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.