scholarly journals Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells

2002 ◽  
Vol 76 (6) ◽  
pp. 2890-2898 ◽  
Author(s):  
Regan N. Theiler ◽  
Teresa Compton
Keyword(s):  
Secretory Pathway ◽  
Viral Envelope ◽  
The Novel ◽  
Host Cell Membrane ◽  
Virus Maturation ◽  
Golgi Compartment ◽  
Infected Cells ◽  
Processed Form ◽  
Golgi Network

ABSTRACT Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in transfected cells as it does in infected cells. We further determined that gO is modified by O-linked glycosylation and that this terminally processed form is highly enriched in virions. However, during studies of gO processing, novel gO complexes were discovered in CMV virions. The newly identified gO complexes, including gO-gL heterodimers, were not readily detected in CMV-infected cells. Further characterization of the trafficking of gO through the secretory pathway of infected cells localized gH, gL, and gO primarily to the Golgi apparatus and trans-Golgi network, supporting the conclusion that the novel virion-associated gO complexes arise in a post-Golgi compartment of infected cells.

scholarly journals The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

1999 ◽  
Vol 73 (4) ◽  
pp. 3430-3437 ◽  
Author(s):  
Alexandra Meindl ◽  
Nikolaus Osterrieder
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Viral Envelope ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Amino Terminal ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an M r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M r Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-M r Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment

1993 ◽  
Vol 106 (3) ◽  
pp. 815-822
Author(s):  
N.J. Bryant ◽  
A. Boyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
High Yield ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Terminal Domain ◽  
Golgi Compartment ◽  
Functional Equivalent ◽  
Golgi Network

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

scholarly journals Identification and characterization of a novel envelope protein in Rana grylio virus

2008 ◽  
Vol 89 (8) ◽  
pp. 1866-1872 ◽  
Author(s):  
Zhe Zhao ◽  
Fei Ke ◽  
You-Hua Huang ◽  
Jiu-Gang Zhao ◽  
Jian-Fang Gui ◽  
...  
Keyword(s):  
Western Blot ◽  
Envelope Protein ◽  
Virus Assembly ◽  
Structural Features ◽  
Envelope Proteins ◽  
Current Data ◽  
Viral Envelope ◽  
Viral Envelope Protein ◽  
Infected Cells

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R–GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

scholarly journals Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

2012 ◽  
Vol 23 (12) ◽  
pp. 2327-2338 ◽  
Author(s):  
Amy J. Curwin ◽  
Julia von Blume ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Calcium Atpase ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Reduced Rate ◽  
Golgi Network

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca2+ ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H+ ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.

scholarly journals The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

2005 ◽  
Vol 79 (18) ◽  
pp. 11943-11951 ◽  
Author(s):  
Alec J. Hirsch ◽  
Guruprasad R. Medigeshi ◽  
Heather L. Meyers ◽  
Victor DeFilippis ◽  
Klaus Früh ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Cellular Protein ◽  
Viral Envelope ◽  
Src Family Kinase ◽  
West Nile ◽  
Virus Particles ◽  
Cellular Genes ◽  
Infected Cells

ABSTRACT The role of cellular genes in West Nile virus (WNV) replication is not well understood. Examination of cellular transcripts upregulated during WNV infection revealed an increase in the expression of the src family kinase (SFK) c-Yes. WNV-infected cell lines treated with the SFK inhibitor PP2 demonstrated a 2- to 4-log decrease in viral titers, suggesting that SFK activity is required for completion of the viral replication cycle. RNA interference mediated knock-down of c-Yes, but not c-Src, and similarly reduced virus yield, specifically implicating c-Yes in WNV production. Interestingly, PP2 treatment did not reduce intracellular levels of either viral RNA or protein, suggesting that the drug does not act on the early stages of replication. However, endoglycosidase H (endoH) digestion of the viral envelope (E) glycoprotein revealed that the acquisition of endoH-resistant glycans by E, but not endogenous major histocompatibility complex class I, was reduced in PP2-treated cells, demonstrating that E specifically does not traffic beyond the endoplasmic reticulum in the absence of SFK activity. Electron microscopy further revealed that PP2-treated WNV-infected cells accumulated an increased number of virions in the ER compared to untreated cells. Therefore, we conclude that inhibition of SFK activity did not interfere with virus assembly but prevented transit of virions through the secretory pathway. These results identify c-Yes as a cellular protein that is involved in WNV assembly and egress.

scholarly journals A New Flavivirus and a New Vector: Characterization of a Novel Flavivirus Isolated from Uranotaenia Mosquitoes from a Tropical Rain Forest

