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

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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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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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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The Lack of an Inherent Membrane Targeting Signal Is Responsible for the Failure of the Matrix (M1) Protein of Influenza A Virus To Bud into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.02306-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4673-4681 ◽

Cited By ~ 54

Author(s):

Dan Wang ◽

Aaron Harmon ◽

Jing Jin ◽

David H. Francis ◽

Jane Christopher-Hennings ◽

...

Keyword(s):

Influenza Virus ◽

Plasma Membrane ◽

Influenza A Virus ◽

Influenza A ◽

Envelope Proteins ◽

Viral Envelope ◽

Membrane Targeting ◽

Virus Like Particles ◽

The Matrix ◽

Targeting Signal

ABSTRACT The matrix protein (M1) of influenza A virus is generally viewed as a key orchestrator in the release of influenza virions from the plasma membrane during infection. In contrast to this model, recent studies have indicated that influenza virus requires expression of the envelope proteins for budding of intracellular M1 into virus particles. Here we explored the mechanisms that control M1 budding. Similarly to previous studies, we found that M1 by itself fails to form virus-like-particles (VLPs). We further demonstrated that M1, in the absence of other viral proteins, was preferentially targeted to the nucleus/perinuclear region rather than to the plasma membrane, where influenza virions bud. Remarkably, we showed that a 10-residue membrane targeting peptide from either the Fyn or Lck oncoprotein appended to M1 at the N terminus redirected M1 to the plasma membrane and allowed M1 particle budding without additional viral envelope proteins. To further identify a functional link between plasma membrane targeting and VLP formation, we took advantage of the fact that M1 can interact with M2, unless the cytoplasmic tail is absent. Notably, native M2 but not mutant M2 effectively targeted M1 to the plasma membrane and produced extracellular M1 VLPs. Our results suggest that influenza virus M1 may not possess an inherent membrane targeting signal. Thus, the lack of efficient plasma membrane targeting is responsible for the failure of M1 in budding. This study highlights the fact that interactions of M1 with viral envelope proteins are essential to direct M1 to the plasma membrane for influenza virus particle release.

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Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

Viruses ◽

10.3390/v8090249 ◽

2016 ◽

Vol 8 (9) ◽

pp. 249 ◽

Cited By ~ 4

Author(s):

Naoki Takizawa ◽

Fumitaka Momose ◽

Yuko Morikawa ◽

Akio Nomoto

Keyword(s):

Influenza A Virus ◽

Lipid Raft ◽

Influenza A ◽

Viral Components

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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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Apical Budding of a Recombinant Influenza A Virus Expressing a Hemagglutinin Protein with a Basolateral Localization Signal

Journal of Virology ◽

10.1128/jvi.76.7.3544-3553.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3544-3553 ◽

Cited By ~ 40

Author(s):

Rosalia Mora ◽

Enrique Rodriguez-Boulan ◽

Peter Palese ◽

Adolfo García-Sastre

Keyword(s):

Plasma Membrane ◽

Influenza A ◽

Mdck Cells ◽

Viral Envelope ◽

Apical Plasma Membrane ◽

Major Protein ◽

Mutant Form ◽

Apical Surface ◽

Cytoplasmic Domains ◽

Viral Budding

ABSTRACT Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.

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Lipid Raft Disruption by Cholesterol Depletion Enhances Influenza A Virus Budding from MDCK Cells

Journal of Virology ◽

10.1128/jvi.00835-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12169-12178 ◽

Cited By ~ 78

Author(s):

Subrata Barman ◽

Debi P. Nayak

Keyword(s):

Lipid Rafts ◽

Influenza A Virus ◽

Lipid Raft ◽

Influenza A ◽

Infectious Virus ◽

Virus Yield ◽

Cholesterol Depletion ◽

Virus Budding ◽

Particle Release ◽

Virus Particles

ABSTRACT Lipid rafts play critical roles in many aspects of the influenza A virus life cycle. Cholesterol is a critical structural component of lipid rafts, and depletion of cholesterol leads to disorganization of lipid raft microdomains. In this study, we have investigated the effect of cholesterol depletion by methyl-β-cyclodextrin (MβCD) treatment on influenza virus budding. When virus-infected Madin-Darby canine kidney cells were treated with MβCD at the late phase of infection for a short duration, budding of virus particles, as determined by protein analysis and electron microscopy, increased with increasing concentrations and lengths of treatment. However, infectious virus yield varied, depending on the concentration and duration of MβCD treatment. Low concentrations of MβCD increased infectious virus yield throughout the treatment period, but higher concentrations caused an initial increase of infectious virus titer followed by a decrease with a longer duration. Relative infectivity of the released virus particles, on the other hand, decreased with increasing concentrations and durations of MβCD treatment. Loss of infectivity of virus particles is due to multiple effects of MβCD-mediated cholesterol depletion causing disruption of lipid rafts, changes in structural integrity of the viral membrane, leakage of viral proteins, a nick or hole on the viral envelope, and disruption of the virus structure. Exogenous cholesterol increased lipid raft integrity, inhibited particle release, and partially restored the infectivity of the released virus particles. These data show that disruption of lipid rafts by cholesterol depletion caused an enhancement of virus particle release from infected cells and a decrease in the infectivity of virus particles.

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Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus

Viruses ◽

10.3390/v10110650 ◽

2018 ◽

Vol 10 (11) ◽

pp. 650 ◽

Cited By ~ 17

Author(s):

Dileep Verma ◽

Dinesh Gupta ◽

Sunil Lal

Keyword(s):

Lipid Rafts ◽

Influenza A Virus ◽

Lipid Raft ◽

Influenza A ◽

Virus Entry ◽

Critical Role ◽

Ganglioside Gm1 ◽

Raft Fraction ◽

Infected Cells ◽

Host Cell Surface

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-β-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.

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A Defect in Influenza A Virus Particle Assembly Specific to Primary Human Macrophages

10.1101/213447 ◽

2017 ◽

Author(s):

Sukhmani Bedi ◽

Takeshi Noda ◽

Yoshihiro Kawaoka ◽

Akira Ono

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cell Line ◽

Influenza A Virus ◽

Influenza A ◽

Actin Polymerization ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Particle Assembly ◽

Human Macrophages

AbstractThe primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In contrast to epithelial cells, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that primary human monocyte-derived macrophages (MDM) are inefficient in IAV release even when compared to a monocytic cell line differentiated to macrophage-like cells, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that IAV particle assembly is blocked in human MDM. Using anin situproximity ligation assay, we further determined that association between HA and the viral ion channel protein M2 is defective at the plasma membrane of MDM. In contrast, HA and another glycoprotein neuraminidase (NA) associate with each other on the MDM surface efficiently. Notably, the defect in association between HA and M2 in MDM was reversed upon inhibition of actin polymerization by cytochalasin D. Altogether, these results suggest that HA-M2 association on the plasma membrane is a discrete step in the IAV assembly process, which is separable from the association between HA and NA and susceptible to suppression by actin cytoskeleton. Overall, our study revealed the presence of a cell-type-specific mechanism negatively regulating IAV assembly at the plasma membrane.ImportanceIdentification of host cell determinants promoting or suppressing replication of many viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM are not permissive to IAV replication due to a defect at the virus particle formation step. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association in MDM is rescued by disruption of the actin cytoskeleton, revealing a previously unknown, negative role for actin polymerization, which is generally thought to play positive roles in IAV assembly. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

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