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1.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
2.
Allergol. immunopatol ; 47(5): 484-490, sept.-oct. 2019. tab, graf
Artigo em Inglês | IBECS | ID: ibc-186523

RESUMO

Introduction and objectives: Preschool-aged group is frequently affected by urticaria, and infections are the most frequently documented factors that cause acute urticaria in children. This prospective study was designed to investigate the underlying factors of acute urticaria in under five-year-old children and to describe predictive factors for progression to chronicity or recurrence after the first attack. Patients and methods: Children younger than five years of age with acute urticaria were recruited between July 2015 and July 2016. Patients (n = 83) were grouped into those below and above two years of age. In order to assess the risk factors for progression to chronicity or recurrence, logistic regression analysis was performed. Results: Upper respiratory tract infection was the most common detectable reason for acute urticaria (49.4%). Herpes Simplex Virus type 1 was significantly isolated in the cases with the manifestation of an acute single-episode urticaria (p = 0.042). Angioedema and food allergy were predominantly observed under two years old (p = 0.001, p = 0.006 respectively). A positive relationship was determined between the duration of urticaria and chronicity (r = 0.301, p = 0.006). The absence of atopic dermatitis (OR: 6.95, 95% CI: 1.35-35.67, p = 0.020), negative Herpes virus serology (OR: 4.25, 95% CI: 0.83-21.56, p = 0.040), and unknown etiology (OR: 3.30, 95% CI: 1.12-9.71, p = 0.030) were the independent risk factors for recurrent urticaria. Conclusions: Preschool-aged children with acute urticaria should be evaluated for infections at the time of admission. Patients with unknown etiology, negative Herpes virus serology, absence of atopic dermatitis, and long lasting urticaria should be followed up for chronicity and recurrence


No disponible


Assuntos
Humanos , Masculino , Feminino , Lactente , Anticorpos Antivirais/sangue , Pré-Escolar , Hipersensibilidade Alimentar/epidemiologia , Herpes Simples/epidemiologia , Herpesvirus Humano 1/fisiologia , Infecções Respiratórias/epidemiologia , Urticária/epidemiologia , Doença Aguda , Doença Crônica , Progressão da Doença , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Risco
3.
Invest Ophthalmol Vis Sci ; 60(12): 3952-3962, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560369

RESUMO

Purpose: γδ T cells offer an important early immune defense against many different pathogens, both bacterial and viral. Herein, we examined the capacity of γδ T cell subsets to provide protection in the cornea against herpes simplex virus-1 (HSV-1). Methods: C57Bl/6 (wild-type [WT]), γδ T-cell deficient (TCRδ-/-) and CCR6-deficient (CCR6-/-) mice were infected intracorneally with HSV-1. At multiple time points following infection, corneas were excised, and cells were immunostained for surface markers, intracellular cytokines, and analyzed using flow cytometry. WT and CCR6-/- γδ T cells were adoptively transferred into TCRδ-/- mice and corneal scores and survival were measured. Results: Intracorneal infection of mice lacking γδ T cells exhibited increased corneal opacity scores, elevated viral titers, and higher mortality compared with WT mice. Both CCR6+ and CCR6neg γδ T cell subsets were observed in corneas after virus infection. CCR6+ γδ T cells produced IL-17A and were predominantly CD44+CD62L+, consistent with natural IL-17+ γδ T cells. In contrast IL-17A production by CCR6neg γδ T cells was infrequent, and this subset was largely single positive for CD62L or CD44. The CCR6+ subset appeared to provide protection against HSV-1 as follows: (1) CCR6-/- mice had more severe corneal opacity compared with WT mice; and (2) adoptive transfer of γδ T cells from WT mice restored protection in TCRδ-/- mice whereas transfer of γδ T cells from CCR6-/- mice did not. Conclusions: γδ T cells in the cornea can be divided into CCR6+ and CCR6neg subsets with the former conferring protection early after intracorneal HSV-1 infection.


