Article Figures & Data
Figures
- Fig. 1.
(A) Gene organization of Tapa, a SIN cDNA. (B) 3′ sequence of the TT21qa; NS, nonstructural genes; S, structural genes; (A)n, poly(A) tail. It is important to note that linearization of TT21qa with XhoI cleaves the DNA between A and G. Therefore, the RNA transcribed from the TT21qa/Xho construct may contain 1 to 5 bases of the XhoI motif. (C) 3′ sequence of the SIN genome recovered from BHK cells transfected with TT21qa/Xho RNA. The 11 PCR products corresponding to the viral RNA (Fig. 3A) were sequenced with JC1000-1H. The size of the poly(A) tail ranged from 17 to 39 nt. All 11 virus isolates were identical with respect to the 3′ terminal 73 nt.
- Fig. 2.
Electrophoretic analysis of the in vitro-synthesized RNA transcripts. Two percent of the total RNA synthesized in a 30-μl reaction mixture was denatured with glyoxal and dimethyl sulfoxide and separated on a 1.25% gel. The gel was soaked in methanol–2,5-diphenyloxazole, dried, and fluorographed. The mock transcription reaction mixture contained no DNA template.
- Fig. 3.
Expression of genomic and subgenomic SIN RNAs from representative virus isolates. Cultures of BHK cells were infected (MOI of 0.1 to 2.4) with the indicated virus isolates and labelled with [3H]uridine in the presence of dactinomycin. Approximately 4 to 6 μg of [3H]uridine-labelled cytoplasmic RNA was denatured with glyoxal, separated on a 1.25% agarose gel, and fluorographed. The upper band in each lane corresponds to the genomic RNA (49S), and the lower band corresponds to the subgenomic RNA (26S). The identity of the virus isolate is indicated at the top of each lane. UI, uninfected cell RNA. (A) Isolates 29-1 to 29-11, viral plaques derived from TT21qa/Xho; (B) plaques derived from T3′18(A)n, T3′17(A)n, and T3′6(A)n; (C) plaques derived from T3′15(A)n and T3′0(A)n. It is important to note that the amount of RNA made by each isolate is due mostly to the MOI, although base changes in recovered viruses could also be partly responsible.
- Fig. 4.
Polyadenylation of the SIN genome carrying a G residue at the −1 position of the 3′CSE. (A) Sequence of the 3′CSE in Tapa. (B) Sequence of the 3′ terminus in T3′18/Xho. Insertion of a G residue in the −1 position of the 3′CSE and of a XhoI site at the 3′ terminus results in the introduction of a C residue in the +1 position. (C) Proposed intermediate during the formation of RV-T3′18 type revertant viruses. (D) Sequence of the 3′ terminus of all six viral isolates indicating the loss of the G residue and polyadenylation at the C residue. (E) Proposed polymerase jumping event during negative-strand synthesis and the loss of the G residue.
- Fig. 5.
Terminal repair of a 3′ truncated SIN genome. (A) Wild-type sequence of the 3′CSE in Tapa; (B) sequence of the 3′ terminus of T3′15/Xho indicating the deletion of the −1 to −4 positions and the insertion of a XhoI site at the terminus; (C) names and sequences of viral isolates generated from T3′15/Xho. The single underline indicates the AU-rich sequences and the poly(A) tail added at the 3′ terminus. The remnants of the XhoI motif are highlighted with a double underline. The number in parentheses at the end of each sequence corresponds to the number of viral isolates containing the same sequence. The sequences for isolates 15.1, 15.2, 15.3, and 15.7 (two separate isolates) were from experiment 1, those for isolates 15.4, 15.5, 15.7, 15.8, and 15.9 were from experiment 2, and those for isolates 15.6 and 15.7 were from experiment 3.
- Fig. 6.
Addition of AU-rich motifs to 3′ mutants of polyadenylated SIN genome. The 3′ sequence of the template used for transfection of BHK cells and the 3′ sequence of the viral isolates recovered from the cells are given for each group. The positions of the base deletions introduced in the parental template are indicated as filled squares. The circled nucleotides highlight the base changes encountered in the revertant virus. The AU-rich sequences and poly(A) tail added to the 3′ end are marked with an underline. (A) T3′18(A)n and virus isolates derived from it. Isolates 18A-1 to 18A-3 correspond to experiment 1 and isolates 18A-4 to 18A-7 correspond to experiment 2. (B) T3′17(A)n and virus isolates derived from it. Isolates 17A-1 to 17A-4, 17A-7, and 17A-10 were derived from experiment 1. Isolates 17A-5, 17A-6, 17A-8, and 17A-9 were derived from transfection experiment 2. (C) T3′6(A)n and virus isolates derived it. The first three isolates were derived from experiment 1, and the rest were derived from experiment 2.
- Fig. 7.
