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.

scholarly journals Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production

2006 ◽  
Vol 80 (16) ◽  
pp. 8178-8189 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Virus Particles ◽  
M1 Protein

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.

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

2016 ◽  
Vol 91 (1) ◽  
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.

scholarly journals Effects of Antibody to the Influenza A Virus M2 Protein on M2 Surface Expression and Virus Assembly

Virology ◽  
1995 ◽  
Vol 212 (2) ◽  
pp. 411-421 ◽  
Author(s):  
Patsy G. Hughey ◽  
Paul C. Roberts ◽  
Leslie J. Holsinger ◽  
Suzanne L. Zebedee ◽  
Robert A. Lamb ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Surface Expression ◽  
M2 Protein

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

2007 ◽  
Vol 81 (22) ◽  
pp. 12169-12178 ◽  
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.

scholarly journals Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are Critical for Production of Infectious Virus Particles

2010 ◽  
Vol 84 (17) ◽  
pp. 8765-8776 ◽  
Author(s):  
Michael L. Grantham ◽  
Shaun M. Stewart ◽  
Erin N. Lalime ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Alanine Scanning Mutagenesis ◽  
Particle Assembly ◽  
Virus Particles

ABSTRACT The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.

scholarly journals The Cysteine Residues of the M2 Protein Are Not Required for Influenza A Virus Replication

Virology ◽  
1997 ◽  
Vol 238 (1) ◽  
pp. 128-134 ◽  
Author(s):  
Maria R Castrucci ◽  
Mark Hughes ◽  
Laura Calzoletti ◽  
Isabella Donatelli ◽  
Krisna Wells ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Cysteine Residues ◽  
M2 Protein
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