The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes

1996 ◽  
Vol 109 (8) ◽  
pp. 2121-2131 ◽  
Author(s):  
E.B. Cluett ◽  
C.E. Machamer
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
High Performance ◽  
Normal Component ◽  
Phosphatidyl Inositol ◽  
Subcellular Fractionation ◽  
Trans Golgi Network ◽  
Enveloped Virus ◽  
Lipid Species ◽  
Golgi Network

We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.

scholarly journals Entry of the Two Infectious Forms of Vaccinia Virus at the Plasma Membane Is Signaling-Dependent for the IMV but Not the EEV

2000 ◽  
Vol 11 (7) ◽  
pp. 2497-2511 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Annett Kuehn ◽  
Sibylle Schleich ◽  
Gaby Rutter ◽  
Heinrich Hohenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Hela Cells ◽  
Signaling Cascade ◽  
Enveloped Virus ◽  
Kinase C ◽  
Mature Virus

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

scholarly journals Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network

2014 ◽  
Vol 206 (5) ◽  
pp. 609-618 ◽  
Author(s):  
Josse van Galen ◽  
Felix Campelo ◽  
Emma Martínez-Alonso ◽  
Margherita Scarpa ◽  
José Ángel Martínez-Menárguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Transmembrane Proteins ◽  
Short Chain ◽  
Membrane Domains ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Specific Membrane

Do lipids such as sphingomyelin (SM) that are known to assemble into specific membrane domains play a role in the organization and function of transmembrane proteins? In this paper, we show that disruption of SM homeostasis at the trans-Golgi network (TGN) by treatment of HeLa cells with d-ceramide-C6, which was converted together with phosphatidylcholine to short-chain SM and diacylglycerol by SM synthase, led to the segregation of Golgi-resident proteins from each other. We found that TGN46, which cycles between the TGN and the plasma membrane, was not sialylated by a sialyltransferase at the TGN and that this enzyme and its substrate TGN46 could not physically interact with each other. Our results suggest that SM organizes transmembrane proteins into functional enzymatic domains at the TGN.

scholarly journals Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

2013 ◽  
Vol 24 (6) ◽  
pp. 832-847 ◽  
Author(s):  
Quyen L. Aoh ◽  
Chao-wei Hung ◽  
Mara C. Duncan
Keyword(s):  
Energy Metabolism ◽  
Intracellular Signaling ◽  
Phosphatidyl Inositol ◽  
Glucose Starvation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Permeabilized Cells ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Energy Dependent ◽  
Adp Ribosylation Factor

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.

scholarly journals An investigation of incorporation of cellular antigens into vaccinia virus particles

2002 ◽  
Vol 83 (10) ◽  
pp. 2347-2359 ◽  
Author(s):  
Oliver Krauss ◽  
Ruth Hollinshead ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Specific Cell ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Membrane Compartments ◽  
Specific Proteins ◽  
Intermediate Compartment ◽  
Low Levels ◽  
Golgi Network ◽  
Exocytic Pathway

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC), cis/medial-Golgi, trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC and cis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.

Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network.

1994 ◽  
Vol 68 (1) ◽  
pp. 130-147 ◽  
Author(s):  
M Schmelz ◽  
B Sodeik ◽  
M Ericsson ◽  
E J Wolffe ◽  
H Shida ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Plasma Membrane Budding as an Alternative Release Mechanism of the Extracellular Enveloped Form of Vaccinia Virus from HeLa Cells

2003 ◽  
Vol 77 (18) ◽  
pp. 9931-9942 ◽  
Author(s):  
Andrea Meiser ◽  
Carmen Sancho ◽  
Jacomine Krijnse Locker
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Hela Cells ◽  
International Health ◽  
Health Department ◽  
Release Mechanism ◽  
Enveloped Virus ◽  
Modified Vaccinia Virus Ankara ◽  
Membrane Budding

