RESUMO
Herpes simplex virus 1 and 2 infections cause high unmet disease burdens worldwide. Mainly HSV-2 causes persistent sexually transmitted disease, fatal neonatal disease and increased transmission of HIV/AIDS. Thus, there is an urgent requirement to develop effective vaccines. We developed nucleic acid vaccines encoding a novel virus entry complex stabilising cell membrane fusion, 'virus-like membranes', VLM. Two dose intramuscular immunisations using DNA expression plasmids in a guinea pig model gave 100% protection against acute disease and significantly reduced virus replication after virus intravaginal challenge. There was also reduced establishment of latency within the dorsal root ganglia and spinal cord, but recurrent disease and recurrent virus shedding remained. To increase cellular immunity and protect against recurrent disease, cDNA encoding an inhibitor of chemokine receptors on T regulatory cells was added and compared to chemokine CCL5 effects. Immunisation including this novel human chemokine gene, newly defined splice variant from an endogenous virus genome, 'virokine immune therapeutic', VIT, protected most guinea pigs from recurrent disease and reduced recurrent virus shedding distinct from a gD protein vaccine similar to that previously evaluated in clinical trials. All DNA vaccines induced significant neutralising antibodies and warrant evaluation for new therapeutic treatments.
Assuntos
Herpes Genital , Vacinas de DNA , Recém-Nascido , Cobaias , Humanos , Animais , Herpesvirus Humano 2/genética , Vacinas de DNA/genética , Internalização do Vírus , Proteínas do Envelope Viral/genética , Imunização , Quimiocinas , Modelos Animais de Doenças , Anticorpos AntiviraisRESUMO
Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.
Assuntos
Genoma Viral , Herpesviridae , Animais , Evolução Molecular , Herpesviridae/classificação , Herpesviridae/genética , Herpesviridae/fisiologia , Herpesviridae/ultraestrutura , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Adaptação ao Hospedeiro , Vírion/química , Vírion/ultraestrutura , Latência Viral , Replicação ViralRESUMO
BACKGROUND: In developed countries, human cytomegalovirus (HCMV) is a major pathogen in congenitally infected and immunocompromised individuals, where multiple-strain infection appears linked to disease severity. The situation is less documented in developing countries. In Zambia, breast milk is a key route for transmitting HCMV and carries higher viral loads in human immunodeficiency virus (HIV)-infected women. We investigated HCMV strain diversity. METHODS: High-throughput sequence datasets were generated from 28 HCMV-positive breast milk samples donated by 22 mothers (15 HIV-infected and 7 HIV-negative) at 4-16 weeks postpartum, then analyzed by genome assembly and novel motif-based genotyping in 12 hypervariable HCMV genes. RESULTS: Among the 20 samples from 14 donors (13 HIV-infected and one HIV-negative) who yielded data meeting quality thresholds, 89 of the possible 109 genotypes were detected, and multiple-strain infections involving up to 5 strains per person were apparent in 9 HIV-infected women. Strain diversity was extensive among individuals but conserved compartmentally and longitudinally within them. Genotypic linkage was maintained within hypervariable UL73/UL74 and RL12/RL13/UL1 loci for virus entry and immunomodulation, but not between genes more distant from each other. CONCLUSIONS: Breast milk from HIV-infected women contains multiple HCMV strains of high genotypic complexity and thus constitutes a major source for transmitting viral diversity.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Variação Genética , Infecções por HIV/complicações , Leite Humano/virologia , Biologia Computacional , DNA Viral/genética , Feminino , Genes Virais , Genoma Viral , Genótipo , Ensaios de Triagem em Larga Escala , Humanos , Carga Viral , ZâmbiaRESUMO
The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.
Assuntos
Sequência de Bases , Infecções por Citomegalovirus/virologia , Citomegalovirus/genética , Genoma Viral , Recombinação Genética , DNA Viral/genética , Bases de Dados de Ácidos Nucleicos , Conjuntos de Dados como Assunto , Evolução Molecular , Genes Virais , Variação Genética , Genoma Viral/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Análise de Sequência de DNA , Sequenciamento Completo do GenomaRESUMO
New antivirals are required to prevent rising antimicrobial resistance from replication inhibitors. The aim of this study was to analyse the range of emerging mutations in herpesvirus by whole genome deep sequencing. We tested human herpesvirus 6 treatment with novel antiviral K21, where evidence indicated distinct effects on virus envelope proteins. We treated BACmid cloned virus in order to analyse mechanisms and candidate targets for resistance. Illumina based next generation sequencing technology enabled analyses of mutations in 85 genes to depths of 10,000 per base detecting low prevalent minority variants (<1%). After four passages in tissue culture the untreated virus accumulated mutations in infected cells giving an emerging mixed population (45-73%) of non-synonymous SNPs in six genes including two envelope glycoproteins. Strikingly, treatment with K21 did not accumulate the passage mutations; instead a high frequency mutation was selected in envelope protein gQ2, part of the gH/gL complex essential for herpesvirus infection. This introduced a stop codon encoding a truncation mutation previously observed in increased virion production. There was reduced detection of the glycoprotein complex in infected cells. This supports a novel pathway for K21 targeting virion envelopes distinct from replication inhibition.
Assuntos
Antivirais/farmacologia , Herpesviridae/genética , Compostos de Amônio Quaternário/farmacologia , Silanos/farmacologia , Proteínas do Envelope Viral/genética , Linhagem Celular , Farmacorresistência Viral/genética , Variação Genética/efeitos dos fármacos , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Proteínas do Envelope Viral/metabolismo , Sequenciamento Completo do GenomaRESUMO
Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis.
Assuntos
Adaptação Fisiológica/genética , Herpesvirus Humano 6/genética , Sequenciamento de Nucleotídeos em Larga Escala , Infecções por Roseolovirus/virologia , Proteínas do Envelope Viral/genética , Internalização do Vírus , Sequenciamento Completo do Genoma , Linhagem Celular , Genoma Viral/genética , Herpesvirus Humano 6/fisiologia , Humanos , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Infecções por Roseolovirus/genética , Seleção Genética , Deleção de Sequência , Proteínas do Envelope Viral/químicaRESUMO
BACKGROUND: Breastfeeding imparts beneficial immune protection and nutrition to infants for healthy growth, but it is also a route for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV) infection. In previous studies, we showed that HCMV adversely affects infant development in Africa, particularly with maternal HIV exposure. In this study, we analyzed infants risks for acquisition of HCMV infection from breastfeeding and compared HIV-positive and HIV-negative mothers. METHODS: Two cohorts were studied in Zambia. (1) Two hundred sixty-one HIV-infected and HIV-uninfected mothers were compared for HCMV deoxyribonucleic acid (DNA) loads and genotypes (glycoprotein gO) in milk from birth to 4 months postpartum. (2) Maternally HIV-exposed and HIV-unexposed infants were compared for HCMV infection risk factors. The second cohort of 460 infants, from a trial of micronutrient-fortified complementary-food to breastfeeding, were studied between 6 and 18 months of age. Human cytomegalovirus seroprevalence was assayed, and logistic regression was used to calculate risk factors for HCMV infection, including maternal HIV exposure and breastfeeding duration. RESULTS: Human cytomegalovirus was detected in breast milk from 3 days to 4 months postpartum, with significantly raised levels in HIV-positive women and independent of genotype. In infants, HCMV antibody seroprevalence was 83% by 18 months age. Longer breastfeeding duration increased infection risk in maternally HIV-unexposed (odds ratio [OR] = 2.69 for 18 months vs <12 months; 95% confidence interval [CI], 0.84-8.59; P = .03) and HIV-exposed infants (OR = 20.37 for >6 months vs never; 95% CI, 3.71-111.70; P < .001). CONCLUSIONS: Prolonged breastfeeding, which is common in Africa, increased risk of HCMV infection in infants. Both HIV-positive and HIV-negative women had extended milk HCMV secretion. Women who were HIV-positive secreted higher HCMV levels, and for longer duration, with their children at increased infection risk. Human cytomegalovirus control is required to maintain health benefits of breastfeeding.
