RNA-protein interactions at the 3' end of the hepatitis A virus RNA.
The regulative cis-acting terminal RNA structures and the proteins involved in the amplification of the hepatitis A virus (HAV) genome are unknown. By UV cross-linking/label transfer experiments, we have analyzed sequences of the 3'-nontranslated region (3'NTR) and preceding domains of the viral genome for their ability to interact with host proteins. A series of cDNA constructs were used to create genomic- and antigenomic-sense transcripts. The results show that the 3'-NTR-poly(A) interacted with host cell proteins with molecular masses of 38, 45, 57, 84, and 110 kDa only weakly, compared with RNA structures also consisting of 3D-coding regions. Protein p38 was most efficiently labeled after interaction with secondary-structure elements located at the 3' end of the HAV RNA, p38 also interacted with a 5'-terminal RNA probe. Optimal RNA binding was found to be dependent on the salt concentration. The specificity of the RNA-protein interaction was proven by competition assays. These data might indicate that a higher-order structure formed at the junction of the 3Dpol-coding sequence and the 3'-NTR of the HAV genome (putative RNA pseudoknot) significantly improves binding of host proteins and thus suggests that this structure might be essential for the formation of the replication complex initiating minus-strand RNA synthesis.
Kusov
Y
Y
Institute of Clinical Microbiology and Immunology, St. Gallan, Switzerland, koussov@molbio.mu-luebeck.de
Weitz
M
M
Dollenmeier
G
G
Gauss-Müller
V
V
Siegl
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006507:Hepatovirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010761:Phosphorus Radioisotopes; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D015347:RNA Probes; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014709:Vero Cells
D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011189:Potassium Chloride