RESUMEN
The innate immune system relies on a variety of pathogen recognition receptors (PRRs) as the first line of defense against pathogenic invasions. Viruses have evolved multiple strategies to evade the host immune system through coevolution with hosts. The CRISPR-Cas system is an adaptive immune system in bacteria or archaea that defends against viral reinvasion by targeting nucleic acids for cleavage. Based on the characteristics of Cas proteins and their variants, the CRISPR-Cas system has been developed into a versatile gene-editing tool capable of gene knockout or knock-in operations to achieve genetic variations in organisms. It is now widely used in the study of viral immune evasion mechanisms. This chapter will introduce the use of the CRISPR-Cas9 system for editing herpes simplex virus 1 (HSV-1) genes to explore the mechanisms by which HSV-1 evades host innate immunity and the experimental procedures involved.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Herpesvirus Humano 1 , Evasión Inmune , Inmunidad Innata , Sistemas CRISPR-Cas/genética , Inmunidad Innata/genética , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/genética , Evasión Inmune/genética , Humanos , Edición Génica/métodos , Animales , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genética , Herpes Simple/inmunología , Herpes Simple/virología , Herpes Simple/genéticaRESUMEN
Eczema herpeticum (EH) is a disseminated severe herpes simplex virus type 1 (HSV-1) infection that mainly occurs in a subset of patients suffering from atopic dermatitis (AD). EH is complex and multifaceted, involving immunological changes, environmental influences, and genetic aberrations. Certain genetic variants of the thymic stromal lymphopoietin (TSLP) may predispose to develop severe HSV-1-induced eczema. Therefore, we investigated the impact of TSLP on HSV-1 infection. TSLP encodes for two distinct forms: a long-form (lfTSLP), primarily associated with type 2 immunity, and a short-form (sfTSLP) with anti-inflammatory and antimicrobial properties. While sfTSLP reduced HSV-1 infectibility in human primary keratinocytes (HPK), lfTSLP did not. In HPK treated with sfTSLP, HSV-1 gene expression, and replication decreased, while virion binding to cells and targeting of incoming capsids to the nucleus were not diminished compared to untreated cells. sfTSLP caused only minor changes in the expression of innate immunity cytokines, and its inhibition of HSV-1 infection did not require de novo protein synthesis. Time window experiments indicated a different antiviral mechanism than LL-37. sfTSLP showed the strongest antiviral effect when administered to HPK before or after inoculation with HSV-1, and outperformed the inhibitory potential of LL-37 under these conditions. Our data show that sfTSLP has antiviral functions and promotes repression of the HSV-1 infection in HPK.
Asunto(s)
Citocinas , Herpesvirus Humano 1 , Queratinocitos , Linfopoyetina del Estroma Tímico , Humanos , Citocinas/metabolismo , Queratinocitos/virología , Queratinocitos/inmunología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/genética , Células Cultivadas , Replicación Viral , Erupción Variceliforme de Kaposi/virología , Erupción Variceliforme de Kaposi/inmunología , Herpes Simple/virología , Herpes Simple/inmunología , Herpes Simple/genética , Inmunidad InnataRESUMEN
OBJECTIVE: To verify the use and identify advantages of molecular methods for congenital infections diagnosis in cerebrospinal fluid of neonates. DATA SOURCE: The review was registered in the International Prospective Register of Systematic Reviews (PROSPERO), under CRD42021274210. The literature search was performed in databases: PubMed, Virtual Health Library/ Latin American and Caribbean Center on Health Sciences Information (VHL/BIREME), Scopus, Web of Science, Excerpta Medica database (EMBASE), Cochrane, ProQuest, and EBSCOhost. The search was carried out from August to October 2021 and updated in December 2022, respecting the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The selection sequence was: 1) Duplicate title removal; 2) Examination of titles and abstracts; 3) Full-text retrieval of potentially relevant reports; and 4) Evaluation of the full text according to eligibility criteria by two independent authors. Inclusion criteria considered randomized and non-randomized control trials, longitudinal, cross-sectional, and peer-reviewed studies in humans, published in English, Spanish, Italian, and Portuguese, with newborns up to 28 days old who had congenital neuroinfections by toxoplasmosis, rubella, cytomegalovirus, herpes simplex (TORCH), and others such as Treponema pallidum, Zika, parvovirus B-19, varicella zoster, Epstein-Barr, and SARS-CoV2, diagnosed by polymerase chain reaction (PCR). Two evaluators extracted the following information: author, year of publication, nationality, subjects, study type, methods, results, and conclusion. DATA SYNTHESIS: The most studied pathogen was herpes simplex. Several articles reported only nonspecific initial symptoms, motivating the collection of cerebrospinal fluid and performing PCR for etiological investigation. CONCLUSIONS: Molecular methods are effective to detect pathogen genomes in cerebrospinal fluid, which can impact clinical evolution and neurological prognosis.
