The Rab11 Pathway Is Required for Influenza A Virus Budding and Filament Formation

ABSTRACT Influenza A virus buds through the apical plasma membrane, forming enveloped virus particles that can take the shape of pleomorphic spheres or vastly elongated filaments. For either type of virion, the factors responsible for separation of viral and cell membranes are not known. We find that cellular Rab11 (a small GTP-binding protein involved in endocytic recycling) and Rab11-family interacting protein 3 ([FIP3] which plays a role in membrane trafficking and regulation of actin dynamics) are both required to support the formation of filamentous virions, while Rab11 is additionally involved in the final budding step of spherical particles. Cells transfected with Rab11 GTP-cycling mutants or depleted of Rab11 or FIP3 content by small interfering RNA treatment lost the ability to form virus filaments. Depletion of Rab11 resulted in up to a 100-fold decrease in titer of spherical virus released from cells. Scanning electron microscopy of Rab11-depleted cells showed high densities of virus particles apparently stalled in the process of budding. Transmission electron microscopy of thin sections confirmed that Rab11 depletion resulted in significant numbers of abnormally formed virus particles that had failed to pinch off from the plasma membrane. Based on these findings, we see a clear role for a Rab11-mediated pathway in influenza virus morphogenesis and budding.

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Release of filamentous and spherical influenza A virus is not restricted by tetherin

Journal of General Virology ◽

10.1099/vir.0.038778-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 963-969 ◽

Cited By ~ 22

Author(s):

Emily A. Bruce ◽

Truus E. Abbink ◽

Helen M. Wise ◽

Ruth Rollason ◽

Rui Pedro Galao ◽

...

Keyword(s):

Plasma Membrane ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Infection ◽

Cellular Protein ◽

Spherical Particles ◽

Enveloped Viruses ◽

Specific Antagonist ◽

Multiple Strains ◽

Scanning Electron

The cellular protein tetherin is thought to act as a ‘leash’ that anchors many enveloped viruses to the plasma membrane and prevents their release. We found that replication of multiple strains of influenza A virus was generally insensitive to alteration of tetherin levels, as assessed by output titre or scanning electron microscopy of cell-associated virions. This included human, swine, avian and equine isolates, strains that form filamentous or spherical particles and viruses that lack the M2 or NS1 proteins. Levels of cell-surface tetherin were not reduced by influenza infection, but tetherin and the viral haemagglutinin co-localized on the plasma membrane. However, tetherin could not be detected in filamentous virions, suggesting that influenza may possess a mechanism to exclude it from virions. Overall, if influenza does encode a specific antagonist of tetherin, it is not M2 or NS1 and we find no evidence for a role in host range specificity.

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Influenza A Virus M2 Protein Apical Targeting Is Required for Efficient Virus Replication

Journal of Virology ◽

10.1128/jvi.01425-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 7

Author(s):

Nicholas Wohlgemuth ◽

Andrew P. Lane ◽

Andrew Pekosz

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Infectious Virus ◽

Virus Assembly ◽

Apical Plasma Membrane ◽

M2 Protein ◽

Particle Release

ABSTRACTThe influenza A virus (IAV) M2 protein is a multifunctional protein with critical roles in virion entry, assembly, and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to those of wild-type virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type-independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells.IMPORTANCEInfluenza A virus assembly and particle release occur at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus, HA, NA, and M2, are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release, while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.

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Lipid Raft-Dependent Targeting of the Influenza A Virus Nucleoprotein to the Apical Plasma Membrane

Traffic ◽

10.1111/j.1600-0854.2004.00237.x ◽

2004 ◽

Vol 5 (12) ◽

pp. 979-992 ◽

Cited By ~ 60

Author(s):

Marlene Carrasco ◽

Maria Joao Amorim ◽

Paul Digard

Keyword(s):

Plasma Membrane ◽

Influenza A Virus ◽

Lipid Raft ◽

Influenza A ◽

Apical Plasma Membrane

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Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking

Journal of Virology ◽

10.1128/jvi.01757-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 13

Author(s):

Pengyang Zhu ◽

Libin Liang ◽

Xinyuan Shao ◽

Weiyu Luo ◽

Shuitao Jiang ◽

...

