RESUMO
BACKGROUND: Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1-associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. METHODS: We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. RESULTS: PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1- and HTLV-1-infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. CONCLUSIONS: HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1- and HIV-1-associated disease.
Assuntos
Coinfecção , Infecções por HIV/complicações , HIV-1 , Infecções por HTLV-I/complicações , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical/epidemiologia , Contagem de Linfócito CD4 , Infecções por HIV/epidemiologia , HIV-1/genética , HIV-1/isolamento & purificação , Infecções por HTLV-I/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Humanos , Paraparesia Espástica Tropical/diagnóstico , Provírus/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. RESULTS: We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. CONCLUSIONS: The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.
Assuntos
Fosfatos de Dinucleosídeos/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Provírus/genética , Proteínas de Ligação a RNA/imunologia , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Processamento Pós-Transcricional do RNA , RNA Interferente PequenoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células/fisiologia , Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Proteínas/metabolismo , Proteínas dos Retroviridae/metabolismo , Linfócitos T/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Encefalomielite Autoimune Experimental/virologia , Proteína Adaptadora GRB2/metabolismo , Infecções por HTLV-I/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/metabolismo , Proteínas dos Retroviridae/genética , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1006120.].
RESUMO
Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in â¼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.
Assuntos
Epigênese Genética , Genoma Humano , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Mutagênese Insercional/genética , Provírus/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Proteínas Virais Reguladoras e Acessórias/genética , Integração Viral/genética , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Fator de Ligação a CCCTC , Linfócitos T CD4-Positivos/virologia , Cromatina/ultraestrutura , Imunoprecipitação da Cromatina , Sequência Consenso , DNA/genética , DNA/metabolismo , Metilação de DNA , DNA Viral/genética , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HTLV-I/virologia , Código das Histonas , Humanos , Ligação Proteica , Proteínas dos Retroviridae/biossíntese , Proteínas dos Retroviridae/genética , Transcrição GênicaRESUMO
HTLV-1 inserts its viral genome into the host cellular DNA in the form of a provirus. The proviral DNA is a key to understand the persistence and pathogenesis of HTLV-1 infection. There has been a significant progress in proviral research due to technological advances on DNA sequencing.Next generation sequencing technology revolutionized our understanding of the human genome,showing how it is organized and regulated, not only by the nucleotide sequence itself but also by epigenetic features and higher-order chromatin structure. We will review recent findings regarding the role of HTLV-1 provirus in HTLV-1 infection.
RESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases in a small percentage of infected individuals. Host immune responses, in particular cytotoxic T lymphocytes (CTLs), influence the proliferation and survival of ATL cells and HTLV-1-infected cells. We generated recombinant vaccinia viruses (rVVs) expressing HTLV-1 basic leucine zipper (bZIP) factor (HBZ) or Tax to study the immunogenic potential of these viral proteins. Vaccination with rVV expressing Tax or HBZ induced specific T-cell responses, although multiple boosters were needed for HBZ. HBZ-stimulated T cells killed HBZ peptide-pulsed T cells and CD4(+) T cells from HBZ transgenic (HBZ-Tg) mice. The anti-lymphoma effect of the CTLs targeting HBZ was tested in mice inoculated with a lymphoma cell line derived from an HBZ-Tg mouse. Transfer of splenocytes from HBZ-immunized mice increased the survival of the lymphoma cell-inoculated mice, suggesting that the anti-HBZ CTLs have a protective effect. The rVV could also induce specific T-cell responses to HBZ and Tax in HTLV-1-infected rhesus monkeys. On the basis of the results of rVV-vaccinated mice and macaques, we identified a candidate peptide (HBZ157-176) for vaccine development. Dendritic cells pulsed with this peptide could generate HBZ-specific CTLs from human CD8(+) T cells. This study demonstrates that HBZ could be a target for immunotherapy of patients with ATL.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Produtos do Gene tax/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Leucemia-Linfoma de Células T do Adulto/prevenção & controle , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Linhagem Celular Tumoral , Humanos , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/virologia , Macaca mulatta , Camundongos Endogâmicos C57BL , Camundongos SCID , Dados de Sequência Molecular , Vacinas Sintéticas/química , Vaccinia virus/imunologia , Proteínas Virais/químicaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4âºFoxp3⺠T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4⺠T cells were enhanced in these mice. Foxp3â»CD4⺠T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3â»T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4⺠T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Linfócitos T Reguladores/imunologia , Proteínas Virais/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Adesão Celular , Movimento Celular , Células Cultivadas , Metilação de DNA , Fatores de Transcrição Forkhead/genética , Infecções por HTLV-I/patologia , Infecções por HTLV-I/fisiopatologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Masculino , Camundongos Transgênicos , Paraparesia Espástica Tropical/etiologia , Proteínas Recombinantes/metabolismo , Proteínas dos Retroviridae , Baço/imunologia , Baço/metabolismo , Baço/patologia , Baço/virologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/virologia , Timo/imunologia , Timo/metabolismo , Timo/patologia , Timo/virologia , Proteínas Virais/genéticaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of adult T-cell leukemia (ATL) and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis, uveitis and infective dermatitis. However, it remains to be elucidated how HTLV-1 induces both neoplastic and inflammatory diseases. A critical component in the Treg-cell machinery is the transcription factor Forkhead box P3 (Foxp3), which is expressed in ~5% of CD4(+) T cells of healthy individuals. Foxp3 is expressed in around 80% of ATL cases. Recent studies point to the capacity of Treg cells to convert to other cell types, even to those with an inflammatory phenotype. These characteristics might indicate that Treg cells might be playing a critical role in HTLV-1 infection, either by being targeted by the virus or by regulating and modulating the immune response. In this review, we will discuss the interplay between Foxp3 expression and HTLV-1, focusing on important viral proteins that might help the virus to trigger the development of such diverse pathologies.