Asunto(s)
COVID-19 , Humanos , Recién Nacido , COVID-19/diagnóstico , COVID-19/líquido cefalorraquídeo , SARS-CoV-2/genética , Herpes Simple/diagnóstico , Herpes Simple/líquido cefalorraquídeo , Herpes Simple/congénito , Toxoplasmosis Congénita/diagnóstico , Toxoplasmosis Congénita/líquido cefalorraquídeoRESUMEN
BACKGROUND: Herpes simplex virus type 1 (HSV-1) is a major cause of viral encephalitis, genital mucosal infections, and neonatal infections. Lactococcus lactis (L. lactis) has been proven to be an effective vehicle for delivering protein antigens and stimulating both mucosal and systemic immune responses. In this study, we constructed a recombinant L. lactis system expressing the protective antigen glycoprotein D (gD) of HSV-1. RESULTS: To improve the stability and persistence of antigen stimulation of the local mucosa, we inserted the immunologic adjuvant interleukin (IL)-2 and the Fc fragment of IgG into the expression system, and a recombinant L. lactis named NZ3900-gD-IL-2-Fc was constructed. By utilizing this recombinant L. lactis strain to elicit an immune response and evaluate the protective effect in mice, the recombinant L. lactis vaccine induced a significant increase in specific neutralizing antibodies, IgG, IgA, interferon-γ, and IL-4 levels in the serum of mice. Furthermore, in comparison to the mice in the control group, the vaccine also enhanced the proliferation levels of lymphocytes in response to gD. Moreover, recombinant L. lactis expressing gD significantly boosted nonspecific immune reactions in mice through the activation of immune-related genes. Furthermore, following the HSV-1 challenge of the murine lung mucosa, mice inoculated with the experimental vaccine exhibited less lung damage than control mice. CONCLUSION: Our study presents a novel method for constructing a recombinant vaccine using the food-grade, non-pathogenic, and non-commercial bacterium L. lactis. The findings indicate that this recombinant vaccine shows promise in preventing HSV-1 infection in mice.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1 , Lactococcus lactis , Ratones Endogámicos BALB C , Lactococcus lactis/genética , Animales , Ratones , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/genética , Herpes Simple/prevención & control , Herpes Simple/inmunología , Femenino , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas Sintéticas/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunologíaRESUMEN
BACKGROUND: To investigate the mechanism of Golgi matrix protein 130(GM130) regulating the antiviral immune response of TLR3 after herpes simplex virus type 1(HSV-1) infection of microglia cells. We explored the regulatory effects of berberine on the immune response mediated by GM130 and TLR3. METHODS: An in vitro model of HSV-1 infection was established by infecting BV2 cells with HSV-1. RESULTS: Compared to the uninfected group, the Golgi apparatus (GA) fragmentation and GM130 decreased after HSV-1 infection; TLR3 increased at 6 h and began to decrease at 12 h after HSV-1 infection; the secretion of interferon-beta(IFN-ß), tumour necrosis factor alpha(TNF-α), and interleukin-6(IL-6) increased after infection. Knockdown of GM130 aggravated fragmentation of the GA and caused TLR3 to further decrease, and the virus titer also increased significantly. GM130 knockdown inhibits the increase in TLR3 and inflammatory factors induced by TLR3 agonists and increases the viral titer. Overexpression of GM130 alleviated fragmentation of the GA induced by HSV-1, partially restored the levels of TLR3, and reduced viral titers. GM130 overexpression reversed the reduction in TLR3 and inflammatory cytokine levels induced by TLR3 inhibitors. Therefore, the decrease in GM130 levels caused by HSV-1 infection leads to increased viral replication by inhibiting TLR3-mediated innate immunity. Berberine can protect the GA and reverse the downregulation of GM130, as well as the downregulation of TLR3 and its downstream factors after HSV-1 infection, reducing the virus titer. CONCLUSIONS: In microglia, one mechanism of HSV-1 immune escape is disruption of the GM130/TLR3 pathway. Berberine protects the GA and enhances TLR3-mediated antiviral immune responses.
