Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus–like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1–specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y13) of VP40, and mutation of Y13 to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1–specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy.
Residues 386 -423 of the rat brain serotonin transporter (SERT) are predicted to form a hydrophilic loop connecting transmembrane spans 7 and 8 (extracellular loop 4 or EL4). EL4 has been hypothesized to play a role in conformational changes associated with substrate translocation. To more fully investigate EL4 structure and function, we performed cysteine-scanning mutagenesis and methanethiosulfonate (MTS) accessibility studies on these 38 residues. Four EL4 mutants (M386C, R390C, G402C, and L405C) showed very low transport activities, low cell surface expression, and strong inhibition by MTS reagents, indicating high structural and functional importance. Twelve mutants were sensitive to very low MTS concentrations, indicating positions highly exposed to the aqueous environment. Eleven mutants were MTS-insensitive, indicating positions that were either buried in EL4 structure or functionally unimportant. The patterns of sensitivity to mutation and MTS reagents were used to produce a structural model of EL4. Positions 386 -399 and 409 -421 are proposed to form ␣-helices, connected by nine consecutive MTS-sensitive positions, within which four positions, 402-405, may form a turn or hinge. The presence of serotonin changed the MTS accessibility of cysteines at nine positions, while cocaine, a non-transportable blocker, did not affect accessibility. Serotonin-induced accessibility changes required both Na ؉ and Cl ؊ , indicating that they were associated with active substrate translocation. With the exception of a single mutant, F407C, neither mutation to cysteine nor treatment with MTS reagents affected SERT affinities for serotonin or the cocaine analog -CIT. These studies support the role of EL4 in conformational changes occurring during translocation and show that it does not play a direct role in serotonin binding.During chemical neurotransmission, neurotransmitters are released into the synaptic cleft, then taken back up into the presynaptic cell. This re-uptake is catalyzed by a large, highly homologous family of membrane transport proteins known as the neurotransmitter/sodium symporter (NSS) 1 family (1). The NSS transporters couple the uptake of specific neurotransmitters and/or amino acids to the transmembrane gradients of Na ϩ , Cl Ϫ , and in some cases, K ϩ or H ϩ (for a recent review, see Ref.2). Within this family, the closely related biogenic amine subfamily is responsible for the re-uptake of serotonin, dopamine, and norepinephrine. These transporters are the targets of antidepressants, cocaine, and amphetamines in the brain (3, 4).The high sequence homology among the NSS transporters suggests a common architecture, predicted to consist of 12 membrane spanning ␣-helices. This general topology has been confirmed experimentally for the serotonin transporter (SERT) (5-8). However, the three-dimensional packing of these membrane spans is still largely unknown, as is the molecular mechanism by which these transporters bind and translocate their substrates.Much recent work has focused on the TM7-EL4-TM8 ...
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