RESUMEN
The human immunodeficiency virus (HIV)-1 transactivator protein Tat is known to play a key role in HIV infection, integrally related to its role in the host cell nucleus/nucleolus. Here we show for the first time that Tat localisation can be modulated by specific methylation, whereby overexpression of active but not catalytically inactive PRMT6 methyltransferase specifically leads to exclusion of Tat from the nucleolus. An R52/53A mutated Tat derivative does not show this redistribution, implying that R52/53, within Tat's nuclear/nucleolar localisation signal, are the targets of PRMT6 activity. Analysis using fluorescence recovery after photobleaching indicate that Tat nucleolar accumulation is largely through binding to nucleolar components, with methylation of Tat by PRMT6 preventing this. To our knowledge, this is the first report of specific protein methylation inhibiting nucleolar retention.
Asunto(s)
Nucléolo Celular/metabolismo , VIH-1/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Arginina/genética , Arginina/metabolismo , Células COS , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/genética , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Metilación , Microscopía Confocal , Mutación , Señales de Localización Nuclear/genética , Proteínas Nucleares/genética , Unión Proteica , Proteína-Arginina N-Metiltransferasas/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Proteína Fluorescente RojaRESUMEN
The human retrovirus human T-cell leukemia virus type I (HTLV-1) infects human T cells by vertical transmission from mother to child through breast milk or horizontal transmission through blood transfusion or sexual contact. Approximately 5% of infected individuals develop adult T-cell leukemia/lymphoma (ATL) with a poor prognosis, while 95% of infected individuals remain asymptomatic for the rest of their lives, during which time the infected cells maintain a stable immortalized latent state in the body. It is not known why such a long latent state is maintained. We hypothesize that the role of functional proteins of HTLV-1 during early infection influences the phenotype of infected cells in latency. In eukaryotic cells, a mRNA quality control mechanism called nonsense-mediated mRNA decay (NMD) functions not only to eliminate abnormal mRNAs with nonsense codons but also to target virus-derived RNAs. We have reported that HTLV-1 genomic RNA is a potential target of NMD, and that Rex suppresses NMD and stabilizes viral RNA against it. In this study, we aimed to elucidate the molecular mechanism of NMD suppression by Rex using various Rex mutant proteins. We found that region X (aa20-57) of Rex, the function of which has not been clarified, is required for NMD repression. We showed that Rex binds to Upf1, which is the host key regulator to detect abnormal mRNA and initiate NMD, through this region. Rex also interacts with SMG5 and SMG7, which play essential roles for the completion of the NMD pathway. Moreover, Rex selectively binds to Upf3B, which is involved in the normal NMD complex, and replaces it with a less active form, Upf3A, to reduce NMD activity. These results revealed that Rex invades the NMD cascade from its initiation to completion and suppresses host NMD activity to protect the viral genomic mRNA.
Asunto(s)
Productos del Gen rex/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas Portadoras/metabolismo , Línea Celular , Productos del Gen rex/genética , Genoma Viral/genética , Humanos , Carioferinas/metabolismo , Mutación , Fosforilación , Unión Proteica , Dominios Proteicos , ARN Helicasas/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/metabolismo , Proteína Exportina 1RESUMEN
After integration to the human genome as a provirus, human T-cell leukemia virus type 1 (HTLV-1) utilizes host T cell gene expression machinery for viral replication. The viral RNA-binding protein, Rex, is known to transport unspliced/incompletely spliced viral mRNAs encoding viral structural proteins out of the nucleus to enhance virus particle formation. However, the detailed mechanism of how Rex avoids extra splicing of unspliced/incompletely spliced viral mRNAs and stabilizes them for effective translation is still unclear. To elucidate the underlying molecular mechanism of Rex function, we comprehensively analyzed the changes in gene expression and splicing patterns in Rex-overexpressing T cells. In addition, we identified 81 human proteins interacting with Rex, involved in transcription, splicing, translation, and mRNA quality control. In particular, Rex interacts with NONO and SFPQ, which play important roles in the regulation of transcription and splicing. Accordingly, expression profiles and splicing patterns of a wide variety of genes are significantly changed in Rex-expressing T cells. Especially, the level of vPD-L1 mRNA that lacks the part of exon 4, thus encodes soluble PD-L1 was significantly increased in Rex-expressing cells. Overall, by integrated analysis of these three datasets, we showed for the first time that Rex intervenes the host gene expression machinery throughout the pathway, probably to escort viral unstable mRNAs from transcription (start) to translation (end). Upon exerting its function, Rex may alter the expression level and splicing patterns of various genes, thus influencing the phenotype of the host cell.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Productos del Gen rex/metabolismo , Infecciones por HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Proteínas de Unión al ARN/metabolismo , Replicación Viral/genética , Antígeno B7-H1/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , Humanos , Factor de Empalme Asociado a PTB/metabolismo , Empalme del ARN , ARN Mensajero , Proteínas de Unión al ARN/genéticaRESUMEN
HTLV-1 is an oncovirus causing ATL and other inflammatory diseases such as HAM/TSP and HU in about 5% of infected individuals. It is also known that HTLV-1-infected cells maintain a disease-free, immortalized, latent state throughout the lifetimes of about 95% of infected individuals. We believe that the stable maintenance of disease-free infected cells in the carrier is an intrinsic characteristic of HTLV-1 that has been acquired during its evolution in the human life cycle. We speculate that the pathogenesis of the virus is ruled by the orchestrated functions of viral proteins. In particular, the regulation of Rex, the conductor of viral replication rate, is expected to be closely related to the viral program in the early active viral replication followed by the stable latency in HTLV-1 infected T cells. HTLV-1 and HIV-1 belong to the family Retroviridae and share the same tropism, e.g., human CD4+ T cells. These viruses show significant similarities in the viral genomic structure and the molecular mechanism of the replication cycle. However, HTLV-1 and HIV-1 infected T cells show different phenotypes, especially in the level of virion production. We speculate that how the activity of HTLV-1 Rex and its counterpart HIV-1 Rev are regulated may be closely related to the properties of respective infected T cells. In this review, we compare various pathological aspects of HTLV-1 and HIV-1. In particular, we investigated the presence or absence of a virally encoded "regulatory valve" for HTLV-1 Rex or HIV-1 Rev to explore its importance in the regulation of viral particle production in infected T cells. Finally, wereaffirm Rex as the key conductor for viral replication and viral pathogenesis based on our recent study on the novel functional aspects of Rex. Since the activity of Rex is closely related to the viral replication rate, we hypothesize that the "regulatory valve" on the Rex activity may have been selectively evolved to achieve the "scenario" with early viral particle production and the subsequent long, stable deep latency in HTLV-1 infected cells.
Asunto(s)
VIH-1 , Virus Linfotrópico T Tipo 1 Humano , Productos del Gen rex/genética , Productos del Gen rex/metabolismo , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , VIH-1/genética , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Human T-cell leukemia virus type-1 (HTLV-1) Rex is a viral RNA binding protein. The most important and well-known function of Rex is stabilizing and exporting viral mRNAs from the nucleus, particularly for unspliced/partially-spliced mRNAs encoding the structural proteins essential for viral replication. Without Rex, these unspliced viral mRNAs would otherwise be completely spliced. Therefore, Rex is vital for the translation of structural proteins and the stabilization of viral genomic RNA and, thus, for viral replication. Rex schedules the period of extensive viral replication and suppression to enter latency. Although the importance of Rex in the viral life-cycle is well understood, the underlying molecular mechanism of how Rex achieves its function has not been clarified. For example, how does Rex protect unspliced/partially-spliced viral mRNAs from the host cellular splicing machinery? How does Rex protect viral mRNAs, antigenic to eukaryotic cells, from cellular mRNA surveillance mechanisms? Here we will discuss these mechanisms, which explain the function of Rex as an organizer of HTLV-1 expression based on previously and recently discovered aspects of Rex. We also focus on the potential influence of Rex on the homeostasis of the infected cell and how it can exert its function.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen rex/metabolismo , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Replicación Viral , Humanos , Biosíntesis de Proteínas , Empalme del ARN , ARN Viral/metabolismoRESUMEN
The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus causing an aggressive T-cell malignancy, adult T-cell leukemia (ATL). Although HTLV-1 has a compact RNA genome, it has evolved elaborate mechanisms to maximize its coding potential. The structural proteins Gag, Pro, and Pol are encoded in the unspliced form of viral mRNA, whereas the Env protein is encoded in singly spliced viral mRNA. Regulatory and accessory proteins, such as Tax, Rex, p30II, p12, and p13, are translated only from fully spliced mRNA. For effective viral replication, translation from all forms of HTLV-1 transcripts has to be achieved in concert, although unspliced mRNA are extremely unstable in mammalian cells. It has been well recognized that HTLV-1 Rex enhances the stability of unspliced and singly spliced HTLV-1 mRNA by promoting nuclear export and thereby removing them from the splicing site. Rex specifically binds to the highly structured Rex responsive element (RxRE) located at the 3' end of all HTLV-1 mRNA. Rex then binds to the cellular nuclear exporter, CRM1, via its nuclear export signal domain and the Rex-viral transcript complex is selectively exported from the nucleus to the cytoplasm for effective translation of the viral proteins. Yet, the mechanisms by which Rex inhibits the cellular splicing machinery and utilizes the cellular pathways beneficial to viral survival in the host cell have not been fully explored. Furthermore, physiological impacts of Rex against homeostasis of the host cell via interactions with numerous cellular proteins have been largely left uninvestigated. In this review, we focus on the biological importance of HTLV-1 Rex in the HTLV-1 life cycle by following the historical path in the literature concerning this viral post-transcriptional regulator from its discovery to this day. In addition, for future studies, we discuss recently discovered aspects of HTLV-1 Rex as a post-transcriptional regulator and its use in host cellular pathways.