Assuntos
Opacidade da Córnea/prevenção & controle , Herpesvirus Humano 1/fisiologia , Linfócitos Intraepiteliais/imunologia , Ceratite Herpética/prevenção & controle , Receptores CCR6/imunologia , Transferência Adotiva , Animais , Córnea/virologia , Opacidade da Córnea/imunologia , Opacidade da Córnea/virologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-17/metabolismo , Ceratite Herpética/imunologia , Ceratite Herpética/virologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gânglio Trigeminal/virologia , Ensaio de Placa Viral
4.
Elife ; 82019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393262

RESUMO

Many viruses previously have been shown to have pressurized genomes inside their viral protein shell, termed the capsid. This pressure results from the tight confinement of negatively charged viral nucleic acids inside the capsid. However, the relevance of capsid pressure to viral infection has not been demonstrated. In this work, we show that the internal DNA pressure of tens of atmospheres inside a herpesvirus capsid powers ejection of the viral genome into a host cell nucleus. To our knowledge, this provides the first demonstration of a pressure-dependent mechanism of viral genome penetration into a host nucleus, leading to infection of eukaryotic cells.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , DNA Viral/metabolismo , Células Eucarióticas/virologia , Herpesvirus Humano 1/fisiologia , Pressão Hidrostática , Internalização do Vírus , Animais , Linhagem Celular
5.
Invest Ophthalmol Vis Sci ; 60(10): 3398-3406, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31387116

RESUMO

Purpose: We previously have reported that ICP22, an immediate early gene of herpes simplex virus type 1 (HSV-1), binds to the CD80 promoter to suppress CD80 expression in antigen-presenting cells, leading to reduced T-cell function and protection. In contrast, overexpression of CD80 exacerbates corneal scarring (CS) in ocularly infected mice. In this study we tested the hypothesis that the absence of ICP22 could increase disease severity. Methods: To test our hypothesis, BALB/c mice were ocularly infected after corneal scarification with a recombinant HSV-1 lacking the ICP22 gene with its parental wild-type (WT) virus (KOS) as a control. Virus replication in the eye, CS, angiogenesis, latency, and reactivation between ICP22 null virus and WT KOS were determined. In addition, expression of IL-2, IL-4, IFN-γ, IFN-α, granzyme A, granzyme B, and perforin by CD4 and CD8 T cells in corneas of infected mice on days 3, 5, 7, 10, 14, 21, and 28 postinfection were determined by flow cytometry. Results: We found similar levels of eye disease and angiogenesis in mice following corneal scarification and ocular infection with the ICP22 null virus or parental WT virus despite reduced virus replication in the eye and reduced latency and reactivation in mice ocularly infected with ICP22 null virus. The similar level of eye disease in ICP22 null virus- and WT virus-infected mice correlated with expression of various proinflammatory cytokines that infiltrated the eye after HSV-1 infection. Conclusions: Our study identified a critical role for ICP22 in HSV-1 pathogenicity and suggests that HSV-1-associated CS is more dependent on host immune responses to infection than to virus replication in the eye. Thus, HSV-1 as means of survival uses ICP22 as a mechanism of immune escape that protects the host from increased pathology.


Assuntos
Substância Própria/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Ceratite Herpética/imunologia , Latência Viral/fisiologia , Animais , Antígeno B7-1/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Imediatamente Precoces/deficiência , Ceratite Herpética/patologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Lágrimas/virologia , Replicação Viral/fisiologia
6.
Adv Virus Res ; 104: 225-281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31439150

RESUMO

Membrane fusion is a fundamental biological process that allows different cellular compartments delimited by a lipid membrane to release or exchange their respective contents. Similarly, enveloped viruses such as alphaherpesviruses exploit membrane fusion to enter and infect their host cells. For infectious entry the prototypic human Herpes simplex viruses 1 and 2 (HSV-1 and -2, collectively termed HSVs) and the porcine Pseudorabies virus (PrV) utilize four different essential envelope glycoproteins (g): the bona fide fusion protein gB and the regulatory heterodimeric gH/gL complex that constitute the "core fusion machinery" conserved in all members of the Herpesviridae; and the subfamily specific receptor binding protein gD. These four components mediate attachment and fusion of the virion envelope with the host cell plasma membrane through a tightly regulated sequential activation process. Although PrV and the HSVs are closely related and employ the same set of glycoproteins for entry, they show remarkable differences in the requirements for fusion. Whereas the HSVs strictly require all four components for membrane fusion, PrV can mediate cell-cell fusion without gD. Moreover, in contrast to the HSVs, PrV provides a unique opportunity for reversion analyses of gL-negative mutants by serial cell culture passaging, due to a limited cell-cell spread capacity of gL-negative PrV not observed in the HSVs. This allows a more direct analysis of the function of gH/gL during membrane fusion. Unraveling the molecular mechanism of herpesvirus fusion has been a goal of fundamental research for years, and yet important mechanistic details remain to be uncovered. Nevertheless, the elucidation of the crystal structures of all key players involved in PrV and HSV membrane fusion, coupled with a wealth of functional data, has shed some light on this complex puzzle. In this review, we summarize and discuss the contemporary knowledge on the molecular mechanism of entry and membrane fusion utilized by the alphaherpesvirus PrV, and highlight similarities but also remarkable differences in the requirements for fusion between PrV and the HSVs.


