The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Virology (Impact Factor: 3.32). 09/2010; 405(2):530-8. DOI: 10.1016/j.virol.2010.06.035
Source: PubMed

ABSTRACT Influenza A virus particles assemble and bud from plasma membrane domains enriched with the viral glycoproteins but only a small fraction of the total M2 protein is incorporated into virus particles when compared to the other viral glycoproteins. A membrane proximal cholesterol recognition/interaction amino acid consensus (CRAC) motif was previously identified in M2 and suggested to play a role in protein function. We investigated the importance of the CRAC motif on virus replication by generating recombinant proteins and viruses containing amino acid substitutions in this motif. Alteration or completion of the M2 CRAC motif in two different virus strains caused no changes in virus replication in vitro. Viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection, suggesting that this motif is a virulence determinant which may facilitate virus replication in vivo but is not required for basic virus replication in tissue culture.

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Available from: Andrew Pekosz, Sep 29, 2015
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    • "There, raft-phase partitioning of M2 depends on acylation, but not on the cholesterol-binding motifs [29]. Surprisingly, however , mutating M2's raft-targeting features acylation and/or cholesterol binding does not compromise virus growth in cell culture [30] [31] [32] [33] [34]. Since virus budding is likely to be a redundant process, we assumed that in the viral context, other proteins might compensate the targeting failure of M2. "
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    ABSTRACT: 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.
    FEBS letters 02/2014; 588(6). DOI:10.1016/j.febslet.2014.02.014 · 3.17 Impact Factor
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    • "However, attenuation of virus infectivity was observed in mice both for virus with non-acylated (Grantham et al., 2009) and CRAC-disrupted M2 (Stewart et al., 2010). "
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    ABSTRACT: Influenza virus is thought to assemble in raft-domains of the plasma membrane, but many of the conclusions were based on (controversial) Triton-extraction experiments. Here we review how sophisticated methods of fluorescence microscopy, such as FPALM, FRET and FRAP, contributed to our understanding of lipid domain association of the viral proteins HA and M2. The results are summarized in light of the current model for virus assembly and lipid domain organization. Finally, it is described how the signals that govern domain association in transfected cells affect replication of influenza virus. For a more comprehensive treatment of raft-association of influenza virus proteins and budding of viral particles the reader is referred to several recent reviews (Chen et al., 2008a, Nayak et al., 2009, Nayak et al., 2004, Rossman et al., 2011, Veit et al., 2011).
    Cellular Microbiology 10/2012; 15(2). DOI:10.1111/cmi.12045 · 4.92 Impact Factor
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    • "However, the proportions of the major structural proteins, especially the ratio of HA : M1, was the same in wild-type WSN and the mutants, as illustrated with radioactively labelled virions by SDS-PAGE, fluorography and densitometric quantification (Fig. 4a). This is in line with other reports investigating similar mutations (Grantham et al., 2009; Rossman et al., 2010a; Stewart et al., 2010). M2 could not be discerned in the fluorograms in Fig. 4(a) as only minor amounts are incorporated into virions. "
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    ABSTRACT: Influenza virus assembly and budding occur in the 'budozone', a coalesced raft domain in the plasma membrane. The viral transmembrane protein M2 is implicated in virus particle scission, the ultimate step in virus budding, probably by wedge-like insertion of an amphiphilic helix into the membrane. In order to do this, M2 is hypothesized to be targeted to the edge of the budozone, mediated by acylation and cholesterol binding. It was recently shown that acylation and cholesterol binding affect the membrane association of the cytoplasmic tail of M2 and targeting of the protein to coalesced rafts. This study tested whether combined removal of the acylation site (C50) and the cholesterol recognition/interaction amino acid consensus motifs (key residues Y52 and Y57) in the amphiphilic helix of M2 influenced virus formation. Recombinant influenza viruses were generated in the influenza strain A/WSN/33 background with mutations in one or both of these features. In comparison with the wild-type, all mutant viruses showed very similar growth kinetics in various cell types. Wild-type and mutant viruses differed in their relative M2 content but not regarding the major structural proteins. The morphology of the viruses was not affected by mutating M2. Moreover, wild-type and mutant viruses showed comparable competitive fitness in infected cells. Lastly, a global comparison of M2 sequences revealed that there are natural virus strains with M2 devoid of both lipid-association motifs. Taken together, these results indicate that the acylation and cholesterol-binding motifs in M2 are not crucial for the replication of influenza virus in cell culture, indicating that other factors can target M2 to the budding site.
    Journal of General Virology 02/2012; 93(Pt 2):282-92. DOI:10.1099/vir.0.038554-0 · 3.18 Impact Factor
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