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M2 proton channel

(Redirected from Influenza virus matrix protein 2)

The Matrix-2 (M2) protein is a proton-selective viroporin, integral in the viral envelope of the influenza A virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds, and is activated by low pH. The M2 protein is encoded on the seventh RNA segment together with the M1 protein. Proton conductance by the M2 protein in influenza A is essential for viral replication.

3D model of the flu virion. (M2 labeled in white.)

Influenza B and C viruses encode proteins with similar function dubbed "BM2" and "CM2" respectively. They share little similarity with M2 at the sequence level, despite a similar overall structure and mechanism.[1]

Structure

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Flu_M2
 
the closed state structure of m2 protein h+ channel by solid state nmr spectroscopy
Identifiers
SymbolFlu_M2
PfamPF00599
InterProIPR002089
SCOP21mp6 / SCOPe / SUPFAM
TCDB1.A.19
OPM superfamily185
OPM protein2kqt
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

In influenza A virus, M2 protein unit consists of three protein segments comprising 97 amino acid residues: (i) an extracellular N-terminal domain (residues 1–23); (ii) a transmembrane segment (TMS) (residues 24–46); (iii) an intracellular C-terminal domain (residues 47–97). The TMS forms the pore of the ion channel. The important residues are the imidazole of His37 (pH sensor) and the indole of Trp41 (gate).[2] This domain is the target of the anti influenza drugs, amantadine and its ethyl derivative rimantadine, and probably also the methyl derivative of rimantadine, adapromine. The first 17 residues of the M2 cytoplasmic tail form a highly conserved amphipathic helix.[3]

The amphipathic helix residues (46–62) within the cytoplasmic tail play role in virus budding and assembly. The influenza virus utilizes these amphipathic helices in M2 to alter membrane curvature at the budding neck of the virus in a cholesterol dependent manner.[4] The residues 70–77 of cytoplasmic tail are important for binding to M1 and for the efficient production of infectious virus particles. This region also contains a caveolin binding domain (CBD). The C-terminal end of the channel extends into a loop (residues 47–50) that connects the trans membrane domain to the C-terminal amphipathic helix. (46–62). Two different high-resolution structures of truncated forms of M2 have been reported: the crystal structure of a mutated form of the M2 transmembrane region (residues 22–46),[5] as well as a longer version of the protein (residues 18–60) containing the transmembrane region and a segment of the C-terminal domain as studied by nuclear magnetic resonance (NMR).[6]

The two structures also suggest different binding sites for the adamantane class of anti-influenza drugs. According to the low pH crystal structure a single molecule of amantadine binds in the middle of the pore, surrounded by residues Val27, Ala30, Ser31 and Gly34. In contrast, the NMR structure showed four rimantadine molecules bind to the lipid facing outer surface of the pore, interacting with residues Asp44 and Arg45. However, a recent solid state NMR spectroscopy structure shows that the M2 channel has two binding sites for amantadine, one high affinity site is in the N terminal lumen, and a second low affinity site on the C terminal protein surface.[7]

Proton conductance and selectivity

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The M2 ion channel of both influenza A is highly selective for protons. The channel is activated by low pH and has a low conductance.[8] Histidine residues at position 37 (His37) are responsible for this proton selectivity and pH modulation. When His37 is replaced with glycine, alanine, glutamic acid, serine or threonine, the proton selective activity is lost and the mutant can transport Na+ and K+ ions also. When imidazole buffer is added to cells expressing mutant proteins, the ion selectivity is partially rescued.[9]

Acharya et al. suggested that the conduction mechanism involves the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior.[10] Water molecules within the pore form hydrogen-bonded networks or 'water wires' from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium ions via second-shell interactions involving bridging water molecules. A collective switch of hydrogen bond orientations may contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.[11] The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms near by. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters.

Function

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The M2 channel protein is an essential component of the viral envelope because of its ability to form a highly selective, pH-regulated, proton-conducting channel. The M2 proton channel maintains pH across the viral envelope during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. As virus enters the host cell by receptor-mediated endocytosis, endosomal acidification occurs. This low pH activates the M2 channel, which brings protons into the virion core. Acidification of virus interior leads to weakening of electrostatic interaction and leads to dissociation between M1 and viral ribonucleoprotein (RNP) complexes. Subsequent membrane fusion releases the uncoated RNPs into the cytoplasm which is imported to the nucleus to start viral replication.

After its synthesis within the infected host cell, M2 is inserted into the endoplasmic reticulum (ER) and transported to the cell surface via trans-Golgi network (TGN). Within the acidic TGN, M2 transports H+ ions out of the lumen, and maintains hemagglutinin (HA) metastable configuration.[12] At its TGN localization, M2 protein's ion channel activity has been shown to effectively activate the NLRP3 inflammasome pathway.[13]

Other important functions of M2 are its role in formation of filamentous strains of influenza, membrane scission and the release of the budding virion. M2 stabilizes the virus budding site, and mutations of M2 that prevent its binding to M1 can impair filament formation at the site of budding.

