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1.
Front Immunol ; 13: 864898, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35444636

RESUMO

Human roseolovirus U20 and U21 are type I membrane glycoproteins that have been implicated in immune evasion by interfering with recognition of classical and non-classical MHC proteins. U20 and U21 are predicted to be type I glycoproteins with extracytosolic immunoglobulin-like domains, but detailed structural information is lacking. AlphaFold and RoseTTAfold are next generation machine-learning-based prediction engines that recently have revolutionized the field of computational three-dimensional protein structure prediction. Here, we review the structural biology of viral immunoevasins and the current status of computational structure prediction algorithms. We use these computational tools to generate structural models for U20 and U21 proteins, which are predicted to adopt MHC-Ia-like folds with closed MHC platforms and immunoglobulin-like domains. We evaluate these structural models and place them within current understanding of the structural basis for viral immune evasion of T cell and natural killer cell recognition.


Assuntos
Herpesvirus Humano 6 , Herpesvirus Humano 7 , Infecções por Roseolovirus , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Humanos , Modelos Estruturais , Proteínas Virais/metabolismo
2.
J Virol ; 95(14): e0162820, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33952641

RESUMO

Like all herpesviruses, the roseoloviruses (HHV6A, -6B, and -7) establish lifelong infection within their host, requiring these viruses to evade host antiviral responses. One common host-evasion strategy is the downregulation of host-encoded, surface-expressed glycoproteins. Roseoloviruses have been shown to evade the host immune response by downregulating NK-activating ligands, class I MHC, and the TCR/CD3 complex. To more globally identify glycoproteins that are differentially expressed on the surface of HHV6A-infected cells, we performed cell surface capture of N-linked glycoproteins present on the surface of T cells infected with HHV6A, and compared these to proteins present on the surface of uninfected T cells. We found that the protein tyrosine phosphatase CD45 is downregulated in T cells infected with HHV6A. We also demonstrated that CD45 is similarly downregulated in cells infected with HHV7. CD45 is essential for signaling through the T cell receptor and, as such, is necessary for developing a fully functional immune response. Interestingly, the closely related betaherpesviruses human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) have also separately evolved unique mechanisms to target CD45. While HCMV and MCMV target CD45 signaling and trafficking, HHV6A acts to downregulate CD45 transcripts. IMPORTANCE Human herpesviruses-6 and -7 infect essentially 100% of the world's population before the age of 5 and then remain latent or persistent in their host throughout life. As such, these viruses are among the most pervasive and stealthy of all viruses. Host immune cells rely on the presence of surface-expressed proteins to identify and target virus-infected cells. Here, we investigated the changes that occur to proteins expressed on the cell surface of T cells after infection with human herpesvirus-6A. We discovered that HHV-6A infection results in a reduction of CD45 on the surface of infected T cells and impaired activation in response to T cell receptor stimulation.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Antígenos Comuns de Leucócito/genética , Linfócitos T/virologia , Linhagem Celular , Regulação para Baixo , Células HEK293 , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Humanos , Estabilidade Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
3.
Mol Biol Cell ; 31(3): 196-208, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31851583

RESUMO

The human herpesvirus-7 (HHV-7) U21 glycoprotein binds to class I major histocompatibility complex (MHC) molecules in the endoplasmic reticulum (ER) and reroutes them to lysosomes. How this single viral glycoprotein efficiently redirects the U21/class I MHC complex to the lysosomal compartment is poorly understood. To investigate the trafficking of HHV-7 U21, we followed synchronous release of U21 from the ER as it traffics through the secretory system. Sorting of integral membrane proteins from the trans-Golgi network (TGN) has been shown to occur through tubular carriers that emanate from the TGN or through vesicular carriers that recruit GGA (Golgi-localized, γ-ear-containing, ARF-binding protein), clathrin adaptors, and clathrin. Here, we present evidence for the existence of a third type of Golgi-derived carrier that is vesicular, yet clathrin independent. This U21-containing carrier also carries a Golgi membrane protein engineered to form inducible oligomers. We propose that U21 employs the novel mechanism of forming oligomeric complexes with class I MHC molecules that result in sorting of the oligomeric U21/class I MHC complexes to Golgi--derived quality control carriers destined for lysosomes.


