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 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 The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals Mutations in the Influenza A Virus M1 Protein Enhance Virus Budding To Complement Lethal Mutations in the M2 Cytoplasmic Tail

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Hsuan Liu ◽  
Michael L. Grantham ◽  
Andrew Pekosz
Keyword(s):  
Virus Particle ◽  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Filament Formation ◽  
Virus Budding ◽  
Virus Particles

ABSTRACTThe influenza A virus M1 and M2 proteins play important roles in virus assembly and in the morphology of virus particles. Mutations in the distal cytoplasmic tail region of M2, and in particular a tyrosine-to-alanine mutation at residue 76 (Y76A), were essential for infectious virus production and filament formation while having limited effects on total virus particle budding. Using a novel selection method, mutations at seven different M1 amino acids (residue 73, 94, 135, 136, or 138 or a double mutation, 93/244) that are not found in circulating influenza virus strains or have not been previously identified to play a role in influenza A virus assembly were found to complement the lethal M2Y76A mutation. These M1 suppressor mutations restored infectious virus production in the presence of M2Y76A and mediated increased budding and filament formation even in the absence of M2. However, the efficiency of infectious virus replication was still dependent on the presence of the distal region of the M2 cytoplasmic tail. The data suggest that influenza A virus budding and genome incorporation can occur independently and provide further support for complementary roles of the M1 and M2 proteins in virus assembly.IMPORTANCEInfluenza virus particle assembly involves the careful coordination of various viral and host factors to optimally produce infectious virus particles. We have previously identified a mutation at position 76 of the influenza A virus M2 protein that drastically reduces infectious virus production and filament formation with minimal effects on virus budding. In this work, we identified suppressor mutations in the M1 protein which complement this lethal M2 mutation by increasing the efficiency with which virus particles bud from infected cells and restoring filament formation at the infected-cell surface. M2 distal cytoplasmic domain sequences were still required for optimal infectivity. This indicates that M1 and M2 can functionally replace each other in some, but not all, aspects of virus particle assembly.

scholarly journals Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

2009 ◽  
Vol 83 (17) ◽  
pp. 8655-8661 ◽  
Author(s):  
Michael L. Grantham ◽  
Wai-Hong Wu ◽  
Erin N. Lalime ◽  
Maria E. Lorenzo ◽  
Sabra L. Klein ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Mdck Cells ◽  
Cysteine Residue ◽  
M2 Protein ◽  
Recombinant Viruses ◽  
Virus Particles

ABSTRACT The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

scholarly journals The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding

2008 ◽  
Vol 82 (20) ◽  
pp. 10059-10070 ◽  
Author(s):  
Benjamin J. Chen ◽  
George P. Leser ◽  
David Jackson ◽  
Robert A. Lamb
Keyword(s):  
Influenza A ◽  
Mdck Cells ◽  
Virus Assembly ◽  
Direct Interaction ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Virus Budding ◽  
Mutant Proteins ◽  
Virus Like Particle ◽  
Growth Analyses

ABSTRACT The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.

Aloe vera and its Components Inhibit Influenza A Virus-Induced Autophagy and Replication

2019 ◽  
Vol 47 (06) ◽  
pp. 1307-1324 ◽  
Author(s):  
Jang-Gi Choi ◽  
Heeeun Lee ◽  
Young Soo Kim ◽  
Youn-Hwan Hwang ◽  
You-Chang Oh ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Mdck Cells ◽  
Aloe Vera ◽  
Post Treatment ◽  
M2 Protein ◽  
Mrna Synthesis ◽  
Catechin Hydrate

Aloe vera ethanol extract (AVE) reportedly has significant anti-influenza virus activity, but its underlying mechanisms of action and constituents have not yet been completely elucidated. Previously, we have confirmed that AVE treatment significantly reduces the viral replication of green fluorescent protein-labeled influenza A virus in Madin-Darby canine kidney (MDCK) cells. In addition, post-treatment with AVE inhibited viral matrix protein 1 (M1), matrix protein 2 (M2), and hemagglutinin (HA) mRNA synthesis and viral protein (M1, M2, and HA) expressions. In this study, we demonstrated that AVE inhibited autophagy induced by influenza A virus in MDCK cells and also identified quercetin, catechin hydrate, and kaempferol as the active antiviral components of AVE. We also found that post-treatment with quercetin, catechin hydrate, and kaempferol markedly inhibited M2 viral mRNA synthesis and M2 protein expression. A docking simulation suggested that the binding affinity of quercetin, catechin hydrate, and kaempferol for the M2 protein may be higher than that of known M2 protein inhibitors. Thus, the inhibition of autophagy induced by influenza virus may explain the antiviral activity of AVE against H1N1 or H3N2. Aloe vera extract and its constituents may, therefore, be potentially useful for the development of anti-influenza agents.

scholarly journals Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus

2008 ◽  
Vol 82 (23) ◽  
pp. 11869-11879 ◽  
Author(s):  
Edward C. Hutchinson ◽  
Martin D. Curran ◽  
Eliot K. Read ◽  
Julia R. Gog ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genome Packaging ◽  
Virus Growth ◽  
Virus Particles ◽  
Synonymous Mutations ◽  
Coding Regions

ABSTRACT The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

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.

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