Assuntos
Herpesvirus Humano 1/fisiologia , Herpesvirus Suídeo 1/fisiologia , Herpesvirus Humano 2/fisiologia , Internalização do Vírus , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Proteínas do Envelope Viral/metabolismo , Ligação Viral
7.
Molecules ; 24(16)2019 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-31405197

RESUMO

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 µM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 µM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 µM; SI: 114.8) compared with that of ACV (EC50: 2.8 µM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 µM; inhibition constant (Ki): 0.3 µM) compared with reference drugs aphidicolin (IC50: 0.8 µM; Ki: 0.4 µM) and ACV triphosphate (ACV-TP) (IC50: 0.9 µM; Ki: 0.5 µM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.


Assuntos
Antivirais , Benzoxepinas , Ácidos Carboxílicos , DNA Polimerase Dirigida por DNA , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Líquens/química , Simulação de Acoplamento Molecular , Proteínas Virais , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacologia , Benzoxepinas/química , Benzoxepinas/farmacologia , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Inibidores da Síntese de Ácido Nucleico/química , Inibidores da Síntese de Ácido Nucleico/farmacologia , Células Vero , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteínas Virais/metabolismo
8.
Oncogene ; 38(34): 6159-6171, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31289361

RESUMO

Malignant tumors of the central nervous system (CNS) continue to be a leading cause of cancer-related mortality in both children and adults. Traditional therapies for malignant brain tumors consist of surgical resection and adjuvant chemoradiation; such approaches are often associated with extreme morbidity. Accordingly, novel, targeted therapeutics for neoplasms of the CNS, such as immunotherapy with oncolytic engineered herpes simplex virus (HSV) therapy, are urgently warranted. Herein, we discuss treatment challenges related to HSV virotherapy delivery, entry, replication, and spread, and in so doing focus on host anti-viral immune responses and the immune microenvironment. Strategies to overcome such challenges including viral re-engineering, modulation of the immunoregulatory microenvironment and combinatorial therapies with virotherapy, such as checkpoint inhibitors, radiation, and vaccination, are also examined in detail.


Assuntos
Neoplasias Encefálicas/terapia , Resistencia a Medicamentos Antineoplásicos , Herpesvirus Humano 1/fisiologia , Terapia Viral Oncolítica/métodos , Terapias em Estudo , Adulto , Neoplasias Encefálicas/genética , Criança , Resistencia a Medicamentos Antineoplásicos/imunologia , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Terapia Viral Oncolítica/efeitos adversos , Vírus Oncolíticos/fisiologia , Terapias em Estudo/métodos , Terapias em Estudo/tendências , Resultado do Tratamento
9.
Int J Biol Macromol ; 137: 54-61, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31226380

RESUMO

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 µg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 µg/mL/>51.4 and 80.5 µg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 µg/mL. Synthesis of viral protein showed a dose-dependent response and the nucleic acid synthesis was inhibited up to 25 µg/mL, by P1 and P1S and up to 50 µg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.