Assuntos
Aleitamento Materno , Infecções por Citomegalovirus/transmissão , Infecções por HIV/complicações , Transmissão Vertical de Doenças Infecciosas , África Subsaariana , Estudos de Coortes , Citomegalovirus/isolamento & purificação , Feminino , Humanos , Lactente , Leite Humano/virologia , Mães , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Estudos Soroepidemiológicos , Carga ViralRESUMO
Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated "CiHHV-6A/B". These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections.
Assuntos
Cromossomos Humanos/virologia , Insuficiência Cardíaca/virologia , Herpesvirus Humano 6/fisiologia , Infecções por Roseolovirus/virologia , Integração Viral , Sequência de Bases , Cromossomos Humanos/genética , Insuficiência Cardíaca/genética , Herpesvirus Humano 6/genética , Humanos , Dados de Sequência Molecular , Filogenia , Estudos Prospectivos , Infecções por Roseolovirus/genéticaRESUMO
Shortly after the discovery of human herpesvirus 6 (HHV-6), two distinct variants, HHV-6A and HHV-6B, were identified. In 2012, the International Committee on Taxonomy of Viruses (ICTV) classified HHV-6A and HHV-6B as separate viruses. This review outlines several of the documented epidemiological, biological, and immunological distinctions between HHV-6A and HHV-6B, which support the ICTV classification. The utilization of virus-specific clinical and laboratory assays for distinguishing HHV-6A and HHV-6B is now required for further classification. For clarity in biological and clinical distinctions between HHV-6A and HHV-6B, scientists and physicians are herein urged, where possible, to differentiate carefully between HHV-6A and HHV-6B in all future publications.
Assuntos
Variação Genética , Herpesvirus Humano 6/classificação , Herpesvirus Humano 6/genética , Infecções por Roseolovirus/virologia , Humanos , Infecções por Roseolovirus/epidemiologia , Infecções por Roseolovirus/imunologiaRESUMO
BACKGROUND: Maternally HIV-exposed (mHIV-EU) infants have poor health even without HIV-1 infection. The responses to vaccination are less well defined. Immunity to oral Poliovirus vaccine (OPV) was studied in Zambian infants participating in a randomised controlled trial of micronutrient fortification to improve child health. METHOD: Maternally HIV-unexposed and mHIV-EU infants were recruited at 6 months age and randomised to basal or enriched micronutrient-fortified diets for 12 months. HIV-exposed mother-infant pairs had received perinatal nevirapine to prevent mother-to-child-transmission. In the cohort of 597 infants, neutralising-antibody titres to OPV were analysed at 18 months with respect to micronutrient fortification, maternal or infant HIV-1 infection, and human cytomegalovirus (HCMV) infection detected by antibodies and viraemia (serum DNA). Vaccine protection was defined as log2 titre>3. RESULTS: Compared to uninfected children, HIV-1-infected children had reduced neutralising antibody titres to OPV, irrespective of diet: log2 titre difference (95% confidence interval) -3.44 (-2.41; -4.46), P<0.01. OPV antibody titres were lower in HIV-infected children with HCMV viraemia compared to those without viraemia at 18 months, but did not reach significance: difference -2.55 (-6.10; 1.01), P=0.14. Breast-feeding duration was independently associated with increasing OPV titre (P-value<0.01). In mHIV-EU children there were reduced neutralising antibody titres to Poliovirus compared with maternally HIV-unexposed, irrespective of diet, maternal education and socioeconomic status: log2 titre difference (95% confidence interval) -0.56 (-0.98; -0.15), P<0.01. This difference was noticeably decreased after adjusting for breast-feeding duration, suggesting that in our study population less breast-feeding by HIV-positive mothers could explain the reduced OPV titres in mHIV-EU infants. CONCLUSION: The mHIV-EU infants had reduced polio vaccine antibody titres which were associated with reduced breast-feeding duration. This has important implications for polio eradication and control of vaccine-preventable diseases, in countries where childhood HIV-1 infection and maternal exposure are public health threats.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/imunologia , Infecções por HIV/transmissão , Soropositividade para HIV/imunologia , Transmissão Vertical de Doenças Infecciosas , Vacinas contra Poliovirus/imunologia , Poliovirus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Aleitamento Materno , Infecções por Citomegalovirus/diagnóstico , Feminino , Infecções por HIV/imunologia , Soropositividade para HIV/sangue , Humanos , Lactente , Poliomielite/imunologia , Poliomielite/prevenção & controle , VacinaçãoRESUMO
BACKGROUND: Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked. METHODS: Cell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation. RESULTS: U83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4. CONCLUSION: U83A diverts human chemokines from signalling, but not regulatory or scavenger, receptors facilitating their clearance, while occupying signalling receptors at the cell surface. This can enhance virus specific inflammation, facilitating dissemination to replication sensitive leukocytes while evading clearance; this has implications for linked neuro-inflammatory pathologies.