Assuntos
Fatores de Transcrição Forkhead/genética , Expressão Gênica , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfoma de Células T/genética , Linfoma de Células T/virologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Infecções por HTLV-I/imunologia , Humanos , Imunomodulação , Linfoma de Células T/imunologia , Provírus , Proteínas dos Retroviridae , Transcrição Gênica , Proteínas Virais/metabolismo , Tropismo Viral , Integração ViralRESUMO
Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.
Assuntos
RNA Viral , Replicação Viral , RNA Viral/genética , Animais , Replicon/genética , Pseudouridina/metabolismo , Vírus de RNA de Cadeia Positiva/genética , Humanos , Vírus da Floresta de Semliki/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Estabilidade de RNA , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologiaRESUMO
BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed. RESULTS: We identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-ß signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1⺠cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration. CONCLUSIONS: STLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals.
Assuntos
Infecções por Deltaretrovirus/veterinária , Modelos Animais de Doenças , Leucemia de Células T/veterinária , Doenças dos Primatas/patologia , Doenças dos Primatas/virologia , Vírus Linfotrópico T Tipo 1 de Primatas/isolamento & purificação , Infecções Tumorais por Vírus/veterinária , Animais , Infecções por Deltaretrovirus/patologia , Infecções por Deltaretrovirus/virologia , Humanos , Leucemia de Células T/patologia , Leucemia de Células T/virologia , Macaca , Vírus Linfotrópico T Tipo 1 de Primatas/crescimento & desenvolvimento , Vírus Linfotrópico T Tipo 1 de Primatas/patogenicidade , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologiaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of a neoplastic disease of CD4+ T cells, adult T-cell leukemia (ATL), and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis, dermatitis, and inflammatory lung diseases. ATL cells, which constitutively express CD25, resemble CD25+CD4+ regulatory T cells (T(reg)). Approximately 60% of ATL cases indeed harbor leukemic cells that express FoxP3, a key transcription factor for T(reg) cells. HTLV-1 encodes an antisense transcript, HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases. In this study, we show that transgenic expression of HBZ in CD4+ T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals. In HBZ-transgenic mice, CD4+Foxp3+ T(reg) cells and effector/memory CD4+ T cells increased in vivo. As a mechanism of increased T(reg) cells, HBZ expression directly induced Foxp3 gene transcription in T cells. The increased CD4+Foxp3+ T(reg) cells in HBZ transgenic mice were functionally impaired while their proliferation was enhanced. HBZ could physically interact with Foxp3 and NFAT, thereby impairing the suppressive function of T(reg) cells. Thus, the expression of HBZ in CD4+ T cells is a key mechanism of HTLV-1-induced neoplastic and inflammatory diseases.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Inflamação/virologia , Linfoma de Células T/virologia , Adulto , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Transformação Celular Viral/genética , Transformação Celular Viral/imunologia , Transformação Celular Viral/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Inflamação/etiologia , Inflamação/genética , Inflamação/patologia , Linfoma de Células T/etiologia , Linfoma de Células T/genética , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-ß (TGF-ß)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-ß signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-ß response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-ß. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-ß signaling. Our results suggest that HBZ, by enhancing TGF-ß signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteína p300 Associada a E1A/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Virais/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Regulação Leucêmica da Expressão Gênica , Regulação Viral da Expressão Gênica , Infecções por HTLV-I/complicações , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Proteínas dos Retroviridae , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
We recently discovered the antisense protein of human T-cell leukemia virus (HTLV) type 2 (APH-2), whose messenger RNA is encoded by the antisense strand of the HTLV-2 genome. We quantified proviral load, level of tax, and APH-2 in a series of blood samples obtained from a cohort of HTLV-2 carriers. We determined whether APH-2 promotes cell proliferation. APH-2 was detectable in most samples tested and was correlated with proviral load. APH-2 levels were not correlated with lymphocyte count in vivo, consistent with the inability of APH-2 to promote cell proliferation in vitro. APH-2 does not promote cell proliferation and does not cause lymphocytosis.
