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1 Institute of Virology – Slovaks Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic
2 National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
Correspondence
Tatiana Betakova
virubeta{at}savba.sk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The 374 aa precursor protein p42 is prototypically cleaved at an internal signal peptidase cleavage site, giving rise to the p31 and CM2 proteins (Hongo et al., 1997; Pekosz & Lamb, 1998). CM2 is a type III integral membrane protein (nomenclature of von Heijne) with a 24 aa N-terminal extracellular domain, a 22 aa transmembrane domain and a 69 aa C-terminal cytoplasmic domain (Hongo et al., 1999; Pekosz & Lamb, 1998).
The CM2 protein shares several structural features in common with the M2 protein of influenza A virus. These small integral membrane proteins, M2 (97 aa) and CM2 (115 aa), are incorporated into the virion (Zebedee & Lamb, 1988; Hongo et al., 1997). Both M2 and CM2 proteins are post-translationally modified by phosphorylation (Sugrue et al., 1990a; Tada et al., 1998) and by palmitoylation (Sugrue et al., 1990a; Hongo et al., 1997). The CM2 protein is also modified by the addition of an N-linked polylactosaminoglycan side chain, attached to N11 (Pekosz & Lamb, 1997; Hongo et al., 1997). The native forms of the CM2 and M2 proteins are disulfide-link homotetramers (Sugrue & Hay, 1991; Holsinger & Lamb, 1991; Hongo et al., 1997; Pekosz & Lamb, 1997).
The M2 protein forms an ion channel (Pinto et al., 1992; Hay, 1992) that plays a role in the uncoating of influenza virions in endosomes (Martin & Helenius, 1991; Helenius, 1992) and reduction of the acidity of the trans-Golgi network (TGN). Thus, during virus infection the intralumenal pH of the TGN is kept above the threshold that triggers the haemagglutinin (HA) to undergo a conformational change to the low pH form, which is involved in promoting membrane fusion (Sugrue et al., 1990a; Ciampor et al., 1992; Grambas et al., 1992). The proton-activated, proton-selective channel activity of M2 (Chizhmakov et al., 1996) is specifically blocked by the anti-influenza drugs amantadine and rimantadine.
The HA esterase glycoprotein (HEF) of influenza C virus has the ability to cause low pH-dependent haemolysis and fusion (Ohuchi et al., 1982), and the virion is presumably uncoated in the acidic endosomal compartment. The CM2 protein might, therefore, have a function similar to that of the M2 protein in virus uncoating. Electrophysiological studies have shown that the CM2 protein forms a voltage-activated ion channel permeable to Cl– (Hongo et al., 2004) and have indicated that CM2 forms an ion channel with properties similar to those of the NB protein of influenza B virus (Sunstrom et al., 1996; Hongo et al., 2004). The CM2-associated ion channel is permeable to Cl– but not to cations (Na+ or K+) (Hongo et al., 2004). Comparison of electrophysiological properties indicate that the BM2 channel of influenza B is similar to the M2 channel (Mould et al., 2003) and that the NB protein has some features in common with the CM2 protein.
To gain further insights as to the function of the protein in virus replication, we have investigated the ability of CM2 to modify the pH within the trans Golgi by co-expressing the protein with the pH-sensitive HA from influenza A virus. The CM2 protein was able to protect the co-expressed HA against acid activation in the TGN, but its activity was much lower than that of the M2 protein.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The viruses B/Johannesburg/26/94 and C/Taylor/1233/47 were grown in 10-day-old fertile hen eggs.
Antibodies.
Anti-HA monoclonal antibodies HC2, HC58 and H9 were described previously (Sugrue et al., 1990a). Rabbit antisera were produced to the peptide corresponding to the C-terminal sequence SAVDVDDGHFVNIELE of the M2 protein conjugated to keyhole limpet haemocyanin. A rabbit polyclonal antibody HA.11 (anti-tag) recognizing the influenza virus HA epitope YPYDVPDYAS was obtained from Covance.
Plasmid construction.
The plasmids encoding the HA and M2 genes of A/chicken/Germany/34 (H7N1, Rostock strain) were described previously (Betakova et al., 2005). CV-1 cells were infected with C/Taylor/1233/47 or B/Johannesburg/26/94 viruses for 8 h. Total RNA from the cells was purified using a Nucleospin nucleic acid purification kit (Clontech). The cDNAs of the viruses were synthesized by reverse transcription of the viral RNAs using an oligonucleotide primer complementary to the common 3'-terminal 12 nt sequence. The NB gene was modified by PCR to contain an NcoI restriction endonuclease site at the initiation codon and a sequence encoding the influenza virus HA-epitope tag sequence YPYDVPDYAS, followed by a termination signal and a BamHI site. A copy of the CM2 gene was modified to contain an NdeI site at the initiation codon followed by sequences encoding the 24 aa signal peptide, and the epitope tag sequence (as above) followed by a termination signal and BamHI site. The PCR products were cut with appropriate restriction enzymes and inserted into either pVOTE.1 or pVOTE.2 (kindly provided by Dr B. Moss) to generate pVOTE.1-NB and pVOTE.2-CM2, respectively. All constructs were sequenced to check for unwanted mutations. Plasmid DNA was purified using Plasmid Maxi kit (Qiagen).
Transfection protocol.
Confluent CV-1 cells were infected with 10 p.f.u. per cell of recombinant vaccinia virus vTF7.3 (Fuerst et al., 1986) in Optimem containing 40 µg cytosine arabinose (AraC) ml–1. After 1 h incubation, the infected cells were transfected with plasmids encoding the proteins mixed with Lipofectine (Life Technologies).
Immunofluorescence assay.
CV-1 cells were grown on glass coverslips and transfected with 3 µg pVOTE.1-HA mixed with 0.5 µg pVOTE.2-CM2, pVOTE.1-M2 or pVOTE.1-NB, as described above. Four hours after transfection, the cells were overlaid with 1 ml MEM containing 20 % fetal calf serum (FCS) and 40 µg AraC ml–1. After an additional 20 h, the cells were fixed with 3 % paraformaldehyde, permeabilized with 0.05 % saponine in PBS and immunolabelled with anti-M2, anti-tag or HC2 antibodies diluted in PBS containing 1 % BSA. Primary antibodies were visualized using fluorescein- or rhodamine-conjugated secondary antibodies diluted in PBS containing 1 % BSA. The nuclei were labelled for 10 min with Hoechst 33342. The cells were viewed using an Olympus I x70 microscope and images were captured using Silicon Graphics Delta vision.
Western blot analysis.
The transfected cells were lysed in extraction buffer (1 % Triton X-100, 1 mM EDTA, 20 mM Tris/HCl, pH 7.4) containing Complete Mini Protease inhibitor (Roche). After 10 min on ice, the lysates were clarified by microcentrifugation for 1 min, and the supernatants were analysed by electrophoresis on 12.5 % SDS-polyacrylamide gels. Immunoblotting was done as described by Grambas et al. (1992) by using rabbit anti-M2 or polyclonal anti-tag serum, protein A–horseradish peroxidase conjugate and enhanced chemiluminescence (ECL) reagent (Amersham).
Metabolic labelling and immunoprecipitation.
Confluent CV-1 cells were transfected with 3 µg pVOTE.1-HA and 0, 0.1 or 1 µg pVOTE.2-CM2, pVOTE.1-M2 or pVOTE.1-NB. Four hours after transfection, the medium was removed and the cells were overlaid with cysteine–methionine-free MEM containing [35S]Trans label (1.85 MBq ml–1). After 18 h incubation, the cells were washed once with PBS, lysed in extraction buffer and immunoprecipitated with monoclonal antibodies: HC2 (which recognizes all forms of HA), HC58 (which recognizes only the neutral form of HA) and H9 (which recognizes only the low pH form of HA). After SDS-PAGE, gels were fixed, dried and then analysed using a phosphoimager (Storm 860; Molecular Dynamics) by exposure to BioMax MS films (Kodak) at –70 °C.
ELISA on transfected cells.
CV-1 cells grown on 96-well plates were transfected with 0.25 µg pVOTE.1-HA with increasing concentration of plasmids encoding the ion channels, as mention above. Four hours after transfection, the cells were overlaid with MEM containing 20 % FCS, 40 µg AraC ml–1, with or without 5 µM amantadine. Transfected cells were incubated for 20 h and fixed with 0.05 % glutaraldehyde in PBS and ELISA was carried out on duplicate wells using anti-HA antibodies HC2, HC58 or H9, as described by Betakova et al. (2004, 2005).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Anti-M2 and HC2 antibodies immunolabelled the membranes and cytoplasm of CV-1 cells co-expressing the HA and M2 proteins (Fig. 1). A similar pattern of labelling was observed with anti-tag and HC2 antibodies in cells co-expressing HA and CM2 (Fig. 1). The NB protein was included in this study because of its electrophysiological similarity to the CM2 protein. M2, CM2 and NB proteins all co-localized with HA and no consistent differences in localization of these proteins in the CV-1 cells were observed.
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, lanes 1), but also in a dramatic increase (24–39 %) in the relative proportion of the native form of HA, recognized by the HC58 antibody (Fig. 2, lanes 2), and a corresponding decrease (8–18 %) in the low pH form of HA, recognized by the H9 antibody, depending on the amount of M2 plasmid (Fig. 2, lanes 3; Table 1). This specific effect of co-expression of the M2 protein on HA conformation was inhibited by amantadine (data not shown). A relative increase (8 %) in native HA and decrease (5 %) in low pH HA was also observed when HA was co-expressed with the CM2 protein (Fig. 2, lanes 1, 2 and 3; Table 1). In contrast, co-expression of the NB protein reduced somewhat the synthesis of HA, but did not affect the ratio of native and low pH forms of the protein. The accumulation of co-expressed M2, CM2 and NB proteins was checked by Western blot analysis with anti-M2 and anti-tag antibodies. The amount of protein detected increased substantially with the higher concentration of plasmid used for transfection (Fig. 2, lanes 4 and 5), indicating that the cells remained viable, although the specific effect on HA was not significantly altered.
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. The ability of CM2 to elevate trans-Golgi pH and protect HA against low pH-induced changes depended on the ratio of expressed HA and CM2 proteins and was indicated by corresponding increases in the proportion of native HA (recognized by HC58 antibody) and decreases in the proportion of low pH HA (recognized by H9 antibody) (Fig. 3a). On this basis, the activity of M2 was about three times higher than the activity of CM2. No significant changes in the relative proportions of native and low pH forms of HA were detected when co-expressed with the NB protein (Fig. 3a). Several different concentrations of HA and NB proteins as well as ratios of HA/NB were used to enhance the sensitivity of the co-expression assay. However, no changes due to NB expression were detected. The maximum percentage change in OD450 obtained with HC58 and H9 antibodies relative to HC2 was recorded at the optimum HA/X (X=CM2, M2 or NB) ratio. Further increase in the expression of the ion channel proteins reduced expression of total HA. The optimum HA/X ratio was similar for all three proteins. To compare the pH modulating activities of these proteins, the average values from six experiments were obtained (Fig. 3b). Co-expression of HA with the CM2 protein resulted in an increase of 8.4±3.6 % in native HA and a corresponding decrease of 6.7±2.5 % in low pH HA, compared with HA expressed in the absence of CM2 (Fig. 3b). The changes caused by the M2 protein were about three times higher than those caused by the CM2 protein and were specifically inhibited by amantadine (5 µM).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sakaguchi et al., 1996; Henkel et al., 1999), and the intracellular co-localization of these two proteins (Hughey et al., 1992). Our results of indirect fluorescence microscopy indicated that the transport of HA in CV-1 cells co-transfected with CM2 or NB was similar and that the HA co-localized with CM2 and NB proteins in a manner similar to that with M2. Data from indirect fluorescence microscopy have also provided evidence for the ability of the CM2 protein to modify the acidity of the trans Golgi (Betakova & Kollerova, 2006); however, this method did not allow quantification of the activity. The ability of the CM2 protein to reduce conversion of the acid-sensitive Rostock HA to its low pH form in co-transfected cells was evident in both radioimmunoprecipitation and ELISA experiments, which gave comparable results. It is apparent therefore that the protein has the ability to modulate trans-Golgi pH, but to a much lesser extent than the M2 proton channel of influenza A viruses. The ELISA experiments were easier to perform and gave reproducible results. Co-expression of HA with the CM2 protein, averaged from six experiments, resulted in an increase of 8.4±3.6 % in the native, neutral pH form of HA and a corresponding decrease of 6.7±2.5 % in the low pH form, whereas the (maximum) changes conferred by M2 were plus 28±4.6 % and minus 22.4±5.4 %, respectively.
The M2 protein forms an H+-activated, H+-selective channel, which has low permeability for other physiological ions (Chizhmakov et al., 1996; Mould et al., 2000). On the contrary, the CM2 protein forms a channel permeable to Cl– but not to cations (Na+ or K+), which exhibits little response to pH above 5.5 (Hongo et al., 2004). Preliminary studies of the electrophysiological properties of the CM2 protein expressed in mouse erythroleukaemia cells have identified a Na+-activated proton permeability (in addition to the Cl– permeability), smaller than the proton conductance of M2, but similar to that associated with NB expression (I. Chizhmakov, D. Ogden & A. Hay, unpublished observations). It would appear that this activity, either independently or together with the Cl– permeability, is responsible for the pH modulating activity; however, it is not clear why the CM2 channel has this capacity while the NB protein does not. It is likely that the influenza B virus BM2 protein, which has an ion channel activity analogous to that of M2 (Mould et al., 2003), performs the equivalent function to that of the influenza A virus protein. Furthermore, the NB protein, in contrast to the BM2 protein, has been shown not to be essential for influenza B virus replication in cell culture (Hatta & Kawaoka, 2003).
Site-directed infrared dichroism and molecular modelling of a CM2 transmembrane peptide predicted a 19–20 aa long 
-helix, with residues L31, L34, M41 and L44 pointing inwards towards the pore of the left-handed coiled-coil tetramer, in accordance with other ion channel structures forming mostly hydrophobic pores, e.g. the K+ channel (Kukol & Arkin, 2000). The putative transmembrane pore of CM2 was occluded by residue M41. A motif of hydrophilic residues, T30, S33 and T40, and Y43, located to the outer surface of the CM2 channel, is homologous to a similar sequence in the NB protein, in accordance with the similarities in the activities of the two channels.
The existence of a specific inhibitor of CM2 would be useful for studying the specific effects and functions of the CM2 protein. Mutations in 5 aa in the M2 protein of influenza A virus, L26, I27, A30, S31 or G34, confer resistance to the anti-influenza drugs amantadine and rimantadine (Grambas et al., 1992; Shuck et al., 2000). L31 in CM2 appears analogous to L26 of the M2 protein and it was predicted that amantadine/rimantadine might block the CM2 channel (Lamb & Pinto, 1997). However, inhibition of the CM2 activity, reported here, required a high concentration of amantadine (data not shown), indicating lack of specificity, and amantadine failed to attenuate the inward currents of CM2-expressing oocytes induced by hyperpolarization (Hongo et al., 2004).
In conclusion, we have shown that the CM2 protein is capable of modifying the pH within the TGN, whereas the NB protein lacked any such activity. However, CM2 activity was only a fraction of that of the M2 protein, half that of a human virus protein (Betakova et al., 2005) and one third that of the Rostock M2, corresponding to the probe HA. Whereas the relationship between the activity of the M2 proton channel and its role in the replication of influenza A viruses is clear, the relationship is less evident in the case of CM2 and, although consistent with a role in virus uncoating, the actual functions of the CM2, and NB, proteins remain to be determined.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 December 2006;
accepted 20 April 2007.
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