Transport reaction

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The generalized transport reaction catalyzed by the M2 channel is:

H+ (out) ⇌ H+ (in)

Inhibition and resistance

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The transmembrane helical tetramer of the influenza A virus M2 protein in complex with the channel-blocking drug amantadine (shown in red). Highly conserved tryptophan and histidine residues known to play key roles in mediating proton transport are shown as sticks. From PDB: 3C9J​.[14]

The anti-influenza virus drug, amantadine, is a specific blocker of the M2 H+ channel. The drug binds in and occludes the central pore.[14] In the presence of amantadine, viral uncoating and disassembly is incomplete.[15] Mutations conferring resistance to adamantane drugs, including amantadine and rimantadine, occur in the transmembrane region and are widespread. The large majority of resistant viruses carry the S31N mutation.[16] Resistance to adamantanes among circulating influenza A viruses varies by region but has globally increased significantly since the early 2000s.[16][17] The US CDC has released information stating that most circulating strains are now resistant to the two drugs available, and as of June 2021, their use is not recommended.[18]

Influenza B and C M2 proteins

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M2 proton channel
Identifiers
SymbolFlu_B_M2
PfamPF04772
InterProIPR006859
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDBPDB: 2KJ1​
CM2
Identifiers
SymbolCM2
PfamPF03021
InterProIPR004267
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

Influenza B and C viruses encode virion proteins with similar proton-transducing function dubbed "BM2" and "CM2" respectively. They share little similarity with M2 at the sequence level, despite a similar overall structure and mechanism.[1][19]

BM2

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The M2 protein of influenza B is 109 residue long, homo-tetramer and is a functional homolog of influenza A protein. There is almost no sequence homology between influenza AM2 and BM2 except for the HXXXW sequence motif in the TMS that is essential for channel function. Its proton conductance pH profile is similar to that of AM2. However, the BM2 channel activity is higher than that of AM2, and the BM2 activity is completely insensitive to amantadine and rimantadine.[1] The structure of the influenza B channel at resolutions of 1.4–1.5 Å, published in 2020, revealed that the channel opening mechanism is different from that of the influenza A channel.[20]

CM2

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CM2 may play a role in genome packaging in virions.[21] CM2 adjusts intracellular pH, and is able to replace influenza A M2 in this capacity.[22]

