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Phosphorylation of the M2 Protein of Influenza a Virus Is Not Essential for Virus Viability

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Phosphorylation of the M2 Protein of Influenza a Virus Is Not Essential for Virus Viability VIROLOGY 252, 54±64 (1998) ARTICLE NO. VY989384 Phosphorylation of the M2 Protein of Influenza A Virus Is Not Essential for Virus Viability Joanne M. Thomas, Mark P. Stevens, Neil Percy,1 and Wendy S. Barclay2 School of Animal and Microbial Sciences, University of Reading, Whiteknights, UK RG6 6AJ Received May 14, 1998; returned to author for revision June 12, 1998; accepted August 18, 1998 M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel. Using an exogenous incorporation assay, we have shown that incorporation of M2 into virus particles is type-specific and does not require phosphorylation of the cytoplasmic tail. In addition, phosphorylation of the cytoplasmic tail is not required for the directional transport of M2 in polarized MDCK cells. Using a reverse genetics and reassortment procedure, we generated a virus (Ra) specifically mutated in segment 7 such that the M2 cytoplasmic tail could no longer be phosphorylated. The virus was found to grow as well as wild-type virus in tissue culture and in eggs, was stable on passage in these systems, and possessed no second-site mutations in the engineered RNA segment. In vivo Ra replicated in Balb/c mice at least as well as the parent strain A/WSN/33. These studies indicate that phosphorylation of the M2 cytoplasmic tail is not required for in vitro or in vivo replication of influenza A virus. © 1998 Academic Press INTRODUCTION tion for the cytoplasmic tail of 54 amino acids has yet to The M2 protein of influenza A virus is a small integral be assigned, although the length and sequence of this membrane protein and a minor component of the virion region are well conserved among influenza A virus iso- envelope (Lamb et al., 1985). M2 possesses an ion chan- lates (Fig. 1a), suggesting that an evolutionary pressure nel activity that is activated in the low pH environment of exists to maintain them. In particular, this region contains the endosome after the virus enters the cell (Hay et al., a number of serine residues, which serve as sites for 1992; Pinto et al., 1992). The consequent acidification of phosphorylation, and a cysteine at residue 50, which the virion interior stimulates the dissociation of the inter- receives fatty acid modification (see Fig. 1a). However, nal virus components in preparation for replication (Mar- neither of these posttranslational modifications is re- tin and Helenius, 1991). Influenza B and C viruses pos- quired for the activity of the ion channel (Holsinger et al., sess similar small membrane proteins, termed NB and 1995; Pinto et al., 1992). The extreme carboxyl-terminal CM2, respectively (Betakova et al., 1996; Brassard et al., four residues are completely conserved, and deletion of 1996; Hongo et al., 1997; Pekosz and Lamb, 1997), which a single amino acid from this terminus results in virus may be functionally homologous to M2. However, these attenuation. Furthermore, it has not been possible to proteins share no sequence homology and are encoded recover viruses with five or 10 residues deleted from the by different strategies (Shaw and Choppin, 1984; Shih C terminus (Castrucci and Kawaoka, 1995). In contrast, and Krug, 1996; Yamashita et al., 1988). the cytoplasmic tails of the other two virion envelope M2 is translated from a spliced message derived from proteins, haemagglutinin (HA) and neuraminidase (NA), RNA segment 7, which also encodes the matrix protein can be deleted both individually or simultaneously with- M1 (Lamb et al., 1981). The M2 protein has an amino- out loss of virus viability (Garcia-Sastre and Palese, 1995; terminal ectodomain of 25 residues, which was recently Jin et al., 1997). Incorporation of HA into influenza parti- shown to be important for incorporation of the protein cles is not a type-specific event because influenza A into the influenza virus envelope (Park et al., 1998). The virus HA proteins are incorporated into influenza B virus transmembrane domain of 19 hydrophobic residues pos- particles after double infection of cells (Sklyanskaya et sesses the ion channel activity that can be blocked by al., 1985). Whether the NA proteins also show phenotypic the antiviral agent amantadine (Duff et al., 1994). A func- mixing is not known, although it is noteworthy that an influenza A virus has been recovered in which the se- quence of the cytoplasmic tail of NA was exchanged for 1 Present address: Celsis Limited, Cambridge Science Park, Milton that of an influenza B virus NA protein (Bilsel et al., 1993). Road, Cambridge, UK CB4 4FX. 2 To whom reprint requests should be addressed. Fax: 44 (1189) The incorporation of M2 or NB proteins into heterotypic 316671. E-mail: [email protected]. particles has not previously been investigated. 