Interaction of woodchuck hepatitis virus surface antigen with hepatocyte plasma membrane in woodchuck chronic hepatitis

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Abstract
The extent of association between woodchuck hepatitis virus surface antigen and host hepatocyte plasma membrane in chronic hepatitis was studied. Purified membranes containing the antigen were treated with various agents which perturb plasma membrane constituents to elute woodchuck hepatitis virus surface antigen. The products from disrupted membranes were analyzed by sedimentation in sucrose gradients and tested to identify the antigen reactivity. The results indicated that membrane-bound wood-cauck hepatitis virus surface antigen was partially released by 4M potassium chloride, potassium thiocyanate and guanidine, 6M urea or 0.1 N sodium hydroxide (pH 13.5), but not in the presence of low concentrations of these reagents or by 10% 2-mercaptoethanol and 1% sodium dodecyl sulfate. No more than 15% of the total membrane-associated woodchuck hepatitis virus surface antigen was eluted by 0.1 N NaOH, which was found to be the most effective eluent among tested agents at the antigen removal. The remaining woodchuck hepatitis virus surface antigen was resistant to further extraction with sodium hydroxide, as expected for an integral membrane protein. Treatment of the infected membranes with 1% Triton X-100 or 50 mM deoxy-cholic acid, that solubilize the membrane lipid bilayer releasing most of the integral membrane proteins, resulted in the sedimentation of almost all detectable woodchuck hepatitis virus surface antigen reactivity with the detergent-insoluble membrane residues, suggesting a firm interaction of the antigen with the plasma membrane matrix. Partition of the membrane peripheral and integral proteins in 1% Triton X-114 into hy-drophilic and hydrophobic protein fractions, respectively, provided additional evidence that most of the detectable woodchuck hepatitis virus surface antigen reactivity within the infected membranes was associated with their integral proteins. The results demonstrate that woodchuck hepatitis virus surface antigen found in hepatocyte plasma membrane behaves as a peripheral and an integral membrane protein. It was shown that most of the woodchuck hepatitis virus surface antigen behaving as an integral membrane protein interacts extremely extensively with the membrane. This unexpectedly strong membrane binding of woodchuck hepatitis virus surface antigen may be irreversible in naturally occurring conditions and could be eliminated only by lysis of infected hepatocyte membranes. Viral envelope material firmly associated with hepatocyte plasma membrane may constitute an important element of a complex mechanism leading to persistence of the virus infection that could also be responsible for preservation of hepatocytes with integrated viral genome.