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Functional studies on the influenza A virus M2 protein

Geraghty, Finola Maria; (1996) Functional studies on the influenza A virus M2 protein. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The M2 protein of influenza A viruses is a homotetramer composed of four 97 amino acid subunits which form an ion permeable channel. It plays an important role both in the process of virus uncoating and in modulating the pH of the transport pathway which is necessary for haemagglutinin (HA) maturation. M2 was stably expressed in mouse erythroleukaemia (MEL) cells under the control of the inducible β-globin locus control region (LCR). The production of M2 peaked at 4-6 days post induction and was at a level comparable to that in virus-infected MDCK cells. The expressed M2 was structurally similar to that produced in virus-infected MDCK cells, in particular with regard to the formation and stability of the tetramer and was also phosphorylated and palmitoylated. The function of the M2 protein expressed in MEL cells was studied using three assays, 1) co-expression of HA and M2, 2) determination of intracellular pH and 3) measurements of ion conductance. Analysis of MEL cells showed that they provided a suitable environment for the co-expression of M2 and HA. Expression of M2 resulted in a decrease in intracellular pH, indicating that the M2 protein is responsible for the decrease in cytoplasmic pH during virus infection. Voltage clamp measurements showed that the expressed M2 formed a proton-selective channel which was specifically inhibited by rimantadine. Two features of the results indicated that the current was due to a proton conductance. At zero membrane potential, both the direction and magnitude of the current were dependent on the proton gradient and secondly the reversal potential was equal to the proton equilibrium potential. Neither the reversal potential nor the amplitude of the current were influenced by the presence of other small ions, including Na+, or K+ or CI-. These results are consistent with the biochemical role that M2 is perceived to play during virus infection. Structure/activity studies were also undertaken to identify residues in the transmembrane domain of the M2 protein which affected both the activation and thermal stability of the tetramer. In particular amino acid differences between the Weybridge and Rostock M2 proteins were studied.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Functional studies on the influenza A virus M2 protein
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Influenza A; Mouse erythroleukaemia cells
URI: https://discovery.ucl.ac.uk/id/eprint/10119646
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