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 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 Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans

2005 ◽  
Vol 86 (5) ◽  
pp. 1279-1290 ◽  
Author(s):  
Gemma C. Carter ◽  
Mansun Law ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Lipid Membrane ◽  
Surface Proteins ◽  
Enveloped Virus ◽  
Virus Core ◽  
Cell Type Specific ◽  
Mature Virus ◽  
Entry Mechanism

Vaccinia virus (VACV) produces two distinct enveloped virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), but the entry mechanism of neither virion is understood. Here, the binding and entry of IMV particles have been investigated. The cell receptors for IMV are unknown, but it was proposed that IMV can bind to glycosaminoglycans (GAGs) on the cell surface and three IMV surface proteins have been implicated in this. In this study, the effect of soluble GAGs on IMV infectivity was reinvestigated and it was demonstrated that GAGs affected IMV infectivity partially in some cells, but not at all in others. Therefore, binding of IMV to GAGs is cell type-specific and not essential for IMV entry. By using electron microscopy, it is demonstrated that IMV from strains Western Reserve and modified virus Ankara enter cells by fusion with the plasma membrane. After an IMV particle bound to the cell, the IMV membrane fused with the plasma membrane and released the virus core into the cytoplasm. IMV surface antigen became incorporated into the plasma membrane and was not left outside the cell, as claimed in previous studies. Continuity between the IMV membrane and the plasma membrane was confirmed by tilt-series analysis to orientate membranes perpendicularly to the beam of the electron microscope. This analysis shows unequivocally that IMV is surrounded by a single lipid membrane and enters by fusion at the cell surface.

scholarly journals Regulation of hepatocyte plasma membrane α1-adrenergic receptors by 4β-phorbol 12-myristate 13-acetate

1995 ◽  
Vol 305 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J F Beeler ◽  
R H Cooper
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Plasma Membranes ◽  
Complete Recovery ◽  
Maximal Inhibition ◽  
Kinase C

The effect of phorbol 12-myristate 13-acetate (PMA) on hepatocyte alpha 1-adrenergic receptors was determined by [3H]prazosin binding to plasma membranes from control and PMA-treated hepatocytes. Membranes from hepatocytes incubated with PMA (1 microgram/ml) for 1 h exhibited a 40% decrease in alpha 1-adrenergic receptors (481 +/- 10 fmol/mg of protein; mean +/- S.E.M. for three separate experiments) relative to vehicle-treated (dimethylformamide) hepatocytes (802 +/- 91 fmol/mg of protein; n = 3), with no significant effect on the KD. The PMA-induced decrease in alpha 1-adrenergic receptors was maximal by 30 min and half-maximal inhibition of [3H]prazosin binding occurred with a PMA concentration of approx. 15 ng/ml. Pretreatment of hepatocytes with staurosporine (5 microM) blocked the effect of PMA, and 4 beta-phorbol 13-monoacetate was ineffective, suggesting the involvement of protein kinase C (PKC). Treatment of hepatocytes with primaquine (300 microM) for 15 min decreased hepatocyte plasma membrane alpha 1-adrenergic receptors by 34.0 +/- 2.4% (mean +/- S.E.M. of three experiments). Removal of primaquine allowed essentially complete recovery (98 +/- 4%; mean +/- S.E.M. for five separate experiments) of plasma membrane [3H]prazosin binding within 20 min, suggesting that the alpha 1-adrenergic receptor undergoes endocytotic recycling. Addition of PMA (1 microgram/ml) to hepatocytes immediately after removal of primaquine, completely inhibited the increase in plasma membrane alpha 1-adrenergic receptors relative to control cells, but had no effect on hepatocytes whose cell surface alpha 1-receptors remaining after primaquine treatment had been inactivated by alkylation. These observations suggested that activation of PKC may facilitate the internalization of the alpha 1-adrenergic receptor in hepatocytes.

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