Asunto(s)
Regulación hacia Abajo , Herpesvirus Humano 1 , Inmunidad Innata , Microglía , Receptor Toll-Like 3 , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 3/genética , Microglía/virología , Microglía/inmunología , Microglía/efectos de los fármacos , Animales , Ratones , Línea Celular , Evasión Inmune , Berberina/farmacología , Citocinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Herpes Simple/inmunología , Herpes Simple/virologíaRESUMEN
Protein expression is regulated through multiple mechanisms, including post-translational modifications (PTMs), which can alter protein structure, stability, localization, and function. Among these, citrullination stands out due to its ability to convert arginine residues into citrulline, altering protein charge and mass. This modification is catalyzed by calcium-dependent protein arginine deiminases (PADs), enzymes implicated in various inflammatory diseases. We have recently shown that human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) exploit these enzymes to enhance their replication capabilities. Although the role of PADs in HCMV and HSV-1 infections is well documented, their involvement in HSV-2 infection has not yet been thoroughly investigated. Here, we demonstrate that HSV-2 manipulates the overall protein citrullination profile by activating three PAD isoforms: PAD2, PAD3, and PAD4. However, as previously observed during HSV-1 infection, PAD3 is the most significantly upregulated isoform, both at the mRNA and protein levels. Consistently, we demonstrate that inhibiting PAD3, either through the specific inhibitor CAY10727 or via CRISPR/Cas9-mediated gene silencing, markedly reduces HSV-2 replication and viral protein expression. Lastly, we show that CAY10727 displays an IC50 value of 0.3 µM, which is extremely close to what was previously observed for HSV-1. Overall, our findings highlight the crucial role of PAD3 in the life cycle of HSV-2 and suggest that the targeted inhibition of PAD3 may represent a promising approach for treating HSV-2 infections, especially in cases resistant to existing antiviral therapies.
Asunto(s)
Herpesvirus Humano 2 , Arginina Deiminasa Proteína-Tipo 3 , Humanos , Herpesvirus Humano 2/fisiología , Herpesvirus Humano 2/genética , Arginina Deiminasa Proteína-Tipo 3/metabolismo , Citrulinación , Herpes Simple/virología , Herpes Simple/metabolismo , Replicación Viral/efectos de los fármacos , Animales , Herpes Genital/metabolismo , Herpes Genital/virología , Herpes Genital/tratamiento farmacológico , Arginina Deiminasa Proteína-Tipo 2/metabolismo , Antivirales/farmacologíaRESUMEN
Static well plates remain the gold standard to study viral infections in vitro, but they cannot accurately mimic dynamic viral infections as they occur in the human body. Therefore, we established a dynamic cell culture platform, based on centrifugal microfluidics, to study viral infections in perfusion. To do so, we used human primary periodontal dental ligament (PDL) cells and herpes simplex virus-1 (HSV-1) as a case study. By microscopy, we confirmed that the PDL cells efficiently attached and grew in the chip. Successful dynamic viral infection of perfused PDL cells was monitored using fluorescent imaging and RT-qPCR-based experiments. Remarkably, viral infection in flow resulted in a gradient of HSV-1-infected cells gradually decreasing from the cell culture chamber entrance towards its end. The perfusion of acyclovir in the chip prevented HSV-1 spreading, demonstrating the usefulness of such a platform for monitoring the effects of antiviral drugs. In addition, the innate antiviral response of PDL cells, measured by interferon gene expression, increased significantly over time in conventional static conditions compared to the perfusion model. These results provide evidence suggesting that dynamic viral infections differ from conventional static infections, which highlights the need for more physiologically relevant in vitro models to study viral infections.