Addition of long AU-rich sequences of 3′ mutants of the SIN genome. (A) Sequences of T3′15(A)n and virus isolates generated from it. Isolates 15A-1, 15A-2, and 15A-3 were derived from experiment 1. Isolate 15A-4 was derived from experiment 2. (B) Sequences of T3′0(A)n and the virus isolates recovered from it. Isolates ZA-1, -2, and -3 were derived from experiment 1, and isolate ZA-4 was derived from experiment 2. The identification of residues and motifs is as described in the legend to Fig. 6.
Tables
- Table 1.
List of oligonucleotides used in this study
Name Sequence Locationa Polarity T11750 AAAGGGAATTCCTCGAGGGGA 3′Vec + JC 3259 AATCAGCAGGGTCATCGC 3′Vec − JC1000-1H CTGCAGAAGCTTGCTGACTAGCACACGAAG 3′S + T11350 TAGTCAGCATCATGCTGC 3′S + T11150 GCGAACTTTATCGTA 3′S + T11600B GCAGCGTCTGCATAACTT 3′NTR + T11200H CTGCAGAAGCTTATGTAAACCACCAGCTGA 3′S − 18TSac CCTAGAGCTCAGTTTTTTTTTTTTTTTTTT Poly(A) − 3′18Xho CTAGCACTCGAGCAAATGTTAAAAACAAAAT 3′CSE − 3′15Xho CTAGCACTCGAGTGTTAAAAACAAAATTTT 3′CSE − 3′18PA TTTTTTTTTTAAATGTTAAAAAC 3′CSE − 3′17PA TTTTTTTTTTAATGTTAAAAAC 3′CSE − 3′15PA TTTTTTTTTTGTTAAAAACAAAAT 3′CSE − 3′6PA TTTTTTTTTTCAAAATTTTGTTG 3′CSE − 3′0PA TTTTTTTTTTTTGTTGATTAATAAA 3′CSE − 3′PAA CTAGCACTCGAGTTTTTTTTTTTTTTT Poly(A) − ↵a The region of the SIN genome to which the oligonucleotide belongs: 3′S, 3′ region of the S-coding region; poly(A), 3′ poly(A) tail; 3′CSE, 19-nt 3′CSE; 3′Vec, 3′ vector sequences downstream of the SIN genome in Tapa.
- Table 2.
Structure and properties of SIN mutantsa
Expt No. Construct 3′NTR Poly(A) tail Relative infectivityb In vivo 3′ modificationsc Reference 1 Tapa/Xho −1 to −310 Yes 6,000 None 22 2 TT21g/Xhod −290 to −310 No 0 None 22 3 TT21h/Xho −290 to −310 Yes 0 None 22 4 TT21i/Xho −1 to −5 Yes 0 None 22 5 TT21j/Xho −1 to −10 Yes 0 None 22 6 TT211/Xho −1 to −14 Yes 79 None 22 7 TT21m/Xho −1 to −16 Yes 74 None 22 8 TT21n/Xho −1 to −20 Yes 328 None 22 9 TT21q/Xho −1 to −28 Yes 1,252 None 22 10 TT21r/Xho −1 to −63 Yes 3,671 None 22 11 TTk/Xho −1 to −24 No 0 None 22 12 TT21s/Xho −1 to −310 No 36 PA 22 13 TT21ra/Xho −1 to −63 No 41 PA 22 14 TT21qa/Xho −1 to −29 No 20 PA 22 15 TT21Bgl-2 −1 to −310 Yes 0 None 22 16 T3′18/Xho −2 to −310 No 47 PA Present work 17 T3′15/Xho −5 to −310 No 24 PA, R Present work 18 T3′18(A)n/Xho −2 to −310 Yes 19 PA Present work 19 T3′17(A)n/Xho −3 to −310 Yes 11 PA, R Present work 20 T3′15(A)n/Xho −5 to −310 Yes 26 PA, R Present work 21 T3′6(A)n/Xho −14 to −310 Yes 43 PA, R Present work 22 T3′0(A)n/Xho −20 to −310 Yes 37 PA, R Present work 23 Tapa/Xho (DNA control)e −1 to −130 Yes 0 None Present work ↵a Duplicate cultures of BHK cells were transfected with 50 ng, 250 ng, or 2 μg of in vitro-synthesized RNA, overlaid with agarose, incubated at 37°C, and monitored for plaque formation over a period of 4 days. The number of plaques per microgram of RNA varied up to twofold among dilutions, suggesting an alteration in transfection efficiencies between diluted and undiluted RNA samples. Irrespective of the transfection efficiencies of individual test RNAs, the representative plaque-purified viruses tested yielded high-titer virus stocks (107 to 109 PFU/ml).
↵b The number of plaques per microgram of test RNA relative to the value for Tapa/Xho. The value for the Tapa/Xho was set at 6,000 to depict relative infectivities of test RNA in whole numbers. The value represents the average of two experiments.
↵c PA, polyadenylation; R, 3′ repair by AU additions.
↵d TT21g/Xho carries a poly(U) at its 3′ terminus.
↵e Mock transcription reaction carried out in the absence of SP6 RNA polymerase.
