Amino acid variations in HCV p7 and sensitivity to antiviral combination therapy with amantadine in chronic hepatitis C

Poster number: 12

C. Welsch (1,2), M. Albrecht (2), U. Mihm (1), N. Grigorian (1), E. Herrmann (1,3), B. Kronenberger (1), G. Teuber (4), T. Lengauer (2) , S. Zeuzem (1), and C. Sarrazin (1)

  1. Klinik für Innere Medizin II, Universitätsklinikum des Saarlandes, Kirrberger Str., 66421 Homburg/Saar, Germany
  2. Max Planck Institut für Informatik, Bioinformatik und angewandte Algorithmik, Stuhlsatzenhausweg 85, 66123 Saarbrücken, Germany
  3. Fachbereich Mathematik der Technischen Universität, Schlossgartenstr. 7, 64289 Darmstadt, Germany
  4. Medizinische Klinik I, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany.

Introduction:

Hepatitis C is an important cause for liver cirrhosis and cancer. A possible benefit of amantadine addition to an interferon-α/ribavirin combination therapy in improving sustained viral response rates has been discussed controversially. In vitro data suggest a direct interaction of amantadine with HCV p7, abrogating ion conductivity. Similarly, amantadine interacts with the M2 ion channel in influenza. HCV p7 is a small hydrophobic protein comprising two transmembrane domains (TMDs). HCV p7 homo-oligomerize into a circular hexamer forming an ion channel. The importance of p7 amino acid variations for amantadine sensitivity has been investigated in the present study.



Material and Methods:

We investigated 67 HCV genotype 1 infected patients who underwent antiviral therapy with or without amantadine. The resulting p7 sequence data were used for in silico analysis to characterize functionally relevant sites and to evaluate the potential impact of specific amino acid substitutions.



Results and Discussion:

HCV subtype 1a showed no association of amino acid variations with antiviral response. Non-responders with HCV-1b infection receiving amantadine showed the substitution L20F within TMD1 more frequently than responders. Moreover, non-responders treated with amantadine also showed more often the L20F substitution than non-responders treated with IFN-αribavirin alone. During antiviral treatment, the emergence of L20F has not been observed. In silico analysis of p7 using a helical wheel representation revealed phenylalanine- and leucine-rich sides oriented towards each other. A predominantly hydrophobic side remains for the membrane lipid phase. Six amino acids of TMD1, comprising H17 and V/L20, appear to be oriented towards the pore lining. A histidine oriented towards the lumen is of functional importance in influenza M2. We observed a corresponding luminal H17, which was strictly conserved in 66 of 67 patients. The functional role of H17 in HCV p7 remains to be elucidated. The substitution L20F is oriented towards the lumen and may lead to changes in size and shape of the p7 ion channel pore. The interaction of amantadine with HCV p7 could be hampered as a consequence.



Conclusions:

We identified the genotypic variation L20F of p7 TMD1 in HCV-1b, associated with phenotypic non-response to antiviral combination therapy comprising amantadine. A helical wheel model of HCV p7 TMDs locate L20F oriented towards the channel lumen. L20F may cause relevant changes in size and shape inside the p7 ion channel pore and may thus impair amantadine action.