Asunto(s)
Herpesvirus Humano 1 , Ligamento Periodontal , Humanos , Ligamento Periodontal/citología , Ligamento Periodontal/virología , Técnicas de Cultivo de Célula , Microfluídica , Herpes Simple/virología , Células Cultivadas , Antivirales/farmacologíaRESUMEN
The periodontal ligament (PDL) is a complex connective tissue that connects the tooth root to the dental alveolar bone and plays crucial mechanical roles. PDL also exhibits regenerative roles and regulatory functions to maintain periodontium integrity and homeostasis. While PDL exposure to oral microbial pathogens is common, virtually nothing is known regarding viral infections of PDL. In particular, human herpes simplex virus type 1 (HSV-1) persistently infects the oral cavity through infections of the oral epithelium, connective tissue and neurons. While the oral spread of HSV-1 is generally asymptomatic, this virus has also been implicated in various oral pathologies. In this study, using a primary cell model derived from PDL (PDL cells), and whole surgical fragments of PDL, we provide evidence supporting the efficient infection of PDL by HSV-1 and the promotion of cytopathic effects. Infection of PDL by HSV-1 was also associated with an acute innate inflammatory response, as illustrated by the production of antiviral interferons and pro-inflammatory cytokines. Furthermore, this inflammatory response to HSV-1 was exacerbated in the presence of bacterial-derived products, such as peptidoglycans. This work therefore highlights the ability of HSV-1 to infect mesenchymal cells from PDL, suggesting that PDL may serve as a viral reservoir for the periodontal spread of HSV-1. Moreover, this raises questions about HSV-1 oral pathogenesis, as HSV-1-associated cytopathic and inflammatory effects may contribute to profound alterations of PDL integrity and functioning.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1 , Ligamento Periodontal , Humanos , Ligamento Periodontal/virología , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/patogenicidad , Herpes Simple/virología , Citocinas/metabolismo , Células CultivadasRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl- channel, is closely associated with multiple pathogen infections, such as SARS-CoV-2. However, whether the function of the CFTR is involved in herpes simplex virus (HSV) infection has not been reported. To evaluate the association of CFTR activity with HSV infection, the antiviral effect of CFTR inhibitors in epithelial cells and HSV-infected mice was tested in this study. The data showed that treatment with CFTR inhibitors in different concentrations, Glyh-101 (5-20 µM), CFTRi-172 (5-20 µM) and IOWH-032 (5-20 µM), or the gene silence of the CFTR could suppress herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) replication in human HaCaT keratinocytes cells, and that a CFTR inhibitor, Glyh-101 (10-20 µM), protected mice from HSV-1 and HSV-2 infection. Intracellular Cl- concentration ([Cl-]i) was decreased after HSV infection via the activation of adenylyl cyclase (AC)-cAMP signaling pathways. CFTR inhibitors (20 µM) increased the reduced [Cl-]i caused by HSV infection in host epithelial cells. Additionally, CFTR inhibitors reduced the activity and phosphorylation of SGK1 in infected cells and tissues (from the eye and vagina). Our study found that CFTR inhibitors can effectively suppress HSV-1 and HSV-2 infection, revealing a previously unknown role of CFTR inhibitors in HSV infection and suggesting new perspectives on the mechanisms governing HSV infection in host epithelial cells, as well as leading to potential novel treatments.
