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1.  Endosome-to-cytosol transport of viral nucleocapsids 
Nature Cell Biology  2005;7(7):653-664.
During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus, they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. The latter step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1 and is regulated by PI3P signaling via the PI3P-binding protein SNX16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PI3P, and by their effectors.
doi:10.1038/ncb1269
PMCID: PMC3360589  PMID: 15951806
Animals; Biological Transport; physiology; Cattle; Cell Line; Cricetinae; Cytosol; metabolism; ultrastructure; Endosomal Sorting Complexes Required for Transport; Endosomes; metabolism; ultrastructure; Epithelial Cells; virology; Fibroblasts; virology; Hela Cells; Humans; Lysophospholipids; physiology; Membrane Fusion; drug effects; physiology; Microscopy, Electron; Microscopy, Fluorescence; Monoglycerides; Nucleocapsid; metabolism; Phosphatidylinositol Phosphates; physiology; Phosphoproteins; genetics; physiology; RNA, Viral; biosynthesis; metabolism; Signal Transduction; physiology; Sorting Nexins; Time Factors; Transport Vesicles; metabolism; ultrastructure; Vesicular Transport Proteins; genetics; physiology; Vesicular stomatitis Indiana virus; physiology; Virus Replication; genetics
2.  Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking 
PLoS ONE  2007;2(9):e851.
Background
Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics.
Methodology/Principal Findings
Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus.
Conclusions/Significance
These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.
doi:10.1371/journal.pone.0000851
PMCID: PMC1952175  PMID: 17786222
3.  The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity†  
Journal of Virology  2005;79(17):11403-11411.
The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.
doi:10.1128/JVI.79.17.11403-11411.2005
PMCID: PMC1193635  PMID: 16103191
4.  The PPPY Motif of Human T-Cell Leukemia Virus Type 1 Gag Protein Is Required Early in the Budding Process 
Journal of Virology  2002;76(19):10024-10029.
Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.
doi:10.1128/JVI.76.19.10024-10029.2002
PMCID: PMC136533  PMID: 12208980
5.  The Y-S-L-I Tyrosine-Based Motif in the Cytoplasmic Domain of the Human T-Cell Leukemia Virus Type 1 Envelope Is Essential for Cell-to-Cell Transmission 
Journal of Virology  1999;73(11):9659-9663.
The human T-cell leukemia virus type 1 (HTLV-1) transmembrane glycoprotein has a 24-amino-acid cytoplasmic domain whose function in the viral life cycle is poorly understood. We introduced premature-stop mutations and 18 single-amino-acid substitutions into this domain and studied their effects on cell-to-cell transmission of the virus. The results show that the cytoplasmic domain is absolutely required for cell-to-cell transmission of HTLV-1, through amino acids which cluster in a Y-S-L-I tyrosine-based motif. The transmission defect in two motif mutants did not result from a defect in glycoprotein incorporation or fusion. It appears that the Y-S-L-I tyrosine-based motif of the HTLV-1 glycoprotein cytoplasmic domain has multiple functions, including involvement in virus transmission at a postfusion step.
PMCID: PMC113006  PMID: 10516080
6.  Early Assembly Step of a Retroviral Envelope Glycoprotein: Analysis Using a Dominant Negative Assay  
The Journal of Cell Biology  1999;145(1):57-68.
As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.
PMCID: PMC2148214  PMID: 10189368
protein processing; posttranslational; viral envelope proteins; genes, dominant; human T cell leukemia virus
7.  Multiple Functions for the Basic Amino Acids of the Human T-Cell Leukemia Virus Type 1 Matrix Protein in Viral Transmission 
Journal of Virology  1999;73(3):1860-1867.
We studied the involvement of the human T-cell leukemia virus type 1 (HTLV-1) Gag matrix protein in the cell-to-cell transmission of the virus using missense mutations of the basic amino acids. These basic amino acids are clustered at the N terminus of the protein in other retroviruses and are responsible for targeting the Gag proteins to the plasma membrane. In the HTLV–bovine leukemia virus genus of retroviruses, the basic amino acids are distributed throughout the matrix protein sequence. The HTLV-1 matrix protein contains 11 such residues. A wild-type phenotype was obtained only for mutant viruses with mutations at one of two positions in the matrix protein. The phenotypes of the other nine mutant viruses showed that the basic amino acids are involved at various steps of the replication cycle, including some after membrane targeting. Most of these nine mutations allowed normal synthesis, transport, and cleavage of the Gag precursor, but particle release was greatly affected for seven of them. In addition, four mutated proteins with correct particle release and envelope glycoprotein incorporation did not however permit cell-to-cell transmission of HTLV-1. Thus, particle release, although required, is not sufficient for the cell-to-cell transmission of HTLV-1, and the basic residues of the matrix protein are involved in steps that occur after viral particle release.
PMCID: PMC104426  PMID: 9971764

Results 1-7 (7)