Assuntos
Proteínas de Transporte/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/virologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Humanos , Lisossomos/metabolismo , Lisossomos/fisiologia , Ligação Proteica , Transporte Proteico , Rede trans-Golgi/metabolismo
4.
J Neurovirol ; 22(4): 488-97, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26727906

RESUMO

In this autopsy-based study, human herpesvirus-6 (HHV-6) and -7 (HHV-7) genomic sequence frequency, HHV-6 variants, HHV-6 load and the expression of HHV-6 antigens in brain samples from the individuals, with and without unspecified encephalopathy (controls), using nested and real-time polymerase chain reactions, restriction endonuclease, and immunohistochemical analysis were examined. GraphPad Prism 6.0 Mann-Whitney nonparametric and chi-square test and Fisher's exact test were used for statistical analysis. The encephalopathy diagnoses were shown by magnetic resonance imaging made during their lifetime and macro- and microscopically studied autopsy tissue materials. Widespread HHV-6 and/or HHV-7 positivity was detected in the brain tissue of various individuals with encephalopathy, as well as in controls (51/57, 89.4 % and 35/51, 68.6 %, respectively; p = 0.009). Significantly higher detection frequency of single HHV-6 and concurrent HHV-6 + HHV-7 DNA was found in pia mater meninges, frontal lobe, temporal lobe, and olfactory tract DNAs in individuals with encephalopathy compared to the control group. HHV-6 load and higher frequency of the viral load >10 copies/10(6) cells significantly differed in samples from individuals with and without encephalopathy. The expression of HHV-6 antigens was revealed in different neural cell types with strong predominance in the encephalopathy group. In all HHV-6-positive autopsy samples of individuals with and without encephalopathy, HHV-6B was revealed. Significantly higher detection frequency of beta-herpesvirus DNA, more often detected HHV-6 load >10 copies/10(6) cells, as well as the expression of HHV-6 antigens in different brain tissue samples from individuals with encephalopathy in comparison with control group indicate on potential involvement of these viruses in encephalopathy development.


Assuntos
Encefalopatias/virologia , DNA Viral/genética , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Infecções por Roseolovirus/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Encefalopatias/diagnóstico , Encefalopatias/patologia , Estudos de Casos e Controles , DNA Viral/metabolismo , Feminino , Lobo Frontal/patologia , Lobo Frontal/virologia , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Humanos , Pessoa de Meia-Idade , Neurônios/patologia , Neurônios/virologia , Bulbo Olfatório/patologia , Bulbo Olfatório/virologia , Pia-Máter/patologia , Pia-Máter/virologia , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/patologia , Lobo Temporal/patologia , Lobo Temporal/virologia , Carga Viral
5.
Biochemistry ; 53(38): 6092-102, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25225878

RESUMO

The putative membrane protein U24 from HHV-6A shares a seven-residue sequence identity (which includes a PxxP motif) with myelin basic protein (MBP), a protein responsible for the compaction of the myelin sheath in the central nervous system. U24 from HHV-6A also shares a PPxY motif with U24 from the related virus HHV-7, allowing them both to block early endosomal recycling. Recently, MBP has been shown to have protein-protein interactions with a range of proteins, including proteins containing SH3 domains. Given that this interaction is mediated by the proline-rich segment in MBP, and that similar proline-rich segments are found in U24, we investigate here whether U24 also interacts with SH3 domain-containing proteins and what the nature of that interaction might be. The implications of a U24-Fyn tyrosine kinase SH3 domain interaction are discussed in terms of the hypothesis that U24 may function like MBP through molecular mimicry, potentially contributing to the disease state of multiple sclerosis or other demyelinating disorders.


