scholarly journals Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

2021 ◽  
Author(s):  
Annett Petrich ◽  
Valentin Dunsing ◽  
Sara Bobone ◽  
Salvatore Chiantia
Keyword(s):  
Plasma Membrane ◽  
Influenza A ◽  
Microscopy Study ◽  
Fluctuation Microscopy

scholarly journals Influenza A M2 recruits M1 to the plasma membrane: a fluorescence fluctuation microscopy study

2021 ◽  
Author(s):  
Annett Petrich ◽  
Valentin Dunsing ◽  
Sara Bobone ◽  
Salvatore Chiantia
Keyword(s):  
Plasma Membrane ◽  
Influenza A ◽  
Cross Correlation ◽  
Matrix Protein ◽  
Viral Proteins ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Respiratory Pathogen ◽  
Oligomeric State ◽  
Fluctuation Microscopy

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics and occasional pandemics of severe illnesses with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the structural stability of the virus. M1 organizes virion assembly and budding at the plasma membrane (PM), where it can interact with other viral components and cellular membrane factors (i.e. lipids and host proteins). Of interest, the recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin (HA), neuraminidase, matrix protein 2 (M2)) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e. (cross-correlation) number and brightness, and scanning fluorescence cross-correlation spectroscopy) to quantify the oligomeric state of M1 and its interaction with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the case that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.

IFITM proteins that restrict the early stages of respiratory virus infection do not influence late-stage replication

2021 ◽  
Author(s):  
Tina Meischel ◽  
Svenja Fritzlar ◽  
Fernando Villalon-Letelier ◽  
Melkamu B. Tessema ◽  
Andrew G. Brooks ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Antiviral Activity ◽  
Influenza A ◽  
Respiratory Viruses ◽  
Parainfluenza Virus ◽  
Early Stages ◽  
Infected Cells ◽  
Cellular Entry ◽  
Overexpression System ◽  
Late Stages

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2 and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus (PIV)-3 infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilise distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours post-infection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. siRNA-mediated knockdown of endogenous IFITM1, IFITM2 and IFITM3 expression, in the presence or absence of pre-treatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2 or IFITM3 immediately after infection did not impact titres of infectious virus released from IAV or PIV-3 infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways, but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses, however their potential to modulate the later stages of virus replication has not been explored. In this study we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early or late stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2 and IFITM3 against influenza A virus (IAV) but not parainfluenza virus (PIV)-3 during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways, yet do not influence the late stages of replication for either virus.

scholarly journals 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

2010 ◽  
Vol 84 (9) ◽  
pp. 4673-4681 ◽  
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.

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

2012 ◽  
Vol 93 (5) ◽  
pp. 963-969 ◽  
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.

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

2002 ◽  
Vol 76 (7) ◽  
pp. 3544-3553 ◽  
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.

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

2010 ◽  
Vol 84 (12) ◽  
pp. 5848-5859 ◽  
Author(s):  
Emily A. Bruce ◽  
Paul Digard ◽  
Amanda D. Stuart
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Influenza A Virus ◽  
Membrane Trafficking ◽  
Influenza A ◽  
Spherical Particles ◽  
Actin Dynamics ◽  
Apical Plasma Membrane ◽  
Thin Sections ◽  
Virus Particles

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.

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

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Sukhmani Bedi ◽  
Takeshi Noda ◽  
Yoshihiro Kawaoka ◽  
Akira Ono
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Influenza A ◽  
Cytochalasin D ◽  
Virus Release ◽  
Cell Type ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Particle Assembly ◽  
Human Macrophages

ABSTRACTInfluenza A virus (IAV) propagates efficiently in epithelial cells, its primary target in the respiratory tract. In contrast, 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 even compared to a monocytic cell line differentiated to macrophage-like cells, primary human monocyte-derived macrophages (MDM) are inefficient in IAV production, 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 a step in IAV particle assembly is blocked in MDM. Using anin situproximity ligation assay, we further determined that HA associates with neuraminidase (NA) but fails to associate with another viral transmembrane protein, M2, at the MDM plasma membrane. Notably, the defects in HA-M2 association and particle assembly in MDM were reversed upon cytochalasin D treatment that inhibits actin polymerization. These results suggest that HA-M2 association on the plasma membrane is a discrete step in IAV production, which is susceptible to suppression by actin cytoskeleton in MDM. Virus release remained inefficient in MDM upon cytochalasin D treatment, suggesting the presence of an additional defect(s) in virus release in this cell type. Overall, our study revealed the presence of multiple cell-type-specific mechanisms negatively regulating IAV production at the plasma membrane in MDM.IMPORTANCEIdentification of host cell determinants promoting or suppressing replication of 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, which are not permissive to IAV replication, fail to support virus particle formation. 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 and particle budding, but not virus release, in MDM are rescued by disruption of actin cytoskeleton, revealing a previously unknown, negative role for actin, which specifically targets an early step in the multistep IAV production. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

scholarly journals EPR and Electron Microscopy Study of the Influenza a M2 Transmembrane Domain Assembly and Drug Response

2016 ◽  
Vol 110 (3) ◽  
pp. 445a-446a
Author(s):  
Elka R. Georgieva ◽  
Peter P. Borbat ◽  
Kirill Grushin ◽  
Svetla Stoilova-McPhie ◽  
Nichita J. Kulkarni ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Influenza A ◽  
Drug Response ◽  
Transmembrane Domain ◽  
Microscopy Study ◽  
Electron Microscopy Study

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

2018 ◽  
Vol 92 (22) ◽  
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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