ABSTRACT In HeLa cells the assembly of modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus (VV) strain, is blocked. No intracellular mature viruses (IMVs) are made and instead, immature viruses accumulate, some of which undergo condensation and are released from the cell. The condensed particles may undergo wrapping by membranes of the trans-Golgi network and fusion with the plasma membrane prior to their release (M. W. Carroll and B. Moss, Virology 238:198-211, 1997). The present study shows by electron microscopy (EM), however, that the dense particles made in HeLa cells are also released by a budding process at the plasma membrane. By labeling the plasma membrane with antibodies to B5R, a membrane protein of the extracellular enveloped virus, we show that budding occurs at sites that concentrate this protein. EM quantitation revealed that the cell surface around a budding profile was as strongly labeled with anti-B5R antibody as were the extracellular particles, whereas the remainder of the plasma membrane was significantly less labeled. To test whether budding was a characteristic of MVA infection, HeLa cells were infected with the replication competent VV strains Western Reserve strain (WR) and International Health Department strain-J (IHD-J) and also prepared for EM. EM analyses, surprisingly, revealed for both virus strains IMVs that evidently budded at the cell surface at sites that were significantly labeled with anti-B5R. EM also indicated that budding of MVA dense particles was more efficient than budding of IMVs from WR- or IHD-J-infected cells. This was confirmed by semipurifying [35S]methionine-labeled dense particles or extracellular enveloped virus (EEVs) from the culture supernatant of MVA- or IHD-J-infected HeLa cells, respectively, showing that threefold more labeled dense particles were secreted than EEVs. Finally, although the released MVA dense particles contain some DNA, they are not infectious, as assessed by plaque assays.

scholarly journals Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

1993 ◽  
Vol 120 (1) ◽  
pp. 5-13 ◽  
Author(s):  
T Nilsson ◽  
M Pypaert ◽  
M H Hoe ◽  
P Slusarewicz ◽  
E G Berger ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Hela Cell ◽  
Cell Line ◽  
Hela Cells ◽  
Hela Cell Line ◽  
Frozen Sections ◽  
Specific Antibodies ◽  
Trans Golgi Network ◽  
Endogenous Protein ◽  
Golgi Network

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N-acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.

scholarly journals Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein

2001 ◽  
Vol 82 (11) ◽  
pp. 2747-2760 ◽  
Author(s):  
María M. Geada ◽  
Inmaculada Galindo ◽  
María M. Lorenzo ◽  
Beatriz Perdiguero ◽  
Rafael Blasco
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Virus Transport ◽  
Enveloped Virus ◽  
Vaccinia Viruses ◽  
Butanedione Monoxime ◽  
Golgi Network ◽  
Dependent Mechanism

Vaccinia virus produces several forms of infectious virions. Intracellular mature virions (IMV) assemble in areas close to the cell nucleus. Some IMV acquire an envelope from intracellular membranes derived from the trans-Golgi network, producing enveloped forms found in the cytosol (intracellular enveloped virus; IEV), on the cell surface (cell-associated enveloped virus) or free in the medium (extracellular enveloped virus; EEV). Blockage of IMV envelopment inhibits transport of virions to the cell surface, indicating that enveloped virus forms are required for virion movement from the Golgi area. To date, the induction of actin tails that propel IEV is the only well-characterized mechanism for enveloped virus transport. However, enveloped virus transport and release occur under conditions where actin tails are not formed. In order to study these events, recombinant vaccinia viruses were constructed with GFP fused to the most abundant protein in the EEV envelope, P37 (F13L). The P37–GFP fusion, like normal P37, accumulated in the Golgi area and was incorporated efficiently into enveloped virions. These recombinants allowed the monitoring of enveloped virus movements in vivo. In addition to a variety of relatively slow movements (<0·4 μm/s), faster, saltatory movements both towards and away from the Golgi area were observed. These movements were different from those dependent on actin tails and were inhibited by the microtubule-disrupting drug nocodazole, but not by the myosin inhibitor 2,3-butanedione monoxime. Video microscopy (5 frames per s) revealed that saltatory movements had speeds of up to, and occasionally more than, 3 μm/s. These results suggest that a second, microtubule-dependent mechanism exists for intracellular transport of enveloped vaccinia virions.

scholarly journals The Plant Vesicle-associated SNARE AtVTI1a Likely Mediates Vesicle Transport from theTrans-Golgi Network to the Prevacuolar Compartment

1999 ◽  
Vol 10 (7) ◽  
pp. 2251-2264 ◽  
Author(s):  
Haiyan Zheng ◽  
Gabriele Fischer von Mollard ◽  
Valentina Kovaleva ◽  
Tom H. Stevens ◽  
Natasha V. Raikhel
Keyword(s):  
Transport Process ◽  
Subcellular Fractionation ◽  
Amino Acid Identity ◽  
Yeast Protein ◽  
Alternative Pathways ◽  
Trans Golgi Network ◽  
Cell Extracts ◽  
Transport Vesicles ◽  
Prevacuolar Compartment ◽  
Golgi Network

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, twoArabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.

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