Translational control of synthesis of simian virus 40 late proteins from polycistronic 19S late mRNA.
The simian virus 40 (SV40) 19S late mRNA is polycistronic, encoding multiple late proteins: agnoprotein, VP2, and VP3. We constructed a chloramphenicol acetyltransferase (CAT) transient expression vector in which the SV40 sequences between nucleotides 5171 and 1046 (via the SV40 origin of replication and including the late promoter) were inserted 5' to the cat gene; therefore, the AUG for CAT expression occurs after the AUGs for agnoprotein, VP2, and VP3. CAT enzyme activity assayed after transfection of these constructions indicates the level of CAT AUG utilization and, therefore, can be used as a measure of the ability of prior AUGs to intercept scanning ribosomes. Specifically, deletions and point mutations of the viral AUGs resulted in increased CAT enzyme activity owing to increased utilization of the downstream CAT AUG. To compare a variety of mutants, we used the levels of increase to calculate the translational efficiency of the viral AUGs. Some of our data agree with predictions of the modified scanning model (MSM). Little variation in downstream CAT AUG utilization was noted regardless of whether the VP2 AUG (in a weak MSM sequence context) was intact or removed. Hence, a scanning ribosome may easily bypass it. Similar analysis of the VP3 AUG (in a favorable MSM sequence context) demonstrated that it could efficiently intercept ribosomes prior to the downstream AUG. Overall, these data indicate that the structure of the 19S late mRNA and the relative efficiency of translational start codon utilization can account for the VP3/VP2 ratio found in infected cells. The agnoprotein reading frame, depending on how the mRNA precursor is spliced, is either not contained in the mRNA or is terminated near the VP2 AUG. Under these conditions, the ability of the agnoprotein AUG to block downstream CAT AUG utilization was found to be minimal in our assay. However, we directly tested the blocking ability of the agnoprotein AUG under conditions in which the reading frame terminated well after the CAT AUG. Although the agnoprotein AUG lies in a very good sequence context, this direct analysis showed that it interfered minimally with utilization of the CAT AUG when under the control of the SV40 late promoter. However, expected high levels of interference were regained when the late promoter was replaced with the Rous sarcoma virus long terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)
Dabrowski
C
C
Graduate Group of Molecular Biology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6076.
Alwine
J C
JC
D000123:Acetyltransferases / Q000235:genetics; D000818:Animals; D002461:Cell Line, Transformed; D015500:Chloramphenicol O-Acetyltransferase; D003062:Codon / Q000235:genetics; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins; D000086082:Viroporin Proteins
D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D003062:Codon; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C042559:agnoprotein, polyomavirus; D000086082:Viroporin Proteins