Keyword(s):

Plasma Membrane ◽

Influenza A Virus ◽

Influenza A ◽

Secretory Pathway ◽

Host Factors ◽

Apical Plasma Membrane ◽

M2 Protein ◽

Internal Deletion

ABSTRACT Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice. IMPORTANCE The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

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Synthesis of Gold Nanoparticles Conjugated with Protein A: Towards the Application in Biosensors for Virus Detection

Communications in Physics ◽

10.15625/0868-3166/21/4/359 ◽

2011 ◽

Vol 21 (4) ◽

pp. 333

Author(s):

Tran Quang Huy ◽

Mai Anh Tuan

Keyword(s):

Electron Microscopy ◽

Gold Nanoparticles ◽

Influenza A Virus ◽

Influenza A ◽

Protein A ◽

Virus Detection ◽

Sodium Ascorbate ◽

Virus Particles ◽

Transmission Electron ◽

Vis Spectroscopy

In this article, a facile and effective technique is described to prepare a complex of gold nanoparticles (GNPs)/protein A (PrA) for biosensors in virus detection. GNPs were synthesized by the reduction of tetrachloroauric (III) acid trihydrate using sodium ascorbate, and then coated with PrA via ultracentrifugation. The complex of GNPs/PrA was characterized using UV-vis spectroscopy and transmission electron microscopy. The immunogold labeling method of scanning electron microscopy was also used to verify the capacity for the detection and binding of GNPs/PrA to H$_{1}$N$_{1}$ influenza A virus particles. The results showed that GNPs were spherical, uniform shape, and approximately 10 nm in size. Noticeably, the complex of GNPs/PrA could detect and bind effectively to H$_{1}$N$_{1}$ influenza A virus particles by a large number of GNPs surrounded. The advantage of the complex of GNPs/PrA showed a highly potential application in biosensors with the improvement of the sensitivity and transducing signal for virus detection.

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Evidence for specific packaging of the influenza A virus genome from conditionally defective virus particles lacking a polymerase gene

Vaccine ◽

10.1016/j.vaccine.2006.06.001 ◽

2006 ◽

Vol 24 (44-46) ◽

pp. 6647-6650 ◽

Cited By ~ 25

Author(s):

Emmie de Wit ◽

Monique I.J. Spronken ◽

Guus F. Rimmelzwaan ◽

Albert D.M.E. Osterhaus ◽

Ron A.M. Fouchier

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Virus Genome ◽

Polymerase Gene ◽

Virus Particles ◽

Defective Virus

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The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

Journal of Cell Science ◽

10.1242/jcs.109.6.1215 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1215-1227 ◽

Cited By ~ 2

Author(s):

I. Hemery ◽

A.M. Durand-Schneider ◽

G. Feldmann ◽

J.P. Vaerman ◽

M. Maurice

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Apical Membrane ◽

Rat Hepatocytes ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Plasma Membrane Protein ◽

Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

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Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

The Journal of Cell Biology ◽

10.1083/jcb.94.3.613 ◽

1982 ◽

Vol 94 (3) ◽

pp. 613-623 ◽

Cited By ~ 127

Author(s):

J Aggeler ◽

Z Werb

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Plasma Membranes ◽

Cell Attachment ◽

High Resolution Electron Microscopy ◽

Peritoneal Macrophages ◽

Coated Pits ◽

Thin Sections ◽

Particle Uptake ◽

Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

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Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus.

The Journal of Cell Biology ◽

10.1083/jcb.83.2.338 ◽

1979 ◽

Vol 83 (2) ◽

pp. 338-347 ◽

Cited By ~ 29

Author(s):

M Büechi ◽

T Bächi

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Plasma Membranes ◽

Sendai Virus ◽

Light And Electron Microscopy ◽

Distal Portion ◽

Double Labeling ◽

Virus Particles ◽

Viral Antigens ◽

Cytoplasmic Surface

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double-labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.

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The fine structure of Sphaerotilus nutans

Canadian Journal of Microbiology ◽

10.1139/m73-051 ◽

1973 ◽

Vol 19 (3) ◽

pp. 309-313 ◽

Cited By ~ 20

Author(s):

Judith F. M. Hoeniger ◽

H.-D. Tauschel ◽

J. L. Stokes

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Fine Structure ◽

Lateral Position ◽

Thin Sections ◽

Liquid Cultures ◽

Sphaerotilus Natans ◽

Stationary Liquid ◽

Basal Disk ◽

Polar Organelle

Sphaerotilus natans developed sheathed filaments in stationary liquid cultures and motile swarm cells in shaken ones. Electron microscopy of negatively stained preparations and thin sections showed that the sheath consists of fibrils. When the filaments were grown in broth with glucose added, the sheath was much thicker and the cells were packed with granules of poly-β-hydroxybutyrate.Swarm cells possess a subpolar tuft of 10 to 30 flagella and a polar organelle which is usually inserted in a lateral position and believed to be ribbon-shaped. The polar organelle consists of an inner layer joined by spokes to an accentuated plasma membrane. The flagellar hook terminates in a basal disk, consisting of two rings, which is connected by a central rod to a second basal disk.

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