Palmitoylation of CM2 is dispensable to influenza C virus replication

Virus Research 157 (2011) 99–105

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Palmitoylation of CM2 is dispensable to influenza C virus replication Yasushi Muraki a,∗ , Takako Okuwa a , Takatoshi Furukawa b , Yoko Matsuzaki b , Kanetsu Sugawara b , Toshiki Himeda a , Seiji Hongo b , Yoshiro Ohara a a b

Department of Microbiology, Kanazawa Medical University School of Medicine, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan Department of Infectious Diseases, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan

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Article history: Received 14 December 2010 Received in revised form 16 February 2011 Accepted 17 February 2011 Available online 23 February 2011 Keywords: Influenza C virus CM2 protein Palmitoylation

a b s t r a c t 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. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Influenza C virus has seven single-stranded RNA segments of negative polarity, encoding PB2, PB1, P3, hemagglutinin-esterasefusion (HEF), nucleoprotein (NP), matrix (M1) protein, CM2, respectively, as well as the non-structural proteins NS1 and NS2 (Palese and Shaw, 2007; Muraki and Hongo, 2010). RNA segment 6 (M gene) of C/Ann Arbor/1/50 is 1180 nucleotides in length and contains a single open reading frame (positions 26–1150) capable of encoding a polypeptide (P42) of 374 amino acids (Hongo et al., 1994, 1998; Muraki et al., 2007). The predominant mRNA transcript of this RNA segment lacks a region from nucleotides 754–981 and encodes a 242-amino-acid matrix (M1) protein (Hongo et al., 1994; Yamashita et al., 1988), whereas unspliced mRNA, synthesized in an amount 1/10 that of the spliced mRNA, is first translated into P42 and then cleaved by a signal peptidase at an internal cleavage site, generating M1 and CM2, which are composed of the N-terminal 259 amino acids and the C-terminal 115 amino acids of P42, respectively (Hongo et al., 1994, 1999; Pekosz and Lamb, 1998). CM2 is the second membrane protein of the virus and is composed of three distinct domains: a 23-residue N-terminal extracellular domain, a 23-residue transmembrane domain, and

∗ Corresponding author. Tel.: +81 76 218 8097; fax: +81 76 286 3961. E-mail address: [email protected] (Y. Muraki). 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.02.013

a 69-residue cytoplasmic domain (Hay, 1998; Hongo et al., 1994; Pekosz and Lamb, 1997). It is abundantly expressed at the plasma membranes of infected cells (Hongo et al., 1997; Pekosz and Lamb, 1997) and is incorporated in a small amount into virions (Hongo et al., 1997). CM2 synthesized in virus-infected cells forms disulfide-linked dimers and tetramers, and is posttranslationally modified by N-glycosylation, palmitoylation and phosphorylation (Hongo et al., 1997; Pekosz and Lamb, 1997; Tada et al., 1998). Three forms of CM2 with different electrophoretic mobilities (CM2o, CM2a and CM2b) are detected in infected cells (Hongo et al., 1997; Pekosz and Lamb, 1997). A mannose-rich oligosaccharide core is added to unglycosylated CM2o (Mr ∼16,000) to form CM2a (Mr ∼18,000), and the maturation of the carbohydrate chain from the high mannose type to the complex type converts CM2a into CM2b (Mr 22,000–30,000). Analyses of a number of CM2 mutants expressed in COS-1 and HeLa cells revealed the positions of the amino acids involved in the posttranslational modifications (Li et al., 2001; Pekosz and Lamb, 1997): an asparagine at residue 11 is an N-glycosylation site, cysteines at residue 1, 6 and 20 are involved in disulfide bond formation, a cysteine residue at 65 is palmitoylated, and serine residues at 78, 103, 108 and a proline at 104 are phosphorylation sites. Evidence was obtained that the N-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface (Pekosz and Lamb, 1997). Li et al. (2001) showed that none of dimer- or tetramer-formation, palmitoylation

