Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the highly contagious diseases of swine with high morbidity and mortality, that negatively affects the pig industry worldwide, in particular China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. Endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological, metabolic processes including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT regulates the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in Clathrin-mediated endocytosis of CSFV and also involved in CSFV trafficking. Tsg101 assisted the virus in entering the host cell through the late endosome (Rab7 and Rab9), and finally to reach the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and dsRNA or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not Golgi in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection.IMPORTANCE CSF, caused by CSFV, is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genus Flavivirus and Hepacivirus within the family Flaviviridae, such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in Pestivirus infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both Clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for Pestivirus inhibitors.
Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.
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