The isolated RSV RdRp adds nts to the 3′ end of the TrC template RNA.

<p>(A) A GTP label is incorporated into products of 26–28 nts in length. Wt or mutant (L_N812A) RdRp was incubated with 0.2 µM TrC RNA template, or its complement Tr 1–25, as indicated, in a reaction containing 200 µM of each NTP and [α-³²P]GTP. (B) GTP incorporation into the 26 nt product is independent of RNA synthesis. Reactions were performed as described for panel A, except that in lanes 3–5, the only NTP in the reaction was [α-³²P]GTP. Lane 2 is a control containing all four NTPs and [α-³²P]GTP. (C) Generation of the 26–28 nt products is dependent on the TrC RNA template containing a 3′-hydroxyl group. TrC RNA templates containing either a 3′-hydroxyl (OH; lane 2) or a 3′-puromycin (PMN; lanes 3 and 4) group were tested at a concentration of 2 µM in reactions containing 1 mM of each NTP and [α-³²P]GTP. In each panel, lane 1 shows the molecular weight ladder.</p

The +3 initiation site is utilized during RSV infection.

<p>(A) Primer extension analysis of Tr sense RNA generated during RSV infection. Two primers were utilized hybridizing to positions 13–35 or 32–55 relative to the 5′ terminus of RSV genome RNA (left and right panels, respectively). Lanes 3 and 4 show cDNAs generated from RNA isolated from mock or RSV infected cells, respectively. The sizes of the products were determined by co-migration of ³²P end-labeled DNA oligonucleotides consisting of Tr sequence 3–35 or 1–35 (left panel, lanes 1 and 2, respectively), or 3–55 or 1–55 (right panel, lanes 1 and 2, respectively) to indicate the lengths of products initiated at +3 or +1. It should be noted that lanes 1–4 of the left panel are all from the same gel, but lanes 1 and 2 required a longer exposure to be detected. (B) Northern blot analysis of small genome sense RNA transcripts generated from the TrC promoter. Lanes 1 and 2 contain RNA isolated from mock or RSV infected cells, respectively. The blot was hybridized with a locked nucleic acid DNA oligonucleotide probe designed to anneal to nts 5–32 relative to the 5′ end of the RSV Tr sequence. (C) Alignment of the sequences from the 3′ terminus of the RSV TrC promoter and the ten nt L gene start (GS) signal. Identical nts are underlined and dashes indicate nts at the −1 and −2 positions relative to the L GS sequence, which are not part of the signal.</p

Analysis of the effect of the 3′ extension on TrC promoter activity <i>in vitro</i>.

<p>RNA synthesis reactions were performed using 2 µM of TrC template RNA either lacking (lanes 2 and 4) or containing (lane 3) a 3′ CUG addition at the 3′ terminus, 1 mM of each NTP and [α-³²P]ATP, and wt (lanes 2 and 3) or mutant (lane 4) RdRp. Both RNA oligonucleotide templates contained a PMN group at the 3′ end. Lane 1 shows the molecular weight ladder.</p

Sequence analysis of the 3′ termini of RSV antigenome and genome RNA isolated from RSV infected cells.

<p>(A) Putative structures formed by the terminal sequences of the TrC and Le promoter regions. Nts 1–25 of the TrC and Le promoter sequences are shown (left and right panels, respectively), with potential secondary structures indicated. In the case of the TrC sequence, the nts added to the 3′ end of the TrC RNA are underlined. (B) Sequence analysis of the antigenome and genome termini. The traces show the sequence of the population of cDNAs representing the antigenome and genome terminal sequences (left and right panels, respectively). In each case, the upper panel shows the sequence of RNA tailed with ATP, and the lower panel shows the sequence of RNA tailed with CTP. Note that any 3′ nt addition matching the base used to tail the RNA would not be detected. (C) Representative traces of different cDNA clone sequences obtained that represent antigenome termini. The relative frequency of each clone of the 19 clones sequenced is indicated. Two clone traces that were obtained are not shown; these contained a deletion of position 1U (or substitution with an A) with no nt additions, and the sequence 3′ <u>CCG</u>CGCUCUUU, in which position 1 appears to have been substituted with a C, and a GCC sequence (underlined) has been added. In panels B and C, all sequences are presented as RNA and positions +1U, +5C, and +10U of the TrC or Le promoter are indicated. The A or C residues at the right hand side of each trace represent the sequence added by the E. coli poly A polymerase, and the additional nts lying between nt +1U of the promoter and the A or C tail are underlined.</p

RNA products are generated from the +3 site on the TrC template.