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or phosphorylation was essential to the transport of CM2 to the cell surface. On the other hand, all of the above-mentioned amino acids were found to be evolutionally conserved among influenza C virus strains examined to date (Matsuzaki et al., 2003; Tada et al., 1997), suggesting the significance of the modifications in viral replication. Palmitoylation is a common posttranslational modification that can influence protein trafficking and protein–protein and protein–membrane interactions. The hydrophobic acyl chains are attached to cysteine residues located in the cytoplasmic and/or transmembrane regions of viral integral membrane proteins, such as hemagglutinin (HA) of influenza A and B viruses, HEF of influenza C virus, and M2 and CM2 of influenza A and C viruses, respectively (Veit and Schmidt, 2006). Palmitoylation of HA is required for efficient membrane fusion in H7 and H1, but not H3 influenza A virus subtypes (Chen et al., 2005; Sakai et al., 2002; Wagner et al., 2005), whereas palmitoylation of HA is required for virus assembly in H3 but not in H1 subtypes (Chen et al., 2005). 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. Using M2 palmitoylation-deficient recombinants of A/WSN/33 (H1N1) or A/Udorn/72 (H3N2) viruses, Grantham et al. (2009) showed that M2 palmitoylation did not contribute significantly to virus replication in vitro and that A/WSN/33 mutant exhibited a slightly reduced virulence in infected mice. The discrepancy in the results between the two studies could be attributable to differences in the virus strains analyzed. Thus the role of M2 palmitoylation in virus replication requires further study. In the present study, in order to investigate the effect of CM2 palmitoylation on influenza C virus replication, we generated a CM2 palmitoylation-deficient influenza C virus, in which a cysteine at residue 65 of CM2 was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. As a result, evidence was obtained that the palmitoylation of CM2 is dispensable to influenza C virus replication. 2. Materials and methods 2.1. Cells and antibodies 293T cells, the HMV-II line of human malignant melanoma cells, and LLC-MK2 cells were maintained at 37 ◦ C in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), RPMI 1640 medium with 10% calf serum, and minimal essential medium (MEM) with 5% FBS and 5% calf serum, respectively (Muraki et al., 2004; Nishimura et al., 1989). Monoclonal antibodies (MAbs) against the HEF (J14, D37), NP (H27) and M1 (L2) proteins of C/Ann Arbor/1/50 (AA/50), and antisera against the AA/50 virion and the CM2 protein were prepared as described previously (Hongo et al., 1994; Sugawara et al., 1991, 1993; Yokota et al., 1983). 2.2. Plasmid DNAs The seven Pol I plasmids for the expression of viral RNAs (vRNAs) of AA/50 (pPolI/PB2, pPolI/PB1, pPolI/P3, pPolI/HEF, pPolI/NP, pPolI/M and pPolI/NS) were described previously (Muraki et al., 2007). The nine plasmid DNAs for the expression of the influenza

C viral proteins (pcDNA/PB2-AA, pcDNA/PB1-AA, pcDNA/P3AA, pME18S/HEF-AA, pCAGGS.MCS/NP-AA, pCAGGS.MCS/M1-AA, pME18S/Met-CM2-YA, pME18S/NS1-YA, and pME18S/NS2-YA) were also reported previously (Muraki et al., 2004). Plasmid DNA, pPolI/CM2-Acy(−), in which 995-TGT-997 of the M gene was replaced with 995-GCT-997, was constructed based on pPolI/M. Details of the primers and PCR protocols will be provided on request. 2.3. Determination of infectious titers of viruses The infectious titers of the recombinant viruses, the supernatants of recombinant-infected HMV-II cells, and the amniotic fluids recovered from recombinant-infected chicken eggs were determined according to the procedure reported by Matrosovich et al. (2006). Briefly, viruses were serially 10-fold diluted and inoculated onto LLC-MK2 monolayers cultured in a 6-well plate. After incubation at 33 ◦ C for 60 min, 0.9% (w/v) of a microcrystalline cellulose (Avicel RC/CLTM , FMC BioPolymer) suspension in MEM containing 1 ␮g/ml trypsin was added onto the cells and incubated at 33 ◦ C. At 6 days postinfection (p.i.), the cells were fixed with 4% paraformaldehyde for 120 min at 4 ◦ C, followed by incubation in 0.5% Triton-X-100 and 20 mM glycine in PBS for 20 min at room temperature. The cells were then reacted with anti-HEF MAb D37 (primary antibody) and anti-mouse IgG conjugated with HRP (secondary antibody) (BioRad). The virus-infected cells were visualized using True Blue® (KPL) substrate and the number of virus plaques was counted. 2.4. Radioimmunoprecipitation HMV-II cells infected with recombinant viruses were labeled with [35 S]methionine (ARC) (30 ␮Ci/35 mm dish) for 4 h at 48 h p.i. in RPMI 1640 medium lacking methionine, or [3 H]palmitic acid (ARC) (500 ␮Ci/35 mm dish) in RPMI1640 supplemented with 5 mM sodium pyruvate. Cells were then disrupted and subjected to immunoprecipitation with anti-CM2 serum as described previously. (Hongo et al., 1994). The immunoprecipitates obtained were then analyzed by SDS–PAGE on 17.5% gels containing 4 M urea, and processed for fluorography (Yokota et al., 1983). In the pulse-chase experiments, the recombinant virus-infected-HMV-II cells were pulse-labeled with [35 S]methionine for 20 min at 26 h p.i., chased for the indicated periods, and then immunoprecipitated with MAbs against HEF (J14), NP (H27) and M1 (L2), and anti-CM2 serum, followed by SDS–PAGE and fluorography as described above. Flotation analysis was performed according to the procedure described previously (Muraki et al., 2007). Briefly, the virus infected-HMV-II cells were pulse-labeled (500 ␮Ci/35 mm dish) for 15 min at 26 h p.i. and chased for 2.5 h. The cells were disrupted and the resultant postnuclear supernatant was divided into 10 fractions using sucrose equilibrium centrifugation at 180,000 × g for 18 h at 4 ◦ C. The fractions were each immunoprecipitated with anti-AA/50 virion or anti-CM2 serum, followed by SDS–PAGE and fluorography. Band intensities were measured using ImageJ 1.42 q software. 2.5. Calculation of the inner surface area of the amniotic cavity of the chicken eggs Eight- or 9-day-old embryonated chicken eggs were subjected to magnetic resonance (MR) imaging using a 3.0-T MR unit (Philips Healthcare, the Netherlands) with an 8-channel head. The eggs, whose amniotic cavity was inoculated with 0.2 ml of gadopentetate dimeglumine (Gd-DTPA) (Bayer Healthcare, Germany), were incubated at 33 ◦ C for 4 h and analyzed by MR imaging. The scanning was performed at 0.8-mm intervals, resulting in 25–30 images for each egg. On each image, the margin of the amniotic region enhanced

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with the Gd-DTPA was traced freehand using ImageJ 1.42q software, and the length of the margin (mm) was multiplied by 0.8. This value was obtained for each image for the egg and the values were summed according to the total number of scans (25–30). 2.6. Statistical analysis Data between groups were analyzed using a paired t-test. A p value of less than 0.05 was considered statistically significant. 3. Results and discussion 3.1. Generation of recombinant influenza C viruses To generate a recombinant parental (rPA) virus, which has the consensus sequences of AA/50, the above-mentioned 16 plasmids were transfected into 293T cells as described previously (Muraki et al., 2007). To rescue a mutant virus, rCM2-C65A, in which the cysteine at residue 65 of CM2 is mutated to alanine, the pPolI/CM2Acy(−) was transfected together with the other six Pol I plasmids (pPolI/PB2, pPolI/PB1, pPolI/P3, pPolI/NP, pPolI/HEF and pPolI/NS) and the nine virus protein-expressing plasmids described above. At 90 h posttransfection (p.t.), the respective culture medium was inoculated into the amniotic cavity of 9-day-old embryonated chicken eggs. After incubation at 33 ◦ C for 72 h, the amniotic fluids were recovered from the eggs. The recombinants in the amniotic fluids were subsequently propagated using 9-day-old fresh eggs and a stock of the recombinant viruses was prepared. The M gene of the rCM2-C65A virus was sequenced to ensure that no unwanted mutations were present. 3.2. Synthesis of CM2 in rCM2-C65A-infected HMV-II cells To examine first whether the CM2 protein without palmitoylation is synthesized in rCM2-C65A-infected cells, HMV-II cells, known to be a susceptible cell line to influenza C virus (Nishimura et al., 1989), were infected with the recombinants at a multiplicity of infection (m.o.i.) of 5 and then subjected to radioimmunoprecipitation as described in Section 2. As shown in Fig. 1A, the CM2 protein was synthesized both in the rPA- and rCM2-C65A-infected cells, but no incorporation of [3 H]palmitic acid into the CM2 proteins synthesized in the rCM2-C65A-infected cells was observed, indicating that CM2 in the rCM2-C65A-infected cells was not palmitoylated. Thus we concluded that the virus stock was available for the subsequent analyses. The amount of CM2 precipitated from the rCM2-C65A-infected cells is greater than that from the rPA-infected cells (Fig. 1A). We confirmed that the amount of CM2 precipitated from HMVII cells infected with AA/50 at an m.o.i. of 5 was equal to that from the rCM2-C65A-infected cells in the same experiment (data not shown). Furthermore, the possibility is ruled out that the half life of CM2 is affected by the lack of palmitoylation, since the pulsechase experiments of virus-infected cells showed that the stability of palmitoylation-deficient CM2 is virtually identical to that of authentic CM2 (Fig. 2B, see below). Taken together, the reason(s) for the difference in the amount of the precipitated CM2 protein between rPA- and rCM2-C65A-infected cells (Fig. 1A) is unclear. 3.3. Growth kinetics of the recombinants in HMV-II cells We next compared the growth kinetics of the recombinant viruses. The rPA or rCM2-C65A viruses were infected to HMV-II cells at an m.o.i. of 5 and incubated at 33 ◦ C for up to 72 h. Under this experimental condition, a single-step replication cycle of the virus requires approximately 24 h (Nishimura et al., 1990). The viruses

Fig. 1. (A) Synthesis of CM2 in virus-infected cells. HMV-II cells infected with rPA or rCM2-C65A were pulse-labeled with [35 S]methionine or [3 H]palmitic acid at 48 h p.i. The lysates of the cells were immunoprecipitated with anti-CM2 serum and analyzed by SDS–PAGE. (B) Growth kinetics of recombinant viruses. HMV-II cells were infected with rPA or rCM2-C65A at an m.o.i. of 5 and then incubated for 72 h. The culture media harvested at the indicated times were used for the determination of infectious titers. Results from three independent experiments are shown.

in the culture media at 12–72 h p.i. were subjected to plaque titration on LLC-MK2 cells. Three independent experiments revealed no significant differences in the infectious titers between the two populations of infected cells (Fig. 1B). In addition, the rPA or rCM2-C65A viruses were infected to HMV-II cells at an m.o.i. of 0.001 and incubated at 33 ◦ C for up to 6 days in the presence of trypsin (10 ␮g/ml), which is a method adopted in our previous report (Muraki et al., 2007). Also, no significant difference was observed in the yield of

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anti-CM2 serum, and analyzed by SDS–PAGE. No significant differences in the synthesis or maturation of the HEF, NP and M1 proteins were observed between the rPA- and rCM2-C65A-infected cells (Fig. 2A). In both populations of infected cells, the amount of CM2a was gradually reduced during the chase, with a concomitant increase in the amount of CM2b (Fig. 2B), a finding consistent with that in C/Yamagata/1/88-infected HMV-II cells (Hongo et al., 1997). These data indicate that the stability and N-glycosylation of palmitoylation-deficient CM2 is virtually identical to those of authentic CM2 in virus-infected cells. To examine the ability of the palmitoylation-deficient CM2 to form dimer and tetramer, the infected cells were pulse-labeled for 20 min and chased for 2 h. The cell lysates were immunoprecipitated with anti-CM2 serum and analyzed by SDS–PAGE in non-reducing condition as reported previously (Hongo et al., 1997). In both populations of infected cells, several bands corresponding to CM2a-monomer (M), CM2a-dimer (D) and CM2a-tetramer (T), and CM2b-dimer (D) and CM2b-tetramer (T) were detected (Fig. 2C), a finding consistent with that in C/Yamagata/1/88-infected HMV-II cells (Hongo et al., 1997). Taken together, these results demonstrate an absence of any significant differences between palmitoylationdeficient CM2 and authentic CM2 in terms of conformational maturation and transport in infected cells. 3.5. Membrane flotation analysis of virus-infected cells To investigate the effect of defect in CM2 palmitoylation on the membrane affinity of viral proteins, we examined the infected cells by membrane flotation analysis according to the procedure described previously (Muraki et al., 2007). In three independent experiments, no significant differences in the affinity of viral proteins were detected between rPA- and rCM2-C65A-infected cells; e.g. 63.8 ± 0.056% (rPA) and 69.3 ± 0.057% (rCM2-C65A) of NP was, respectively, recovered in the bottom fractions (fraction Nos. 9 and 10) just after the pulse period, and 28.8 ± 0.014% (rPA) and 26.3 ± 0.045% (rCM2-C65A) of NP floated up to the membrane fractions (fraction Nos. 3 and 4) during the chase period (Fig. 3A). The ratio of the NP protein recovered in the membrane fractions did not reach statistical significance between rPA- and rCM2-C65Ainfected cells (data not shown). For the membrane affinity of HEF and M1, essentially same results were obtained (Fig. 3A, data not shown). Also, no significant difference in the kinetics of CM2 was observed between the two cell populations (Fig. 3B). These observations confirmed that in rCM2-C65A-infected cells the affinity of viral proteins to the plasma membrane is virtually identical to that in rPA-infected cells.

Fig. 2. Pulse-chase experiments of virus-infected cells. HMV-II cells infected with rPA or rCM2-C65A were pulse-labeled with [35 S]methionine at 26 h p.i. and chased for the indicated number of hours (h). The lysates were immunoprecipitated with MAbs against HEF, NP and M1 (A) or anti-CM2 serum (B, C), and analyzed by SDS–PAGE in the presence (A, B) or absence (C) of 2-ME.

progeny viruses between the two populations of infected cells (data not shown). Thus palmitoylation of CM2 did not appear to have significant effect on the generation of infectious virions in cultured cells. 3.4. Viral proteins synthesized in HMV-II cells The HMV-II cells infected with rPA or rCM2-C65A at an m.o.i. of 5 were pulse-labeled with [35 S]methionine for 20 min at 26 h p.i. and chased for 1, 2, 4 and 8 h. The cell lysates were prepared either immediately after the pulse or after the chase, immunoprecipitated with MAbs against HEF (J14), NP (H27) and M1 (L2) or

3.6. Growth of the recombinants in embryonated chicken eggs M2 palmitoylation of A/WSN/33 did not contribute significantly to virus replication in vitro, whereas the palmitoylation-deficient A/WSN/33 virus displayed a reduced virulence after infection in mice (Grantham et al., 2009). This finding suggests that the significance of viral protein palmitoylation varies according to the host analyzed. Therefore, we attempted to examine the growth kinetics of the recombinants in an embryonated chicken egg, another representative host for the propagation of influenza viruses. Empirically, 0.1 ml of 10−3 to 10−4 -diluted virus stock was inoculated into the allantoic or amniotic cavity of chicken eggs in order to avoid the production of defective interfering particles. In the experiment, however, the m.o.i. is not known since the number of cells lining the inner surface of the cavities is difficult to count. In the present study, to estimate the m.o.i. more accurately, we attempted to measure the inner surface area of the amniotic cavity by visualizing the cavity using MR imaging.

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Fig. 3. Flotation analysis of virus-infected cells. The virus-infected cells were pulse-labeled with [35 S]methionine at 26 h p.i. and chased for 2.5 h prior to flotation analysis. Viral proteins in each fraction were immunoprecipitated with antiserum against the AA/50 virion (A) or anti-CM2 serum (B), and analyzed by SDS–PAGE. A representative result from three independent experiments is shown.

Two 8-day-old eggs and three 9-day-old eggs were examined by MR imaging. As shown in the left panel of Fig. 4A (9-day-old egg), the fetus was clearly observed and the amniotic cavity was recognized as the surrounding dark area. As shown in the right panel of Fig. 4A (9-day old egg), inoculation of Gd-DTPA into the amniotic cavity showed that the cavity was pleomorphic rather than spherical. Using the Gd-DTPA-enhanced eggs, the inner surface area of the amniotic cavities were estimated to be 7.03 and 8.67 cm2 (8-day old eggs), and 13.95 and 8.42 cm2 (9-day old eggs). Taking the average cell size into account, we estimate that there are approximately 5 × 106 cells lining the inner surface of the amniotic cavity. Based on the calculation, we inoculated the respective recombinants into the amniotic cavity of twelve fresh eggs (9-day-old) so that the m.o.i. was approximately 0.001 PFU/cell, and incubated the eggs at 33 ◦ C. The amniotic fluids were harvested at 1, 2 and 3 days postinoculation and the viruses in the fluids were titrated on LLC-MK2 cells. As shown in Fig. 4B, there were no significant differences in virus yields at 1–3 days postinoculation between the two recombinants, suggesting the generation of infectious virions in eggs is not affected by CM2 palmitoylation. Studies demonstrate that the influenza A virus M2 protein has a role in infectious virus production and virion morphology (Chen et al., 2008; Iwatsuki-Horimoto et al., 2006; McCown and Pekosz, 2005, 2006; Rossman et al., 2010a, 2010b). Truncations of the M2 cytoplasmic tail resulted in a decrease in infectious virus production. The tyrosine at residue 76 on the M2 cytoplasmic tail was shown to be critical for NP incorporation into virions (Grantham et al., 2010). These findings suggest the direct (or indirect) interaction between NP and the cytoplasmic tail of M2 during the vRNP incorporation process. Since the influenza C virus CM2 protein is structurally similar to M2 and the cysteine at residue 65 of CM2 is evolutionally con-

served, we first hypothesize that the defect of CM2 palmitoylation on its cytoplasmic tail is involved in the genome packaging through interaction with vRNP; e.g. palmitoylation contributes to proper regional structure formation in the CM2 cytoplasmic tail, which is competent to recruit vRNP efficiently into virions. In the present study, however, we could not show any apparent differences in the viral growth as well as in the synthesis, maturation and membrane affinity of viral proteins between rPA- and rCM2-C65A-infected HMV-II cells. These findings indicate that defect in CM2 palmitoylation did not influence any characteristics of viral proteins, and that CM2 palmitoylation is dispensable to virus replication. Grantham et al. (2009) showed that A/WSN/33 lacking M2 palmitoylation exhibited a slightly reduced virulence in infected mice. We, therefore, investigated the growth of the recombinants in another host, embryonated chicken eggs, since an animal experimental model for influenza C virus is not available to date. To compare the growth more exactly, we first measured the inner surface area of the amniotic cavity and the m.o.i. was estimated, which is, to our knowledge, a method that has not been reported before. The result revealed that there were no significant differences in the growth of the two recombinants, also confirming that CM2 palmitoylation is not essential in the replication of influenza C virus in embryonated eggs. Using the established reverse-genetics system (Muraki et al., 2007), we have attempted to generate recombinant influenza C viruses without CM2. However, no infectious recombinants have been rescued to date (data not shown). On the other hand, we have recently shown that CM2 is involved in genome packaging and uncoating processes in the virus replication cycle by generating CM2-deficient virus-like particles (Furukawa et al., 2011). Taken together, these findings suggest that CM2 is critical to virus replication. The obtained data in the present study, therefore, suggest

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Acknowledgments We thank Dr. Nami Watanabe (Yamagata Saisei Hospital), Ms Yukiko Ito, Ms Yuka Takahashi and Mr. Toshimitsu Sato (Department of Radiology, Yamagata University Hospital) for their assistance with the MR imaging. We also thank Ms Sumie Saito (Kanazawa Medical University) for her excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Takeda Science Foundation, Terumo Life Science Foundation, and a Grant-in-Aid from the Global COE program of the Japan Society for the Promotion of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2011.02.013. References

Fig. 4. (A) An MR image of embryonated chicken eggs. A 9-day-old egg (left panel), or a 9-day-old egg whose amniotic cavity was inoculated with Gd-DTPA (right panel) were subjected to MR imaging. Representative images of each egg are shown. In the right panel, black line indicates the margin of the amniotic cavity traced freehand. (B) Growth of the recombinants in embryonated chicken eggs. The respective recombinant viruses were inoculated into the amniotic cavity of 12 eggs and the eggs were incubated at 33 ◦ C for 1, 2 or 3 days. The viruses grown in the cavity were titrated on LLC-MK2 cells and the titers from the respective eggs are depicted as circles. The data were initially log-transformed and then statistically compared between groups using a paired t-test. A p value of less than 0.05 was considered statistically significant. NS; not significant.

that another posttranslational modification(s) rather than palmitoylation, ion channel function and/or a specific domain(s) of CM2 are essential to influenza C virus replication.

4. Conclusion A recombinant influenza C virus lacking CM2 palmitoylation was successfully generated by reverse genetics, and it was demonstrated that palmitoylation of CM2 is dispensable to influenza C virus replication in cultured cells as well as in embryonated chicken eggs.

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