2009 ◽  
Vol 83 (9) ◽  
pp. 4462-4468 ◽  
Author(s):  
Sandra Junglen ◽  
Anne Kopp ◽  
Andreas Kurth ◽  
Georg Pauli ◽  
Heinz Ellerbrok ◽  
...  
Keyword(s):  
Rain Forest ◽  
Phylogenetic Analyses ◽  
Cell Culture Supernatant ◽  
The Novel ◽  
Glycosylation Sites ◽  
Ns3 Protein ◽  
Distinct Cluster ◽  
Infected Cells ◽  
Novel Virus

ABSTRACT A novel flavivirus was isolated from Uranotaenia mashonaensis, a mosquito genus not previously known to harbor flaviviruses. Mosquitoes were caught in the primary rain forest of the Taï National Park, Côte d'Ivoire. The novel virus, termed nounané virus (NOUV), seemed to grow only on C6/36 insect cells and not on vertebrate cells. Typical enveloped flavivirus-like particles of 60 to 65 nm in diameter were detected by electron microscopy in the cell culture supernatant of infected cells. The full genome was sequenced, and potential cleavage and glycosylation sites and cysteine residues were identified, suggesting that the processing of the NOUV polyprotein is similar to that of other flaviviruses. Phylogenetic analyses of the whole polyprotein and the NS3 protein showed that the virus forms a distinct cluster within the clade of mosquito-borne flaviviruses. Only a distant relationship to other known flaviviruses was found, indicating that NOUV is a novel lineage within the Flaviviridae.

scholarly journals Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

2021 ◽  
Author(s):  
Dana A. Dahhan ◽  
Gregory D. Reynolds ◽  
Jessica J. Cárdenas ◽  
Dominique Eeckhout ◽  
Alexander Johnson ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Mass Spectrometry Analysis ◽  
Chemical Labeling ◽  
Spectrometry Analysis ◽  
Trans Golgi Network ◽  
Evolutionarily Conserved ◽  
Golgi Network

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 968-969
Author(s):  
K. McCammon ◽  
M. Segal ◽  
J. Sambrook ◽  
M. J. Gething ◽  
A. McDowall
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Cysteine Residue ◽  
Homologous Sequence ◽  
Golgi Compartment ◽  
Golgi Network

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

scholarly journals Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins

1997 ◽  
Vol 327 (3) ◽  
pp. 625-635 ◽  
Author(s):  
Kazuhisa NAKAYAMA
Keyword(s):  
Secretory Pathway ◽  
Serum Proteins ◽  
Mammalian Species ◽  
Viral Envelope ◽  
Biologically Active ◽  
Proprotein Convertases ◽  
Bioactive Materials ◽  
Precursor Proteins ◽  
And Function ◽  
Golgi Network

Limited endoproteolysis of inactive precursor proteins at sites marked by paired or multiple basic amino acids is a widespread process by which biologically active peptides and proteins are produced within the secretory pathway in eukaryotic cells. The identification of a novel family of endoproteases homologous with bacterial subtilisins and yeast Kex2p has accelerated progress in understanding the complex mechanisms underlying the production of the bioactive materials. Seven distinct proprotein convertases of this family (furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6, LPC/PC7/PC8/SPC7) have been identified in mammalian species, some having isoforms generated via alternative splicing. The family has been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms. Furin, the first proprotein convertase to be identified, has been most extensively studied. It has been shown to be expressed in all tissues and cell lines examined and to be mainly localized in the trans-Golgi network, although some proportion of the furin molecules cycle between this compartment and the cell surface. This endoprotease is capable of cleaving precursors of a wide variety of proteins, including growth factors, serum proteins, including proteases of the blood-clotting and complement systems, matrix metalloproteinases, receptors, viral-envelope glycoproteins and bacterial exotoxins, typically at sites marked by the consensus Arg-Xaa-(Lys/Arg)-Arg sequence. The present review covers the structure and function of mammalian subtilisin/Kex2p-like proprotein convertases, focusing on furin (EC 3.4.21.85)

scholarly journals Characterization of Herpes Simplex Virus-Containing Organelles by Subcellular Fractionation: Role for Organelle Acidification in Assembly of Infectious Particles

2001 ◽  
Vol 75 (3) ◽  
pp. 1236-1251 ◽  
Author(s):  
Carol A. Harley ◽  
Anindya Dasgupta ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Subcellular Fractionation ◽  
Sequence Of Events ◽  
Cell Extracts ◽  
Viral Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Golgi Network

ABSTRACT The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSV-infected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with thetrans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.

Sign in / Sign up

Export Citation Format

Share Document