RESUMEN
HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.
Asunto(s)
Proteínas de la Cápside , Cápside , Glicina , VIH-1 , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Fenilalanina , Humanos , Transporte Activo de Núcleo Celular , Cápside/química , Cápside/metabolismo , Glicina/metabolismo , VIH-1/química , VIH-1/genética , VIH-1/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/virología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Permeabilidad , Fenilalanina/metabolismo , Solubilidad , Internalización del Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismoRESUMEN
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel phlebovirus in the Bunyaviridae family. Most patients infected by SFTSV present with fever and thrombocytopenia, and up to 30% die due to multiple-organ dysfunction. The mechanisms by which SFTSV enters multiple cell types are unknown. SFTSV contains two species of envelope glycoproteins, Gn (44.2 kDa) and Gc (56 kDa), both of which are encoded by the M segment and are cleaved from a precursor polypeptide (about 116 kDa) in the endoplasmic reticulum (ER). Gn fused with an immunoglobulin Fc tag at its C terminus (Gn-Fc) bound to multiple cells susceptible to the infection of SFTSV and blocked viral infection of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation assays following mass spectrometry analysis showed that Gn binds to nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cellular protein with surface expression in multiple cell types. Small interfering RNA (siRNA) knockdown of NMMHC-IIA, but not the closely related NMMHC-IIB or NMMHC-IIC, reduced SFTSV infection, and NMMHC-IIA specific antibody blocked infection by SFTSV but not other control viruses. Overexpression of NMMHC-IIA in HeLa cells, which show limited susceptivity to SFTSV, markedly enhanced SFTSV infection of the cells. These results show that NMMHC-IIA is critical for the cellular entry of SFTSV. As NMMHC-IIA is essential for the normal functions of platelets and human vascular endothelial cells, it is conceivable that NMMHC-IIA directly contributes to the pathogenesis of SFTSV and may be a useful target for antiviral interventions against the viral infection.
Asunto(s)
Fiebre/virología , Cadenas Pesadas de Miosina/fisiología , Phlebovirus/patogenicidad , Trombocitopenia/virología , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN , Humanos , Microscopía Confocal , Cadenas Pesadas de Miosina/genética , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genéticaRESUMEN
Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.
Asunto(s)
Atelinae/virología , Hepadnaviridae/patogenicidad , Hepatocitos/virología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Receptores Virales/metabolismo , Simportadores/metabolismo , Tupaia/virología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Hepadnaviridae/genética , Hepadnaviridae/metabolismo , Infecciones por Hepadnaviridae/virología , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Humanos , Datos de Secuencia Molecular , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMEN
Common vetch (Vicia sativa L.) is widely planted as forage, green manure and food. Phosphate (Pi) deficiency is an important constraint for legume crop production. In this study, P-deficiency tolerance in 40 common vetch collections was evaluated under hydroponic condition. The collections were clustered into three groups based on the tolerance level. Physiological responses to P-deficiency in two tolerant collections (418 and 426) in comparison with one sensitive collection (415) were investigated. Greater growth inhibition was observed in sensitive collection compared with two tolerant collections, although the inorganic phosphorus (P) content in sensitive collection was higher than those in tolerant collections. The internal and external purple acid phosphatase activity in plants showed no significant difference between 418 and 415 under low phosphate condition. Transcriptomic analysis in the tolerant collection 426 in response to Pi starvation showed that many common adaptive strategies were applied and PHOSPHATE STARVATION RESPONSE (PHR)-related Pi signaling and transporter genes were altered. VsPHT1.2 had the highest expression level in root among all VsPHT1s, and it was remarkably upregulated after short time of P-deficiency treatment in tolerant collections compared with sensitive collection. In conclusion, common vetch response to P starvation by altering the expressions of core genes involved in Pi transport and signaling, and the elevated expression of VsPHT1.2 gene might contribute to higher Pi acquisition efficiency in P-deficiency tolerant collections.
RESUMEN
Mechanical forces regulate multiple essential pathways in the cell. The nuclear translocation of mechanoresponsive transcriptional regulators is an essential step for mechanotransduction. However, how mechanical forces regulate the nuclear import process is not understood. Here, we identify a highly mechanoresponsive nuclear transport receptor (NTR), Importin-7 (Imp7), that drives the nuclear import of YAP, a key regulator of mechanotransduction pathways. Unexpectedly, YAP governs the mechanoresponse of Imp7 by forming a YAP/Imp7 complex that responds to mechanical cues through the Hippo kinases MST1/2. Furthermore, YAP behaves as a dominant cargo of Imp7, restricting the Imp7 binding and the nuclear translocation of other Imp7 cargoes such as Smad3 and Erk2. Thus, the nuclear import process is an additional regulatory layer indirectly regulated by mechanical cues, which activate a preferential Imp7 cargo, YAP, which competes out other cargoes, resulting in signaling crosstalk.