Assuntos
Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Deleção de Genes , Regulação Viral da Expressão Gênica , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fyn/química
6.
PLoS One ; 9(6): e99139, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24901711

RESUMO

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the µ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining µ subunits in the cells are eventually able to reroute class I molecules to lysosomes.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Complexo 1 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 3 de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Muromegalovirus/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas do Envelope Viral/metabolismo
7.
J Virol ; 88(6): 3298-308, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24390327

RESUMO

UNLABELLED: The U21 gene product from human herpesvirus 7 binds to and redirects class I major histocompatibility complex (MHC) molecules to a lysosomal compartment. The molecular mechanism by which U21 reroutes class I MHC molecules to lysosomes is not known. Here, we have reconstituted the interaction between purified soluble U21 and class I MHC molecules, suggesting that U21 does not require additional cellular proteins to interact with class I MHC molecules. Our results demonstrate that U21, itself predicted to contain an MHC class I-like protein fold, interacts tightly with class I MHC molecules as a tetramer, in a 4:2 stoichiometry. These observations have helped to elucidate a refined model describing the mechanism by which U21 escorts class I MHC molecules to the lysosomal compartment. IMPORTANCE: In this report, we show that the human herpesvirus 7 (HHV-7) immunoevasin U21, itself a class I MHC-like protein, binds with high affinity to class I MHC molecules as a tetramer and escorts them to lysosomes, where they are degraded. While many class I MHC-like molecules have been described in detail, this unusual viral class I-like protein functions as a tetramer, associating with class I MHC molecules in a 4:2 ratio, illuminating a functional significance of homooligomerization of a class I MHC-like protein.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Infecções por Roseolovirus/metabolismo , Infecções por Roseolovirus/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas de Transporte/genética , Herpesvirus Humano 7/química , Herpesvirus Humano 7/genética , Humanos , Ligação Proteica , Multimerização Proteica , Proteínas Virais/genética
8.
Am J Transplant ; 13 Suppl 3: 67-77; quiz 77-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23347215

RESUMO

Human herpesviruses (HHV) 6 and 7 are ubiquitous infections that reactivate commonly in transplant recipients. However, clinical diseases due to these viruses are reported only in 1% of solid organ transplant recipients. Fever, rash and bone marrow suppression are the most common manifestations, but symptoms of tissue invasive disease may be observed. Treatment of HHV-6 and HHV-7 disease includes antiviral therapy and cautious reduction in immunosuppression. HHV-8 is an oncogenic gamma-herpesvirus that causes Kaposi's sarcoma, Castleman's disease and primary effusion lymphomas in transplant recipients. Nonmalignant diseases such as bone marrow suppression and multiorgan failure have also been associated with HHV-8. Reduction in immunosuppression is the first line treatment of HHV-8 infection. Other alternatives for treatment, especially for HHV-8 diseases not responsive to immuno-minimization strategies, are surgery and chemotherapy. Sirolimus has been shown to be a beneficial component for the treatment of Kaposi's sarcoma and the role of antivirals for HHV-8 infection is being investigated.


Assuntos
Infecções por Herpesviridae/complicações , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Herpesvirus Humano 8/metabolismo , Transplante de Órgãos/efeitos adversos , Antivirais/uso terapêutico , Infecções por Herpesviridae/etiologia , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/uso terapêutico , Sarcoma de Kaposi/complicações , Sarcoma de Kaposi/tratamento farmacológico , Sirolimo/uso terapêutico
9.
PLoS Pathog ; 7(11): e1002362, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22102813

RESUMO

Herpesviruses have evolved numerous immune evasion strategies to facilitate establishment of lifelong persistent infections. Many herpesviruses encode gene products devoted to preventing viral antigen presentation as a means of escaping detection by cytotoxic T lymphocytes. The human herpesvirus-7 (HHV-7) U21 gene product, for example, is an immunoevasin that binds to class I major histocompatibility complex molecules and redirects them to the lysosomal compartment. Virus infection can also induce the upregulation of surface ligands that activate NK cells. Accordingly, the herpesviruses have evolved a diverse array of mechanisms to prevent NK cell engagement of NK-activating ligands on virus-infected cells. Here we demonstrate that the HHV-7 U21 gene product interferes with NK recognition. U21 can bind to the NK activating ligand ULBP1 and reroute it to the lysosomal compartment. In addition, U21 downregulates the surface expression of the NK activating ligands MICA and MICB, resulting in a reduction in NK-mediated cytotoxicity. These results suggest that this single viral protein may interfere both with CTL-mediated recognition through the downregulation of class I MHC molecules as well as NK-mediated recognition through downregulation of NK activating ligands.


