RNA-PROTEIN INTERACTIONS AT THE 3'-END OF THE HEPATITIS-A VIRUS-RNA

The regulative cis-acting terminal RNA structures and the proteins inv olved in the amplification of the hepatitis A virus (HAV) genome are u nknown. By UV cross-linking/label transfer experiments, we have analyz ed sequences of the 3'-nontranslated region (3'-NTR) and preceding dom ains of the viral genome for their ability to interact with host prote ins, A series of cDNA constructs were used to create genomic- and anti genomic-sense transcripts. The results show that the 3'-NTR-poly(A) in teracted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consist ing of 3D-coding regions, Protein p38 was most efficiently labeled aft er interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe, Opt imal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competiti on assays, These data might indicate that a higher-order structure for med at the junction of the 3D(pol)-coding sequence and the 3'-NTR of t he HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might he essen tial for the formation of the replication complex initiating minus-str and RNA synthesis.