Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses.

Mutations in the influenza M2 membrane protein which confer resistance to the antiviral drug amantadine are exclusively located within the transmembrane region of the molecule. The influence of specific amino acid substitutions on the activity of the M2 protein in influenza A virus-infected cells is assessed in this report by their effects upon haemagglutinin (HA) stability and virus growth. A number of amino acid substitutions, e.g., L26H, A30T, S31N and G34E reduced the activity of the M2 protein of A/chicken/Germany/34 (Rostock) and caused a substantial increase in expression of the low-pH form of HA. The adverse effects of the mutations on virus replication were evident from changes selected during subsequent passage of the mutant viruses in the presence or absence of amantadine: reversion to wt, the acquisition of a second suppressor mutation in M2, or the appearance of a complementary mutation in HA which increased its pH stability. In contrast, 127T and 127S, mutations which were most readily selected following passage of the wt virus in the presence of drug, caused an increase in M2 activity. Furthermore, in double mutants the 127T mutation suppressed the attenuating effects of the A30T and S31N mutations on M2 activity. The influence of primary structure on the consequences of particular amino acid changes was further emphasized by the contrasting effects of the G34E mutation on the activities of two closely related proteins, causing an increase in the activity of the M2 of A/chicken/Germany/27 (Weybridge) as opposed to the decrease in activity of the Rostock protein. Estimates of differences in trans Golgi pH based on the degree of conversion of HA to the low-pH form, or complementation of differences in pH stability of mutant HAs, indicate that changes in M2 may influence pH within the transport pathway by as much as 0.6. The results thus provide further evidence that M2 regulates transmembrane pH gradients in the trans Golgi. Incompatibility between particular HA and M2 components and the selection of M2 mutants with suboptimal activity stresses the essential relationship between the structures and functions of these two virus proteins.

Maturation of influenza A virus hemagglutinin--estimates of the pH encountered during transport and its regulation by the M2 protein.

The susceptibility of H7 influenza A viruses to the M2-mediated alteration in HA resulting from treatment with amantadine or rimantadine depends both on the pH stability of HA and the pH encountered during transport to the plasma membrane of the particular virus-infected cell. pH stabilities of a range of virus mutant HAs exhibited an inverse correlation with drug sensitivity and the proportion of low-pH HA expressed on the surface of infected cells in the absence of drug. The lowest pH encountered during transport was thus estimated from the proportion of HA expressed in its native conformation and its pH stability profile. Lower drug sensitivity and improved HA maturation of MDCK cells compared to that in CEF reflect the higher pH within the appropriate compartment of the former. Differences in apparent pH resulting from infection with two closely related virus strains, Rostock or Weybridge, e.g., approximately 6.0 and 5.4, respectively, in CEF, were abrogated by rimantadine treatment (pH approximately 5.2 in CEF) and were attributable to intrinsic properties of their respective M2 proteins. The greater activity of the Rostock M2, which was estimated to be capable of increasing vesicular pH by as much as 0.8 pH units, correlates with the lower pH stability of the HA. This emphasizes the essential relationship between the characteristics of the two virus proteins as well as the subtle role of M2 in regulating the pH of the transport pathway to protect the structural integrity of the hemagglutinin glycoprotein.

Regulation of pH by the M2 protein of influenza A viruses.

Inhibition of the function of the M2 protein by amantadine can cause a conformational change in the haemagglutinin (HA) of H7 influenza A viruses and the consequent expression of the low pH form of the glycoprotein on the surface of virus-infected cells. Immunofluorescence studies showed that this conversion occurs shortly after HA exists from the Golgi complex apparently during its transport through the trans Golgi network and using the pH probe, DAMP/anti-DNP, that it is the direct result of reduced vesicular pH. The lowest pHs encountered were estimated using mutant HAs differing in pH stability to be approximately 5.2 and 5.6 in virus-infected CEF or MDCK cells, respectively, in the absence of functional M2. Depending on the particular M2, this protein was responsible for increases in vesicular pH of up to 0.8 units. The influence of mutations in both HA and M2 on the maturation of native HA illustrates the important relationship between the structural and functional properties of these two proteins. Using the fluorescent probe SNARF-1 the M2 protein was also shown to be largely responsible for the 0.3-0.4 unit reduction in intracellular pH of virus-infected cells. The data thus provide further evidence for the pH regulatory function of M2 and its importance for the maturation of the HA glycoprotein.

Virulence of rimantadine-resistant human influenza A (H3N2) viruses in ferrets.

The influence of rimantadine-resistance mutations on the virulence of human H3N2 viruses in ferrets was examined. The similarities in virulence of the drug-resistant mutants with single amino acid substitutions at three different locations, 27, 30, and 31, within the M2 sequence and their corresponding sensitive wild-type isolates contrasted with differences in virulence between the three pairs of viruses. These data provide further evidence that rimantadine-resistant viruses that emerge during treatment of patients with the drug are unaltered both in their growth characteristics and virulence.