Electrophoretic analysis of the ribonucleoproteins of hepatitis delta virus

Replication of hepatitis delta virus (HDV) is dependent on delta antigen (delta Ag), an HDV-encoded protein, which binds to HDV RNA and is capable of multimerization. To characterize HDV-specific ribonucleoprotein complexes (RNP) we used electrophoresis into non-denaturing agarose gels followed by northern analysis, to detect HDV RNA, and immunoblot, to detect delta Ag. We studied RNP from three sources: (i) vRNP, disrupted virions obtained from infected woodchuck serum; (ii) sRNP, disrupted particles secreted from transfected cultured cells; and (iii) cRNP, isolated from cells in which HDV genome replication was occurring, sRNP were approximately 28% smaller than vRNP. Treatment of vRNP with aurin tricarboxylic acid disrupted both delta Ag-delta Ag and delta Ag-RNA interactions while vanadyl ribonucleosides released the RNA without causing detectable disruption of the multimeric delta Ag complex. cRNP were smaller and more heterogeneous than VRNP and sRNP, and probably contained host components. The application of these electrophoretic procedures, and especially the use of prior treatments with vanadyl ribonucleoside complexes have provided valuable information on the RNP of HDV, and we expect they should find applicability in RNP studies of other RNA viruses. (C) 1998 Elsevier Science B.V. All rights reserved.