See also

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References

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  1. ^ a b c Pielak RM, Chou JJ (February 2011). "Influenza M2 proton channels". Biochimica et Biophysica Acta (BBA) - Biomembranes. 1808 (2): 522–9. doi:10.1016/j.bbamem.2010.04.015. PMC 3108042. PMID 20451491.
  2. ^ Tang Y, Zaitseva F, Lamb RA, Pinto LH (October 2002). "The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue". The Journal of Biological Chemistry. 277 (42): 39880–6. doi:10.1074/jbc.M206582200. PMID 12183461.
  3. ^ Holsinger LJ, Nichani D, Pinto LH, Lamb RA (March 1994). "Influenza A virus M2 ion channel protein: a structure-function analysis". Journal of Virology. 68 (3): 1551–63. doi:10.1128/JVI.68.3.1551-1563.1994. PMC 236612. PMID 7508997.
  4. ^ Rossman JS, Jing X, Leser GP, Lamb RA (September 2010). "Influenza virus M2 protein mediates ESCRT-independent membrane scission". Cell. 142 (6): 902–13. doi:10.1016/j.cell.2010.08.029. PMC 3059587. PMID 20850012.
  5. ^ Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, Tereshko V, Nanda V, Stayrook S, DeGrado WF (January 2008). "Structural basis for the function and inhibition of an influenza virus proton channel". Nature. 451 (7178): 596–9. Bibcode:2008Natur.451..596S. doi:10.1038/nature06528. PMC 3889492. PMID 18235504.
  6. ^ Schnell JR, Chou JJ (January 2008). "Structure and mechanism of the M2 proton channel of influenza A virus". Nature. 451 (7178): 591–5. Bibcode:2008Natur.451..591S. doi:10.1038/nature06531. PMC 3108054. PMID 18235503.
  7. ^ Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrado WF, Hong M (February 2010). "Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers". Nature. 463 (7281): 689–92. Bibcode:2010Natur.463..689C. doi:10.1038/nature08722. PMC 2818718. PMID 20130653.
  8. ^ Mould JA, Li HC, Dudlak CS, Lear JD, Pekosz A, Lamb RA, Pinto LH (March 2000). "Mechanism for proton conduction of the M(2) ion channel of influenza A virus". The Journal of Biological Chemistry. 275 (12): 8592–9. doi:10.1074/jbc.275.12.8592. PMID 10722698.
  9. ^ Venkataraman P, Lamb RA, Pinto LH (June 2005). "Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus". The Journal of Biological Chemistry. 280 (22): 21463–72. doi:10.1074/jbc.M412406200. PMID 15784624.
  10. ^ Acharya R, Carnevale V, Fiorin G, Levine BG, Polishchuk AL, Balannik V, Samish I, Lamb RA, Pinto LH, DeGrado WF, Klein ML (August 2010). "Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus". Proceedings of the National Academy of Sciences of the United States of America. 107 (34): 15075–80. Bibcode:2010PNAS..10715075A. doi:10.1073/pnas.1007071107. PMC 2930543. PMID 20689043.
  11. ^ Thomaston JL, Alfonso-Prieto M, Woldeyes RA, Fraser JS, Klein ML, Fiorin G, DeGrado WF (November 2015). "High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction". Proceedings of the National Academy of Sciences of the United States of America. 112 (46): 14260–5. Bibcode:2015PNAS..11214260T. doi:10.1073/pnas.1518493112. PMC 4655559. PMID 26578770.
  12. ^ Sakaguchi T, Leser GP, Lamb RA (May 1996). "The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus". The Journal of Cell Biology. 133 (4): 733–47. doi:10.1083/jcb.133.4.733. PMC 2120830. PMID 8666660.
  13. ^ Ichinohe T, Pang IK, Iwasaki A (May 2010). "Influenza virus activates inflammasomes via its intracellular M2 ion channel". Nature Immunology. 11 (5): 404–10. doi:10.1038/ni.1861. PMC 2857582. PMID 20383149.
  14. ^ a b Thomaston JL, Alfonso-Prieto M, Woldeyes RA, Fraser JS, Klein ML, Fiorin G, DeGrado WF (November 2015). "High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction". Proceedings of the National Academy of Sciences of the United States of America. 112 (46): 14260–5. Bibcode:2015PNAS..11214260T. doi:10.1073/pnas.1518493112. PMC 4655559. PMID 26578770.
  15. ^ Greber UF, Singh I, Helenius A (February 1994). "Mechanisms of virus uncoating" (PDF). Trends in Microbiology. 2 (2): 52–6. doi:10.1016/0966-842X(94)90126-0. PMID 8162442.
  16. ^ a b Dong G, Peng C, Luo J, Wang C, Han L, Wu B, et al. (2015-03-13). "Adamantane-resistant influenza a viruses in the world (1902-2013): frequency and distribution of M2 gene mutations". PLOS ONE. 10 (3): e0119115. Bibcode:2015PLoSO..1019115D. doi:10.1371/journal.pone.0119115. PMC 4358984. PMID 25768797.
  17. ^ Deyde VM, Xu X, Bright RA, Shaw M, Smith CB, Zhang Y, et al. (July 2007). "Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide". The Journal of Infectious Diseases. 196 (2): 249–57. doi:10.1086/518936. PMID 17570112.
  18. ^ "Influenza Antiviral Medications: Summary for Clinicians". Centers for Disease Control and Prevention. 6 May 2021. Retrieved 14 June 2021.
  19. ^ Kukol A, Arkin IT (February 2000). "Structure of the influenza C virus CM2 protein transmembrane domain obtained by site-specific infrared dichroism and global molecular dynamics searching". The Journal of Biological Chemistry. 275 (6): 4225–9. doi:10.1074/jbc.275.6.4225. PMID 10660588.
  20. ^ Mandala VS, Loftis AR, Shcherbakov AA, Pentelute BL, Hong M (February 2020). "Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism". Nature Structural & Molecular Biology. 27 (2): 160–167. doi:10.1038/s41594-019-0371-2. PMC 7641042. PMID 32015551. S2CID 211017938.
  21. ^ Furukawa T, Muraki Y, Noda T, Takashita E, Sho R, Sugawara K, Matsuzaki Y, Shimotai Y, Hongo S (February 2011). "Role of the CM2 protein in the influenza C virus replication cycle". Journal of Virology. 85 (3): 1322–9. doi:10.1128/JVI.01367-10. PMC 3020500. PMID 21106743.
  22. ^ Stewart SM, Pekosz A (January 2012). "The influenza C virus CM2 protein can alter intracellular pH, and its transmembrane domain can substitute for that of the influenza A virus M2 protein and support infectious virus production". Journal of Virology. 86 (2): 1277–81. doi:10.1128/JVI.05681-11. PMC 3255851. PMID 21917958.
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