<p>(A and B). Effect of omitting UTP from the RNA synthesis reaction. RNA synthesis reactions were performed with all four NTPs (lane 1) or with UTP omitted (lane 2). Reactions contained 2 µM TrC template RNA, wt RdRp, and 1 mM each NTP including either [α-³²P]ATP (A), or [α-³²P]GTP (B). (C) The 21 nt product is initiated with GTP. RNA synthesis reactions were performed with either [α-³²P]GTP (lane 1) or [γ-³²P]GTP (lanes 2 and 3) as a label. The reactions contained 2 µM TrC template RNA, 10 µM cold GTP and 1 mM ATP, CTP and UTP, and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. (D) [γ-³²P]GTP is incorporated into 11 and 13 nt products if UTP is omitted from the reaction. RNA synthesis reactions were performed with either [α-³²P]GTP (lane 1) or [γ-³²P]GTP (lanes 2 and 3) as a label. The reactions included 2 µM TrC template RNA, 50 µM cold GTP and 1 mM ATP, and CTP and either wt (lanes 1 and 2) or mutant (lane 3) RdRp. Note that the 25 nt bands in panel D, lanes 2 and 3 could be due to kinase activity (either in the RSV RdRp or a contaminant of the preparation) phosphorylating the TrC template RNA. The long products detected with [α-³²P]GTP in lanes 1 of panels C and D might be due to extensive 3′ nt addition, or repeated stuttering of the RdRp on the U tracts in the template.</p

Analysis of the role of internal sequences of the TrC RNA in 3′ nt addition.

<p>(A) Schematic diagram showing the two putative hairpin loop structures formed by the TrC RNA. Nts 1, 14 and 16, which were subjected to mutagenesis are underlined. (B) Effect of mutation of nt 1, or nts 14 and 16 of the TrC RNA on 3′ nt addition. Reactions were performed containing 25 nt TrC RNA that was of wt sequence (lanes 1 and 4), or containing a 1U/A substitution (lanes 2 and 5), or substitution of nts 14A and 16A with U residues (lanes 3 and 6). Reactions were performed using 0.2 µM RNA and 500 µM of each NTP. Lanes 1–3 show RNAs labeled with [α-³²P]GTP, and lanes 4–6 show RNAs labeled with [α-³²P]ATP.</p

Reconstitution of RSV RNA synthesis <i>in vitro</i>.

<p>(A) Sequence of the 25 nt TrC RNA used in the <i>in vitro</i> assay and the expected complementary Tr sense product. The first A residue the RdRp would encounter at position +14 of the template and the corresponding U residue in the product are underlined. The 3′ nt of the template is marked +1, reflecting the numbering system used throughout the paper, and the +3 initiation site identified in this study is indicated by a black dot. (B) Page Blue stained gel showing isolated wt L/P (lane 1) and mutant L_N812A/P (lane 2) complexes. The bands correlating to the expected migration patterns for L (250 kDa) and P (27 kDa) are indicated. Mass spectrometry of a representative gel showed that the bands indicated with asterisks and dots contain L and P specific polypeptides, respectively. (C) Hsp70 and/or HSC70 co-purifies with RSV L/P complexes. Total insect cell lysates (lane 1) and isolated wt L/P complexes (lane 2) were subjected to SDS-PAGE and Western blot analysis, and probed with an anti-Hsp70/HSC70 antibody. (D) Substitution of asparagine 812 to alanine in the GDNQ motif in L abolishes RSV RNA synthesis in a minigenome assay. Northern blot analysis of RSV transcription and replication products (CAT mRNAs 1/2 and anti-minigenome, respectively) generated from a dicistronic CAT minigenome in cells transfected with plasmids expressing minigenome RNA together with N, P, M2-1 and either wt L or mutant (L_N812A), as indicated. (E) RNA products synthesized by the RSV RdRp. Isolated wt (lanes 2 and 4) or mutant (L_N812A) (lane 3) RdRp was incubated with 0.2 µM template RNA consisting of either TrC 1–25 (lanes 2 and 3) or its complement Tr 1–25 (lane 4), with 200 µM of each NTP in the presence of [α-³²P]ATP. The labeled products were separated by denaturing gel electrophoresis and visualized by autoradiography. Lane 1 shows the molecular weight ladder, representing nts 1–25 of the anticipated Tr product.</p

A semi-specific sequence of nts is added to the 3′ terminus of the TrC RNA.

