Sequences involved in determining the locations of the 5' ends of the late RNAs of simian virus 40

Abstract

The 5' ends of the simian virus 40 (SV40) late RNAs are heterogeneous in location, spanning a 300-nucleotide region from residues 28 to 325. To examine whether upstream or downstream measuring functions analogous to the TATA box play roles in positioning the 5' ends of these RNAs, we determined by S1 and primer extension mapping the locations of the 5' ends of the late viral RNAs made in monkey cells infected with: (i) three wild-type strains of SV40 that contain tandem duplications of the enhancer region that are 64, 85, and 91, rather than 72, base pairs in length; (ii) four viable mutants that contain alterations in the 21-base-pair tandem repeats; and (iii) four viable mutants that possess small deletions or insertions at or near the major cap site at residue 325. Most of the 5' ends of the RNAs were identical in location to those seen with wild-type strain 776. The only exceptions were the absence of RNAs whose 5' ends mapped to within three bases upstream or downstream of a sequence alteration. In addition, the sequences within residues 251 to 277 that function as transcriptional initiation sites in wild-type strain 776 also did so in their second locations in the wild-type strains in which these sequences are duplicated. Differences were noted in the relative abundances of the numerous 5' ends of the late RNAs, even among the wild-type strains. These findings indicate that many (and likely all) of the approximately two dozen locations of 5' ends of SV40 late RNAs are each determined largely by sequences within their immediate vicinity. However, sequences somewhat removed from these transcriptional initiation sites may modulate the efficiencies with which they are utilized.

The late spliced 19S and 16S RNAs of simian virus 40 can be synthesized from a common pool of transcripts

“…The heterogeneity of the 5' ends of the RNAs is indicated (----) at the left ends of the RNAs. The levels of the different RNA species in the cytoplasm of infected cells are listed at the right and were determined from Somasekhar and Mertz (39) and quantification of experiments similar to those presented in Fig. 2.…”

“…The 5' ends of these RNAs map to numerous locations within the late leader region from nucleotide residue (nt) 28 to 400, with the predominant 5' end, referred to as the major cap site, mapping to nt 325 (13,31,39). Precursor RNAs are either unspliced or spliced within the leader region such that they lack the intron from nt 295 through 434 (RNA species A and B, respectively [ Fig.…”

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40

“…Plasmids containing cDNA copies of each of the late 19S a The SV40 nucleotide residues deleted, inclusive, were determined either previously (4; Somasekhar et al, in preparation) or for this report. WT800 and WT830 differ in the precise size of their duplicated enhancer region (54).…”

Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap

“…1D), were constructed which, when transfected into COS cells, resulted in the synthesis of SV40 major late 16S mRNAs identical in sequence except for a difference in the lengths of their 5' untranslated regions (UTRs). Because transcription initiates from the SV40 late promoter at numerous sites (7,21), the Rous sarcoma virus (RSV) promoter was used to generate mRNAs with unique 5' ends. The mRNAs expressed from the RSV promoter differed from the 16S mRNAs of wild-type SV40 in that they contained (i) a substitution of the sequence GGTCGACC in place of SV40 nucleotides (nt) 502 through 521, removing the LP1 termination codon, and (ii) a substitution of the sequence GGTCG in place of SV40 nt 1464 through 1492, removing two translation termination codons between the LP1 and VP1 open reading frames (ORFs).…”