RESUMO
Human herpesvirus 6, HHV-6, commonly infects children, causing febrile illness and can cause more severe pathology, especially in an immune compromised setting. There are virulence distinctions between variants HHV-6A and B, with evidence for increased severity and neurotropism for HHV-6A. While HHV-6B is the predominant infant infection in USA, Europe and Japan, HHV-6A appears rare. Here HHV-6 prevalence, loads and variant genotypes, in asymptomatic compared to symptomatic infants were investigated from an African region with endemic HIV-1/AIDS. DNA was extracted from blood or sera from asymptomatic infants at 6 and 18 months age in a population-based micronutrient study, and from symptomatic infants hospitalised for febrile disease. DNA was screened by qualitative and quantitative real-time PCR, then genotyped by sequencing at variable loci, U46 (gN) and U47 (gO). HIV-1 serostatus of infants and mothers were also determined. HHV-6 DNA prevalence rose from 15% to 22% (80/371) by 18 months. At 6 months, infants born to HIV-1 positive mothers had lower HHV-6 prevalence (11%, 6/53), but higher HCMV prevalence (25%, 17/67). HHV-6 positive febrile hospitalized infants had higher HIV-1, 57% (4/7), compared to asymptomatic infants, 3% (2/74). HHV-6A was detected exclusively in 86% (48/56) of asymptomatic HHV-6 positive samples genotyped. Co-infections with both strain variants were linked with higher viral loads and found in 13% (7/56) asymptomatic infants and 43% (3/7) HIV-1 positive febrile infants. Overall, the results show HHV-6A as the predominant variant significantly associated with viremic infant-infections in this African population, distinct from other global cohorts, suggesting emergent infections elsewhere.
Assuntos
Exantema Súbito/epidemiologia , Exantema Súbito/virologia , Variação Genética , Infecções por HIV , Herpesvirus Humano 6/classificação , Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/fisiopatologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Adulto , África Subsaariana/epidemiologia , DNA Viral/análise , DNA Viral/isolamento & purificação , Doenças Endêmicas , Exantema Súbito/complicações , Exantema Súbito/fisiopatologia , Feminino , Genótipo , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1 , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/isolamento & purificação , Humanos , Lactente , Dados de Sequência Molecular , Prevalência , Análise de Sequência de DNA , Zâmbia/epidemiologiaRESUMO
Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCR1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology.
