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

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, USA.
Journal of Virology (Impact Factor: 4.44). 09/2011; 86(2):1277-81. DOI: 10.1128/JVI.05681-11
Source: PubMed


The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmembrane domain
were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter cytosolic pH
to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious virus production
to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to that of M2, sequences
in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.

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    • "CM2 is an ion channel protein, which is also involved in packaging of vRNPs during virus assembly, and release of vRNPs during virus uncoating [86,87]. CM2 is believed to be structurally and functionally equivalent to proteins M2 of influenza A and NB of influenza B [88,89]. "
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    ABSTRACT: Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection. All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure. When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3[prime] splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.
    Full-text · Article · Jan 2014 · BMC Research Notes
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    • "A recombinant influenza C virus lacking CM2 palmitoylation had no defects in growth properties [14], whereas the growth of a CM2 glycosylation-deficient influenza C virus was impaired [15]. A chimeric influenza A virus M2 protein containing the CM2 transmembrane domain, not authentic CM2, could partially restore the infectious virus production of an M2-deficient influenza A virus [16]. Taken together, the role(s) of CM2 in virus replication remains to be fully elucidated, particularly in terms of the contribution of proton and Cl– permeabilities to the virus replication. "
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    ABSTRACT: CM2 is the second membrane protein of influenza C virus and possesses three conserved cysteines at residue 1, 6 and 20 in its extracellular domain, all of which are involved in the formation of disulfide-linked oligomers of the molecule. In the present study, to examine the effect of CM2 oligomerization on virus replication, we generated a mutant recombinant virus, rC1620A, in which all three cysteines on CM2 were substituted to alanines. The rC1620A virus was more attenuated than the recombinant wild-type (rWT) virus in cultured cells. The CM2 protein synthesized in rC1620A-infected cells could not apparently be detected as a tetramer and was transported to the cell surface less efficiently than was authentic CM2. The amount of CM2 protein incorporated into the rC1620A virions was comparable to that into the rWT virions, although the main CM2 species in the rC1620A virions was in the form of a dimer. Analyses of one-step grown virions and virus-infected cells could not provide evidence for any difference in growth between rC1620A and rWT. On the other hand, the amount of genome present in VLPs possessing the mutant CM2 (C1620A-VLPs) was approximately 31% of that in VLPs possessing wild-type CM2 (WT-VLPs). The incoming genome from VLPs was less efficiently transported to the nucleus in the C1620A-VLP-infected cells than in WT-VLP-infected cells, leading to reduced reporter gene expression in the C1620A-VLP-infected cells. Taken together, these findings demonstrate that CM2 oligomerization affects the packaging and uncoating processes. Thus, we concluded that disulfide-linked CM2 oligomers facilitate virus growth by affecting the replication processes.
    Preview · Article · Apr 2013 · PLoS ONE
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    • "Moreover, the fact that the incoming GFP-vRNA was transported to the nucleus less efficiently in N11A-VLP-infected cells than WT-VLP-infected cells (Fig. 5D) supports the idea that the uncoating of N11A-VLPs is impaired. The glycosylation-deficient CM2 has recently shown to possess the ability to reduce pH of cytoplasm (Stewart and Pekosz, 2012) although it remains unknown whether the activity of the glycosylation-deficient CM2 is similar to that of authentic CM2. A number of studies showed that glycosylation of channel proteins directly influences the gating characteristics and pH sensitivity of the channel (Freeman et al., 2000; Montpetit et al., 2009; Wirkner et al., 2008). "
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    ABSTRACT: CM2 is the second membrane protein of influenza C virus and possesses a conserved motif for N-glycosylation. To investigate the role(s) of CM2 glycosylation in the virus replication, we generated rN11A, a recombinant influenza C virus lacking the glycosylation site. The rN11A virus grew less efficiently than the wild-type (WT) virus, although the biochemical characteristics of the mutant CM2 were similar to those of authentic CM2. The amount of the genome (GFP-vRNA) in the CM2-N11A-virus-like particles (VLPs) was 13% of that found in WT-VLPs. The incoming GFP-vRNA was less efficiently transported to the nucleus in CM2-N11A-VLP-infected cells than WT-VLP-infected cells, leading to the reduced reporter gene expression in CM2-N11A-VLP-infected cells. Thus the glycosylation of CM2 is required for efficient replication of influenza C virus, and the obtained findings confirmed and extended the previous observation that CM2 is involved in the genome packaging and uncoating processes.
    Preview · Article · Aug 2012 · Virology
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