Asunto(s)
Antivirales , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Herpes Simple , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Replicación Viral , Animales , Ratones , Antivirales/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Humanos , Herpes Simple/tratamiento farmacológico , Herpes Simple/virología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Replicación Viral/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Herpesvirus Humano 2/fisiología , Femenino , Línea Celular , Células Epiteliales/virología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células HaCaT , Queratinocitos/virología , Queratinocitos/efectos de los fármacos , Ratones Endogámicos BALB C , Chlorocebus aethiops , Simplexvirus/efectos de los fármacos , Simplexvirus/fisiologíaRESUMEN
BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening mucocutaneous autoimmune disease that affects desmoglein-1 and desmoglein-3, leading to intraepithelial vesiculobullous lesions. In the oral mucosa, PV lesions can mimic other diseases such as mucous membrane pemphigoid, other forms of pemphigus, recurrent aphthous stomatitis, erythema multiforme, Stevens-Johnson syndrome, and virus-induced ulcers like herpes simplex virus (HSV), making diagnosis challenging. The co-occurrence of PV with Crohn's disease is rare and predominantly seen in younger patients. The therapeutic mainstay for both PV and Crohn's disease usually involves systemic corticosteroids combined with immunosuppressants and immunobiological drugs. Literature indicates that the use of these drugs, particularly TNF-alpha inhibitors, for managing autoimmune diseases like Crohn's can potentially induce other autoimmune diseases known as autoimmune-like syndromes, which include episodes of lupus-like syndrome and inflammatory neuropathies. There are few cases in the literature reporting the development of PV in individuals with CD undergoing infliximab therapy. CASE REPORT: A young female with severe Crohn's disease, treated with the TNF-alpha inhibitor infliximab, developed friable pseudomembranous oral ulcerations. Histopathological and immunofluorescence analyses confirmed these as PV. The treatment included clobetasol propionate and low-level photobiomodulation, which resulted in partial improvement. The patient later experienced severe intestinal bleeding, requiring intravenous hydrocortisone therapy, which improved both her systemic condition and oral lesions. Weeks later, new ulcerations caused by herpes virus and candidiasis were identified, leading to treatment with oral acyclovir, a 21-day regimen of oral nystatin rinse, and photodynamic therapy, ultimately healing the oral infections. To manage her condition, the gastroenterologists included methotrexate (25 mg) in her regimen to reduce the immunogenicity of infliximab and minimize corticosteroid use, as the patient was in remission for Crohn's disease, and the oral PV lesions were under control. CONCLUSION: Young patients with Crohn's disease should be referred to an oral medicine specialist for comorbidity investigation, as oral PV and opportunistic infections can arise during immunosuppressive therapy. The use of TNF-alpha inhibitors in patients treated for inflammatory bowel disease, such as Crohn's, should be carefully evaluated for potential side effects, including oral PV.
Asunto(s)
Enfermedad de Crohn , Herpes Simple , Factores Inmunológicos , Infliximab , Pénfigo , Humanos , Pénfigo/tratamiento farmacológico , Pénfigo/complicaciones , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/tratamiento farmacológico , Femenino , Herpes Simple/complicaciones , Herpes Simple/tratamiento farmacológico , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/uso terapéutico , Infliximab/uso terapéutico , Infliximab/efectos adversos , Adulto , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Enfermedades de la Boca/tratamiento farmacológico , Enfermedades de la Boca/complicacionesRESUMEN
Bloodstream HSV-1 and HSV-2 infections can cause devastating outcomes with high morbidity and mortality, especially in neonates or immunocompromised individuals. Proper patient management for herpes simplex virus (HSV) bloodstream infections is time-sensitive and requires a rapid, accurate, and definitive diagnosis. The absence of the U.S. Food and Drug Administration (FDA)-approved molecular assays for HSV detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the unmet need for improved diagnostics. We prospectively compared the cycle threshold values in paired samples including whole blood (WB), plasma, serum, and peripheral blood mononuclear cells (PBMCs) from patients with bloodstream HSV infections. This analysis employed a modified use of the FDA-cleared Simplexa HSV-1 & 2 Direct assay. The clinical performance in serum was assessed by comparing the results of 247 remnant specimens on this sample-to-answer platform to established laboratory-developed tests in a blinded fashion. Serum samples exhibited significantly lower cycle thresholds than whole blood samples [2.6 cycle threshold (Ct) bias, P < 0.001]. The modified Simplexa assay demonstrated 100% positive percent agreement for the detection of HSV-1 and HSV-2 DNA in serum samples and yielded an overall agreement of 95% (95% CI, 0.92 to 0.97), with a κ statistic of 0.75 (95% CI, 0.62 to 0.86) compared to the composite reference method. Discordance rates were 5.20% for HSV-1 and 0.81% for HSV-2. This investigation demonstrates that serum is an optimal specimen type for HSV detection when compared to several blood compartments. Serum offers a promising sample type for rapid and accurate diagnosis of HSV bloodstream infections using the modified Simplexa assay. IMPORTANCE: Rapid, accurate, and definitive diagnosis of herpes simplex virus (HSV) infections is crucial in clinical settings for patient management. The absence of FDA-authorized molecular assays for HSV-1/2 detection in blood, coupled with a lack of consensus on the optimal sample type, underscores the need for improved diagnostic methods. Furthermore, rapid diagnosis of HSV bloodstream infections enables timely administration of antiviral treatment, influences patient management decisions for those at high risk, and can contribute to shorter hospital stays, thereby reducing healthcare costs.