Assuntos
Quimiocina CCL5/genética , Proteínas de Ligação a DNA/genética , Leucócitos Mononucleares/metabolismo , Receptores CCR7/genética , Receptores de Quimiocinas/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Virais/fisiologia , Fatores de Transcrição/genética , Ligação Competitiva , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL11/farmacologia , Quimiocina CCL19/metabolismo , Quimiocina CCL19/farmacologia , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacologia , Quimiocina CCL5/farmacologia , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiocinas C/metabolismo , Quimiocinas C/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Regulação para Baixo/genética , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Herpesvirus Humano 6/imunologia , Humanos , Fosfatos de Inositol/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Mimetismo Molecular , RNA Interferente Pequeno/genética , Receptores de Quimiocinas/agonistas , Receptores Virais/agonistasRESUMO
HIV-1 strains use C-C-chemokine receptor 5, CCR5, as a coreceptor for host transmission. Human CCR5 chemokine ligands inhibit binding and infection, whereas CCR5 mutations also inhibit infection by preventing surface expression, resulting in delayed progression to AIDS. Here, we describe a human herpesvirus 6 (HHV-6A) chemokine, U83A, which binds CCR5 with higher affinity than human chemokines, displacing their binding and leading to inhibition of chemotaxis of human leukocytes. Similarly, U83A inhibits infection by HIV-1 strains which use CCR5, but not the CXCR4, coreceptor. Unlike human CCR5 chemokine ligands which induce rapid CCR5 internalization mediated via clathrin, treatment with U83A prevents internalization. A spliced truncated U83A isoform, U83A-N, also binds CCR5 albeit with lower affinity, and this correlates with lower HIV-1 infection inhibition, whereas further truncation abolishes binding and any inhibition. Confocal microscopy confirms CCR5 internalization inhibition by U83A treatment, whereas labeled transferrin uptake shows that endocytosis via clathrin is unaltered. Previous results show that, although U83A-N is an antagonist, U83A is an agonist for CCR1, CCR4, CCR6, and CCR8 present on immune effector and antigen-presenting cells and here also shown for CCR5. Thus, U83A could act as a novel inhibitor of HIV-1 infection while also stimulating local immunity to the virus.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Antagonistas dos Receptores CCR5 , Quimiocinas/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , HIV-1/imunologia , Receptores CCR5/agonistas , Proteínas Virais/farmacologia , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Processamento Alternativo/genética , Animais , Células COS , Quimiocinas/genética , Quimiocinas/imunologia , Chlorocebus aethiops , Clatrina/imunologia , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/farmacologia , Receptores CCR5/imunologia , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The human herpes virus 6 (HHV-6)-encoded chemokine receptor U51 constitutively activates phospholipase C (PLC) and inhibits cAMP-responsive element (CRE)-mediated gene transcription via the activation of G(q/11) proteins. Yet, chemokines known to bind U51 differentially regulate U51 coupling to G proteins. CCL5/RANTES induced pertussis toxin (PTX)-insensitive increases in PLC activity and changes in intracellular free calcium concentration ([Ca2+]i), whereas both CCL2/MCP-1 and CCL11/eotaxin failed to stimulate PLC activity or increase [Ca2+]i. In contrast, all three chemokines counteracted the effects of U51 on CRE activity via the activation of PTX-sensitive G(i/o) proteins. For each of the tested chemokines, coexpression of U51 with a variety of G alpha subunits, however, revealed a distinct profile for preferred G-protein coupling, which could be shifted by modulation of the relative expression of G proteins. These findings are consistent with a chemokine-selective trafficking of receptor stimulus to distinct G proteins and suggest that the constitutive activity of U51 and the chemokine-induced signaling involve different active states of the receptor. By virtue of its ability to constitutively activate signaling pathways, its G-protein promiscuity, and the chemokine-directed trafficking of receptor stimulus, U51 can be considered a sensitive and versatile virally encoded signaling device, potentially of importance in HHV-6-related pathologies.