Assuntos
Antivirais/química , Antivirais/farmacologia , Azadirachta/química , Herpesvirus Humano 1/efeitos dos fármacos , Polissacarídeos/química , Polissacarídeos/farmacologia , Sulfatos/química , Linhagem Celular Tumoral , Herpesvirus Humano 1/fisiologia , Humanos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
10.
PLoS Pathog ; 15(6): e1007884, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206552

RESUMO

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Poliadenilação , Precursores de RNA/metabolismo , Processamento de RNA , RNA Viral/metabolismo , Latência Viral/fisiologia , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Precursores de RNA/genética , RNA Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
12.
Int J Biol Macromol ; 136: 521-530, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158418

RESUMO

The provisioning of compound libraries with a high degree of diversity and attractive pharmacological properties is a limiting step in drug development. This study reports the production of highly bioactive sulfated polysaccharides, originally present in a nonsulfated, dormant state in natural sources, and demonstrates their antiviral activity (human cytomegalovirus EC50 values of 2.34-7.77 µg/mL) at a low degree of cytotoxicity. Furthermore, data strongly suggested the inhibition of virus entry as the main mode of antiviral action. Remarkably, the utilized oleum-DMF reagent was able to generate a range of sulfated polysaccharides from various natural sources, possessing varying saccharide compositions, degrees of sulfation (0.4-1.7) and molecular masses (38-94,000 g/mol). Typically, in a matter of minutes, this reagent not only solubilized polysaccharides but also chemically converted their hydroxyl functionality into sulfates. The most active sulfated polysaccharide (EC50 of 2.62 µg/mL) proved to be a 94,000 g/mol branched glucan with sulfates at C-6/C-3,6/C-2,3,6 positions. In conclusion, the important determinants of such compounds' antiviral activity are: (i) degree of sulfation, (ii) molecular mass and (iii) structural features. Thus, our approach offers a huge prospect for the improvement of natural source-derived libraries based on biologically active polysaccharides with diversified chemical profiles.


Assuntos
Antivirais/química , Antivirais/farmacologia , Produtos Biológicos/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Sulfatos/química , Antivirais/isolamento & purificação , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/fisiologia , Glicosilação , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Humanos , Peso Molecular , Plantas/química , Polissacarídeos/isolamento & purificação , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 89-101, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102363

RESUMO

Herpes simplex virus (HSV), including HSV-1 and HSV-2, is an important pathogen that can cause many diseases. Usually these diseases are recurrent and incurable. After lytic infection on the surface of peripheral mucosa, HSV can enter sensory neurons and establish latent infection during which viral replication ceases. Moreover, latent virus can re-enter the replication cycle by reactivation and return to peripheral tissues to start recurrent infection. This ability to escape host immune surveillance during latent infection and to spread during reactivation is a viral survival strategy and the fundamental reason why no drug can completely eradicate the virus at present. Although there are many studies on latency and reactivation of HSV, and much progress has been made, many specific mechanisms of the process remain obscure or even controversial due to the complexity of this process and the limitations of research models. This paper reviews the major results of research on HSV latency and reactivation, and discusses future research directions in this field.


Assuntos
Herpes Simples , Herpesvirus Humano 1 , Ativação Viral , Latência Viral , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Humanos , Ativação Viral/fisiologia , Latência Viral/fisiologia , Replicação Viral
14.
Prep Biochem Biotechnol ; 49(7): 686-694, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31035907

RESUMO

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness-in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120 min), and temperature (25 °C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.


Assuntos
Técnicas de Cultura de Células/métodos , Herpesvirus Humano 1/fisiologia , Inativação de Vírus , Álcoois/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Desenho de Equipamento , Herpesvirus Humano 1/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Projetos de Pesquisa , Temperatura Ambiente , Células Vero , Ensaio de Placa Viral , Inativação de Vírus/efeitos dos fármacos
15.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088925

RESUMO

Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action.IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib's inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.


