Unique features of internal initiation of hepatitis C virus RNA translation.
The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.
Reynolds
J E
JE
Department of Biochemistry, University of Cambridge, UK.
Kaminski
A
A
Kettinen
H J
HJ
Grace
K
K
Clarke
B E
BE
Carroll
A R
AR
Rowlands
D J
DJ
Jackson
R J
RJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005796:Genes / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010849:Picornaviridae / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012314:RNA Cap Analogs; D012367:RNA, Viral / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics
D018387:Codon, Initiator; D018076:DNA, Complementary; D012314:RNA Cap Analogs; D012367:RNA, Viral; D011994:Recombinant Proteins