Assuntos
Proteínas de Transporte/metabolismo , Citotoxicidade Imunológica , Herpesvirus Humano 7/patogenicidade , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Proteínas Virais/metabolismo , Apresentação de Antígeno , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 7/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Lisossomos , Infecções por Roseolovirus/imunologia , Proteínas Virais/imunologia
10.
J Biol Chem ; 285(47): 37016-29, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20833720

RESUMO

The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules.


Assuntos
Proteínas de Transporte/metabolismo , Glioblastoma/metabolismo , Antígeno HLA-A2/metabolismo , Herpesvirus Humano 7/metabolismo , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Diferenciação Celular , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Imunofluorescência , Glicosilação , Antígeno HLA-A2/genética , Humanos , Imunoprecipitação , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Microglobulina beta-2/antagonistas & inibidores , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
11.
Orv Hetil ; 151(16): 645-51, 2010 Apr 18.
Artigo em Húngaro | MEDLINE | ID: mdl-20353917

RESUMO

Human herpesvirus 7 known since 1990 is closely related to herpesvirus 6B. It replicates in human cells only after binding CD4 receptor. It establishes lifelong latency in infected cells, and its frequent reactivations result in asymptomatic virus shedding through saliva. Most children acquire infection by age 3 and 4, but in any later age group seronegative individuals are at risk of infection. Rarely, exanthema subitum or convulsions with fever in children, pityriasis rosea in young adults, lethal complications in immunocompromised persons with concomitant herpesvirus 6B and cytomegalovirus reactivation occur. The most important pathogenic changes are due to the altered cytokine and growth factor secretion from infected lymphocytes with subsequent chain reaction on immune and other cells. Antiviral antibodies are detected by commercial kits (immunofluorescence, ELISA, immunoblot), nucleic acid by nested polymerase chain reaction. The majority of conditions due to infection do not require antiviral medication, but the severe complications are treated with ganciclovir and its derivates or foscarnet and cidofovir.


Assuntos
Herpesvirus Humano 7/metabolismo , Infecções por Roseolovirus , Anticorpos Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Herpesvirus Humano 6/isolamento & purificação , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 7/isolamento & purificação , Humanos , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/tratamento farmacológico , Infecções por Roseolovirus/epidemiologia , Infecções por Roseolovirus/prevenção & controle
12.
J Virol ; 84(3): 1265-75, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19923186

RESUMO

Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport.


Assuntos
Endocitose/fisiologia , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Receptores da Transferrina/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química
13.
Arch Dermatol Res ; 299(4): 213-9, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17404748

RESUMO

The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.


Assuntos
Antígenos Virais/metabolismo , Células Dendríticas/metabolismo , Herpesvirus Humano 7/metabolismo , Líquen Plano/metabolismo , Líquen Plano/terapia , Acitretina/uso terapêutico , Corticosteroides/uso terapêutico , Adulto , Biópsia , Terapia Combinada , DNA Viral/efeitos dos fármacos , DNA Viral/metabolismo , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/patogenicidade , Humanos , Ceratolíticos/uso terapêutico , Líquen Plano/virologia , Masculino , Pessoa de Meia-Idade , Fototerapia/métodos , Indução de Remissão , Infecções por Roseolovirus/metabolismo , Infecções por Roseolovirus/patologia , Pele/metabolismo , Pele/patologia , Pele/virologia , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
14.
Curr Gene Ther ; 6(3): 399-420, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16787191