Assuntos
Infecções por HTLV-II/virologia , Vírus Linfotrópico T Tipo 2 Humano/metabolismo , Linfocitose/virologia , Provírus/metabolismo , Proteínas dos Retroviridae/sangue , Carga Viral , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Assintomáticas , Estudos de Coortes , Feminino , Produtos do Gene tax/sangue , Infecções por HTLV-II/sangue , Infecções por HTLV-II/complicações , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-IdadeRESUMO
An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2022).1.
RESUMO
Parvovirus B19 (B19) belongs to the Erythroparvovirus genus and is known to cause the fifth disease in children. Primary infection of pregnant women is associated with a high risk of hydrops fetalis and stillbirth due to severe fetal anemia. Virus-like particle (VLP) vaccine candidates for B19 have been developed, although none have been approved so far. The B19 phospholipase A2 domain (B19 PLA2), located in the VP1 unique region, is believed to be associated with adverse inflammatory reactions, and previous effective attempts to improve this vaccine modality inserted a mutation to impair the PLA2 activity of VLPs. In this study, we designed VLPs with a deletion mutant of PLA2 (â¿PLA2 B19 VLP), devoid of PLA2 activity, and confirmed their immunogenicity and safe use in vivo. These results were supported by the lack of histological inflammatory reactions at the site of immunization or the production of IL-6 in â¿PLA2 B19 VLP-immunized mice, that were observed in mice immunized with B19 VLPs. CD4+ T cells from mice vaccinated with VLPs and B19-seropositive human samples were not activated by B19 PLA2 stimulation, suggesting that the B19 PLA2 domain does not constitute a major CD4+ T cell epitope. Most importantly, the â¿PLA2 B19 VLPs induced neutralizing antibodies against B19, in levels similar to those found in B19-seropositive human samples, indicating that they could be used as a safe and effective vaccine candidate against B19.
Assuntos
Parvovirus B19 Humano , Vacinas de Partículas Semelhantes a Vírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Criança , Epitopos de Linfócito T , Feminino , Humanos , Interleucina-6 , Camundongos , Parvovirus B19 Humano/genética , Fosfolipases A2/genética , GravidezRESUMO
Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37°C-39°C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.
RESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T do Adulto , DNA , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Provírus/genética , Sequências Reguladoras de Ácido NucleicoRESUMO
BACKGROUND: Parvovirus B19 (B19) is a well-known cause of fifth disease in children, but infection during pregnancy may cause hydrops fetalis and stillbirth. The receptor-binding domain (RBD) of the VP1 unique capsid plays a pivotal role in infection. Here, we aimed to improve the immunogenicity of an RBD-based vaccine by genetically fusing it with Streptococcus pneumoniae surface protein A (PspA). METHODS: Mice were intramuscularly injected with RBD-based vaccines. Antigen-specific antibodies and neutralizing activity against B19 were measured. Protective immunity against S. pneumoniae was evaluated by monitoring the survival of mice nasally challenged with bacteria and determining antigen-specific T cell activation in splenic cells. RESULTS: RBD alone failed to generate neutralizing antibodies against B19, but fusion with PspA induced higher levels of neutralizing IgG compared to B19 virus-like particles. Furthermore, a comparable level of PspA-specific IgG was induced by RBD-PspA and PspA alone, which was sufficient to protect mice against pneumococcal infection. Stimulation with PspA, but not RBD, induced cytokine production in splenic cells from mice immunized with RBD-PspA, suggesting that PspA-specific T cells supported immunoglobulin class switching of both RBD- and PspA-specific B cells. CONCLUSIONS: RBD-PspA should be an effective bivalent vaccine against B19 and S. pneumoniae infections.