Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

Husain, Matloob; Moss, Bernard

doi:10.1128/jvi.77.16.9008-9019.2003

Cited by 34 publications

(37 citation statements)

References 71 publications

(60 reference statements)

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“…(i) Viral stock. Wild-type VACV (strain WR) and the recombinant VACV vF13L-green fluorescent protein (GFP) chimera were propagated in BSC-40 cells and purified by sucrose gradient sedimentation as described previously (15,16). The experiments presented in this study were carried out with the IMV form of VACV, unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

Pereira

Leite

Brasil

et al. 2012

J Virol

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Viral manipulation of transduction pathways associated with key cellular functions such as survival, response to microbial infection, and cytoskeleton reorganization can provide the supportive milieu for a productive infection. Here, we demonstrate that vaccinia virus (VACV) infection leads to activation of the stress-activated protein kinase (SAPK)/extracellular signal-regulated kinase (ERK) 4/7 (MKK4/7)-c-Jun N-terminal protein kinase 1/2 (JNK1/2) pathway; further, the stimulation of this pathway requires postpenetration, prereplicative events in the viral replication cycle. Although the formation of intracellular mature virus (IMV) was not affected in MKK4/7-or JNK1/2-knockout (KO) cells, we did note an accentuated deregulation of microtubule and actin network organization in infected JNK1/2-KO cells. This was followed by deregulated viral trafficking to the periphery and enhanced enveloped particle release. Furthermore, VACV infection induced alterations in the cell contractility and morphology, and cell migration was reduced in the JNK-KO cells. In addition, phosphorylation of proteins implicated with early cell contractility and cell migration, such as microtubule-associated protein 1B and paxillin, respectively, was not detected in the VACV-infected KO cells. In sum, our findings uncover a regulatory role played by the MKK4/7-JNK1/2 pathway in cytoskeleton reorganization during VACV infection.

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Section: Methodsmentioning

confidence: 99%

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

Pereira

Leite

Brasil

et al. 2012

J Virol

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“…HeLa cells were infected with vEH21i in the absence or presence of IPTG as indicated. After 8 h, cells were incubated with TR-Tfn (row 1) or FM4-64 (row 2) for 20 min at 37°C, fixed, and examined by confocal microscopy as described previously (1,16). Alternatively, the cells were fixed, permeabilized, and stained with mouse anti-␣ adaptin MAb (row 3) and mouse anti-p230 MAb (row 4), followed by Alexa 594-conjugated antibody to mouse IgG.…”

Section: Effect Of Gfp-eh21 Expression On Endocytosis Of Transferrin mentioning

confidence: 99%

“…Most of the IEV and CEV/EEV membrane proteins traffic through the secretory pathway and are detected in the endoplasmic reticulum, Golgi apparatus, and plasma membrane to various extents (21). F13 is exceptional in that it is targeted directly to Golgi membranes (16). When COPIImediated transport was inhibited, the EEV membrane proteins examined except for F13 were retained in the endoplasmic reticulum and IEV were not formed (15).…”

mentioning

confidence: 99%

“…A dominant-negative form of Eps15 called EH21 and a green fluorescent protein (GFP)-EH21 fusion protein inhibit receptor-mediated endocytosis (1,3). In a previous study (16), we showed that cotransfection of an expression plasmid encoding GFP-EH21 and a plasmid expressing the vaccinia virus F13 protein resulted in the accumulation of the latter in the plasma mem-brane. This result provided evidence for receptor-mediated endocytosis of F13, which was inhibited by the dominant-negative form of Eps15.…”

mentioning

confidence: 99%

“…Trans-Golgi cisternae (27) and early endosomes (32) each have been proposed as the progenitors of the wrapping membrane for reasons to be discussed later. The retrieval of viral proteins from the plasma membrane to the endocytic compartment, a potentially important step if wrapping of IMV occurs by early endosomes, has thus far been demonstrated only for B5 (37) and F13 (16), and the significance of this is unknown.…”

mentioning

confidence: 99%

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Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

Husain

Moss

2005

J Virol

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Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.Poxviruses are large, enveloped DNA viruses that replicate in the cytoplasm of their host cells (23). The assembly of vaccinia virus, the most intensively studied member of the poxvirus family, can be divided into two phases. The first begins with the formation of crescent-shaped membranes in virus factory areas of the cytoplasm and culminates in the production of infectious intracellular mature virions (IMV) (8). The second phase begins with the wrapping of IMV particles by cisternal membranes to form intracellular enveloped virions (IEV) (13). IEV are transported along microtubules to the periphery, where their outer membrane fuses with the plasma membrane (14,38,39). The majority of the extracellular virus particles, called cell-associated enveloped virions (CEV), adheres to the plasma membrane and mediates direct cell-to-cell spread (4). The released extracellular enveloped virions (EEV) may contribute to the long-range spread of virus (24).Seven proteins are known to be associated with IEV-or EEV-specific membranes. Of these, A36 (35), A56 (28), and B5 (11, 19) are type I integral membrane proteins, A33 (25) and A34 (9) are type II integral membrane proteins, and F13 (17) and probably F12 (34) are peripheral membrane proteins....

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Defective rotavirus particle assembly in lovastatin-treated MA104 cells

2008

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Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in "empty-looking" rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion.

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Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

Cited by 34 publications

References 71 publications

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

A Vaccinia Virus-Driven Interplay between the MKK4/7-JNK1/2 Pathway and Cytoskeleton Reorganization

Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

Defective rotavirus particle assembly in lovastatin-treated MA104 cells

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