RESUMO

Amplicon-6 and Tamplicon-7 are novel non-integrating vectors derived from the lymphotropic Human Herpesviruses 6 and 7 (HHV-6 and HHV-7). In the presence of helper viruses the amplicon vectors replicate to yield packaged defective genomes of size approximately 150 kb and consisting of multiple repeat units containing (i) the oriLyt DNA replication origin (ii) the pac-1 and pac-2 cleavage and packaging signals (iii) bacterial plasmid DNA sequences (iv) the chosen transgene(s). Employing CD46 as a receptor HHV-6 gains entry into varied cells, including lymphocytes and dendritic cells, whereas HHV-7 employs the CD4 receptor to target CD4+ cells. The amplicon-based vectors have facilitated the characterization of viral DNA replication and packaging. Following electroporation and helper virus superinfection, the vectors can be transmitted as cell associated and as cell-free virions secreted into the medium. Analyses by flow cytometry have shown good cell spread and efficient gene expression. Exemplary transgenes have included: (i) The Green Fluorescence Protein (GFP) (ii) Genes for potential use in anti-viral vaccination e.g., the HSV-1 glycoprotein D (gD) with and without the trans-membrane region, expressed intracellularly, at the cell membrane or as secreted proteins. (iii) Tumor cell antigens. (iv) Apoptotic genes for development of oncolytic vectors. Due to their cell tropism, their structure as concatemeric genomes, with less than 1.5 kb of viral DNA sequences, the HHV-6 and 7 amplicons have the potential to become unique vectors for immunization and lymphotropic gene therapy.


Assuntos
Amplificação de Genes , Vetores Genéticos/química , Herpesvirus Humano 6/química , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/química , Herpesvirus Humano 7/genética , Vetores Genéticos/metabolismo , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo
15.
J Histochem Cytochem ; 52(5): 671-81, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15100244

RESUMO

Human fractalkine (CX3CL1), a delta-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.


Assuntos
Quimiocinas CX3C/metabolismo , Herpesvirus Humano 7/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Glândulas Salivares/metabolismo , Adulto , Idoso , Complexo CD3/metabolismo , Quimiocina CX3CL1 , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Feminino , Herpesvirus Humano 7/imunologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Glândula Parótida/metabolismo , Glândula Parótida/patologia , Glândula Parótida/virologia , Glândulas Salivares/patologia , Glândulas Salivares/virologia , Glândula Submandibular/metabolismo , Glândula Submandibular/patologia , Glândula Submandibular/virologia
16.
Traffic ; 4(12): 824-37, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14617346

RESUMO

Like all members of the herpesvirus family, human herpesvirus-7 has evolved mechanisms to evade immune detection. The human herpesvirus-7 gene product U21 encodes an immunoevasin that binds to class I major histocompatibility complex molecules and diverts them to a lysosomal compartment. Here we show that the cytoplasmic tail of U21, although sufficient to sequester a heterologous membrane protein (CD4 chimera), has no effect on U21's ability to redirect class I major histocompatibility complex molecules to lysosomes. Instead, the ER-lumenal domain of U21 is sufficient to redirect class I major histocompatibility complex molecules to the lysosomal compartment. These observations demonstrate a novel viral immunoevasive mechanism for U21, and implicate the ER-lumenal domain of a type I transmembrane protein in lysosomal sorting.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/farmacologia , Retículo Endoplasmático/metabolismo , Herpesvirus Humano 7/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Lisossomos/metabolismo , Proteínas Virais/química , Proteínas Virais/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Antígenos CD4/biossíntese , Antígenos CD4/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Citometria de Fluxo , Humanos , Leucina/química , Manose/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retroviridae/genética
17.
J Virol Methods ; 111(2): 145-56, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12880930

RESUMO

Codon-optimization refers to the alteration of gene sequences, to make codon usage match the available tRNA pool within the cell/species of interest. Codon-optimization has emerged as a powerful tool to increase protein expression by genes from small RNA and DNA viruses, which commonly contain overlapping reading frames as well as structural elements that are embedded within coding regions; these features are not widespread among large DNA viruses. We therefore examined whether codon-optimization might influence protein expression from a herpesvirus gene. We focused on the U51 gene from human herpesviruses-6 and -7, which was cloned in both native and codon-optimized form, with an N-terminal HA epitope tag to allow protein detection. Codon-optimization was associated with a profound (10-100 fold) increase in U51 expression in human (293A, HSG, K562) or hamster (CHO) cell lines, suggesting this may represent a valuable tool to facilitate functional studies on recalcitrant herpesvirus genes. Finally, it is postulated that the suboptimal expression of native U51 may reflect a regulatory mechanism that controls viral gene expression.


Assuntos
Códon/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 6/metabolismo , Herpesvirus Humano 7/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Citometria de Fluxo , Herpesvirus Humano 6/genética , Herpesvirus Humano 7/genética , Humanos , Fases de Leitura Aberta , Receptores de Quimiocinas/genética , Receptores Virais
18.
J Virol ; 75(8): 3925-36, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11264381

RESUMO

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).


Assuntos
Replicação do DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 7 , Origem de Replicação/genética , Proteínas Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Sequência Consenso/genética , DNA Viral/química , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/metabolismo , Análise Heteroduplex , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Viral/análise , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética
19.
J Virol ; 74(10): 4530-40, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10775589

RESUMO

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.


Assuntos
Glicoproteínas/genética , Heparina/metabolismo , Herpesvirus Humano 7/metabolismo , Proteínas do Envelope Viral/genética , Motivos de Aminoácidos , Baculoviridae/genética , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Éxons/genética , Glicoproteínas/química , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Herpesvirus Humano 7/genética , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Testes de Precipitina , Splicing de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Transcrição Gênica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo
20.
J Virol ; 74(4): 2011-6, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10644374

RESUMO

Human herpesvirus 7 (HHV-7) is a T-lymphotropic virus which utilizes the CD4 receptor as its main receptor to enter the target cells. Hence, HHV-7 can interfere with human immunodeficiency virus type 1 (HIV-1) infection in CD4(+) T cells. It was recently suggested that the CXC chemokine receptor 4 (CXCR4), which was found to be a crucial coreceptor for T-tropic HIV-1 strains, may also play a role in the HHV-7 infection process. However, the results presented here demonstrate that CXCR4 is not involved in HHV-7 infection. The natural ligand of CXCR4, SDF-1alpha, was not able to inhibit HHV-7 infection in SupT1 cells or in CD8(+) T-cell-depleted peripheral blood mononuclear cells. Also, AMD3100, a specific CXCR4 antagonist with potent antiviral activity against T-tropic HIV strains (50% inhibitory concentration ¿IC(50), 1 to 10 ng/ml), completely failed to inhibit HHV-7 infection (IC(50), >250 microg/ml). Thus, two different agents known to specifically interact with CXCR4 were not able to inhibit HHV-7 infection. Other T-lymphoid cell lines, expressing both CD4 and CXCR4 (e.g., HUT-78 and MT-4) could not be infected by HHV-7. In addition, the CD4-transfected cell lines HOS. CD4 and U87.CD4 and the CD4/CXCR4 double-transfected cell lines HOS. CD4.CXCR4 and U87.CD4.CXCR4 were not infectable with HHV-7. Also, we found no down-regulation of surface-bound or intracellular CXCR4 in HHV-7-infected CD4(+) T cells. As compared to uninfected SupT1 cells, stromal cell-derived factor 1alpha (SDF-1alpha)/CXCR4-mediated intracellular calcium flux was unchanged in SupT1 cells that were acutely or persistently infected with HHV-7. All these data argue against CXCR4 as a receptor involved in the HHV-7 infection process.


Assuntos
Linfócitos T CD4-Positivos/virologia , Herpesvirus Humano 7/metabolismo , Receptores CXCR4/metabolismo , Receptores Virais/metabolismo , Antígenos Virais/imunologia , Benzilaminas , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Ciclamos , Herpesvirus Humano 7/imunologia , Herpesvirus Humano 7/fisiologia , Compostos Heterocíclicos/farmacologia , Humanos , Receptores CXCR4/antagonistas & inibidores , Células Tumorais Cultivadas
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