Asunto(s)
Herpes Simple , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Técnicas de Diagnóstico Molecular , Sensibilidad y Especificidad , Humanos , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 1/genética , Herpes Simple/diagnóstico , Herpes Simple/virología , Herpesvirus Humano 2/aislamiento & purificación , Herpesvirus Humano 2/genética , Masculino , Femenino , Estudios Prospectivos , Persona de Mediana Edad , Adulto , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Adulto Joven , Anciano , Adolescente , Niño , Factores de Tiempo , Preescolar , Lactante , Anciano de 80 o más AñosRESUMEN
Recently it was discovered that extracellular 2'-3'cGAMP can activate the STING pathway in a cGAS-independent fashion by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that this non-canonical activation of STING pathway would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state that limits herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced inhibition of viral replication. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP. This work presents novel and important findings about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.
Asunto(s)
Herpesvirus Humano 1 , Proteína Portadora de Folato Reducido , Replicación Viral , Humanos , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteína Portadora de Folato Reducido/metabolismo , Proteína Portadora de Folato Reducido/genética , Antivirales/farmacología , Antivirales/metabolismo , Línea Celular , Herpes Simple/virología , Herpes Simple/metabolismo , Células THP-1 , Transporte Biológico , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interferones/metabolismo , Transducción de SeñalRESUMEN
Herpes simplex virus type 1 (HSV-1) is responsible for a wide range of human infections, including skin and mucosal ulcers, encephalitis, and keratitis. The gold standard for treating HSV-1 infections is acyclovir. However, the use of this drug is associated with several limitations such as toxic reactions and the development of drug-resistant strains. So, there is an urgent need to discover and develop novel and effective agents against this virus. For the first time, this study aimed to investigate the antiviral effects of the Thermally Expanded Graphite (TEG)-copper oxide (CuO) nanocomposite against HSV-1 and compare results with its constituent components. After microwave (MW)-assisted synthesis of TEG and CuO nanosheets as well as MW-CuO/TEG nanocomposite and characterization of all these nanomaterials, an MTT assay was used to determine their cytotoxicity. The quantitative real-time PCR was then used to investigate the effects of these nanomaterials on viral load. Three-hour incubation of HSV-1 with TEG nanosheets (500 µg/mL), MW-CuO nanosheets (15 µg/mL), and MW-CuO/TEG nanocomposite (35 µg/mL) resulted in a decrease in viral load with an inhibition rate of 31.4 %, 49.2 %, and 74.4 %, respectively. The results from the post-treatment assay also showed that TEG nanosheets (600 µg/mL), MW-CuO nanosheets (15 µg/mL), and MW-CuO/TEG nanocomposite (10 µg/mL) led to a remarkable decrease in viral load with an inhibition rate of 56.9 %, 63 %, and 99.9 %, respectively. The combination of TEG and MW-CuO nanosheets together and the formation of a nanocomposite structure display strong synergy in their ability to inhibit HSV-1 infection. MW-CuO/TEG nanocomposites can be considered a suitable candidate for the treatment of HSV-1 infection.
