Decreased function of survival motor neuron protein impairs endocytic pathways

This document is the Accepted Manuscript version. The final, definitive version is available online at https://doi.org/10.1073/pnas.1600015113.Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.Peer reviewedFinal Accepted Versio

Independent Segregation of Human Immunodeficiency Virus Type 1 Gag Protein Complexes and Lipid Rafts

Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55(Gag) polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55(Gag) mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55(Gag) interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55(Gag) that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55(Gag) forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains

HIV-1 egress is gated through late endosomal membranes

HIV-1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV-1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live-cell imaging techniques, by immunocytochemistry and by immuno-electron microscopy that HIV-1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage-derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV-1 morphogenesis is thoroughly rooted in the endosomal system

NPXY Motifs in the β1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry▿

Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by β1 integrin. The β1 integrin cytoplasmic tail contains two NPXY motifs, which function in recruitment of adaptor proteins and clathrin for endocytosis and serve as sorting signals for internalized cargo. As reovirus infection requires disassembly in the endocytic compartment, we investigated the role of the β1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (β1+/+ cells), reovirus infectivity was significantly reduced in cells expressing mutant β1 integrin in which the NPXY motifs were altered to NPXF (β1+/+Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to β1+/+ cells and β1+/+Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into β1+/+ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into β1+/+Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in β1+/+ and β1+/+Y783F/Y795F cells, providing further evidence that the β1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the β1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus