Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.
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.
According to the prion hypothesis, atypical phenotypes arise when a prion protein adopts an alternative conformation and persist when that form assembles into self-replicating aggregates. Amyloid formation in vitro provides a model for this protein-misfolding pathway, but the mechanism by which this process interacts with the cellular environment to produce transmissible phenotypes is poorly understood. Using the yeast prion Sup35/[PSI + ], we found that protein conformation determined the size distribution of aggregates through its interactions with a molecular chaperone. Shifts in this range created variations in aggregate abundance among cells due to a size threshold for transmission, and this heterogeneity, along with aggregate growth and fragmentation, induced age-dependent fluctuations in phenotype. Thus, prion conformations may specify phenotypes as population averages in a dynamic system. Prion proteins adopt a spectrum of conformations or strains, which create phenotypes of distinct severity and stability in vivo (1-3). These phenotypes are linked to the assembly of the protein into aggregates that, at unique rates, template the conversion of newly-made prion protein to a similar state and are fragmented (4). But, how do these biochemical events translate into distinct phenotypes? One possibility is an "abundance-based" model, in which phenotypes are linked to an equilibrium between aggregated and soluble prion protein that determines protein activity and the number of heritable prions (propagons) (5,6). However, the conversion and fragmentation reactions also create heterogeneity in aggregate size, raising the possibility of a second, "size-based" model in which a subpopulation of aggregates establishes and propagates phenotypes (7).To distinguish between these models, we focused on the [PSI + ] Weak and [PSI + ] Strong conformations of the yeast prion protein Sup35, which create phenotypes of different stabilities in vivo (8). To sustain these phenotypes in a dividing culture, Sup35 protein in the prion conformation must be inherited (7). To test whether conformational differences impact phenotypic stability by altering protein transmissibility, we monitored Sup35-GFP transfer to daughter cells. Using fluorescence loss in photobleaching (FLIP), a [PSI + ] Weak strain transferred half as much Sup35-GFP (~15% vs. ~30%; Fig. 1A) and contained ~50% fewer ** This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the
Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by 1 integrin. The 1 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 1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (1؉/؉ cells), reovirus infectivity was significantly reduced in cells expressing mutant 1 integrin in which the NPXY motifs were altered to NPXF (1؉/؉Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to 1؉/؉ cells and 1؉/؉Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into 1؉/؉ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into 1؉/؉ Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in 1؉/؉ and 1؉/؉Y783F/Y795F cells, providing further evidence that the 1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the 1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus.
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