1 Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovakia
2 National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK
Correspondence
Tatiana Betakova
virubeta{at}savba.sk
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ABSTRACT |
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Furthermore, electrophysiological studies have shown that the M2 channels of the two viruses differ in two important respects, representing mechanistic changes in the channel (Chizhmakov et al., 2003): (i) the Rostock M2 possesses a sevenfold greater proton conductance, corresponding to its greater pH-modulating activity; and (ii) the two channels differ in activation characteristics. More specifically, they differ in the direction of rectification induced by high pH (>7). Whereas the Weybridge M2, like that of human viruses, deactivates in response to external (but not internal) high pH, the Rostock M2 deactivates in response to internal (but not external) high pH. The latter difference in activation characteristics was shown to be determined by three amino acid differences, V27I, F38L and D44N, within the transmembrane domain, which distinguish the Weybridge and Rostock proteins, respectively. Substitution of all three residues was required to transform the Weybridge phenotype into that of the Rostock M2 and, conversely, single substitutions in Rostock M2 were sufficient to effect the opposite phenotypic change. However, these mutagenesis experiments did not resolve which of the amino acid differences affected the ion flux through the channel. To answer that question, we have used a semi-quantitative HAM2 co-expression assay to assess the effects of single and double mutations at these three positions, aa 27, 38 and 44, on the pH-modulating activity of M2 and to determine whether the differences in ion flux and activation are genetically linked.
DNA copies of coding sequences for the HA of influenza virus strain A/chicken/Germany/34 (H7N1, Rostock strain) and the M2 proteins of the Rostock strain, A/chicken/Germany/27 (H7N7, Weybridge strain) and A/PR/8/34 (H1N1) were inserted into plasmid pVOTE.1 (kindly provided by B. Moss, National Institutes of Health, Bethesda, MD, USA) to generate pVOTE.1-HA and pVOTE.1-M2, respectively. Plasmids encoding the mutant M2 proteins I27V, L38F, 127V+L38F, N44D and D44N (Fig. 1) were prepared by using four-primer PCR. Sequences of oligonucleotide primers and details of cloning are available upon request. CV-1 cells were infected for 1 h with recombinant vaccinia virus vTF7.3 (10 p.f.u. per cell) (Fuerst et al., 1986
), transfected for 4 h with plasmids mixed with Lipofectin (Life Technologies) and incubated for a further 16 h in minimal essential medium containing 20 % fetal calf serum and 40 µg arabinose C ml1, with or without 5 µM amantadine.
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The mutant Rostock proteins containing single amino acid substitutions of I27V or L38F possessed a pH-modulating activity that was indistinguishable from that of the wt protein (Fig. 3b), as did the double mutant I27V+L38F (data not shown). Substitution of aspartic acid for arginine 44 (N44D), however, caused a significant decrease in activity (approximately to the levels found for W-M2), indicating that the change in this residue alone could account for the difference in the pH-modulating activities of the two wt channel proteins. Furthermore, substitution of asparagine for aspartic acid 44 in the M2 PR8-M2 (D44N) caused an increase in activity (resistant to amantadine) to a level comparable to that of the wt Rostock protein, confirming the more general influence of this particular substitution at residue 44 on M2 activity.
Thus, whereas any one of the single amino acid substitutions I27V, L38F or N44D caused a switch in the activation characteristics of Rostock M2 to those of the Weybridge channel (Chizhmakov et al., 2003), only the change in residue 44 between asparagine and aspartic acid was necessary and was sufficient to account for the difference between the pH-modulating activities (proton flux) of the two channel proteins. It was apparent, therefore, that there was no strict genetic correlation between the two phenotypic properties and that they represented separable functional characteristics.
Previous studies of mutant viruses have shown that a number of single amino acid substitutions, at residues 26, 30, 31 and 34, as well as 27, can cause increases or decreases in pH-modulating activity, as assayed by HAM2 co-expression, e.g. I27T and I27S substitutions caused increases in activity of Rostock M2, whereas I27N, like I27V in the present study, caused no detectable difference (Grambas et al., 1992). Furthermore, it was also observed, by passage in cell culture, that mutations that altered the fusion pH of the HA or the pH-modulating activity of M2 could influence selection of changes in the corresponding properties of M2 and HA, respectively. Thus, for example, a Weybridge mutant with an increased HA fusion pH selected a mutation in M2, G34E, that caused a compensatory increase in pH-modulating activity.
As for the significance of the differences in phenotype, there was a clear correlation between the greater pH-modulating activity and proton flux of the M2 channel and the higher HA fusion pH of the Rostock virus. However, there was no clear indication as to the advantage conferred by the unusual activation characteristics of its M2 channel protein, especially with respect to modulation of trans-Golgi pH. As acquisition of the latter property by the Rostock M2 required two unusual amino acid substitutions in residues 27 and 44 (among known M2 sequences), whereas the former alteration was effected by the change in residue 44 (N44D) alone, it appeared likely that the change in activation characteristics was selected for in addition to, and not coincidentally with, the increase in M2 channel activity. We do not know, however, how the Rostock virus with these peculiar properties (in HA and M2) emerged whether in vivo or as a result of extensive passage in vitro or how the sequence in which the (complementary) characteristics of HA and M2 were acquired. That the change in activation characteristics is apparently not associated with the change in modulation of trans-Golgi pH points to its greater significance for the activity of M2 in virus entry. It may be that removal of the strict regulation by pH outside the virion (in the case of Weybridge M2) is necessary to facilitate uncoating of the Rostock virion at a higher endosomal pH, consistent with the higher pH at which fusion between the virus and endosomal membranes is promoted by the Rostock HA. As the change in activation affects the direction of the pH-induced effect (from outside, the N terminus of M2, to inside, the C terminus) and not the intrinsic nature of the pH-induced change in the voltage dependence of channel conductance, this appears to provide a mechanism for switching off the normal activation property. Furthermore, maintenance of the intrinsic conductance characteristics of the channel emphasizes their importance. However, the mechanistic significance of reduced outward H+ flux at high external pH in, for example, promoting H+ transfer into the virion or preserving the integrity of the RNA genome of virus exposed to an alkaline environment, has yet to be elucidated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 June 2004;
accepted 22 September 2004.
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