scholarly journals Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication

2019 ◽  
Author(s):  
Bodan Hu ◽  
Stefanie Siche ◽  
Lars Möller ◽  
Michael Veit
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Reverse Genetics ◽  
Protein Composition ◽  
Surface Expression ◽  
Membrane Curvature ◽  
Proton Channel ◽  
Cellular Proteins ◽  
Amphiphilic Helix ◽  
Assembly Site

AbstractM2 of influenza virus functions as proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularisation of membranes can be performed by a variety of amphiphilic peptides we used reverse genetics to investigate whether they can substitute for M2’s helix.Virus could not be generated if M2’s helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor, from the Epsin N-Terminal Homology domain or the non-natural membrane inducer RW16. Transmission EM of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles and ion channel activity. The protein composition and specific infectivity was also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication, but other helices can replace its basic (curvature-inducing) function.ImportanceInfluenza is unique among enveloped viruses since it does not rely on the cellular ESCRT-machinery for budding. Instead viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 which explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

scholarly journals Amphipathic Helices of Cellular Proteins Can Replace the Helix in M2 of Influenza A Virus with Only Small Effects on Virus Replication

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Bodan Hu ◽  
Stefanie Siche ◽  
Lars Möller ◽  
Michael Veit
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Reverse Genetics ◽  
Protein Composition ◽  
Membrane Curvature ◽  
Proton Channel ◽  
Cellular Proteins ◽  
Amphiphilic Helix ◽  
Assembly Site

ABSTRACT M2 of influenza virus functions as a proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularization of membranes can be performed by a variety of amphiphilic peptides, we used reverse genetics to investigate whether the peptides can substitute for M2’s helix. Virus could not be generated if M2’s helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor from the Epsin N-terminal homology domain or the nonnatural membrane inducer RW16. Transmission electron microscopy of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles, and ion channel activity. The protein composition and specific infectivity were also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication but that other helices can replace its basic (curvature-inducing) function. IMPORTANCE Influenza virus is unique among enveloped viruses since it does not rely on the cellular ESCRT machinery for budding. Instead, viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site, where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 that explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

scholarly journals Improvement of Influenza A/Fujian/411/02 (H3N2) Virus Growth in Embryonated Chicken Eggs by Balancing the Hemagglutinin and Neuraminidase Activities, Using Reverse Genetics

2005 ◽  
Vol 79 (11) ◽  
pp. 6763-6771 ◽  
Author(s):  
Bin Lu ◽  
Helen Zhou ◽  
Dan Ye ◽  
George Kemble ◽  
Hong Jin
Keyword(s):  
Amino Acids ◽  
Virus Replication ◽  
Receptor Binding ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Vaccines ◽  
H3n2 Virus ◽  
Virus Growth ◽  
Chicken Eggs ◽  
Embryonated Chicken

ABSTRACT The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.

scholarly journals Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine

2012 ◽  
Vol 93 (10) ◽  
pp. 2204-2214 ◽  
Author(s):  
Lindomar Pena ◽  
Amy L. Vincent ◽  
Crystal L. Loving ◽  
Jamie N. Henningson ◽  
Kelly M. Lager ◽  
...  
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Reverse Genetics ◽  
Influenza Viruses ◽  
Dependent Manner ◽  
Lung Pathology ◽  
Influenza A Viruses ◽  
Virus Shedding ◽  
The Impact ◽  
Strain Dependent

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

Spotting Differences in Molecular Dynamic Simulations of Influenza A M2 Protein-Ligand Complexes by Varying M2 construct, Lipid Bilayer and Force Field

2019 ◽  
Author(s):  
Dimitrios Kolokouris ◽  
Iris Kalenderoglou ◽  
Panagiotis Lagarias ◽  
Antonios Kolocouris
Keyword(s):  
Molecular Dynamic ◽  
Lipid Bilayer ◽  
Influenza A ◽  
Md Simulations ◽  
Membrane Curvature ◽  
Buffer Size ◽  
M2 Protein ◽  
Dynamic Simulations ◽  
Amphipathic Helices ◽  
Drug Protein Binding

<p>We studied by molecular dynamic (MD) simulations systems including the inward<sub>closed</sub> state of influenza A M2 protein in complex with aminoadamantane drugs in membrane bilayers. We varied the M2 construct and performed MD simulations in M2TM or M2TM with amphipathic helices (M2AH). We also varied the lipid bilayer by changing either the lipid, DMPC or POPC, POPE or POPC/cholesterol (chol), or the lipids buffer size, 10x10 Å<sup>2 </sup>or 20x20 Å<sup>2</sup>. We aimed to suggest optimal system conditions for the computational description of this ion channel and related systems. Measures performed include quantities that are available experimentally and include: (a) the position of ligand, waters and chlorine anion inside the M2 pore, (b) the passage of waters from the outward Val27 gate of M2 S31N in complex with an aminoadamantane-aryl head blocker, (c) M2 orientation, (d) the AHs conformation and structure which is affected from interactions with lipids and chol and is important for membrane curvature and virus budding. In several cases we tested OPLS2005, which is routinely applied to describe drug-protein binding, and CHARMM36 which describes reliably protein conformation. We found that for the description of the ligands position inside the M2 pore, a 10x10 Å<sup>2</sup> lipids buffer in DMPC is needed when M2TM is used but 20x20 Å<sup>2</sup> lipids buffer of the softer POPC; when M2AH is used all 10x10 Å<sup>2</sup> lipid buffers with any of the tested lipids can be used. For the passage of waters at least M2AH with a 10x10 Å<sup>2</sup> lipid buffer is needed. The folding conformation of AHs which is defined from hydrogen bonding interactions with the bilayer and the complex with chol is described well with a 10x10 Å<sup>2</sup> lipids buffer and CHARMM36. </p>

scholarly journals Functional, Dynamic and Structural Understanding of M2 Proton Channel from Influenza A and its Inhibition

2016 ◽  
Vol 110 (3) ◽  
pp. 192a
Author(s):  
Timothy A. Cross ◽  
Riqiang Fu ◽  
E. Vindana Ekanayake ◽  
Yimin Miao ◽  
Joana Paulino ◽  
...  
Keyword(s):  
Influenza A ◽  
Proton Channel ◽  
M2 Proton Channel

scholarly journals A Computational Study of the Closed and Open States of the Influenza A M2 Proton Channel

2005 ◽  
Vol 89 (4) ◽  
pp. 2402-2411 ◽  
Author(s):  
Yujie Wu ◽  
Gregory A. Voth
Keyword(s):  
Influenza A ◽  
Computational Study ◽  
Proton Channel ◽  
M2 Proton Channel ◽  
Open States

MC1568 inhibits HDAC6/8 activity and influenza A virus replication in lung epithelial cells: role of Hsp90 acetylation

2016 ◽  
Vol 8 (17) ◽  
pp. 2017-2031 ◽  
Author(s):  
Simona Panella ◽  
Maria Elena Marcocci ◽  
Ignacio Celestino ◽  
Sergio Valente ◽  
Clemens Zwergel ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Lung Epithelial Cells ◽  
Lung Epithelial

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells

1985 ◽  
Vol 5 (9) ◽  
pp. 2181-2189
Author(s):  
L V Jones ◽  
R W Compans ◽  
A R Davis ◽  
T J Bos ◽  
D P Nayak
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Surface Expression ◽  
African Green Monkey ◽  
Monkey Kidney ◽  
Kidney Cells ◽  
African Green Monkey Kidney ◽  
Amino Terminal ◽  
Green Monkey ◽  
Na Gene

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

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