Asunto(s)
Antivirales , Cobre , Grafito , Herpesvirus Humano 1 , Nanocompuestos , Cobre/farmacología , Cobre/química , Herpesvirus Humano 1/efectos de los fármacos , Grafito/química , Grafito/farmacología , Antivirales/farmacología , Antivirales/química , Nanocompuestos/química , Células Vero , Chlorocebus aethiops , Animales , Carga Viral/efectos de los fármacos , Microondas , Sinergismo Farmacológico , Supervivencia Celular/efectos de los fármacos , Humanos , Herpes Simple/tratamiento farmacológico , Herpes Simple/virologíaRESUMEN
The multifunctional tegument protein pUL21 of HSV-2 is phosphorylated in infected cells. We have identified two residues in the unstructured linker region of pUL21, serine 251 and serine 253, as phosphorylation sites. Both phosphorylation sites are absent in HSV-1 pUL21, which likely explains why phosphorylated pUL21 was not detected in cells infected with HSV-1. Cells infected with HSV-2 strain 186 viruses deficient in pUL21 phosphorylation exhibited reductions in both cell-cell spread of virus infection and virus replication. Defects in secondary envelopment of cytoplasmic nucleocapsids were also observed in cells infected with viruses deficient in pUL21 phosphorylation as well as in cells infected with multiple strains of HSV-2 and HSV-1 deleted for pUL21. These results confirm a role for HSV pUL21 in the secondary envelopment of cytoplasmic nucleocapsids and indicate that phosphorylation of HSV-2 pUL21 is required for this activity. Phosphorylation of pUL21 was substantially reduced in cells infected with HSV-2 strain 186 mutants lacking the viral serine/threonine kinase pUL13, indicating a requirement for pUL13 in pUL21 phosphorylation. IMPORTANCE: It is well known that post-translational modification of proteins by phosphorylation can regulate protein function. Here, we determined that phosphorylation of the multifunctional HSV-2 tegument protein pUL21 requires the viral serine/threonine kinase pUL13. In addition, we identified serine residues within HSV-2 pUL21 that can be phosphorylated. Phenotypic analysis of mutant HSV-2 strains with deficiencies in pUL21 phosphorylation revealed reductions in both cell-cell spread of virus infection and virus replication. Deficiencies in pUL21 phosphorylation also compromised the secondary envelopment of cytoplasmic nucleocapsids, a critical final step in the maturation of all herpes virions. Unlike HSV-2 pUL21, phosphorylation of HSV-1 pUL21 was not detected. This fundamental difference between HSV-2 and HSV-1 may underlie our previous observations that the requirements for pUL21 differ between HSV species.
Asunto(s)
Herpesvirus Humano 2 , Nucleocápside , Replicación Viral , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiología , Fosforilación , Animales , Chlorocebus aethiops , Humanos , Células Vero , Nucleocápside/metabolismo , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Citoplasma/metabolismo , Citoplasma/virología , Línea Celular , Proteínas Estructurales Virales/metabolismo , Proteínas Estructurales Virales/genética , Ensamble de Virus , Herpes Simple/virología , Herpes Simple/metabolismoRESUMEN
Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell's post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.
Asunto(s)
Exocitosis , Aparato de Golgi , Herpesvirus Humano 1 , Vías Secretoras , Liberación del Virus , Proteínas de Unión al GTP rab , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Humanos , Animales , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células Vero , Red trans-Golgi/metabolismo , Red trans-Golgi/virología , Chlorocebus aethiops , Herpes Simple/virología , Herpes Simple/metabolismo , Virión/metabolismo , Células HeLa , Membrana Celular/metabolismo , Membrana Celular/virologíaAsunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Faringitis , Humanos , Masculino , Adulto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/aislamiento & purificación , Herpes Simple/diagnóstico , Faringe/virología , Laringe/virologíaRESUMEN
Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.