0042-6822/98 $25.00 Copyright © 1998 by Academic Press 54 All rights of reproduction in any form reserved. INFLUENZA A VIRUS M2 PHOSPHORYLATION 55 FIG. 1. (a) Amino acid sequence alignment of the cytoplasmic tail domain of a number of M2 proteins. p, The site of fatty acid modification at cysteine 50. Serines that serve as sites for phosphorylation are indicated by arrows. Swiss-prot accession numbers are shown in bold. (b) Amino acid sequences of the cytoplasmic tails of A/WSN/33 M2 and the phosphorylation mutant M2 proteins P-2 and P-1-2. During influenza A virus infection, high levels of M2 tained only unphosphorylated M2 could be isolated us- accumulate at the surface of infected cells, yet the pro- ing a reverse genetics procedure. tein is present at low frequency in the virus envelope (Zebedee and Lamb, 1988). This discrepancy implies that RESULTS only a subset of M2 molecules within the cell are incor- porated into particles. In the current study, we asked Incorporation of exogenous M2 proteins into virus whether phosphorylation of the M2 cytoplasmic tail reg- particles ulates incorporation of M2 molecules into particles and To detect incorporation of M2 protein into virus parti- hence controls viral assembly. We tested whether un- cles, we took advantage of the fact that the A/WSN/33 phosphorylated M2 protein would be incorporated into M2 migrates more slowly than M2 of A/PR/8/34 (Panay- influenza virus particles in an exogenous incorporation otov and Schlesinger, 1992) under nonreducing SDS± assay. In parallel, we examined whether the incorpora- PAGE. We established a Madin±Darby canine kidney tion of M2 is type-specific by asking whether M2 can be (MDCK) cell line that stably expresses the A/WSN/33 M2 incorporated into influenza B virus particles. We have protein. M2 expression at the cell surface was detected also ascertained whether the phosphorylation status of by immunohistochemical staining using the 14C2 mono- M2 affects its directional transport in polarized cells clonal antibody as primary antibody. The levels of ex- because phosphorylation of other proteins has been pression of M2 protein in these cells were similar to implicated in their targeting (Casanova et al., 1990; Jones those found late in infection (Fig. 2a, lanes 6 and 7). et al., 1995). We next infected these cells with the A/PR/8/34 strain Finally, bearing in mind the acute sensitivity of the of influenza A virus at a high m.o.i. and analyzed the virus to deletions of the M2 cytoplasmic tail and the released virus for incorporation of exogenous M2 protein conservation of the posttranslational modifications in by Western blot, also using 14C2 (Fig. 2a). In virus re- this domain, we investigated whether a virus that con- leased from cells expressing the exogenous M2 protein, 56 THOMAS ET AL. tein NB was readily seen (Fig. 2b, lanes 1 and 2). NB is visible as a single species in this experiment because the glycosylated species in released virus have been digested with endo-F and the B/Lee/40 infected cell lysate used as a control was generated in the presence of tunicamycin. These results demonstrate that incorpo- ration of M2 protein into influenza virus particles is a type-specific event. The primary site for phosphate addition on the A/Udorn/72 M2 cytoplasmic tail is at serine residue 64 (Holsinger et al., 1995). This residue, and the surrounding consensus for modification, is conserved in the M2 se- quences accumulated to date (Fig. 1a). It has been dem- onstrated that modified M2 proteins. which are unable to phosphorylate serine 64, use other serines for phosphor- ylation (Holsinger et al., 1995). The A/WSN/33 M2 protein contains two serines in the cytoplasmic domain at posi- tions 64 and 71. To investigate the role of phosphate moieties on the M2 cytoplasmic tail, we established stable and clonal cell lines that expressed M2 proteins mutated (by serine-to-alanine substitutions) at serine 71 (P-2) and at both serines 64 and 71 (P-1±2) (Fig. 1b). It is interesting to note that we were not able to establish a cell line expressing M2 mutated at serine 64 alone (P-1), although we could detect expression of this protein after transient transfection (data not shown). After infection of cells expressing either the P-2 or P-1±2 mutant M2 proteins, we monitored the M2 species in released virus by Western blotting. Only tetrameric FIG. 2. Analysis of purified A/PR/8/34 and B/Lee/40 virus particles to detect incorporation of exogenous A/WSN/33 M2. (a) Purified A/PR8/34 virus (lanes 1 and 2) or B/Lee/40 virus (lanes 3 and 4) were analyzed for incorporation of exogenous M2 by 15% SDS±PAGE under nonreducing conditions followed by Western blot using the 14C2 monoclonal anti- body.
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