Assuntos
Quimiocinas/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Receptores Virais/agonistas , Receptores Virais/metabolismo , Animais , Células COS , Quimiocina CCL11 , Quimiocina CCL2/farmacologia , Quimiocina CCL5 , Quimiocinas CC/farmacologia , Chlorocebus aethiops , AMP Cíclico/metabolismo , Genes Reporter , Herpesvirus Humano 6/patogenicidade , Humanos , Ligantes , Receptores de Quimiocinas/genética , Receptores Virais/genética , Elementos de Resposta , Transdução de Sinais , TransfecçãoRESUMO
Human herpesvirus 6 (HHV-6) is the only human herpesvirus encoding U94/rep, homologue to the parvovirus non-structural gene rep68/78. Results to date suggest that HHV-6 U94/rep might regulate viral gene expression and have a role in viral latency. To determine the effect of U94/REP upon viral replication, the protein was produced. The purified U94/REP retained the characteristic immunological features. It was internalized and localized in the nucleus of human cells, showing marked inhibitory activity on the replication of HHV-6 (both variants A and B). The effect of U94/REP was dose-dependent and sensitive to treatment with single-stranded but not double-stranded DNA. U94/REP inhibited the replication of other betaherpesviruses, HHV-7 and human cytomegalovirus, but had no effect on herpes simplex virus. These results confirm the action of U94/rep latency gene in the regulation of HHV-6 replication with implications for co-reactivations and latency of human betaherpesviruses.
Assuntos
Betaherpesvirinae/fisiologia , Herpesvirus Humano 6/fisiologia , Proteínas Virais/fisiologia , Replicação Viral , Linhagem Celular , Núcleo Celular/química , Citomegalovirus/fisiologia , Herpesvirus Humano 7/fisiologia , Humanos , Transporte Proteico , Simplexvirus/fisiologia , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismoRESUMO
Leukotropic human herpesvirus 6 (HHV-6) establishes a persistent infection associated with inflammatory diseases and encodes chemokines that could chemoattract leukocytes for infection or inflammation. HHV-6 variant A encodes a distant chemokine homolog, U83A, and a polymorphism promoting a secreted form was identified. U83A and three N-terminal modifications were expressed and purified, and activities were compared with a spliced truncated isoform, U83A-Npep. U83A efficiently and potently induced calcium mobilization in cells expressing single human CCR1, CCR4, CCR6, or CCR8, with EC50 values <10 nM. U83A also induced chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8. High-affinity binding, 0.4 nM, was demonstrated to CCR1 and CCR5 on monocytic/macrophage cells, and pretreatment with U83A or modified forms could block responses for endogenous ligands. U83A-Npep acted only as antagonist, efficiently blocking binding of CCL3 to CCR1 or CCR5 on differentiated monocytic/macrophage leukemic cells. Furthermore, CCL3 induction of calcium signaling via CCR1 and CCL1 induced chemotaxis via CCR8 in primary human leukocytes was inhibited. Thus, this blocking by the early expressed U83A-Npep could mediate immune evasion before finishing the replicative cycle. However, late in infection, when full-length U83A is made, chemoattraction of CCR1-, CCR4-, CCR5-, CCR6-, and CCR8-bearing monocytic/macrophage, dendritic, and T lymphocyte cells can facilitate dissemination via lytic and latent infection of these cells. This has further implications for neuroinflammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been linked. Applications also discussed include novel vaccines/immunotherapeutics for cancer and HIV as well as anti-inflammatories.
Assuntos
Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas/metabolismo , Genes Virais/genética , Receptores de Quimiocinas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Animais , Western Blotting , Linhagem Celular , Quimiotaxia de Leucócito , Cromatografia Líquida de Alta Pressão , DNA Recombinante , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Genes Virais/imunologia , Herpesvirus Humano 6/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
A cell fusion assay using fusion-from-without (FFWO) recombinant adenoviruses (RAds) and specific antibody showed a role in fusion modulation for glycoprotein gO, the recently identified third component of the gH/gL gCIII complex of human cytomegalovirus (HCMV). As in HCMV, RAd gO expressed multiple glycosylated species with a mature product of 125 kDa. Coexpression with gH/gL RAds showed gCIII reconstitution in the absence of other HCMV products and stabilisation by intermolecular disulfide bonds. Properties of HCMV clinical isolate, Pt, also implicated gO in cell spread. Compared to laboratory strain AD169, Pt was resistant to gH antibody plaque inhibition, but mature gH was identical. However, the gO sequences were highly divergent (20%), with further variation in laboratory strain Towne gO (34%). Thus, gO forms gCIII with gH/gL, performs in cell fusion, and is a newly identified HCMV hypervariable locus which may influence gCIII's function in mediating infection.