mRNA cap formation by the human respiratory syncytial virus large protein

Figures

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  Figure 1.. The HRSV L–P complex catalyzes RNA capping through PRNTase activity. (A) The negative-strand leader [Le(−)] region at the 3′-end of the HRSV genome serves as a promoter for the HRSV RNA-dependent RNA polymerase (RdRp) composed of L and P to synthesize the full-length anti-genome and the short leader RNA initiated with the 5′-ACGCG and GCGAA sequences, respectively. After the synthesis of the leader RNA, the RdRp initiates the synthesis of NS1 mRNA with the 5′-terminal cap 1 structure (m7GpppGm) from the internal gene-start sequence. (B) Start sequences of 10 HRSV mRNAs are compared. The consensus mRNA-start sequence (R = G or A) is shown. (C) The partially purified HRSV RdRp complex (2 μg), composed of C-tagged L and non-tagged P, was analyzed by 10% SDS-PAGE followed by One-Step Blue staining. Lane M shows marker proteins with the indicated molecular masses. The positions of the origin (ori.), C-tagged L, P, and bromophenol blue (BPB) are shown. (D and E) The L–P complex (200 and 20 ng) was analyzed by Western blotting with anti-C-tag (D) and anti-P (E) antibodies, respectively. (F) The L–P complex (0.2 μg) was incubated with 5′-pppGGGAC and [α-32P]GDP or [α-32P]GTP. The…

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  Figure 2.. The HRSV L–P complex catalyzes de novo RNA synthesis initiation at the genomic Le promoter. (A and B) In vitro transcription was performed with ATP (A) or GTP (B), [α-32P]CTP, Le(−)1–10 (the WT or indicated mutant templates), and L–P complex. CIAP-resistant RNA products were analyzed by 20% urea-PAGE followed by autoradiography. The positions of 5′-hydroxyl oligo-RNA markers (lane M), xylene cyanol FF (XC), and BPB are shown. In panel B, the lower band is 5′-hydroxyl GCG, which migrates faster than 5′-hydroxyl GC [42, 53]. The graphs show the amounts of the indicated transcripts (n = 3). The dot-plots, columns, and error bars indicate the individual values, means, and standard deviations, respectively, of three independent experiments. Statistical significance was determined by one-way ANOVA [ns, not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; compared to control (WT)]. (C) Proposed RNA synthesis initiation sites in the Le promoter using ATP or GTP as an initiator nucleotide are shown. (D and E) The L–P complexes containing the WT or indicated mutant Ls were subjected to the de novo RNA synthesis initiation assay with the WT template using…

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  Figure 3.. PRNTase motifs, except for motif C, are structurally or catalytically required for the capping activity of the HRSV L–P complex. (A) A schematic structure of HRSV L is depicted with the RNA-dependent RNA polymerase (RdRp), GDP PRNTase, and methyltransferase (MTase) domains. Conserved motifs A–E in the PRNTase domain of HRSV are aligned with those of vesicular stomatitis virus (VSV). The numbers indicate the positions of amino acid residues in Ls. In VSV, H1227 in PRNTase motif D serves as the covalent pRNA attachment site [17]. (B–G) The L–P complexes containing the WT or indicated mutant Ls were subjected to the RNA capping (B and E), AC synthesis (C and F), and GC(G) synthesis (D and G) assays as in Figs. 1H, 2D, and 2E, respectively. The graphs show the relative amounts of the indicated products, with the amounts for the no-enzyme control (lane 1) and WT L (lane 2) set to 0% and 100%, respectively (n = 3). Statistical significance was determined by one-way ANOVA [ns, not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; compared to control (WT)].

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  Figure 4.. The HRSV counterpart of the rhabdovirus priming-capping loop plays important, but not critical, roles in capping and de novo RNA synthesis initiation at the genomic Le promoter. (A and B) The three-dimensional structures of the VSV (A, PDB id: 6U1X) and HRSV (B, PDB id: 6PZK) PRNTase domains were visualized as ribbon diagrams using UCSF ChimeraX program (ver. 1.7) [56]. Key amino acid residues in PRNTase motifs A–E (labeled by circled letters), priming-capping loop in VSV L (A) and their HRSV counterparts (B) are shown as stick models [HR motif (motif D), blue; other residues in PRNTase motifs, cornflower blue; T1161 and I1163 in VSV L and E1269, K1270, and K1271 in HRSV L, light sea green; W1167 in VSV L and Y1276 in HRSV L, magenta; P1261 in HRSV L, gray]. The structural backbones of the VSV priming-capping loop and motif B, as well as their HRSV counterparts, are colored gray. The covalent pRNA attachment site in VSV L [17] and the corresponding site in HRSV L are indicated by red arrowheads. In VSV L, two Zn2+ ions are shown as grayish-blue spheres. (C) WebLogos for PRNTase motif B and downstream sequences of 225 alpharhabdoviral [53] and 5 pnuemoviral Ls are shown with amino…

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  Figure 5.. JNJ-8003 inhibits capping catalyzed by the HRSV L–P complex. (A and B) In vitro RNA capping was carried out with the L–P complex in the presence or absence of the indicated compounds (A, 1 µM; B, the indicated concentrations) as in Fig. 1H. In (B), the graph shows percent inhibition of capping with the compounds at various concentrations. Symbols and error bars represent the means and standard deviations, respectively, of three independent experiments. (C) The L–pRNA formation assay was performed with the L–P complex in the presence or absence of the indicated compounds (1 µM) as in Fig. 1J. (D) The three-dimensional structure of the HRSV PRNTase domain complexed with JNJ-8003 (PDB id: 8FU3) is shown as a ribbon diagram, with the key residues in PRNTase motifs A–E [HR motif (motif D), blue; other residues in PRNTase motifs, cornflower blue). JNJ-8003 is shown in gray, with nitrogen, oxygen, and fluorine atoms colored blue, red, and light green, respectively.

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  Figure 6.. The HRSV L–P complex sequentially methylates the cap structure at the guanine-N7 position, followed by the 2′-O position. (A and B) 32P-Labeled GpppGGGAC (A) or m7GpppGGGAC (B) was incubated with the L–P complexes containing the WT or indicated mutant Ls in the presence or absence of 100 µM SAM for 2 h. Nase P1 digests of the products were analyzed by PEI-cellulose TLC followed by autoradiography. Lane M shows a mixture of the indicated cap structures. The graphs show the amounts of the cap structure methylated at the guanine-N7 and 2′-O positions (open and closed columns, respectively). The dot-plots, columns, and error bars indicate the individual values, means, and standard deviations, respectively, of three independent experiments. Statistical significance was determined by one-way ANOVA [ns, not significant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; compared to control (WT)]. (C and D) The cap methylation assay was performed with the L–P complex in the presence of 100 µM SAM for the indicated time periods (C) or in the presence of the indicated concentrations of SAM for 2 h (D) as in panel A. (E) 32P-Labeled GpppGGGAC or GpppGmGGAC was incubated with or without the WT L–P…

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  Figure 7.. A proposed model of mRNA cap formation by HRSV L. The pathway of the formation of the cap 1 structure by HRSV L is schematically represented. GTP, the 5′-terminal end of pre-mRNA, and SAM are colored in red, blue, and, green, respectively. Pi, PPi and SAH denote inorganic phosphate, inorganic pyrophosphate, and S-adenosyl-l-homocysteine, respectively. JNJ-8003 inhibits the PRNTase reaction at the step of the L–pRNA intermediate formation. The step involving the production of GDP from GTP has not been clearly demonstrated in this study.