Asunto(s)
Núcleo Celular , Mecanotransducción Celular , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
BACKGROUND: Circular RNAs have been emerging as biomarkers in diagnosis and prognosis of pancreatic ductal adenocarcinoma (PDAC). The hsa_circ_0013912 (circ_0013912) has been retrieved to be upregulated in PDAC. Here, we further investigated its role in PDAC cells, as well as its mechanism via serving as competing endogenous RNA (ceRNA) for miRNA (miR)-7-5p, which is abundant in pancreas and suppresses the development of PDAC. MATERIALS AND METHODS: The clinical human tissues were harvested from Gene Expression Omnibus (GEO) database and PDAC patients, and expression of circ_0013912 and miR-7-5p was detected by real-time quantitative PCR. The interaction between both was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation and biotin-miRNA pull-down assay. Functional experiments were performed using Cell Counting Kit-8 assay, colony formation assay, fluorescence-activated cell separation method, caspase 3 activity assay kit, Western blotting, transwell assays, and xenograft tumor model. RESULTS: circ_0013912 was upregulated in PDAC tumors and cells; besides, circ_0013912 upregulation was associated with TNM stage and lymph node metastasis. Silencing circ_0013912 inhibited cell viability, colony formation ability, cell cycle entrance, migration and invasion, but facilitated apoptosis rate and caspase 3 activity in PANC-1 and AsPC-1 cells, accompanied with decreased c-myc, cyclin D1 and vimentin, and increased E-cadherin. Furthermore, miR-7-5p was a target of circ_0013912. Blocking miR-7-5p could promote cell growth, migration and invasion of PANC-1 and AsPC-1 cells with circ_0013912 silencing or not. Tumor growth was also restrained by circ_0013912 downregulation. CONCLUSION: Circ_0013912 knockdown could suppress cell growth and metastasis of PDAC cells via sponging miR-7-5p.
RESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is the leading threat of cancer-related death in humans. Increasing studies show that circular RNAs (circRNAs) are important indicators in cancer diagnosis and prognosis. This study intended to explore the function and mechanism of circ_0015756 in HCC, providing the additional opinion for HCC treatment. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of circ_0015756 and miR-610. Cell viability was assessed by cell counting kit-8 (CCK-8) assay, and colony formation capacity was ascertained by colony formation assay. Cell migration and invasion were monitored by transwell assay. Cell cycle progression and apoptosis were analyzed by flow cytometry assay. Circ_0015756 oncogenicity was determined by Xenograft models. The targets of circ_0015756 and miR-610 were predicted by bioinformatics tools and validated using RNA pull-down, RNA immunoprecipitation (RIP) and dual-luciferase reporter assays. The expression level of fibroblast growth factor receptor 1 (FGFR1) was measured by Western blot. RESULTS: The expression of circ_0015756 was increased in HCC tissues, serums and cells. Circ_0015756 downregulation impaired HCC cell viability, colony formation capacity, invasion and migration, induced cell cycle arrest and apoptosis, and inhibited tumor growth in vivo. MiR-610 was ensured as a target of circ_0015756, and miR-610 absence reversed the effects of circ_0015756 downregulation. Further, FGFR1 was targeted by miR-610, and FGFR1 overexpression overturned the effects of miR-610 restoration in HCC cells. Circ_0015756 could regulate FGFR1 expression by targeting miR-610. CONCLUSION: Circ_0015756 played its tumorigenic properties in HCC by activating FGFR1 via sponging miR-610, and circ_0015756 was expected to be a vital indicator in HCC diagnosis and treatment.
RESUMEN
Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for human hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV). Species barriers to HBV/HDV infection are mainly determined at entry level by variations in the sequences of particular NTCP orthologs. In this study, we sought to determine whether the NTCP ortholog in woodchuck (Marmota monax), woodchuck NTCP (wNTCP) supports viral infection. We found that wNTCP is capable of supporting HBV/HDV infection in HepG2 cells, but to much lower extent than human NTCP (hNTCP), which is about 90% reduction of hNTCP. Comprehensive site-directed mutagenesis mapping of hNTCP and wNTCP revealed that the residue at position 263 is a novel site crucial for viral entry. The important role of site 263 in infection is conserved among NTCP orthologs and may therefore be a potential target for blocking the viral entry.
Asunto(s)
Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis Delta/metabolismo , Especificidad del Huésped/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Receptores Virales/genética , Simportadores/genética , Internalización del Virus , Secuencia de Aminoácidos/genética , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Clonación Molecular , Células Hep G2 , Hepatocitos/virología , Humanos , Hígado/metabolismo , Hígado/virología , Masculino , Marmota/virología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Unión Proteica/genética , Receptores Virales/metabolismo , Simportadores/metabolismoRESUMEN
OBJECTIVE: To observe the effect of Yiqi Jianpi plus anticancer herbs on spleen deficiency in colorectal cancer and its anti-tumor role. METHODS: Human intestinal cancer cell HT29 xenograft of nude mice model was established. The expression of EGF, VEGF, gastric cancer tumor growth in mice were observed. RESULTS: Protein kinase C expression in in the Yiqi Jianpi group and Yiqi Jianpi anti-tumor group was significantly better than the model group (P<0.01, P<0.05). There was significantly more apoptotic cells in Yiqi Jianpi anti-tumor group than Yiqi Jianpi group and model group (P<0.01). Epidermal growth factor and vascular endothelial growth factor expression in Yiqi Jianpi group was significantly lower than Yiqi Jianpi group and model group (P<0.05). CONCLUSIONS: Tumor can inhibit the expression of PKC inhibition. Yiqi Jianpi and anticancer treatment can reduce this inhibition. Besides this treatment can also inhibit expression of tumor related genes such as epidermal growth factor and vascular endothelial growth factor.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales , Medicamentos Herbarios Chinos/farmacología , Bazo/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células HT29 , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.
Asunto(s)
Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis Delta/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Receptores Virales/metabolismo , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Células Cultivadas , Susceptibilidad a Enfermedades , Hepatitis B/patología , Hepatitis B/virología , Hepatitis D/patología , Hepatitis D/virología , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Humanos , Ligandos , Datos de Secuencia Molecular , Transportadores de Anión Orgánico Sodio-Dependiente/química , Unión Proteica , Estructura Terciaria de Proteína , Receptores Virales/química , Reproducibilidad de los Resultados , Simportadores/química , Tupaiidae , Virión/metabolismoRESUMEN
Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.DOI:http://dx.doi.org/10.7554/eLife.00049.001.