Palmitoylation of the influenza a virus M2 protein

doi:10.1016/0042-6822(90)90272-S

Virology

Volume 179, Issue 1, November 1990, Pages 51-56

https://doi.org/10.1016/0042-6822(90)90272-S Get rights and content

Abstract

The M2 proteins of a variety of influenza A viruses of different subtypes were shown to possess associated palmitate. Susceptibility to removal by reduction or treatment with hydroxylamine is consistent with attachment via a thioester linkage to cysteine. The absence of the acyl group from the M2 proteins of several equine viruses of the H3N8 subtype correlates with the replacement of cysteine 50 with phenylalanine and points to this as the site of palmitate attachement.

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Cited by (79)

S-acylation of influenza virus proteins: Are enzymes for fatty acid attachment promising drug targets?
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Covalent attachment of saturated fatty acids (palmitate and stearate) to hemagglutinin (HA) of influenza virus is a protein modification essential for viral replication. The enzymes catalysing acylation of viral proteins have not been identified, but likely candidates that acylate cellular substrates are members of a protein family that contain a DHHC (Asp–His–His–Cys) cysteine-rich domain. Since 23 DHHC-proteins with distinct, only partly overlapping substrate specificities are present in humans, only a few of them might acylate HA in airway cells of the lung. We argue here that these DHHC-proteins might be promising drug targets since their blockade should result in suppression of viral replication, while acylation of cellular proteins will not be (or very little) compromised.
Acylation and cholesterol binding are not required for targeting of influenza A virus M2 protein to the hemagglutinin-defined budozone
2014, FEBS Letters
Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells.
Mechanism of Function of Viral Channel Proteins and Implications for Drug Development
2012, International Review of Cell and Molecular Biology
Citation Excerpt :
M2 assembles into cystein‐linked homodimers which noncovalently associate into homotetramers (Holsinger and Lamb, 1991; Sugrue and Hay, 1991). Some of the M2 proteins of various subtypes have been identified to be palmitylated at Cys-50 (Sugrue et al., 1990). Amantadine resistance mutant studies of influenza viruses correlated resistance to mutations in the seventh segment, especially to the TMD of M2 (Hay et al., 1985).
Viral channel-forming proteins comprise a class of viral proteins which, similar to their host companions, are made to alter electrochemical or substrate gradients across lipid membranes. These proteins are active during all stages of the cellular life cycle of viruses. An increasing number of proteins are identified as channel proteins, but the precise role in the viral life cycle is yet unknown for the majority of them. This review presents an overview about these proteins with an emphasis on those with available structural information.
A concept is introduced which aligns the transmembrane domains of viral channel proteins with those of host channels and toxins to give insights into the mechanism of function of the viral proteins from potential sequence identities. A summary of to date investigations on drugs targeting these proteins is given and discussed in respect of their mode of action in vivo.
Palmitoylation of CM2 is dispensable to influenza C virus replication
2011, Virus Research
Citation Excerpt :
Palmitoylation of the influenza B HA is involved in membrane fusion, particularly in fusion pore dilation (Ujike et al., 2004, 2005). The influenza A virus M2 protein is modified by palmitic acid through a labile thioester linkage (Sugrue et al., 1990; Veit et al., 1991), and the cysteine residue 50 of the M2 protein was demonstrated to be the palmitoylation site (Holsinger et al., 1995). Using a recombinant influenza A virus lacking M2 palmitoylation, in which the genomes were composed of seven segments from an H3N8 virus (A/Equine/Miami/63) and the M segment from an H1N1 virus (A/Puerto Rico/8/34), Castrucci et al. (1997) reported that M2 palmitoylation was dispensable to virus replication both in vitro and in vivo.
CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.
Ion channel regulation by protein palmitoylation
2011, Journal of Biological Chemistry
Citation Excerpt :
Little is known about which of the potential palmitoyl acyltransferases (gene family zDHHC) control ion channel palmitoylation, although analysis of both NMDA (22) and BK (51) channels has suggested that distinct palmitoylated domains on the same polypeptide can be regulated by different zDHHCs. It has been more than 20 years since the first palmitoylated ion channels were discovered (18, 52). The recent development of new bioinformatics and proteomics tools, together with the identification of zDHHCs, is starting to revolutionize our understanding of ion channel palmitoylation.
Protein S-palmitoylation, the reversible thioester linkage of a 16-carbon palmitate lipid to an intracellular cysteine residue, is rapidly emerging as a fundamental, dynamic, and widespread post-translational mechanism to control the properties and function of ligand- and voltage-gated ion channels. Palmitoylation controls multiple stages in the ion channel life cycle, from maturation to trafficking and regulation. An emerging concept is that palmitoylation is an important determinant of channel regulation by other signaling pathways. The elucidation of enzymes controlling palmitoylation and developments in proteomics tools now promise to revolutionize our understanding of this fundamental post-translational mechanism in regulating ion channel physiology.
Solution NMR structure of the V27A drug resistant mutant of influenza A M2 channel
2010, Biochemical and Biophysical Research Communications
Citation Excerpt :
Another interesting detail revealed by the V27A structure is the position of the Ser50 side chain. The native residue at position 50 is cysteine, which is palmitoylated in the virus [21]. This cysteine was mutated to serine in our structural investigation to avoid problems associated with potential disulfide bond formation.
The M2 protein of influenza A virus forms a proton-selective channel that is required for viral replication. It is the target of the anti-influenza drugs, amantadine and rimantadine. Widespread drug resistant mutants, however, has greatly compromised the effectiveness of these drugs. Here, we report the solution NMR structure of the highly pathogenic, drug resistant mutant V27A. The structure reveals subtle structural differences from wildtype that maybe linked to drug resistance. The V27A mutation significantly decreases hydrophobic packing between the N-terminal ends of the transmembrane helices, which explains the looser, more dynamic tetrameric assembly. The weakened channel assembly can resist drug binding either by destabilizing the rimantadine-binding pocket at Asp44, in the case of the allosteric inhibition model, or by reducing hydrophobic contacts with amantadine in the pore, in the case of the pore-blocking model. Moreover, the V27A structure shows a substantially increased channel opening at the N-terminal end, which may explain the faster proton conduction observed for this mutant. Furthermore, due to the high quality NMR data recorded for the V27A mutant, we were able to determine the structured region connecting the channel domain to the C-terminal amphipathic helices that was not determined in the wildtype structure. The new structural data show that the amphipathic helices are packed much more closely to the channel domain and provide new insights into the proton transfer pathway.

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Palmitoylation of the influenza a virus M2 protein

Abstract

FEBS Lett.

FEBS Lett.

J. Biol. Chem.

Virology

Biologic potential of amantadine-resistant influenza A virus in an avian model

J. Infect. Dis.

Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection

J. Virol.

Drug resistance and the mechanisms of action on influenza A virus

J. Respir. Dis. Suppl.

Characterisation of a gene coding for M proteins which is involved in host-range restriction of an avian influenza virus in monkeys

J. Virol.

Uncoating of a rimantadine-resistant variant of influenza virus in the presence of rimantadine

J. Gen. Virol.

Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatis virus

J. Virol.

Acylation of viral and eukaryotic proteins

Biochem. J.

The mechanism of action of amantadine and rimantadine against influenza viruses