<p>RNA synthesis reactions were performed with either [α-³²P]ATP, [α-³²P]CTP, [α-³²P]GTP, or [α-³²P]UTP (panels A–D, respectively). In each case, isolated wt or mutant (L_N812A) RdRp was incubated with 0.2 µM TrC RNA template (lanes 2–4), or its complement Tr 1–25 (lane 5), in a reaction containing either all four NTPs (each at 500 µM; lane 2), or a single NTP (at 500 µM, lanes 3–5). Lane 1 of each panel shows the molecular weight ladder. It should be noted that to avoid confusion the marker indicators are aligned to the outermost part of the molecular weight ladder band, which in each case migrated somewhat more slowly than the rest of the gel. The position of bands representing TrC RNA containing an additional 1, 2, or 3 nts at the −1, −2, and −3 positions relative to the template, respectively, are indicated.</p

CRV substitutions inhibited the elongation step of replication.

<p>(A) Schematic diagram of the 3' end of the replication-specific minigenome. The two initiation sites in the <i>tr promoter</i> (+1, +3) are indicated with green arrows, and the small tr (sm tr) and genome RNA products are shown in blue. The regions contained within the 13–35 and 24–48 primers are indicated in red. (B, C) Primer extension analysis of RNA generated by wt and variant L proteins using the 13–35 and 24–48 primers, respectively. The position of the +1 and +3 initiation sites were determined by co-migration of end-labeled DNA oligonucleotides, corresponding in length and sequence to cDNAs representing initiation from those sites (Lanes 1 and 2). Lane 3 shows RNA from a control transfection lacking L expression plasmid. The white lines between lanes 2 and 3 indicate where empty lanes were excised. (D, E) Quantification of replicates of the experiments shown in panels B and C. Panel D shows levels of RNA initiated at +1 and +3, detected by primer extension with primer 13–35. Panel E shows levels of RNA initiated at +1 detected by primer 24–48. In D and E the data are normalized to a mean of the two wt samples included in each experiment at 100%. In panel D, the bars show the mean and range of two independent experiments. In panel E, the bars show the mean and standard deviation of three independent experiments.</p

CRV substitutions inhibited mRNA capping.

<p>(A) Schematic diagram of the 3' end of the minigenome. The initiation sites within the <i>le promoter</i> (+1, +3) and at the first <i>gs</i> (+45) are indicated with green arrows, and the positive-sense small le (sm le), CAT 1 mRNA, and antigenome RNAs that are produced from this region of the minigenome are shown below in blue. The regions contained within the negative sense 56–75 and 91–113 primers and probes are indicated in red. (B) Primer extension analysis of RNA generated by wt and variant L proteins using the 56–75 primer and Sensiscript reverse transcriptase. Lane 1 is a marker prepared by end-labeling a 31 nt DNA oligonucleotide corresponding in sequence to the primer extension product representing initiation at the first <i>gs</i> signal (M 45–75). (C) Quantification of RNA initiated at the <i>gs</i> signal based on replicates of the experiments shown in panel B. The data are normalized to a mean of the two wt samples included in each experiment at 100% and the bars show the mean and standard deviation for three independent experiments. (D) Primer extension analysis of the RNA generated by wt and the R1339A and H1338A/R1339A variant polymerases using the 56–75 primer and Thermoscript reverse transcriptase. RNAs were treated with buffer only, or pyrophosphatase in buffer, as indicated by -/+ symbols. (E and F) Primer extension analysis of RNA generated by wt and variant L proteins, performed with Thermoscript reverse transcriptase with either the 56–75 (E), or 91–113 primer (F). (G) Northern blot analysis of small RNA transcripts produced from the minigenome. The RNA was migrated on a 6% urea-acrylamide gel and detected with an end-labeled 56–75 oligonucleotide probe.</p