacy in vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p = 0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.
Assuntos
Imunocompetência , Mutação , Simplexvirus/genética , Simplexvirus/fisiologia , Células Tumorais Cultivadas/patologia , Animais , Encéfalo/virologia , Neoplasias Encefálicas/patologia , Humanos , Idoxuridina/farmacocinética , Masculino , Ratos , Ratos Sprague-Dawley , Ribonucleotídeo Redutases/genética , Simplexvirus/patogenicidade , Células Tumorais Cultivadas/metabolismo , Tirosina 3-Mono-Oxigenase/deficiênciaRESUMO
Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant- and wild-type virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.
Assuntos
Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Simplexvirus/genética , TATA Box , Transcrição Gênica , Animais , Sequência de Bases , Gânglios/microbiologia , Genoma Viral , Hibridização In Situ , Cinética , Neurônios/microbiologia , Células PC12 , Mapeamento por Restrição , Simplexvirus/fisiologia , Células Vero , Replicação ViralRESUMO
A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Genes Virais , Regiões Promotoras Genéticas , Simplexvirus/genética , Transcrição Gênica , Animais , Sequência de Bases , Bucladesina/farmacologia , Linhagem Celular , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/farmacologia , Mapeamento por Restrição , Simplexvirus/fisiologia , TATA Box , Acetato de Tetradecanoilforbol/farmacologia , Células VeroRESUMO
We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLAT1.8 was replication competent in the eye and in ganglia during acute infection but reactivated from explant cultures of ganglia with reduced efficiency (49%) relative to those of wild-type and marker-rescued viruses (94 and 85%, respectively) despite the fact that levels of mutant viral DNA in ganglia during latent infection were comparable to wild-type levels. The neurovirulence of KOS was not significantly altered by the removal of sequences specifying the LATs, as judged by numbers of animals dying on or before 30 days postinfection. Examination of ganglia latently infected with dlLAT1.8 by in situ hybridization revealed no LAT expression. The genotype of reactivated virus was identical to that of input dlLAT1.8 virus as judged by Southern blot analysis. These studies suggest that although the LATs are not essential for the establishment and reactivation of latency in our model, they may play a role in determining the frequency of reactivation of virus from the latent state.