Assuntos
Antivirais/farmacologia , Bortezomib/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Internalização do Vírus/efeitos dos fármacos , Aciclovir/farmacologia , Animais , Núcleo Celular/metabolismo , Sinergismo Farmacológico , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Prepúcio do Pênis/citologia , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Humanos , Masculino , Nucleocapsídeo/metabolismo , Células Vero
16.
PLoS One ; 14(5): e0215553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071098

RESUMO

BACKGROUND: The prevalence of, and risk factors for, herpes simplex virus type-1 (HSV-1) infection and reactivation in older individuals are poorly understood. METHODS: This is a prospective population-based study among community-dwelling individuals aged 40-79 years, followed from 1993, formed as a random subsample of the UK-based EPIC-Norfolk cohort. HSV-1 seropositivity was derived from immunoglobulin G measurements and frequent oro-labial HSV reactivation was self-reported. We carried out two cross-sectional studies using logistic regression to investigate childhood social and environmental conditions as risk factors for HSV-1 seropositivity and comorbidities as risk factors for apparent HSV oro-labial reactivation. RESULTS: Of 9,929 participants, 6310 (63.6%) were HSV-1 IgG positive, and 870 (of 4,934 seropositive participants with reactivation data) experienced frequent oro-labial reactivation. Being born outside the UK/Ireland, contemporaneous urban living and having ≥4 siblings were risk factors for HSV-1 seropositivity. Ever diagnosed with kidney disease, but no other comorbidities, was associated with an increased risk of frequent HSV reactivation (adjOR 1.87, 95%CI: 1.02-3.40). DISCUSSION: Apparent HSV-1 seropositivity and clinical reactivation are common within an ageing UK population. HSV-1 seropositivity is socially patterned while risk factors for oro-labial HSV reactivation are less clear. Further large studies of risk factors are needed to inform HSV-1 control strategies.


Assuntos
Herpes Simples/epidemiologia , Herpesvirus Humano 1/fisiologia , Imunoglobulina G/metabolismo , Ativação Viral , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/metabolismo , Estudos Transversais , Feminino , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Boca/virologia , Estudos Prospectivos , Reino Unido/epidemiologia
18.
Elife ; 82019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31090537

RESUMO

Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of ß-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.


Assuntos
Antivirais/metabolismo , Herpesvirus Humano 1/fisiologia , Análise de Célula Única , Animais , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Humanos , Mutação/genética , Transdução de Sinais , Transcrição Genética , Replicação Viral , beta Catenina/metabolismo
19.
Exp Eye Res ; 185: 107664, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31085182

RESUMO

HSV-1 infection in corneal epithelium initiates the process of herpes simplex keratitis. We investigated the dynamic change of the host proteins in corneal epithelial cells infected with HSV-1 to understand the virus-host interaction. iTRAQ coupled with LC-MS/MS was applied to quantitatively analyze the protein profiles in HSV-1 infected corneal epithelial cells at 6 and 24 h post-infection (hpi), and the results were validated by multiple reaction monitoring (MRM). We also performed bioinformatic analysis to investigate the potentially important signal pathways and protein interaction networks in the host response to HSV-1 infection. We identified 292 proteins were up-regulated and 168 proteins were down-regulated at 6 hpi, while 132 proteins were up-regulated and 89 proteins were down-regulated at 24 hpi, which were validated by MRM analysis. We found the most enriched GO terms were translational initiation, cytosol, poly(A) RNA binding, mRNA splicing via spliceosome and extracellular exosome for the dysregulated proteins. KEGG pathway analysis revealed significant changes in metabolism pathway characterized by decreased tricarboxylic acid cycle activity and increased glycolysis. Proteins interaction network analysis indicated several proteins including P4HB, ACLY, HSP90AA1 and EIF4A3, might be critical proteins in the host-virus response. Our study for the first time analyzed the protein profile of HSV-1 infected primary corneal epithelial cells by quantitative proteomics. These findings help to better understand the host-virus interaction and the pathogenesis of herpes simplex keratitis.


Assuntos
Epitélio Anterior/virologia , Herpesvirus Humano 1/fisiologia , Western Blotting , Linhagem Celular , Cromatografia Líquida , Biologia Computacional , Regulação para Baixo , Epitélio Anterior/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Proteômica , RNA Mensageiro/metabolismo , Transdução de Sinais , Espectrometria de Massas em Tandem , Regulação para Cima
20.
Virol Sin ; 34(4): 386-396, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31020575

RESUMO

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/genética , MicroRNAs/genética , Proteínas Virais/genética , Replicação Viral , Animais , Linhagem Celular , Herpesvirus Humano 1/fisiologia , Mutação com Perda de Função , Camundongos , RNA Viral/genética , Latência Viral
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